The Structure and Function of the Plasma Membrane

120 4

4.1 An Overview of Membrane Functions The outer walls of a house or car provide a strong, inﬂexible 4.2 A Brief History of Studies on Plasma Membrane barrier that protects its human inhabitants from an unpredictable Structure and harsh external world. You might expect the outer boundary of a living cell to be constructed of an equally tough and impenetra- 4.3 The Chemical Composition of Membranes ble barrier because it must also protect its delicate internal con- 4.4 The Structure and Functions of Membrane Proteins tents from a nonliving, and often inhospitable, environment. Yet 4.5 Membrane Lipids and Membrane Fluidity cells are separated from the external world by a thin, fragile 4.6 The Dynamic Nature of the Plasma Membrane structure called the plasma membrane that is only 5 to 10 nm wide. It would require about ﬁve thousand plasma membranes 4.7 The Movement of Substances Across Cell stacked one on top of the other to equal the thickness of a Membranes single page of this book. 4.8 Membrane Potentials and Nerve Impulses Because it is so thin, no hint of the plasma membrane is THE HUMAN PERSPECTIVE: Defects in Ion Channels and detected when a section of a cell is examined under a light Transporters as a Cause of Inherited Disease microscope. In fact, it wasn’t until the late 1950s that techniques for preparing and staining tissue had progressed to the point EXPERIMENTAL PATHWAYS: The Acetylcholine Receptor where the plasma membrane could be resolved in the electron

Three-dimensional, X-ray crystallographic structure of a ␤2-adrenergic receptor ( ␤2-AR), which is a member of the G protein-coupled receptor (GPCR) superfamily. These integral membrane proteins are characterized as containing seven transmembrane helices. The ␤2-AR is a resident of the plasma membrane of a variety of cells, where it normally binds the ligand epinephrine and mediates such responses as increased heart rate and relaxation of smooth muscle cells. Until recently, GPCRs had been very difﬁcult to crystallize so that high-resolution structures of these important proteins have been lacking. This situation is now rapidly changing as the result of recent advances in crystallization technology. The image shown here depicts two ␤2-ARs, which were crystallized in the presence of cholesterol and palmitic acid (yellow) and a receptor-binding ligand (green). The crystals used to obtain this image were selected from more than 15,000 trials. (F ROM VADIM CHEREZOV ET AL ., COURTESY OF RAYMOND C. S TEVENS , S CIENCE 318:1258, 2007; © 2007, REPRINTED WITH PERMISSION FROM AAAS.) 121

Figure 4.1 The trilaminar appearance of membranes. (a) Electron micrograph showing the three-layered (trilaminar) structure of the plasma membrane of an erythrocyte after staining the tissue with the heavy metal osmium. Osmium binds preferentially to the polar head groups of the lipid bilayer, producing the trilaminar pattern. The arrows denote the inner and outer edges of the membrane. ( b) The outer edge of a differentiated muscle cell grown in culture showing the similar trilaminar structure of both the plasma membrane (PM) and the membrane of the sarcoplasmic reticulum (SR), a calcium-storing compartment of the cytoplasm. ( A: C OURTESY OF J. D. R OBERTSON ; B: FROM ANDREW R. M ARKS ET AL ., J. C ELL BIOL . 114:305, 1991; REPRODUCED WITH PERMISSION OF THE ROCKEFELLER UNIVERSITY PRESS .)

(a) 50 nm

P M S R

(b)

microscope. These early electron micrographs, such as those as to the molecular composition of the various layers of a mem- taken by J. D. Robertson of Duke University (Figure 4.1a), brane, an argument that went to the very heart of the subject of portrayed the plasma membrane as a three-layered structure, membrane structure and function. As we will see shortly, cell consisting of darkly staining inner and outer layers and lightly membranes contain a lipid bilayer, and the two dark-staining staining middle layer. All membranes that were examined layers in the electron micrographs of Figure 4.1 correspond closely—whether they were plasma, nuclear, or cytoplasmic primarily to the inner and outer polar surfaces of the bilayer. membranes (Figure 4.1 b), or taken from plants, animals, or We will return to the structure of membranes below, but first we microorganisms—showed this same ultrastructure. In addition to will survey some of the major functions of membranes in the providing a visual image of this critically important cellular struc- life of a cell (Figure 4.2). ture, these electron micrographs touched off a vigorous debate 4.1 An Overview of Membrane FunctionsMembrane of Overview An 4.1 | An Overview of Membrane 2. Scaffold for biochemical activities. Membranes not only enclose compartments but are also a distinct compart- Functions ment themselves. As long as reactants are present in solu- 1. Compartmentalization. Membranes are continuous, tion, their relative positions cannot be stabilized and their unbroken sheets and, as such, inevitably enclose compart- interactions are dependent on random collisions. Because ments. The plasma membrane encloses the contents of of their construction, membranes provide the cell with an the entire cell, whereas the nuclear and cytoplasmic mem- extensive framework or scaffolding within which compo- branes enclose diverse intracellular spaces. The various nents can be ordered for effective interaction. membrane-bounded compartments of a cell possess 3. Providing a selectively permeable barrier. Membranes markedly different contents. Membrane compartmental- prevent the unrestricted exchange of molecules from one ization allows specialized activities to proceed without side to the other. At the same time, membranes provide external interference and enables cellular activities to be the means of communication between the compartments regulated independently of one another. they separate. The plasma membrane, which encircles a Chapter 4 The Structure and Function of the Plasma Membrane 122 Figure 4.2 membrane ofmembrane the mitochondrion. releasecauses of Ca sion of ADP to ATP association with occurs in close transduc example of the role inof energy membranes into of i onemove cell directly from the cytoplasm plasmodesmata, called adjoining plant cells, between ample of the communi role in cell–cell of membranes chemical message (such as IPas (such message chemical t and triggers of the plasma membrane outer surface abscisic a hormone (e.g., In this case, transduction). of from information in the transfer brane one side (5) An example of the involveme the plasma membrane. space by a trans into the extracellular plant cells are metabolic processes in the cytoplasm, by various wh Hydrogen ions, (4) An example of solute transport. press exert and space available the out fill to cell ca through the plasma membrane, rapidly to penetrate Water barrier. permeable as a selectively membranes (3) An exam of the thylakoidchloroplasts. membranes that is associated with the by an enzyme catalyzed sast fezm oaiain The fixation of CO localization. as a site of enzyme (2) An example of the rolebounded of cytop vacuole. (acid hydrolases) areenzymes sequestered within th h in which example compartmentalization of membrane out of the enclosed living space.out of the enclosed that promote the movement of select elements into a both have gated yet “bridges” barrier, as a general serve both be compared can to a moat around a castle: cell, (6) A summary of membrane functions in a plant cell. plant a in functions membrane of summary A 2 Acid hydrolases ϩ osfo yolsi aeos.(6) An ex- warehouse. ions from a cytoplasmic (1) (2) RuBP PGA CO 3 + notectpam nti ae IP In this case, ) into the cytoplasm. 2 Ca IP 2 ADP + 3 ATP Hormone (5) port protein located in protein located port ure wall. cell its against snihos (7) Ants neighbors. to another (signal (4) (7) outer surface of theouter surface H 2 acid) binds to the e membrane- pumped out of he release of a + molecules are able lasmic membranes the inner by the plant cell is cell plant the by in The conver-tion. allow materials toallow materials ain Openingscation. using the plant ple of the role of ich areich produced nt of a mem- ydrolytic (1) An nd 3 H (3) 2 O ilb icse nCatr ,6 ,ad1,respecti and 12, 8, membr 6, nuclear be discussed in Chapters 5, will and cytoplasmic, membrane chloroplast, cell chondrial, functions all and structure to the of common aspects Specialized are here discussed ples but remember that the tions of the plasma membrane, We will concentrate in this chapter on the structur 5. 4. 7 . 6. tml,a process known as stimuli, role in the response a critical to of aplays cell branes possess possess branes cules ( Responding to external stimuliResponding to external cells. muscle and nerve cr is especially This capability ents across itself. thereby establishing ionic g specific ions, transport is also The ableplasma to membrane macromolecules. to fuel its metabolism and build that are necessary as sugars and amino a such to accumulate substances, allows The membrane’s a machinery transport tration. gion where that solute is present at much higher co where the solute is present at low in concentration from a re often one side of the to membrane another, substances fr transporting for physically machinery Transporting solutes. and mitochondria. versions is contained of chloropla within membranes co for these energy the machinery ATP. In eukaryotes, from and fats to carbohydrates energy fer of chemical are in the Membranes trans- also involved carbohydrates. and stored energy, into chemical converted pigments, by membrane-bou in sunlight is absorbed when energy photosynth occurs transduction during mental energy The most fund transduction). (energy to another type is con of energy in the processes one type by which transduction.Energy cytoskeleton.intracellular and the materials extracellular between interaction withinmay also the facilitate plasma membrane the Proteins and information. and to exchange materials to adhere when appropri and signal one another, nize allows The plasma membrane to recog cells neighbors. a cell organisms between mediates the interactions of multicellu the plasma membrane living cell, every Intercellular interaction. suicide. to commit or possibly from stores, internal calcium to re compound, higher of a concentration particular to move towar to prepare division, for cell glycogen, to manufacture a cell mayplasma tell membrane more a signals generated For example, activities. internal a signal that stimulates to generate membrane or in receptorbrane stimulus with an external may cause of a The plasma interaction mem-environmental stimuli. of recognizing capable and responding to diffe fore, the with differenthave membranes receptors and are, of c Different types tension. as light or mechanical ligands ) or respond to other types of stimuli) or respond such to other types receptors The contains plasma membrane the Membranes are involvedMembranes intimately that combine with specific mole- Situated at the outer edge ofSituated signal transductionsignal . The plasma membrane plasma The external e and func- itical foritical Mem-. vely. f mito- of its to a re- radi-

ells t the om verted ncen- and its hibits princi- rent the d a lar lease sts gion cids, re- ate, a- esis in anes cell n- - nd s. 123 4.2 | A Brief History of Studies ratio of the surface area of water covered by the extracted lipid to the surface area calculated for the red blood cells from on Plasma Membrane Structure which the lipid was extracted varied between 1.8 to 1 and 2.2 The first insights into the chemical nature of the outer bound- to 1. Gorter and Grendel speculated that the actual ratio was ary layer of a cell were obtained by Ernst Overton of the Uni- 2:1 and concluded that the plasma membrane contained a bi- versity of Zürich during the 1890s. Overton knew that molecular layer of lipids, that is, a lipid bilayer (Figure 4.3 b). nonpolar solutes dissolved more readily in nonpolar solvents They also suggested that the polar groups of each molecular than in polar solvents, and that polar solutes had the opposite layer (or leaflet ) were directed outward toward the aqueous en- solubility. Overton reasoned that a substance entering a cell vironment, as shown in Figure 4.3 b,c. This would be the ther- from the medium would first have to dissolve in the outer modynamically favored arrangement, because the polar head boundary layer of that cell. To test the permeability of the groups of the lipids could interact with surrounding water outer boundary layer, Overton placed plant root hairs into molecules, just as the hydrophobic fatty acyl chains would be hundreds of different solutions containing a diverse array of protected from contact with the aqueous environment (Figure solutes. He discovered that the more lipid-soluble the solute, 4.3 c). Thus, the polar head groups would face the cytoplasm the more rapidly it would enter the root hair cells (see p. 149). on one edge and the blood plasma on the other. Even though He concluded that the dissolving power of the outer boundary Gorter and Grendel made several experimental errors (which layer of the cell matched that of a fatty oil. fortuitously canceled one another out), they still arrived at the The first proposal that cellular membranes might contain correct conclusion that membranes contain a lipid bilayer. a lipid bilayer was made in 1925 by two Dutch scientists, E. In the 1920s and 1930s, cell physiologists obtained evi- Gorter and F. Grendel. These researchers extracted the lipid dence that there must be more to the structure of membranes from human red blood cells and measured the amount of sur- than simply a lipid bilayer. It was found, for example, that lipid face area the lipid would cover when spread over the surface of water (Figure 4.3 a). Since mature mammalian red blood cells lack both nuclei and cytoplasmic organelles, the plasma mem- R brane is the only lipid-containing structure in the cell. Conse- OP O – quently, all of the lipids extracted from the cells can be O assumed to have resided in the cells’ plasma membranes. The HCH H HC CH O O OC OC HCH HCH HCH HCH (b) HCH HCH HCH HCH HCH HCH HCH HCH HCH HCH 4.2 CH HCH

CH HCH Struc Membrane Plasma on Studies of History Brief A Stationary Movable HCH HCH barrier barrier HCH HCH Lipids HCH HCH HCH HCH HCH HCH HCH H H

(a) (c) Figure 4.3 The plasma membrane contains a lipid bilayer. (a) Calcu- blood cells contained enough lipid to form a layer over their surface lating the surface area of a lipid preparation. When a sample of phos- that was two molecules thick: a bilayer. ( b) As Gorter and Grendel pholipids is dissolved in an organic solvent, such as hexane, and spread first proposed, the core of a membrane contains a bimolecular layer over an aqueous surface, the phospholipid molecules form a layer over of phospholipids oriented with their water-soluble head groups the water that is a single molecule thick: a monomolecular layer. The facing the outer surfaces and their hydrophobic fatty acid tails facing molecules in the layer are oriented with their hydrophilic groups the interior. The structures of the head groups are given in Figure 4.6 a. bonded to the surface of the water and their hydrophobic chains (c) Simulation of a fully hydrated lipid bilayer composed of the directed into the air. To estimate the surface area the lipids would phospholipid phosphatidylcholine. Phospholipid head groups are cover if they were part of a membrane, the lipid molecules can be orange, water molecules are blue and white, fatty acid chains are compressed into the smallest possible area by means of movable green. ( C: F ROM S.-W. C HIU , T RENDS IN BIOCHEM . S CI . 22:341, barriers. Using this type of apparatus, which is called a Langmuir © 1997, WITH PERMISSION FROM ELSEVIER .) trough after its inventor, Gorter and Grendel concluded that red ture Chapter 4 The Structure and Function of the Plasma Membrane 124 composed of hydrophobic amino acids. The two leaflets The two composed of hydrophobic amino acids. iae.(bilayer. proteinslipid-penetrating that have been studied s emb here depictedpartially a protein that was only Although the o proteins the lipid bilayer. penetrate Unlike previous mod and Nicolsonproposed in 1972. by Singer chains that extend through the lipid bilayer typica o Those portions them glycoproteins and glycolipids. o chains contain short centage of the phospholipids, as w proteins, of most membrane surface The external as that proposedthe same basic organization by Sin oe.(pores. of proteins that extends through to fo the membrane by a is lined which on both surfaces lipid bilayer, ( per selective the for account to 1950s early the in t revised They proteins. globular of layer a by face of a lipid Jamesbilayer that was and lined on both its inner Davson Hugh comp was membrane plasma the that proposed 1935, Danielli In membrane. the in tein presen the by explained be could tension surface in This structures. lipidpure of those thanlower much membranestensionscalculweresurfaceof the larly, membraneplasma thepenetrate couldsubstance a not solubilitywas not the sole determining factor as t Figure 4.4 a ) A revised 1954 version of the Davson-Danielli mod ) A revised 1954 version of the Davson-Danielli b ) The as fluid-mosaicin structure model of membrane c ) A current representation s of the plasma membrane A brief history of the structure of the plasma memb of the structure history A brief monomolecular layer lly occur as lly pan the entire edded in the bilayer, riginal model shownriginal ger and Nicolson. uas makingf sugars, rm protein-linedrm f the polypeptide ell as a small per- as a small ell and outer sur- el showing the of the bilayer owhether or meability of meability heir modelheir ce of pro-of ce ated to beto ated decrease ␣ howing itially Simi-. l,theels, helices rane. osed EMSINFROMPERMISSION .NL. Garth Nicolson of the University of California, San San (Figure 4.4 California, of University the of flu Nicolson Singer Garth Jonathan the in S. by detailed 1972 in as proposed model mosaic structure, membrane of concept duits for polar solutes and ions to enter and exit provi could which pores, protein-lined by penetrated bilaye lipid the layers, protein inner and outer the ure 4.4ure versi revised the In studied. had they membranes the protein at the lower pumps H right with the plasma membrane of the vesicle interaction Most of the proteins a in this membrane is apparent. The high protein densi vesicles. synaptic of purified onwith their information relative numbers obtained using known constructed structures of the vario cle D AAAS. 1972;175:720, D F. J. (head groups. by t of lipids as indicated contain different types C : ELL FROM ICOLSON xeiet cnutd n h lt 16s e t a ne a to led 1960s late the in conducted Experiments 2:4,2006,127:841, ANIELLI B a : S ), Davson and Danielli suggested that, in addition to addition in that, suggested Danielli and Davson ), ERNE IHPRISO FROM PERMISSION WITH REPRINTED HIGEO b S, d .In the ). C, COPYRIGHT ) Molecular model of the membrane of a synaptic ves of a synaptic ) Molecular model of the membrane CIENCE OL LST ON T AKAMORI .J S J. S. ERNE IHPRISO FROM PERMISSION WITH REPRINTED fluid-mosaic model (d) 7:2,1972;175:720, NE ANDINGER 1972 P , APE R S TALET ERNE IHPRISO FROM PERMISSION WITH REPRINTED ϩ ., :,1954A: 7:8, osit h eil.(ions into the vesicle. .L N L. G. OREYOFCOURTESY COPYRIGHT ICOLSON , which which has asserved , he differently coloredhe differently .J S J. S. ERNE WITHREPRINTED us proteins along ty of the membranety from the analysis The large blue. 92 AAAS;1972, re required for the R NE ANDINGER S, EINHARD the cell. CIENCE A E ws also was r F: LSEVIER on (Fig-on de con-de ROM Diego J AHN G. and id- i- .) w , 125 the “central dogma” of membrane biology for more than three decades, the lipid bilayer remains the core of the membrane, but attention is focused on the physical state of the lipid. Un- like previous models, the bilayer of a fluid-mosaic membrane is present in a fluid state, and individual lipid molecules can Myelin move laterally within the plane of the membrane. sheath The structure and arrangement of membrane proteins in the fluid-mosaic model differ from those of previous models in that they occur as a “mosaic” of discontinuous particles that penetrate the lipid sheet (Figure 4.4 b). Most importantly, the Axon fluid-mosaic model presents cellular membranes as dynamic structures in which the components are mobile and capable of coming together to engage in various types of transient or semipermanent interactions. In the following sections, we will examine some of the evidence used to formulate and support this dynamic portrait of membrane structure and look at some of the recent data that bring the model up to date (Figure 4.4 c,d ).

R E V I E W

1. Describe some of the important roles of membranes in Figure 4.5 The myelin sheath. Electron micrograph of a nerve cell the life of a eukaryotic cell. What do you think might be axon surrounded by a myelin sheath consisting of concentric layers of the effect of a membrane that was incapable of per- plasma membrane that have an extremely low protein/lipid ratio. The forming one or another of these roles? myelin sheath insulates the nerve cell from the surrounding 2. Summarize some of the major steps leading to the cur- environment, which increases the velocity at which impulses can travel rent model of membrane structure. How does each new along the axon (discussed on page 167). The perfect spacing between model retain certain basic principles of earlier models? the layers is maintained by interlocking protein molecules (called P 0) that project from each membrane. (F ROM LEONARD NAPOLITANO , FRANCIS LEBARON , AND JOSEPH SCALETTI , J. C ELL BIOL . 34:820, 1967; REPRODUCED WITH PERMISSION OF THE ROCKEFELLER UNIVERSITY PRESS .) 4.3 | The Chemical Composition of Membranes Membranes are lipid–protein assemblies in which the compo- relative to other membranes, lipid is diminished. In contrast, nents are held together in a thin sheet by noncovalent bonds. the myelin sheath acts primarily as electrical insulation for the As noted above, the core of the membrane consists of a sheet nerve cell it encloses, a function that is best carried out by a of lipids arranged in a bimolecular layer (Figure 4.3 b,c). The thick lipid layer of high electrical resistance with a minimal lipid bilayer serves primarily as a structural backbone of the content of protein. Membranes also contain carbohydrates, membrane and provides the barrier that prevents random which are attached to the lipids and proteins as indicated in movements of water-soluble materials into and out of the cell. Figure 4.4 c.

The proteins of the membrane, on the other hand, carry out 4.3 most of the speciﬁc functions summarized in Figure 4.2. Each

Membrane Lipids Membranesof Composition Chemical The type of differentiated cell contains a unique complement of membrane proteins, which contributes to the specialized Membranes contain a wide diversity of lipids, all of which are activities of that cell type (see Figure 4.32 d for an example). amphipathic ; that is, they contain both hydrophilic and hy- The ratio of lipid to protein in a membrane varies, de- drophobic regions. There are three main types of membrane pending on the type of cellular membrane (plasma vs. endo- lipids: phosphoglycerides, sphingolipids, and cholesterol. plasmic reticulum vs. Golgi), the type of organism (bacterium vs. plant vs. animal), and the type of cell (cartilage vs. muscle Phosphoglycerides Most membrane lipids contain a vs. liver). For example, the inner mitochondrial membrane has phosphate group, which makes them phospholipids . Because a very high ratio of protein/lipid in comparison to the red most membrane phospholipids are built on a glycerol back- blood cell plasma membrane, which is high in comparison to bone, they are called phosphoglycerides (Figure 4.6 a). Unlike the membranes of the myelin sheath that form a multilayered triglycerides, which have three fatty acids (page 47) and are not wrapping around a nerve cell (Figure 4.5). To a large degree, amphipathic, membrane glycerides are diglycerides —only two these differences can be correlated with the basic functions of of the hydroxyl groups of the glycerol are esterified to fatty these membranes. The inner mitochondrial membrane con- acids; the third is esterified to a hydrophilic phosphate group. tains the protein carriers of the electron-transport chain, and Without any additional substitutions beyond the phosphate Chapter 4 The Structure and Function of the Plasma Membrane 126 carbohydrate is a simple sugar, the glycolipid is ca is glycolipid the sugar, simple a is carbohydrate a is molecule the carbohydrate, a is tution If backbone. glycerola with built not is that brane is the substitutionphosphorylcholine, theis If moiety. sph the of alcohol terminal the to esterified groups Sphingolipids phipathic character. di phosphoglyceridesa exhibitthe of all end, other chains at one end of the molecule and a polar head R f iue 4.6 Figure of fa (R a to linked sphingosine of consist Sphingolipids ceramide alcohol that contains a long hydrocarbon chain (Fig chain hydrocarbon long a contains that alcohol called the glycolipid is called a a called is glycolipid the broside hydrophobic hydrocarbon tw chains at one end have and in a hyd sphingolipids all differences Since by chains. carbohydrate identified been have gangliosides and the two thefatty moleculeacyl chains, is calle head 4.23).] group (see Figure Figure 4.6 acid structures of phosphoglycerides (see also Figure 2. (see also Figure structures of phosphoglycerides phatidylethanolamine hlseo,wihi hw ntenx gr.(R is shown which in the next figure. cholesterol, A third memb sides and gangliosides are glycolipids. is a phos Sphingomyelin structures of sphingolipids. o h popae ot omny ihr hln (form choline either phosphatidylcholine commonly most phosphate, the to membranephosphoglycerides additionalanhave group tail(s) of the molecule, is actually much longer than the hyd is actually tail(s) of the molecule, represents which t lipid, green of each [The portion PS), or inositol (forming (forming inositol or PS), mg (CH omega carbons 3 situated is bond double last their cause EPA retina. and DHA are described as omega-3 fatty a molecules mostof notablycertain inmembranes, the PE into primarily incorporated are and respectively, b double EPAsix and containfive DHA and oil. fish in rated fatty acids (EPA and DHA) found at high conce apparentfocusedtheon healthhighlbenefits two of in Recent chain. acyl fatty saturatedone and urated Phosphoglyceridesone double often bond). contain on m possess polyunsaturated or(i.e., bond), doubleone monounsaturated lack(i. double bonds), urated (i.e., in A lengthmembrane (Figure fatty 4.6). acid may be unbranched hydrocarbons approximatelybic, 16 to 22 the fatty acyl chains Inar contrast, PE are neutral. PC of those whereas charge, negative overall an have PI the highly water-soluble domain at one end of the molec attache is it which phosphateto chargednegatively together and, hydrophilic and small is groups these sphingomyelin head group hc i vruly bet n ot ebae.Instea membranes. most in absent virtually is which , sphingolipids if it is a small cluster of sugars that includes sialic ; acid, The various sphingosine-based . lipids have addition The chemical structures of membrane lipids. of membrane structures chemical The 3 ed f h fty cl hi.Wt fty acid fatty With chain. acyl fatty the of end ) tpyilgcp,the At headphysiologic groups pH, . of PS and , which is the only phospholipid of the mem-the of phospholipid only the is which , b A less abundant class of membrane lipids,membrane of class abundant less A , PC), ethanolamine (forming (forming ethanolamine PC), , ) by its amino group. This molecule is a a is molecule This group. amino its by ) E,srn (forming serine PE), , r eiaie fshnoie an amino are derivatives of sphingosine, , ganglioside phosphatidylinositol udes f different of Hundreds . ϭ 2.(22). phosphatidylserine glycolipid he hydrophobic hlpd cerebro-pholipid; fatty acyl chain). acyl fatty rane lipid is rane I.Ec of Each PI). , b d ) The e hydropho- group at the phosphatidic the substi-the lled a lled rophilic ( stinct am-stinct d, forms a forms d, . possesse., l,calledule, a molecule ure4.6 terest hasterest y unsatu-y brain and fully sat- rm thefrom n PC and ) The ih thewith ingosine ore thanore ntration e unsat- tty acid acid tty rophilic carbons cids be- long,o . If the If . linked onds, phos- their their an d cere- ing b d, al ). , (b) (a) serine Phosphatidyl- A ganglioside A cerebroside Sphingomyelin Ceramide Sphingosine (cardiolipin) glycerol Diphosphatidyl- inositol Phosphatidyl- (cephalin) ethanolamine Phosphatidyl- choline (lecithin) Phosphatidyl- acid Phosphatidic phosphatidic acid Dioleoyl (G M2 ) (CH (CH G a l N A c HO H 3 H 3 ) H 3 ) 3 3 N CH + 3 N CH N CH OH + + OH H H N CH + SiA COO 2 OH HOHH 2 2 Gal CH CH CH – H CHO H CH CH 2 OC HO H 2 2 2 O 2 2 O O P OC HO CH – O O O O Gal Glu – C O 2 O O O O O O O O CH P P P CH NH – – – – H + O O 2 2 OP O O O 3 H H H CO HC Ceramide Ceramide COCR' C O HC CO HC CO HC NH R C H NH R C H 2 2 2 C H OH C O C H C C C CH CH CH CH 2 2 2 2 2 C O (CH C O C O C H OH C H OH HC (CH CH CH O O O O O O O O (CH C C C HC (CH CH CH HC (CH CH CH R'' R''' R R R' 2 2 ) ) 7 7 CH=CH(CH CH=CH(CH 2 ) 12 2 CH 2 ) 12 ) 12 CH 3 CH 2 2 ) ) 7 7 3 CH CH 3 3 3 127 region at the other, they are also amphipathic and basically similar in overall structure to the phosphoglycerides. The fatty acyl chains of sphingolipids, however, tend to be longer and more highly saturated than those of phosphoglycerides. Glycolipids are interesting membrane components. Rela- tively little is known about them, yet tantalizing hints have emerged to suggest they play crucial roles in cell function. The nervous system is particularly rich in glycolipids. The myelin sheath pictured in Figure 4.5 contains a high content of a particular glycolipid, called galactocerebroside (shown in Figure 4.6 b), which is formed when a galactose is added to ceramide. Mice lacking the enzyme that carries out this reaction exhibit severe muscular tremors and eventual paralysis. Similarly, humans who are unable to synthesize a particular ganglioside (G M3 ) suffer from a serious neurological disease characterized by severe seizures and blindness. Glycolipids also play a role in certain infectious diseases; the toxins that cause cholera and botulism both enter their target cell by first binding to cell- Figure 4.7 The cholesterol molecules (shown in green) of a lipid surface gangliosides, as does the influenza virus. bilayer are oriented with their small hydrophilic end facing the external surface of the bilayer and the bulk of their structure packed in among the fatty acid tails of the phospholipids. The placement of cholesterol Cholesterol Another lipid component of certain mem- molecules interferes with the flexibility of the lipid hydrocarbon chains, branes is the sterol cholesterol (see Figure 2.21), which in which tends to stiffen the bilayer while maintaining its overall fluidity. certain animal cells may constitute up to 50 percent of the Unlike other lipids of the membrane, cholesterol is often rather evenly lipid molecules in the plasma membrane. Plant cells contain distributed between the two layers (leaflets). ( FROM H. L. S COTT , cholesterol-like sterols, but biologists disagree as to whether CURR . O PIN . S TRUCT . B IOL . 12:499, 2002, F IGURE 3; © 2002, WITH or not they completely lack cholesterol. Cholesterol molecules PERMISSION FROM ELSEVIER .) are oriented with their small hydrophilic hydroxyl group toward the membrane surface and the remainder of the molecule embedded in the lipid bilayer (Figure 4.7). The hydrophobic rings of a cholesterol molecule are flat and rigid, and they lipid molecules has remarkable consequences for cell struc- interfere with the movements of the fatty acid tails of the ture and function. Because of thermodynamic considerations, phospholipids (page 138). the hydrocarbon chains of the lipid bilayer are never exposed to the surrounding aqueous solution. Consequently, mem- The Nature and Importance of the Lipid Bilayer Each branes are never seen to have a free edge; they are always con- type of cellular membrane has its own characteristic lipid tinuous, unbroken structures. As a result, membranes form composition, differing from one another in the types of lipids, extensive interconnected networks within the cell. Because of the nature of the head groups, and the particular species of the flexibility of the lipid bilayer, membranes are deformable fatty acyl chain(s). Because of this structural variability, it is and their overall shape can change, as occurs during locomotion estimated that some biological membranes contain hundreds of chemically distinct species of phospholipids, which can be catalogued by mass spectrometry. The biological significance Table 4.1 Lipid Compositions of Some of this remarkable diversity of lipid species remains the subject 4.3 of interest and speculation. Biological Membranes* The Chemical Composition of Membranesof Composition Chemical The The percentages of some of the major types of lipids of a Human Human Beef heart variety of membranes are given in Table 4.1. The lipids of a Lipid erythrocyte myelin mitochondria E. coli membrane are more than simple structural elements; they can have important effects on the biological properties of a mem- Phosphatidic acid 1.5 0.5 0 0 brane. Lipid composition can determine the physical state of Phosphatidylcholine 19 10 39 0 Phosphatidyl- the membrane (page 138) and influence the activity of partic- ethanolamine 18 20 27 65 ular membrane proteins. Membrane lipids also provide the Phosphatidylgycerol 0 0 0 18 precursors for highly active chemical messengers that regulate Phosphatidylserine 8.5 8.5 0.5 0 cellular function (Section 15.3). Cardiolipin 0 0 22.5 12 Various types of measurements indicate that the com- Sphingomyelin 17.5 8.5 0 0 bined fatty acyl chains of both leaflets of the lipid bilayer span Glycolipids 10 26 0 0 a width of about 30 Å and that each row of head groups (with Cholesterol 25 26 3 0 its adjacent shell of water molecules) adds another 15 Å. *The values given are weight percent of total lipid. Thus, the entire lipid bilayer is only about 60 Å (6 nm) thick. Source: C. Tanford, The Hydrophobic Effect , p. 109, copyright 1980, John Wiley The presence in membranes of this thin film of amphipathic & Sons, Inc. Reprinted by permission of John Wiley & Sons, Inc. Chapter 4 The Structure and Function of the Plasma Membrane 128 of the early clinical studies with liposomes met wi met liposomes with studies clinical early the of g to intended is DNA or drug the where cells target o surfaces the to selectively bind to liposomes the t hormones) or antibodies as (such proteins specific constructed are liposomes the of walls the studies, 4.9) (Figure lumen its within or concentration high at drugs The co body. or liposome the of wall the the to linked be can DNA within molecules DNA d or to drugs vehicles as developed been also have Liposomes mem natural a of that than environment simpler much s and function their into liposomes be can inserted h Liposomes membrane. pro Membrane proven in invaluable research. membrane natural a of bilayer lipid the manner same the in organized is that bilayer lipid somes apparent throughout this and subsequent chapters. and in many other cellularplasma membrane, activiti chargeseparatingelectric in cell, a compositionof prope the maintaining in bilayer lipid the of tance i The 8.32). Figure (see sheet continuous one become processes in which two separate membranes come toge where two cells fuse to form a single fertilization, mic vesicles fuse to the plasma membrane (Figure 4. (Figure membrane plasma the to fuse vesicles mic the For events in ofwhichexample, branes. secretion, thought to facilitate the regulated fusion or buddi (Figure (Figure 4.8 to form the walls of fluid-filled spherical vesicles, spherical of to the spontane fluid-filled walls form assemble molecules phospholipid the lution, exampl for is amount in dispersed of an phosphatidylcholine aq If, cell. living a than tube test a within more demonstrated be can which self-assemble, to ity (a) ( Figure 4.8 lsammrn ipasudltn ufls ( undulating ruffles. plasma membrane displays cleavage furrowcleavage of this dividing ctenophore egg beg Unlike most di towardpulled the center of the cell. accompanied by of the the deformation plasma membra a ) contains The leading edge of a moving cell often Another important feature of the lipid bilayer is i is bilayer lipid the of feature important Another . The walls of these liposomes consist of a continuo a of consist liposomes these of walls The . The dynamic properties of the plasma membrane. properties dynamic The a ) or cell division (Figure 4.8 b b .The lipid bilayer is). ) Division of a cell is (b) iigcls theviding cells, ins at one pole and sites where the ne as it is cl,involve cell, ng of mem- called called s across theacrosss f particular f tudied in tudied a to contain to e, a small a e, as that of that as cytoplas- ueous ueous so- internalr . In these In . th failure th hat allow hat es will be 8 .Mosto. ntained c ts abil- ts ), or of or ), ther to mpor- easily brane. eliver ously lipo- teins ave us htc oye ta poet te iooe fo imm from liposomes 4.9). (Figure destruction the protects that polymer thetic o coating outer an contain that Caelyx) stealth (e.g., so-called somes of has development obstacle the This with overcome system. immune the of cells cytic removed rapidly were vesicles injected the because centrated in tendsthe toinner promoteleaflet, the whic Phosphatidylethanolamine, extracellular ligands. r as serve often they where leaflet outer the in are theglycolipids theplasmaof All ferentproperties. an physical different properties. having chemical monolayers independent s b more-or-less two lipid of the composed as that of thought follows be It can 4.10). (Figure PS and PE concentrat low a and sphingomyelin) (and PC of c tion high relatively a has leaflet outer the t to leaflet, compared ner that, indicate data These attacked. are phosphatidylse its of percent 10 than less and (PE) phos- the of about20percent of the membrane’s percent phosphatidyletha 80 phatidylcholine (PC) of the membrane is approximately lipid-dige hydrolyzed, a with treated phospholipase, are cells blood red human bilay the of leaflet outer the in reside that penetrate lipids d to able only are cannot consequently, enzymes and, membrane plasma lipid-digesting that fact advanta of takes line conclusion this to led One has that ments composition. lipid different distinctly leaflets distinct two of consists bilayer lipid The The Asymmetry of Membrane Lipids lsammrn arw) ( (arrows). plasma membrane fusion its contents discharging after granule tory This electron micrograp fusing with other membranes. oe ndrcinlytruhteeg (moves through unidirectionally the egg. B B : URWEN OREYOFCOURTESY The different classes of lipids in Figure 4.10 exhi 4.10 Figure in lipids of classes different The .) G ARY (c) F REEMAN A C: UTS OFOURTESY ; C : OREYOFCOURTESY c ) Membranes are of) Membranes capable J EAN with the overlying P S h shows a secre- AUL USAN eceptors foreceptors uvtr ofcurvature er. If intact If er. membrane that have a have that R by phago-by oncentra- rine (PS) rine nolamine ge of the of ge EVEL h is con- J but only f a syn- a f O experi- bit dif-bit he in- he ion of ion table, ilayer sting lipo- been igest ; une the d 129

Protective layer to the cytoplasm (Section 15.3) and the recruitment of proof polyethylene Antibody teins to the cytosolic face of the plasma membrane. glycol

Membrane Carbohydrates The plasma membranes of eukaryotic cells also contain carbohydrate. Depending on the species and cell type, the carbohydrate content of the plasma membrane ranges between 2 and 10 percent by weight. More than 90 percent of the membrane’s carbohydrate is covalently linked to proteins to form glycoproteins; the remaining carbohydrate is covalently linked to lipids to form glycolipids, which were discussed on page 126. As indicated in Figure 4.4 c, all of the carbohydrate of the plasma membrane Drug crystallized Lipid-soluble faces outward into the extracellular space. 1 The carbohydrate of in aqueous fluid drug in bilayer Lipid bilayer internal cellular membranes also faces away from the cytosol (the basis for this orientation is illustrated in Figure 8.14). Figure 4.9 Liposomes. A schematic diagram of a stealth liposome The modification of proteins was discussed briefly on containing a hydrophilic polymer (such as polyethylene glycol) to pro- page 54.The addition of carbohydrate, or glycosylation , is the tect it from destruction by immune cells, antibody molecules that target most complex of these modifications. The carbohydrate of it to specific body tissues, a water-soluble drug enclosed in the fluid- glycoproteins is present as short, branched hydrophilic filled interior chamber, and a lipid-soluble drug in the bilayer. oligosaccharides , typically having fewer than about 15 sugars per chain. In contrast to most high-molecular-weight carbohydrates (such as glycogen, starch, or cellulose), which are the membrane, which is important in membrane budding and polymers of a single sugar, the oligosaccharides attached to fusion. Phosphatidylserine, which is concentrated in the inner membrane proteins and lipids can display extensive variability leaflet, has a net negative charge at physiologic pH, which in composition and structure. Even the same protein can dis- makes it a candidate for binding positively charged play different chains of sugars in different cells and tissues. lysine and arginine residues, such as those adjacent to the Oligosaccharides may be attached to several different amino membrane-spanning ␣ helix of glycophorin A in Figure 4.18. acids by two major types of linkages (Figure 4.11). These The appearance of PS on the outer surface of aging lympho- 1 cytes marks the cells for destruction by macrophages, whereas It can be noted that even though phosphatidylinositol contains a sugar group (Figure 4.6), it is not considered to be part of the carbohydrate portion of the its appearance on the outer surface of platelets leads to blood membrane in this discussion. coagulation. Phosphatidylinositol (PI), which is concentrated in the inner leaflet, can be phosphorylated at different sites on Asparagine the inositol ring, which converts the lipid into a phospho- inositide. As discussed in Chapter 15, phosphoinositides play a key role in the transfer of stimuli from the plasma membrane CH OH 2 O NH H O H H N C CH 2 CH C O Exoplasmic OH H O H 25 Polypeptide backbone SM PC PS PE PI Cl H NHCOCH3 4.3 N-Acetylglucosamine The Chemical Composition of Membranesof Composition Chemical The Serine (X=H) Threonine (X=CH3)

0 CH OH 2 X NH O O O CH CH

% total membrane lipid OH C O H H

H NHCOCH3 25 Cytosolic N-Acetylgalactosamine Figure 4.10 The asymmetric distribution of phospholipids (and Figure 4.11 Two types of linkages that join sugars to a cholesterol) in the plasma membrane of human erythrocytes. (SM, polypeptide chain. The N-glycosidic linkage between asparagine and sphingomyelin; PC, phosphatidylcholine; PS, phosphatidylserine; PE, N-acetylglucosamine is more common than the O-glycosidic linkage phosphatidylethanolamine; PI, phosphatidylinositol; Cl, cholesterol.) between serine or threonine and N-acetylgalactosamine. Chapter 4 The Structure and Function of the Plasma Membrane 130 tahn ete tria sgr h fnto o the of blood-group antigens remains a mystery. function The sugar. terminal either attaching capab enzymes lack type enzy blood O both with possess people whereas type blood AB with People strates. differ recognize they yet gene, same the of versions alt by encoded are enzymes two These terminus. chain galactose adds that enzyme an has blood B type with whereas to the end of the chain, acetylgalactosamine pro’ bod ye s ,B B r (iue 4.12). (Figure O or adds that enzyme AB, an has A type blood B, having person A, is type blood person’s a whe glycolip determine membrane the plasma cell of blood red carbohydrates the of The 8). (Chapter ments cellular different to proteins membrane of sorting the interactions of a cell with its environment (Ch in role important an play projections carbohydrate GalNAc, fucose; Fuc, glucose; Figure 4.12 n tutr.(a,glcoe GlcNAc, galactose; (Gal, and B structure. AB blood has gangliosides w A person with type here. and O blood typ B, a ganglioside) that produce the A, to membrane attached The oligosaccharides membrane. lipids and proteins to membrane oftached the red b of sug chain by a short or O blood is determined AB, .Draw the basic structure of the major types of li 1. .What is a liposome? How are liposomes used in med 2. .What is an oligosaccharide? How are they linked t 3. W E I V E R brane activities? into a bilayer? How is the bilayer important for me Which are glycolipids? How are these lipids organiz fer from glycerolipids? Which lipids are phospholip found in cellular membranes. How do sphingolipids d therapies? blood types? membrane proteins?Howare they relatedtohuman G a l N A c Blood-group antigens.Blood-group Gal Fuc Fuc Fuc Gal Gal Gal G l c N A c G l c N A c G l c N A c O antigen B antigen A antigen N -acetylgalactosamine.) a Glu Gal a Glu Gal Gal hte esnhstp ,B, A, a person has type Whether N Glu aeyguoaie Glu,-acetylglucosamine; lood cell ars covalently at-ars covalently es are shown ith both the A lipids (forming apter 7) and mediating pids compart- a person n sub-ent m- o ids? to theto ABO ed ernate an if- ical e of le mes, ther ids ids N A A - 3. 2. 1 . These are 4.13). bilayer (Figure distinguished by the intimacy of their relationship be grouped proteinsMembrane into three can distinc th to exposed are molecules cytoplasmic with teract proteins membrane of parts those whereas space, lar the to exposed are substances extracellular with or exampl for membrane, those plasma parts of membrane proteins the that interact with In “sidedness.” brane (as in Figure 4.4 Figure in (as other the of those from different very are membrane so that the properties of one tive to the cytoplasm, orientatio deﬁned a has protein membrane Each teins. hundredscontainmay differ membrane of a cell, that Depending on the cell type and the particular organ 4.4 (Figure 4.14 (Figure prot the and lipid surrounding with contact direct into brought bilayer, the within anchored is preserv protein is membrane the of barrier permeability the As ofthe membrane. theprotein into the lipid “wall” chains of the teractions withbilaye acyl the fatty Waalder van form domains transmembrane in residues transmembr bilayer—the Amino lipid character. hydrophobic a have to domains—tend the within reside that amphipath membrane ofanintegral thoseportions cussedbelow, As also portions. are hydrophobic and hydrophilic proteins both having membrane integral layer, of phospholipids the Like respiration. and synthesis or processes the during electrons transfer membrane, that agents the across solutes and ions of movement as channels substance or involv transporters speciﬁc membrane surface, bind that receptors as capacities: fol the in function proteins membrane integral Most Integral Membrane Proteins ii mlcls ht ae otc wt a transmembr a with contact make that molecules lipid of Membrane Proteins is situated within the bilayer. but are covalently linked to a lipid molecu surface, or cytopl on either the extracellular lipid bilayer, Lipid-anchored proteins by noncovalent bonds. are yet of the mem associated with the surface side, or extr on either the cytoplasmic the lipid bilayer, Peripheral proteins proteins and roughly 60 percent of all current drug gral proteins constitute 25–30 percent of all encod Genome-sequencingtispanning. studies suggest that i one membrane-spanning whereas others segment, are mu sides Some integral of the membrane. proteins have o that protrude from both the extracellular and cytop entirely through the lipid bilayer and thus have do proteins are Integral proteins | The Structure and Functions a ). According to current consensus, most of theof most consensus, current to According ). transmembrane proteins d ). This asymmetry is referred to as mem-as to referred is asymmetry This ). that penetrate the Integral lipid bilayer. that are outsideentirely of located that are outside the located hti,they pass that is, ; r, which which sealsr, surface of a to the lipid extracellu- other cells of photo-of elle elle within asmic molecules e cytosol.e mains ed acellular targets. ed in the a result,a lasmic ent pro-ent le that a the at s h bi- the t classes protein that in-that surface d theed, brane lowing n rela-n nly i is ein nte- dis- in-s acid ane ane ane as ic, l- e, 131

Integral membrane proteins (a)

Peripheral membrane protein

(a)

Lipid bilayer

Peripheral (b) membrane proteins

Etn P (b) GPI- anchored protein I Man GlcNAc Figure 4.14 The interactions between membrane proteins and 0 0 lipids. (a) Aquaporin is a membrane protein containing four subunits 0 Man 0 P (colored differently in the illustration) with each subunit containing an Man C Etn P 0 aqueous channel. Analysis of the protein’s structure revealed the pres- P Etn ence of a surrounding layer of bound lipid molecules. In this case, these lipid molecules are not likely to play a role in the function of aquaporin because the protein retains its function as a water channel in bilayers 4.4 containing nonnative lipids. ( b) Two views of another tetrameric mem- The Structure and Functions of Membrane ProteinsMembrane of Functions and Structure The brane protein, in this case the bacterial K ϩ channel, KcsA. Anionic phosphatidylglycerol molecules (red/gray) are seen in each crevice between the subunits and are thought to be required for normal channel function. A K ϩ ion (purple sphere) is seen in transit through the pore. (A: F ROM CAROLA HUNTE AND SEBASTIAN RICHERS , C URR . O PIN . Cytoplasm STRUCT . B IOL . 18:407, © 2008; B: F ROM ANTHONY G. L EE , TRENDS BIOCHEM . S CI . 36:497, 2011, © BOTH WITH PERMISSION Lipid-anchored protein FROM ELSEVIER SCIENCE .) (c) Figure 4.13 Three classes of membrane protein. (a) Integral proteins typically contain one or more transmembrane helices (see Figure 5.4 for an exception). ( b) Peripheral proteins are noncovalently bonded to domain, such as those shown in Figure 4.14 a, are simply pas- the polar head groups of the lipid bilayer and/or to an integral sive bystanders and are rapidly exchanged with other lipid membrane protein. ( c) Lipid-anchored proteins are covalently bonded molecules in the bilayer. There is increasing evidence, how- to a lipid group that resides within the membrane. The lipid can be ever, that certain sites on the surface of many membrane pro- phosphatidylinositol, a fatty acid, or a prenyl group (a long-chain teins do form important functional interactions with specific hydrocarbon built from five-carbon isoprenoid units). I, inositol; lipid molecules. An example of this is shown in Figure 14.4 b, GlcNAc, N-acetylglucosamine; Man, mannose; Etn, ethanolamine; where anionic lipid molecules are seen to bind in the crevice at GPI, glycosylphosphatidylinositol. the interfaces between the subunits of a tetrameric KcsA K ϩ Chapter 4 The Structure and Function of the Plasma Membrane 132 T (Figure4.15 l theleafletsbetweenpathtwoof thetakes a often tures the block into Astwo this thepieces. occurs, knifeblade,awith struckthenfrozensolidand is fracture replication urudn c.(surrounding ice. (arrow)ridge marks the junction of the particulate approximately 8 nm in dia be covered with particles The P fract of a freeze-fractured human erythrocyte. iesostept ftefatr ln.(line shows the path of the fracture plane. preferentially segregated with the outer half of th molecules (b fractured reveal that glycophorin cell Thin section 4.18). proteingral (Figure glycophorin groupscarbohydrate that project from the external and labeled with a fixed, was thawed, the cell replica, but rather and then fractured, that was frozen cyte rived primarily from the results of a technique cal technique a of resultsthe fromprimarily rived bilaye the to externalremainingsimply than rather h ,o colsi ae n h ,or protoplasmic and the P, or ectoplasmic face, the E, These exposed fa replica. a metallic deposit to form then be coverewithin the center of the bilayer can ofsegregate the bilayer halves with one of the two goes them around the proteins than cracking rather of the lipid bilay that leads it through the middle ofte through a fracture the tissue, plane runs blade, brane structure.brane Figure 4.15 sis DistributionIntegralof Proteins: Freeze-Fracture within the membrane.able to move laterally integral proteins need not be fixed structures later, con hydrophilic As typically will residues at strategic locations. channels these of linings The bilayer. the through passageway aqueous an provides that nel large families of membrane proteins contain an inte S protein’sthe into deep regions. membrane-embedded p to solvent aqueous the allow others whereas cules, transmembrane domains are essentially devoid of wat proteins, membrane some In membrane. the of edge the and other substance prot hormones, hydrophilic molecular-weight substrates, water-soluble have with interact to that tend surfaces domains nonembedded These Sec in discussed proteins globular the like more be project into either the or cytoplasm extracellular these specific lipid molecules.lacks The channel does not open normally in a channel. bil R B C particles a road strewn with which pebbles, are called iue1.7.Asshown in Figure Figure4.15 18.17). owed metalsaredeposited theiron exposed surfaces foto : IOL OCKEFELLER LAKANDILLACK FROM hs prin o a itga mmrn poen that protein membrane integral an of portions Those 347 1982; 93:467, . The concept that proteins penetrate through membran replica . Since the fracture plane passes through the centerfracturethroughpassestheplanethe Since . P EDRO Freeze fracture: a technique for investigating cell cell investigating for technique a Freeze fracture: , which is viewed in the electron microscopeelectron(seethe in viewed is which , a V ). Once the membranes are split in this manner,thismembranesin split theare Once ). U ( INCE NT c P NIVERSITY a ) This micrograph shows of an erythro- the surface ) When a block of frozen tissue is struck by a knif tissue is struck of frozen a block ) When NODAINTO B , C (see InSection this tissue 18.2). procedure, R: .MT. POUE IHPRISO OF PERMISSION WITH EPRODUCED S P LAANDILVA ARCHESI RESS .) .C J. , M B ARIA F: ELL b ROM thereplica resembles , .TR. r The fracture planeer. ebae The rede membrane. membrane-associated B lack particles) have particles) lack The exposed faces. d with a metal face with the n following a path than preparing a surface of the inte-surface ces are referred to as IOL ee.A smallmeter. T s of the labeled, nhl,and theyin half, ure face is seen to marker for the fc.( face. HOMAS ORRISI 569 1970; 45:649, . fracture plane space tend to be discussed but may be whichfrac- ipid bilayeripid led rm a shad-arm b rior rior chan- r, was de- was r, .C J. , ) Replica in 2.5.tion ayer that er mole- tain key key tain W. enetrate (low- s eins) at freeze- Analy- T everal lipid lipid mem- ELL HE the es, e of rl ebae rtis r dfcl t ioae n a Removal of these in proteins form. from the membrane isolate no to difficult are proteins membrane gral domains, transmembrane hydrophobic their of Because of Integral Membrane Proteins Studying the Structure and Properties (b) (a) such differences. in Figure 7.32 identified(asillustrated bythereplica gapofaju replicasan these in outmembranestand the of parts differen Localized membrane. the of croheterogeneity fracturing technique is that it allows an investiga half (see Figure 7.30Figure(see half brane (Figure 4.15(Figurebrane p the ofproteinhalf (particle)separatesone with Consequen half. in it cracking than rather it around fractureparticgiventhereachesplane a When core. throughhalfway least at extend thatproteins brane mostthe bilayer, of these particles correspond to i (c) Fracture faceP Fracture faceE Cytoplasm Cytoplasm Exterior d .Bohmclaaye,i otat average Biochemical in outcontrast, analyses, ). c ), leaving a corresponding pit in the otherthe correspondingin pita leaving ), c ). One of the great values of the freeze-the ofvalues great the of One ). P tion of the mi- ntegral mem- nctionshown lasma mem-lasma h lipid the le, it goes it le, d can becan d l,eachtly, soluble rmally e inces inte- 133

Lipid Aqueous sent prokaryotic versions of a particular protein, which are of- bilayer solution ten smaller than their eukaryotic homologues and easier to obtain in large quantity. Once the structure of one member of a membrane protein family is determined, researchers can Nonionic detergent usually apply a strategy called homology modeling to learn about the structure and activity of other members of the family. For example, solution of the structure of the bacterial K ϩ channel KcsA (shown in Figure 4.39) provided a wealth of data that could be applied to the structure and mechanism of Membrane Detergent-solubilized ϩ protein protein action of the much more complex eukaryotic K channels (Figure 4.42). Figure 4.16 Solubilization of membrane proteins with detergents. One of the first membrane proteins whose entire three- The nonpolar ends of the detergent molecules associate with the non- dimensional structure was determined by X-ray crystallogra- polar residues of the protein that had previously been in contact with phy is shown in Figure 4.17. This protein—the bacterial the fatty acyl chains of the lipid bilayer. In contrast, the polar ends of the detergent molecules interact with the surrounding water molecules, photosynthetic reaction center—consists of three subunits keeping the protein in solution. Nonionic detergents, as shown here, containing 11 membrane-spanning ␣ helices. Most mem- solubilize membrane proteins without disrupting their structure. brane proteins have not been as amenable to crystallization as the photosynthetic reaction center. Among the obstacles, many membrane proteins (1) are present at low numbers per cell, (2) are unstable in the detergent-containing solutions requires the use of a detergent, such as the ionic (charged) required for their extraction, (3) are prone to aggregation, and detergent SDS (which denatures proteins) or the nonionic (4) are heavily modified by glycosylation and cannot be (uncharged) detergent Triton X-100 (which generally does expressed as recombinant proteins in other types of cells. not alter a protein’s tertiary structure). Some of the technical difficulties in preparing membrane protein crystals have been overcome using new methodologies – + CH 3 (CH2)11 OSO3 Na and laborious efforts. In one study, for example researchers Sodium dodecyl sulfate (SDS) were able to obtain high-quality crystals of a bacterial trans- CH 3 CH 3 porter after testing and refining more than 95,000 different conditions for crystallization. In some cases, mutant versions CH 3 CCH 2 C (O CH 2 CH 2)10 OH of a membrane protein are found to be better suited to form- CH 3 CH 3 ing the ordered arrays of molecules that make up a crystal lat- Triton X-100 tice. In many other cases, crystallization has been achieved by covalently linking the membrane protein to other molecules, Like membrane lipids, detergents are amphipathic, being often small soluble proteins. These added elements stabilize composed of a polar end and a nonpolar hydrocarbon chain (see Figure 2.20). As a consequence of their structure, detergents can substitute for phospholipids in stabilizing integral proteins while rendering them soluble in aqueous solution (Figure 4.16). Once the proteins have been solubilized by the 4.4

detergent, various analyses can be carried out to determine the ProteinsMembrane of Functions and Structure The protein’s amino acid composition, molecular mass, amino acid sequence, and so forth. Researchers have had great difficulty obtaining crystals of most integral membrane proteins for use in X-ray crystallography.In fact, fewer than 1 percent of the known high-resolution protein structures represent integral membrane proteins (see http://blanco.biomol.uci.edu/MemPro_resources.html for a discussion of principles of protein structure and http://blanco. biomol.uci.edu/Membrane_Proteins_xtal.html for an up- dated gallery). 2 Furthermore, most of these structures repre- Figure 4.17 An integral protein as it resides within the plasma 2Many integral membrane proteins have a substantial portion that is present in membrane. Tertiary structure of the photosynthetic reaction center of a the cytoplasm or extracellular space. In many cases, this soluble portion has been bacterium as determined by X-ray crystallography. The protein cleaved from its transmembrane domain, crystallized, and its tertiary structure contains three different membrane-spanning polypeptides, shown in determined. While this approach provides valuable data about the protein, it yellow, light blue, and dark blue. The helical nature of each of the fails to provide information about the protein’s orientation within the mem- transmembrane segments is evident. (F ROM G. F EHER , J. P. A LLEN , brane. Another crystallographic approach to the study of membrane proteins, M. Y. O KAMURA , AND D. C. R EES , N ATURE 339:113, © 1989; which uses the electron microscope rather than X-ray diffraction, is discussed in REPRINTED BY PERMISSION FROM MACMILLAN PUBLISHERS LTD .) the Experimental Pathways on page 173. Chapter 4 The Structure and Function of the Plasma Membrane 134 mainsthe as described within are which embedded membrane, protein a of segments Those layer? the in embedded actually are chain polypeptide the segme Which is: askmightquestion onefirst The gene. an of sequence nucleotide the from deduced readily computer-b a from sequence acid amino its bilayer of analysis (computational) lipid the within entation learned about the structure of a membrane protein a Domains Transmembrane Identifying these approaches in the following paragraphs.some examine will We proteins. membrane most of tion or three-dimensional the determining for approaches larg researchers rely still proteincrystallization, su increasing Despite grown. were crystals the which m lipid-phase the in cholesterol of use the and tor to bound ligand specific a of presence the by tated core of the lipid bilayer as an sp that acids amino nonpolar predominantly 20 about commonly found where helicescommonly come inttransmembrane sequence is This Gly-X-X-X-Gly tween residues 79 and 83. helices of the dimer cross over one another in the single transmembrane domain. transmembrane single 3 the of structure crystal the ple, F formation. lattice for required contacts molecular sites on the surface of the protein that can partic increaseproteinth and target the of structure the close proximity.close Figure 4.18 within the erythrocyte membrane (Figure 4.32 (Figure membrane within the erythrocyte molecules are Glycophorin present as hom sition 26). res linked to the asparagine larger oligosaccharide oligosaccharides are oligosaccharides small but one of the All the protein (shown in the inset). to a number of amino acid residues on the outer sur Carbohydrates are lipid head groups. charged atively domain of protein the membrane ionic bonds form wit amino acid residues charged of the cytoplasmic positively The fo hydrophobic residues (brown-colored circles). passes through consists the membrane predominantly o pnteblyr An example is the P helix in F not span the bilayer. proteins have been found to contain loops or helice the hydrocarbon core of a lipid bilayer of 30 Å wid this is the minimum because stretch of polyp length, Transmembrane helices are a with an aqueous solvent. ca thus, and, chains acyl that is surrounded by fatty for a memb important This is particularly figuration. thereby creating a high backbone, of the polypeptide allows for a maximum number of hydrogen bonds to be hg) no n o te nrclua los f h r the of the loops of Crystallization intracellular the of one into phage) a from b (derived T4 molecule a serting T4-lysozyme in the chapter opening image on page 120 was obtain It was noted on page 55 that the ae sml srcue hy oss o a tig o string a of consist they structure; simple a have , Glycophorin A, an integral protein with a an integral A, Glycophorin O ␤ -linked (the exception chains is a 2 -adrenergic receptor was also facili-also was receptor -adrenergic ␣ helix is a favored it because conformation The single ␣ helix. ␤ 2 -adrenergic receptor shownreceptor -adrenergic 3 ␣ The chemical Thestructure chemical th. A few integral membrane integral A few th. nnot form hydrogennnot form bonds s that penetrate but do s that penetrate igure 4.39. helix that get el a be can deal great A eptide capable of spanningeptide capable ly stable (low-energy) con- stable (low-energy) ly d rane-spanning polypeptiderane-spanning transmembrane do- transmembrane t least 20 amino acids in .The two). formed between atoms between formed idue at po- region be- 16 face of attached ur odimers of h neg- ely on indirecton ely ipate in inter- o e number ofnumber e the recep-the nd its ori- , which is which , r exam-or du inedium ed by in- lipid bi-lipid eceptor. acterio- ccess inccess isolated ganiza- n thean nts ofnts ased the of Pro f Ser Lys + – requires distorting the helix to do so. Figure 4.19 Figure so. do to helix the requires distorting ev membrane, the of regions polar innermost the with an out reach to able are chains side whose residues pair a contains figures these in helices the of Each rn.Of the 20 amino acids that make up the lone brane. the major pla integral protein of the erythrocyte A, glyc of Figure structure in two-dimensional the shown depicts which is helix transmembrane single a of ments using a a using ments we can usually identify brane the protein, transmemb chains of it the has bilayer become withinte which hy the with parallel oriented is ring aromatic each tyrosine residues with their hydrophobic aromatic s Figure Figure 4.19 polar residues which (Figu are noncharged, threonine, the case The of exceptions the ar glycine residues). H an (or chains side hydrophobic have three but all a glycophorin monomer (amino acids 73 to 92 of Figu hi hdohbc niomn.Ti i ilsrtd i illustrated 4.19 Figures in is shown helices transmembrane model This environment. hydrophobic their fi to them allow that ways in accommodated be to and of ends the of one near be to tend they but transmemb in helices, appear also may residues charged Fully ba peptide the of atoms oxygen the of one with bond A glycophorin hydroxyl of group of the those residue’s side chain unlike can form not residue, threonine do not alter the profile Hydroof the entire stretch. a in acids amino polar few a or one that guarantees polypep the of sections short of hydrophobicity the ave “running a provides approach This neighbors. its hydrophobicity mea providesa that value a assigned is polypeptide Exterior surface Lys + – Knowing the amino acid sequence of an integral mem-integral an of sequence acid amino the Knowing Ile Leu Arg a + – shows a portion of a transmembrane helix with a of the amino acid at that site as well as that ofthat as well as site that at acid amino the of Arg + yrpty plot hydropathy – 90 Thr Gly Gly Leu Thr lle Leu Phe Ala Gly Val Val IIe Ser Ile Ile Leu Met IIe Ile 80 n hc ec st aog a along site each which in , Interior surface (cytosol) d phobicity phobicity of e serine and a hydrogen of chargedof sma mem- sure of theof sure grated. ide chains; showstwo drocarbon d interactd sequence ␣ the helixthe rane rane seg- ie andtide, atom inatom re 2.26). re 4.18), ophorin ae ofrage” ckbone. b helix of en if itif en and the n . The The . intot Bilayer 4.18, rane rane c . 135

(a) (b) (c) (d) Figure 4.19 Accommodating various amino acid residues within molecules of the polar face of the lipid bilayer. ( c) The side chains of transmembrane helices. (a) In this portrait of a small portion of a the two aspartic acid residues of this transmembrane helix can also transmembrane helix, the hydroxyl group of the threonine side chain reach the polar face of the bilayer but introduce distortion in the helix (arrow) is able to form a (shared) hydrogen bond with a backbone oxy- to do so. ( d ) The aromatic side chains of the two tyrosine residues of gen within the lipid bilayer. Hydrogen bonds are indicated by the this transmembrane helix are oriented perpendicular to the axis of the dashed lines and their distances are shown in angstroms. ( b) The side membrane and parallel to the fatty acyl chains with which they inter- chains of the two lysine residues of this transmembrane helix are sufﬁ- act. (F ROM ANNA C. V. J OHANSSON AND ERIK LINDAHL , B IOPHYS . ciently long and ﬂexible to form bonds with the head groups and water J. 91:4459, 4453, 2006, © 2006, WITH PERMISSION FROM ELSEVIER .)

amino acids can be determined using various criteria, such as transmembrane segment tend to be more positively charged their lipid solubility or the energy that would be required to than those at the extracellular flank. transfer them from a nonpolar medium into an aqueous Not all integral membrane proteins contain transmem- medium. A hydropathy plot for glycophorin A is shown in brane ␣ helices. A number of membrane proteins contain a Figure 4.20. Transmembrane segments are usually identified relatively large channel positioned within a circle of membrane- as a jagged peak that extends well into the hydrophobic side of spanning ␤ strands organized into a barrel as illustrated in the spectrum. A reliable prediction concerning the orientation Figure 5.4. To date, aqueous channels constructed of ␤ barrels of the transmembrane segment within the bilayer can usually have only been found in the outer membranes of bacteria, be made by examining the flanking amino acid residues. In mitochondria, and chloroplasts. most cases, as illustrated by glycophorin in Figure 4.18, those parts of the polypeptide at the cytoplasmic flank of a Experimental Approaches to Identifying Conformational Changes within an Integral Membrane Protein Suppose you have isolated a gene for an integral membrane protein and, based on its nucleotide sequence, determined that it contains 12 apparent membrane-spanning ␣ helices. You might want to know which sites within these transmembrane domains are accessi- 4.4

ble to the aqueous environment and how this changes as the ProteinsMembrane of Functions and Structure The

G protein carries out its function. This type of information is + particularly important when working with a membrane chan- 0 nel or transporter that functions in the movement of hy- G drophilic substances across the membrane. Although these _ Hydrophobicity determinations are difficult to make without detailed structural models, considerable insight can be gained by site- directed mutagenesis, that is, by introducing specific changes into the gene that encodes the protein (Section 18.17). An 50 100 experiment of this type, which was conducted on lactose per- Amino acid residue # mease, a sugar-transporting protein in bacterial cell mem- N-terminus C-terminus branes, is shown in Figure 4.21. In this experiment, Figure 4.20 Hydropathy plot for glycophorin A, a single researchers prepared different versions of the permease in membrane-spanning protein. Hydrophobicity is measured by the free which nearly all of the individual amino acids of the protein energy required to transfer each segment of the polypeptide from a were replaced, one at a time, by a cysteine residue. Each nonpolar solvent to an aqueous medium. Values above the 0 line are mutated membrane protein was then incubated with a water- energy-requiring ( ϩ⌬ Gs), indicating they consist of stretches of amino soluble reagent (NEM) that is capable of adding an alkyl acids that have predominantly nonpolar side chains. Peaks that project group to the cysteine side chain—as long as it has access to above the red-colored line are interpreted as a transmembrane domain. that side chain. The incubations with NEM were carried out Chapter 4 The Structure and Function of the Plasma Membrane 136 (a) cussed further on page 159.cussed further i system transport ion-driven of type This membrane. a sugar the of transport about brings conformations Alternation between space in the other ext conformation. the to open is and conformation one in cell ial of cytoplasm the to open is site sugar-binding the presence of the sugar (sugar the of presence in Figure 4.21 labeled in these two conformations supports the mod t residues the of positions the of analysis Careful of the sugar induces a conformational change that in suggest the words, other in results, The conditions. protein are accessible to the aqueous medium under between the protein in the absence of the sugar (sugar the of absence the in protein the between The difference in NEM reactivity of the transported. t sugar the with incubated is permease the when NEM The gold spheres in to the alkylating agent when the protein is incubat the external The medium (the periplasm in bacteria). ( agent NEMthe alkylating in the absence of a sugar spheres the residues indicate prote of the membrane inaccessible to NEM. maki thus molecule, the of regions packed tightly in face the chains fatty acyl of the phospholipids or to presumed are XII, and VI helices on notably most Those residuescysteine that are n to the cytoplasm. th permease the in hydrophilicinward-facing cavity as gold spheres) that show a significant sugar NEM by alkylated become that protein 4.21 The image sugaror toin beits transported absence. presen the in either conditions: different two under of the protein is at thesurface (lactose permease) and its resultsexperiment are discussed in the tex out its activity.protein as it carries of in the conformation changes about dynamic learn Figure 4.21 b ) The gold spheres denote the residues that are muc The image in Figure 4.21 a Many of these accessible residues are thought to l . hw hs eius(eitda e pee) f t of spheres) red as (depicted residues those shows An experiment employing site-directed mutagenesis employing t An experiment c According to this model of alternating access,. b are seen to cluster in a portion of the protein nea b ) indicates that different parts of theof parts different that indicates ) b The experimental strategy of the strategy The experimental shows those residues (depicted increase tp ( top. nteasneo theof absence the in .The cytoplasmict. ed with the sugar. (b) in that reacted with to be transported. authors concluded h more accessible a membrane a in reactivity to ) Thered to be located hat becomehat the bacter-the a permease. ot labeled, in Figure at is openis at these two ) and theand ) racellular ce of theof ce cross thecross el shown residues g themng addition the two ine an either dis-s beo he o r nance w spectrum called technique a characteristic by a monitored produces which electron, them. that distance the to sensitive are properties whose chemic introduce to is protein a in betwee residues distance lected the about learn to way One function. carrie protein a as occur that events dynamic about to approach another is protein membrane a in acids a model of alternating access to the medium as depi one that is open to the external medium i.e., cavity, that the gold spheres correspond to residues that l the structure that is depicted in parts protein. Figure 4.22 Figure protein. subunit each from one helices, transmembrane four by is channel the to opening cytoplasmic The subunits. intensity of their EPR signals. These results indica results These signals. EPRtheir of intensity decre which pH, this at together closer are subunits t on groups nitroxide the because 6.5 pH at broader spec The another. one to nitroxides the of proximity depends line each of shape The open. is channel the 3. pH at spectrum the shows line blue the and state, shows line red The spectrum obtained at pH 6.5 when the channel helix. is in transmembrane each of end cyt the near introduced was nitroxide a when tained ( which is why these types of labeling experiments re the outward-facing conformation has not been direct conformation as determined by X-ray crystallography bacterial bacterial K respon ques in protein The in medium. the of pH the in changes opened is channel its as protein membrane oc that changes conformational the discover to used techniq this how shows 4.22 Figure residue. cysteine o group —SH the to nitroxide the attaching then and site mutatingthat first protein by a in site any at FROM F © 2007 N A IG – B 1 . F: Determining Determining spatial relationships between specific a C BY ( Nitroxides ROM EPR URRENT .R K R. H. A TI O N A L ϩ .RH. ) spectroscopy hne,is a tetramer composed of four identicalchannel, O ONALD ABACK are chemical groups that contain an unpaired IININPINION A (c) AEYOFCADEMY ats H Lactose a , K IHPRISO FROM PERMISSION WITH shows the EPR spectra that were ob-were that spectra EPR the shows A nitroxide group be introducedcan . ABACK S TRUCTURAL , S TALET CIENCES a External medium + – b (periplasm) lcrn aaantc reso- paramagnetic electron is that of the inward-facing Cytoplasm ,PA 0:9,2007, PNAS 104:492, ., .S A.) S. U. , B I O L OG Y ats H Lactose ine an outward-facing The results support. main important.) cted in part ly ly determined, The structure of. E LSEVIER 444 200414:414, C to a cysteinea to R: te that thethat te the closed separates E P R I N T E D l groupsal oplasmic bounded ot its out s learning ss theases cur in ain cur when5 e fourhe rm is trum c . e wasue (Note. n theon of theof in ation, se-n mino e to se thef + hen the 137

External medium Dehydrated Permeation K+ ion pathway

Plasma membrane

Nitroxide-labeled cysteine residue

pH 3.5 Closed Hydrated Open K+ ion pH 6.5 Cytoplasm

(a) (b)

Figure 4.22 Use of EPR spectroscopy to monitor changes in confor- different subunits. (Nitroxides are described as “spin-labels,” and this ؉ mation of a bacterial K ion channel as it opens and closes. (a) EPR technique is known as site-directed spin labeling.) ( b) A highly spectra from nitroxides that have been attached to cysteine residues schematic model of the ion channel in the open and closed states based near the cytoplasmic end of the four transmembrane helices that line on the data from part a. Opening of the channel is accompanied by the channel. The cysteine residue in each helix replaces a glycine the movement of the four nitroxide groups apart from one another. residue that is normally at that position. The shapes of the spectra de- (A: R EPRINTED BY PERMISSION FROM MACMILLAN PUBLISHERS pend on the distances between unpaired electrons in the nitroxides on LTD : FROM E. P EROZO ET AL ., N ATURE STRUCT . B IOL . 5:468, 1998.)

activation of the channel is accompanied by an increased sep- plasma membrane surface function as enzymes, specialized aration between the labeled residues of the four subunits (Fig- coats (see Figure 8.24), or factors that transmit transmem- ure 4.22 b). An increase in diameter of the channel opening brane signals (see Figure 15.19). Peripheral proteins typically allows ions in the cytoplasm to reach the actual permeation have a dynamic relationship with the membrane, being re- pathway (shown in red) within the channel, which allows only cruited to the membrane or released from the membrane the passage of K ϩ ions (discussed on page 153). An alternate depending on prevailing conditions. technique, called FRET, that can also be used to measure changes in the distance between labeled groups within a pro- 4.4 Lipid-Anchored Membrane Proteins tein, is illustrated in Figure 18.8. ProteinsMembrane of Functions and Structure The Several types of lipid-anchored membrane proteins can be distinguished. Numerous proteins present on the external face Peripheral Membrane Proteins of the plasma membrane are bound to the membrane by a Peripheral proteins are associated with the membrane by weak small, complex oligosaccharide linked to a molecule of phos- electrostatic bonds (refer to Figure 4.13 b). Peripheral proteins phatidylinositol that is embedded in the outer leaflet of the can usually be solubilized by extraction with high-concentration lipid bilayer (refer to Figure 4.13 c). Peripheral membrane pro- salt solutions that weaken the electrostatic bonds holding teins containing this type of glycosyl-phosphatidylinositol peripheral proteins to a membrane. In actual fact, the distinc- linkage are called GPI-anchored proteins . They were discov- tion between integral and peripheral proteins is blurred be- ered when it was shown that certain membrane proteins could cause many integral membrane proteins consist of several be released by a phospholipase that specifically recognized and polypeptides, some that penetrate the lipid bilayer and others cleaved inositol-containing phospholipids. The normal cellu- that remain on the periphery. lar scrapie protein PrP C (page 66) is a GPI-linked molecule, The best studied peripheral proteins are located on the as are various receptors, enzymes, and cell-adhesion proteins. internal (cytosolic) surface of the plasma membrane, where A rare type of anemia, paroxysmal nocturnal hemoglobinuria, they form a fibrillar network that acts as a membrane “skeleton” results from a deficiency in GPI synthesis that makes red (see Figure 4.32 d). These proteins provide mechanical sup- blood cells susceptible to lysis. port for the membrane and function as an anchor for integral Another group of proteins present on the cytoplasmic side membrane proteins. Other peripheral proteins on the internal of the plasma membrane is anchored to the membrane by one Chapter 4 The Structure and Function of the Plasma Membrane 138 iae cmoe o popaiycoie n phos- and If the temperature of the bilayer is kept rel rated. phosphatidylcholine largely are acids fatty of whose phatidylethanolamine, composed bilayer hshtdlhln n hshtdltaoaie ( and phosphatidylethanolamine. phosphatidylcholine tails are free to move in certain directions, even t even tails are free to directions, move in certain the lipid molecules and thei temperature, transition flow, and viscosity is a measure of the resistance and viscosity to flow, 4 viscosity). (or fluidity de its is membrane by a of lipid the of state physical The 4.5 4.13Figure to (refer bilayer lipid the of or more long hydrocarbon chains embedded in the inn perature. Figure 4.23 Membrane Fluidity the transformation of a normal cell to a malignant p the with implic been have associated Ras) and (Src way this inmembrane proteins two least At legend). liiyadvsoiyaeivreyrltd fluidit are related; inversely and viscosity Fluidity .Why are detergents necessary to solubilize membra 1. .Describe the properties of the three classes of m 4. What ismeantbythestatementthatproteinsofa3. How can one determine: (1) the location of transmem- 2. W E I V E R true ofthemembrane’scarbohydrate? membrane are distributed asymmetrically? Is this al to the external medium? relative locations of transmembrane helices with ac brane segmentsintheaminoacidsequenceor(2) fraction? integral proteinsthatresideinapurifiedmembrane proteins? How might one determine the diversity of among themselves. how theydiffer fromoneanother, andhowtheyvary brane proteins (integral, peripheral, and lipid-anc (a) | Membrane Lipids and The bilayer shown here is composed phospholi of two The structure of the lipid bilayer depends on the tem- structure The 4 Consider a simple artificial artificial simple a Consider y is a measure of the ease of flow. c hough they retainhough they a n accompanyingand r hydrophobic a ) Above) the atively warmatively state. hored), em- er leaflet unsatu- scribed scribed td inated pids: so cess ne lasma a edsrbda rsaln e.( gel. as a crystalline be described can and movement of the molecules restricted, is greatly eg,37(e.g., h at cd fteblyr the bilayer, the of acids fatty the contai those than unsatu of degree the greater The tightly chains. unsaturated more together pack chains satu with phospholipids Consequently, 4.23). and 2.19 have crooks in the chain at the sites of a double b osdrbedge fodr (considerable degree of order. most 60 tempera melting the lowers acid stearic bon of double molecule one of introduction The gels. bilayer the eil rod.flexible fatty Saturated acids have the shape of constructed. w of lipids particular the on turn in depends packed which be to molecules lipid the of ability the on 4.23 changes (Figure 4.23 (Figure changes bilayer the wherereached is point a lowered, slowly te the If bilayer. the of plane the within laterally rotate around ax their can phospholipidsindividual arrang parallel a toward tend molecules the of axes c this in orientation; specified a retain still cules crystal a in As crystal. liquid two-dimensional a as h tmeaue t hc ti cag ocr i call is temperaturetransition occurs change this which at temperature The ment of the phospholipid fatty acid chains is great phase crystalline to a frozen gel crystalline in wh drogenates the double bonds of the vegetable oils u by have been saturated acids of margarine the fatty Vegetable oils contain polyuns is a solid. margarine Vegetable oils remain a liqu familiar food products. L 5 4.7), cholesterol molecules disrupt the close packin close the disrupt molecules cholesterol 4.7), bilay the within orientation their of Because terol. The physical state of the membrane is also affected temp melting its lower the phospholipid, a of chains the shorter The length. chain acid fatty is fluidity IHPRISO OF PERMISSION WITH The effect of fatty acid saturation on acid melting saturation temp of fatty The effect IVEL Y The transition temperature of a particular bilayer a ). At this temperature, the lipid bilayer is best des best is bilayer lipid the temperature, this At ). (b) M Њ Њ C (Table 4.2). C), the lipid exists in a relatively fluid state (Fig state fluid relatively a in exists lipid the C), EMBRANES Cis -unsaturated fatty acids, on the other hand,other the on acids, fatty -unsaturated C, C b AMBRIDGE AMBRIDGE ). The lipid is converted from a liquida from converted is lipid The ). 5 . Another factor that influences bilayer b eo h rniintmeaue the) Below temperature, the transition A U U lower – NIV NIV B .N R N. R. : P. P. the temperaturethe before a chemical process that hy- a chemical trtdftyais whereas acids, fatty aturated di h ergrtr whereasid in the refrigerator, sed as the starting material.sed as the starting RESS RESS erature by is illustrated OBERTSON 1983,; .) the entire bilayer ich ich the move- ase, the long the ase, ond (Figures mperature ismperature ly ly restricted. REPRINTED , the mole- the , by choles- a straight, mn,yetement, is or moveor is er (Figureer distinctly g of fattyof g fatty acyl fatty hich it isit hich together, ration of ration T, depends ue al-ture erature. d the ed i a in d cribed HE rated ning ning ure 139 Table 4.2 Melting Points of the Common The initial “emergency” response is mediated by enzymes that 18-Carbon Fatty Acids remodel membranes, making the cell more cold resistant. Re- modeling is accomplished by (1) desaturating single bonds in ؇ Fatty acid cis Double bonds M.p.( C) fatty acyl chains to form double bonds, and (2) reshuffling the Stearic acid 0 70 chains between different phospholipid molecules to produce Oleic acid 1 13 ones that contain two unsaturated fatty acids, which greatly Linoleic acid 2 Ϫ9 lowers the melting temperature of the bilayer. Desaturation of Linolenic acid 3 Ϫ17 single bonds to form double bonds is catalyzed by enzymes Eicosapentanoic acid (EPA)* 5 Ϫ54 called desaturases . Reshuffling is accomplished by phospholi- *EPA has 20 carbons. pases , which split the fatty acid from the glycerol backbone, and acyltransferases , which transfer fatty acids between phospholipids. In addition, the cell changes the types of phospholipids being synthesized in favor of ones containing more acyl chains and interfere with their mobility. The presence of unsaturated fatty acids. As a result of the activities of these cholesterol tends to abolish sharp transition temperatures and various enzymes, the physical properties of a cell’s membranes creates a condition of intermediate fluidity. In physiologic are matched to the prevailing environmental conditions. terms, cholesterol tends to increase the durability while decreas- Maintenance of fluid membranes by adjustments in fatty acyl ing the permeability of a membrane. composition has been demonstrated in a variety of organisms, including hibernating mammals, pond-dwelling fish whose body temperature changes markedly from day to night, cold- The Importance of Membrane Fluidity resistant plants, and bacteria living in hot springs. Prokaryotic What effect does the physical state of the lipid bilayer have on cells that live at very high temperatures have plasma mem- the biological properties of the membrane? Membrane fluid- branes that contain highly unusual lipids. ity provides a perfect compromise between a rigid, ordered structure in which mobility would be absent and a completely Lipid Rafts fluid, nonviscous liquid in which the components of the membrane could not be oriented and structural organization and Every so often an issue emerges that splits the community of mechanical support would be lacking. In addition, fluidity cell biologists into believers and nonbelievers. The issue of allows for interactions to take place within the membrane. For lipid rafts falls into this category. When membrane lipids are example, membrane fluidity makes it possible for clusters of extracted from cells and used to prepare artificial lipid bilayers, membrane proteins to assemble at particular sites within the cholesterol and sphingolipids tend to self-assemble into mi- membrane and form specialized structures, such as intercellular crodomains that are more gelated and highly ordered than junctions, light-capturing photosynthetic complexes, and surrounding regions consisting primarily of phosphoglyc- synapses. Because of membrane fluidity, molecules that inter- erides. Because of their distinctive physical properties, such act can come together, carry out the necessary reaction, and microdomains tend to float within the more fluid and disor- move apart. dered environment of the artificial bilayer (Figure 4.24 a). As a Fluidity also plays a key role in membrane assembly, a result, these patches of cholesterol and sphingolipid are re- subject discussed in Chapter 8. Membranes arise only from ferred to as lipid rafts . When added to these artificial bilayers, preexisting membranes, and their growth is accomplished by certain proteins tend to become concentrated in the lipid the insertion of lipids and proteins into the fluid matrix of the rafts, whereas others tend to remain outside their boundaries. membranous sheet. Many of the most basic cellular processes, GPI-anchored proteins show a particular fondness for the or- including cell movement, cell growth, cell division, formation dered regions of the bilayer (Figure 4.24 a). of intercellular junctions, secretion, and endocytosis, depend The controversy arises over whether similar types of 4.5

on the movement of membrane components and would sphingolipid- and cholesterol-rich lipid rafts, such as that il- FluidityMembrane and Lipids Membrane probably not be possible if membranes were rigid, nonfluid lustrated in Figure 4.24 b, exist within living cells. Most of the structures. evidence in favor of lipid rafts is derived from studies that employ unnatural treatments, such as detergent extraction or cholesterol depletion, which makes the results difficult to in- Maintaining Membrane Fluidity terpret. Attempts to demonstrate the presence of lipid rafts in The internal temperature of most organisms (other than birds living cells have generally been unsuccessful, which can either and mammals) fluctuates with the temperature of the external mean that such rafts do not exist or they are so small (5 to 25 nm environment. Since it is essential for many activities that the diameter) and short-lived as to be difficult to detect with cur- membranes of a cell remain in a fluid state, cells respond to rent techniques. The concept of lipid rafts is very appealing changing conditions by altering the types of phospholipids of because it provides a means to introduce order into a seem- which they are made. Maintenance of membrane fluidity is an ingly random sea of lipid molecules. Lipid rafts are postulated example of homeostasis at the cellular level and can be to serve as floating platforms that concentrate particular pro- demonstrated in various ways. For example, if the temperature teins, thereby organizing the membrane into functional com- of a culture of cells is lowered, the cells respond metabolically. partments (Figure 4.24 b). For example, lipid rafts are thought Chapter 4 The Structure and Function of the Plasma Membrane 140 ttemlclrlvl (at the molecular level. measures which par the height of various microscope, This image is provided raft-associated. exclusively peaks show the positions protein, of a GPI-anchored into the orange-colored ra spontaneously themselves which and sphingomyelin molecules, background, black which lipid bilayer containing phosphatidylcholine, mit signals from space to the cel the extracellular th proteins membrane other with interact to ceptors cell-s for environment local favorable a provide to Figure 4.24 pholipid can move laterally within the same leaflet same the within laterally move can pholipid th that r a As discussion state. fluid relatively a in exist can bilayer previous the from apparent is It of the Plasma Membrane 4.6 concentrate in this region. GPI-anchored proteins GPI-anchored ar in thisconcentrate region. acids (blue head groups) fatty with long saturated Phosphatidylcho and sphingolipids (red head groups). of consists primarily The outer leaflet of the raft .How can the two sides of a lipid bilayer have dif 4. Why is membrane fluidity important to a cell?3. What ismeantbyamembrane’stransitiontemperature, 2. What istheimportanceoffattyacidunsaturationfor 1. W E I V E R important in the formation of lipid rafts? length of fatty acyl chains? How are these properti and howisitaffected bythedegreeofsaturationor rate fatty acids? membrane fluidity? Of enzymes that are able to desatu- ionic charges? | The Dynamic Nature (a) Lipid rafts. b ) Schematic model of a lipid raft within a cell.a within raft lipid a of model Schematic ) ( a ) Image of the upper surface of an artificial) Image of the upper surface cholesterol (yellow) also tend to by an atomic force appears as the t.The yellowfts. e thought to ts of the specimen which is almostwhich line molecules esult, a phos- a esult, organize l interior. rae re- urface ferent with con-with at trans-at es e lipid lipid e and of lipid rafts is discussed in in discussed is rafts lipid of controvers (The signaling. that are in cell involved leaflet lipid-anchored tends to concentrate proteins glycerophospholipids acyl with long fatty saturated lipids consist primari the inner-leaflet raft result, on the lipids of the have an organizing effect raft The lipids in th become in lipid concentrated rafts. .BJ. ties of proteinsmembrane ways.have been revealed in several pr dynamic The model. fluid-mosaic the of formulation cornerstone a was membrane the of plane the within c proteins integral that demonstration The proteins. o mobility the of determinant important an is lipid state physical the embedded, are membrane a of teins sive transmembrane movement. transmembrane sive ing lipid and asymmetry may also reverse the slow r one leaflet to These the enzymes play other. a role i phospholipi certain move actively that enzymes tain cell However, membr the unfavorable. which is thermodynamically of must sheet lipid hydrophobic the internal of the group through head hydrophilic the occur, flip For surprising. not is finding This 4.25). (Figure r most the is membrane the of side other the to flop i make, can phospholipid a that motions possible the Th leaflet. other the to across move to days to hours a molecule phospholipid a takes it contrast, In two. s a in end other the to bacterium a of end one from ca phospholipid a that estimated is It compound fluorescent 4.29). Figure or particles gold to lipids he polar the the linking by microscope the under served mol lipid individual of within the bilayer of the plasma mobility membrane can be di The ease. siderable B MJ. OHMSR ANDIOCHEMISTRY IOL Science ICHAE L Because lipids provide the matrix in which integral which in matrix the provide lipids Because C. (b) HEM 3:06 2011.)( 334:1046, E DWARDSON 277,. protein GPI–anchored OE OFCOVER M OLECULAR Signaling protein Nature Revs. Mol. Cell Biol. Cell Mol. Nature Revs. © 2002 T A F: 3,J #30, ROM B HE ULY IOLO GY .E S E. D. A 6 02 C 2002; 26, ME RIC AN .) ASLOWSKY ly of cholesterol andly inrlae.As a inner leaflet. y over the existence e outer leaflet of the tis The inner tails. , such as Src kinase, as Src such , S UTS OFOURTESY 168 2010 11:688, CEYFOROCIETY n establish- , ate of pas- TALET rectly rectly ob- matter ofmatter f integralf diffusen econd orecond us, of all of us, an movean estricted s fromds flp to-flop s flip-ts of theof ads of of ads s con- n thein ecules s (see (see s oper- pro- ., pass ane, 141

Cell Exterior has played an important role in cell biology and is currently Transverse diffusion (flip-flop) used in an invaluable technique to prepare speciﬁc antibodies 5 (~10 sec) (Section 18.18). The ﬁrst experiments to demonstrate that membrane proteins could move within the plane of the membrane utilized cell fusion, and they were reported in 1970 by Larry Frye

-- 9 Flex (~10 sec) and Michael Edidin of Johns Hopkins University. In their experiments, mouse and human cells were fused, and the locations of specific proteins of the plasma membrane were followed once the two membranes had become continuous. To follow the distribution of either the mouse membrane pro- Lateral shift -- 6 teins or the human membrane proteins at various times after (~10 sec) Cytosol fusion, antibodies against one or the other type of protein were prepared and covalently linked to fluorescent dyes. The antibodies against the mouse proteins were complexed with a Figure 4.25 The possible movements of phospholipids in a membrane. The types of movements in which membrane dye that fluoresces green and the antibodies against human phospholipids can engage and the approximate time scales over which proteins with one that fluoresces red. When the antibodies they occur. Whereas phospholipids move from one leaflet to another at were added to fused cells, they bound to the human or mouse a very slow rate, they diffuse laterally within a leaflet rapidly. Lipids proteins and could be located under a fluorescence light mi- lacking polar groups, such as cholesterol, can move across the bilayer croscope (Figure 4.26 a). At the time of fusion, the plasma quite rapidly. membrane appeared half human and half mouse; that is, the two protein types remained segregated in their own hemisphere (step 3, Figure 4.26 a,b). As the time after fusion increased, the membrane proteins were seen to move laterally The Diffusion of Membrane within the membrane into the opposite hemisphere. By about Proteins after Cell Fusion 40 minutes, each species’ proteins were uniformly distributed Cell fusion is a technique whereby two different types of cells, around the entire hybrid cell membrane (step 4, Figure 4.26 a). or cells from two different species, can be fused to produce one If the same experiment was performed at lower temperature, cell with a common cytoplasm and a single, continuous the viscosity of the lipid bilayer increased, and the mobility of plasma membrane. Cells are induced to fuse with one another the membrane proteins decreased. These early cell fusion ex- by making the outer surface of the cells “sticky” so that their periments gave the impression that integral membrane pro- plasma membranes adhere to one another. Cells can be teins were capable of virtually unrestricted movements. As we induced to fuse by addition of certain inactivated viruses that will see shortly, subsequent studies made it apparent that attach to the surface membrane, by adding the compound membrane dynamics was a much more complex subject than polyethylene glycol, or by a mild electric shock. Cell fusion first envisioned.

1 2 3 4 Human cell Addition of 40 sendai minutes 4.6 (fusing) virus MembranePlasma the of Nature Dynamic The

Mouse cell (a) (b)

Figure 4.26 The use of cell fusion to reveal mobility of membrane respectively. ( b) Micrograph showing a fused cell in which mouse and proteins. (a) Outline of the experiment in which human and mouse human proteins are still in their respective hemispheres (equivalent to cells were fused (steps 1–2) and the distribution of the proteins on the the hybrid in step 3 of part a). ( B: F ROM L. D. F RYE AND MICHAEL surface of each cell were followed in the hybrids over time (steps 3–4). EDIDIN , J. C ELL SCIENCE 7:328, 334, 1970; BY PERMISSION OF THE Mouse membrane proteins are indicated by solid circles, human mem- COMPANY OF BIOLOGISTS , L TD . C OURTESY OF MICHAEL EDIDIN , brane proteins by open circles. Locations of human and mouse proteins JOHNS HOPKINS UNIVERSITY . http://jcs.biologists.org/content/7/2/319. in the plasma membranes of the hybrid cells were monitored by full.pdf+html?sid=d93ae648-abca-4f5d-90a6-a6d9726d7d30) interaction with fluorescent red and fluorescent green antibodies, Chapter 4 The Structure and Function of the Plasma Membrane 142 netgtd For example, protei investigated. particular the on depend result often The studies these 18.1). (Section microscopy video enhanced an movements diameter), of the in labeled molecules nm are followed 40 by (approximately particles gold antibody with usually labeled, are molecules protein et o tee oeue sol poue gradual a 4.27 (Figure produce recovery fluorescence should of molecules circl irradiated the in fluorescence these of reappearance of mo random ments the then mobile, are membrane the in teins l the If fluorescence. of devoid largely is that cell lar spot (typically about 1 about (typically spot lar bea laser leavi focused fluorescentthe path, bleaches its inmolecules sharply a by irradiated and scope cells are placed under t Once labeled, cent antibody. protein such can a be labeled using a specific probe, m particular A dye. fluorescent a to linkage by beled are cells cultured in components membrane integral ficient, measure of the rate of diffusion (expressed as a di that are free to diffuse. providesmeasurea percentagetheof labeledtheof original the of percentage a as (expressed recovery photobleaching (FRAP) In a technique called microscope. using cells living of membranes the in molecules of techniques allow Several researchers to follow the movements Restrictions on Protein and Lipid Mobility I I distances and to determine how they might be restra the movements of individual protein molecules over researcherst allow that developed been have niques altern limitations, Tothesearound get allowed. time dista limited a over diffuse only can that ones and truly are that proteins between distinguish cannot large 1 distance (e.g., ecules(hundreds to thousands) as they diffuse over average movement of a relatively large number of lab FRAP FRAPtechnique can hasonly itsfoll drawbacks. ( and werenotfree todiffuse backinto theirradiated bilayer c lipid pure 70 to (30 proteins a membrane of fraction significant in would they than brane m plasma a in slowly more much moved proteins brane ficient have been debated. of this group.) The reasons for the reduced diffusi ob moiie Fgr .8 protein B). 4.28, (Figure to be immobilized proteins membrane fail to moveSome and are conside coefficients of approximately 10 one would expect proteins with to diffmigrate size, and as lipid viscosity such parameters on physical were (If protein based mobility ficial lipid bilayer. less than would be measured in a rates considerably a though generally protein A), 4.28, (Figure membrane proteins membrane throughout move the Some randomly than 10 al suis tlzn FA sgetd ht 1 mem (1) that suggested FRAP utilizing studies Early In single-particle tracking (SPT) tracking single-particle D ftembl oeue.The ) extentof ofthe fluorescencemobile molecules. Ϫ 10 to 10 Ϫ ␮ 12 ) sarsl,researchers As a usingresult, m). FRAP cm , which which is illustrated in Figure 4.27, ␮ 2 /sec, as is observed for molecules as is observed /sec, m diameter) on the surface of theof surface the on diameter) m fluorescence recovery afterfluorescence recovery Ϫ 8 to 10 , individual membrane individual , b poie a direct a provides ) Ϫ 9 cm rl.Butthe ircle. ffusion coef- 2 /sec rather abeled pro-abeled a relatively e. The rate The e. s a fluores- ng a circu-a ng molecules computer- intensity) nce in thein nce very very short he micro- strictly ined. o observeo eled mol- immobile the light the on coef- protein ate tech-ate embrane percent) n being being n n arti- usion -coated rt la-first that m ow the the d red ) a 2) em- of s t ve- a - , ( eedn ntepoens en olwd The rate depending on the protein(s) being followed. ( o followed is region bleached the in fluorescence of 2 (step molecules dye the bleach to irradiated then regi A small labeled with a fluorescent dye (step 1). I related to the diffusion coefficient of the fluoresce b a ) The rate of fluorescence recovery within the illum nti ehiu,a particular component of the) Inm this technique, elo nte Fgr .8 rti ) For example protein C). 4.28, or another (Figure cell nonrandom) manner toward of t one part rected (i.e., a protein is found to move in a highl In some cases, proteins by fluorescence afterrecovery photobleachi Figure 4.27 (b) (a) Fluorescence Illuminate Measuring the diffusion rates of membrane membrane of rates diffusion the Measuring l 3 2 1 Time Recovery with laserbeam Photobleach spot fluorescent dye Label proteins with ntly labeled protein.ntly on of the surface ison of the surface ), and the recovery the and ), ver time (step 3). (step time ver inated spot can vary of recovery is of recovery embrane is first ng (FRAP). y di- a, he 143 brane skeleton,” consisting of peripheral proteins situated on the cytoplasmic surface of the membrane. A certain proportion of a membrane’s integral protein molecules are either tethered A to the membrane skeleton (Figure 4.28, protein B) or other- D E wise restricted by it. B Information concerning the presence of membrane barriers has been obtained using an innovative technique that allows investigators to trap integral proteins and drag them through the plasma membrane with a known force. This technique, which uses an apparatus referred to as optical tweezers , C takes advantage of the tiny optical forces that are generated by a focused laser beam. The integral proteins to be studied are tagged with antibody-coated beads, which serve as handles that can be gripped by the laser beam. It is generally found F that optical tweezers can drag an integral protein for a limited distance before the protein encounters a barrier that causes it to be released from the laser’s grip. As it is released, the protein typically springs backward, suggesting that the barriers Figure 4.28 Patterns of movement of integral membrane proteins. are elastic structures. Depending on the cell type and the conditions, integral membrane One approach to studying factors that affect membrane proteins can exhibit several different types of mobility. Protein A is protein mobility is to genetically modify cells so that they pro- capable of diffusing randomly throughout the membrane, though its duce altered membrane proteins. Integral proteins whose cy- rate of movement may be limited; protein B is immobilized as the toplasmic portions have been genetically deleted often move result of its interaction with the underlying membrane skeleton; much greater distances than their intact counterparts, indicat- protein C is being moved in a particular direction as the result of its ing that barriers reside on the cytoplasmic side of the mem- interaction with a motor protein at the cytoplasmic surface of the brane. These findings suggest that the membrane’s underlying membrane; movement of protein D is restricted by other integral skeleton forms a network of “fences” around portions of the proteins of the membrane; movement of protein E is restricted by membrane, creating compartments that restrict the distance fences formed by proteins of the membrane skeleton, but it can hop an integral protein can travel (Figure 4.28, protein E). Pro- into adjacent compartments through transient openings in a fence; movement of protein F is restrained by extracellular materials. teins move across the boundaries from one compartment to another through breaks in the fences. Such openings are thought to appear and disappear along with the dynamic dis- assembly and reassembly of parts of the meshwork. Mem- brane compartments may keep specific combinations of particular membrane protein may tend to move toward the proteins in close enough proximity to facilitate their interaction. leading or the trailing edge of a moving cell. Integral proteins lacking that portion that would nor- I In most studies, the largest fraction of protein species ex- mally project into the extracellular space typically move at a hibit random (Brownian) movement within the membrane much faster rate than the wild-type version of the protein. at rates consistent with free diffusion (diffusion coeffi- This finding suggests that the movement of a transmembrane ϫ Ϫ9 2 protein through the bilayer is slowed by extracellular materials cients about 5 10 cm /sec), but the molecules are un- 4.6 able to migrate freely more than a few tenths of a that can entangle the external portion of the protein molecule micrometer. Long-range diffusion occurs but at slower (Figure 4.28, protein F). MembranePlasma the of Nature Dynamic The rates, apparently because of the presence of a system of barriers. These barriers are discussed in the following Membrane Lipid Mobility Proteins are huge molecules, so sections. it isn’t surprising that their movement within the lipid bilayer might be restricted. Phospholipids, by comparison, are small Control of Membrane Protein Mobility It is apparent molecules that make up the very fabric of the lipid bilayer. that plasma membrane proteins are not totally free to drift One might expect their movements to be completely unfet- around randomly on the lipid “sea.” Instead, this class of pro- tered, yet a number of studies have suggested that phospholipid teins is subjected to various influences that affect their mobility diffusion is also restricted. When individual phospholipid and, in doing so, promote intramembrane organization. Some molecules of a plasma membrane are tagged and followed un- membranes are crowded with proteins, so that the random der the microscope using ultra high-speed cameras, they are movements of one molecule can be impeded by its neighbors seen to be confined for very brief periods and then hop from (Figure 4.28, protein D). The strongest influences on an inte- one confined area to another. Figure 4.29 a shows the path gral membrane protein are thought to be exerted from just taken by an individual phospholipid within the plasma mem- beneath the membrane on its cytoplasmic face. The plasma brane over a period of 56 milliseconds. Computer analysis membranes of many cells possess a fibrillar network, or “mem- indicates that the phospholipid diffuses freely within one Chapter 4 The Structure and Function of the Plasma Membrane 144 fake compartments werefake compartments assigned in For the sake of unconfined Brownian movement. simple, now of the phospholipid can be extended trajectory The m present membrane. in a cellular “picket fences” (F are embedded in the underlying soil.are embedded in the underlying unlike not is whos fence picket This a by cows or horses of confinement skeleton. membrane the to tached domains cytoplasmic whoseproteins membrane tegral studies speculate that the fences are constructed o o authors movement? The phospholipid with interfere ho bilayer, lipid the beneath lies skeleton membrane diffusion. phospholipid restrict that fences the of that disrupt the membrane underlying skeleton remov Treatmentwith membrane the of forth. so and green), (sh compartment adjacent an into fence another over and blue) in (shaded compartment neighboring a into the jumps it before purple) in (shaded compartment OF PERMISSION WITH K cially cially in cells whose various surfaces display dist mobi and composition protein in variations distinct e however, of Most membranes, surface residing lower on a culture dish. or upper the at situated membrane homogeneous relatively the on out carried are above, dis those as such dynamics, membrane of studies part, Polarity Cell and Domains Membrane eti ersne yasnl oo.(ment is represented by a single color. The movement of the phospholipid within ea movement. appears slowed the molecule must because hop a barr of diffusion of rate t the overall However, movement. as that expected is by as unhinde rapid compartment of diffusio The rate compartment. into a neighboring lipids diffuse withinfreely a confined compartment as it diffuses withinof a rat the plasma membrane of a single labeled phospholipid unsaturated track in USUMI ROM (a) a Finish scridotfr3 si natfiilblyr wh bilayer, out for 33 ms in an artificial is carried Figure 4.29 phospholipids within the plasma membrane is confined T N, AK AHIR O AGOY A .C J. , F Experimental demonstration that diffusion of diffusion that demonstration Experimental JWR TAL ET UJIWARA T ELL HE B R IOL OCKEFELLER Start 5:03 02 R 2002; 157:1073, . b ., b and indicated by differentand indicated colors. ) shownThe same experiment OREYOFCOURTESY (b) Start U NIVERSITY is followed for 56 ms bols.Phospho-fibroblast. before hopping explained by red Brownian he phospholipid ier to continue its n within a ich lacks the lacks ich EPRODUCED uch moreuch open, A inct functions. o te most the For KIHIRO ch compart-ch comparison, f rows of in- P u i the if But RESS Finish iy espe-lity, w can it can w plasma es some dd inaded . “fence” e postse f thesef ( areat- cussed agents xhibit a .) a cell cell a then ) The the ebae f h priua poen nie recogniz 4.31).that antibody (Figure antigen protein particular the of membrane graphic pattern that reflects the distribution withi tibody combines with the of surface the cell in a u antibodies of variety a with treated are sperm when Fo a mosaic of differentof domains. types localized cons techniques, numerous by revealed as which, brane itntprs a sperm is covered by a distinct parts, itshaving each Although divided into a n own specialized functions. tail, and midpiece, head, into divided be can A mat have the most highly differentiated structure. (see F to facilitate their internalization cialized membran plasma the of patches into concentrated are lip low-density for receptors and 4.57), Figure (see into regions within located of the plasma membrane the receptors for neurotransmitter substances are c cellular substrate (a substrate cellular which adheres to an or underlyin the basal membrane, epithelia neighboring with interacts which membrane, a possesses different the enzymes than the the lumen, late that indicate substance absorbs selectively which Studies membrane, plasma 4.30). (Figure out functions carry surfaces different h whose are cells kidney polarized the of tubules microscopic the up make the epithelial cells that line the inte For example, Figure 4.30 cell with the underlying basement membrane. with the underlying cell containsproteins integral surface that function in proteinsintegral that function in intercellular in the later as sucrose such and lactose; disaccharides, tains integral proteins that function in ion transp epithelial cell. gradients • generationofion contact • cell-substratum membrane Basal • cellcommunication adhesion • cellcontactand membrane Lateral plasma • protection • regulatedsecretion andwaterintake • regulation ofnutrient membrane Apical plasma Of all the various types of mammalian cells, sperm m sperm cells, mammalian of types various the all Of Differentiated functions of the plasma membrane of of the plasma membrane functions Differentiated The apical surface of this intestinal epithelial ce surface The apical basement membranebasement Glucose Na continuous + ). In other examples,other In ). eato;and the basalteraction; Monosaccharide ort and hydrolysis ofort the association of the al surface containsal surface + igure 8.38). plasma mem- stinal wall or n the plasma oncentrated nique topo- r example, ral ral plasma , each an- each , ure sperm Disaccharide oproteins umber of different synapses g extra- ll con-ll s froms l cells,l e spe- e ists ofists d by ed ighly ighly pical an ay 145 sodium dodecyl sulfate (SDS). (The technique of SDS–PAGE is discussed in Section 18.7.) The SDS keeps the integral proteins soluble and, in addition, adds a large number of negative charges to the proteins with which it associates. Because the number of charged SDS molecules per unit weight of protein tends to be relatively constant, the molecules separate from one another according to their molecular weight. The largest (a) (b) proteins move most slowly through the molecular sieve of the gel. The major proteins of the erythrocyte membrane are sep- Anterior head arated into about a dozen conspicuous bands by SDS–PAGE Posterior head (Figure 4.32 c). Among the proteins are a variety of enzymes (including glyceraldehyde 3-phosphate dehydrogenase, one of the enzymes of glycolysis), transport proteins (for ions and sugars), and skeletal proteins (e.g., spectrin). Posterior tail Integral Proteins of the Erythrocyte Membrane A model of the erythrocyte plasma membrane showing its major (c) (d) proteins is seen in Figure 4.32 d. The most abundant integral proteins of this membrane are a pair of carbohydrate-containing, Figure 4.31 Differentiation of the mammalian sperm plasma membrane-spanning proteins, called band 3 and glycophorin membrane as revealed by fluorescent antibodies. (a–c ) Three pairs of A. The high density of these proteins within the membrane is micrographs, each showing the distribution of a particular protein at evident in the freeze-fracture micrographs of Figure 4.15. the cell surface as revealed by a bound fluorescent antibody. The three Band 3, which gets its name from its position in an elec- proteins are localized in different parts of the continuous sperm trophoretic gel (Figure 4.32 c), is present as a dimer composed membrane. Each pair of photographs shows the fluoresence pattern of of two identical subunits (a homodimer ). Each subunit spans the bound antibody and a phase contrast micrograph of the same cell. the membrane at least a dozen times and contains a relatively (d ) Diagram summarizing the distribution of the proteins. ( A–C: FROM DIANA G. M YLES , P AUL PRIMAKOFF , AND ANTHONY small amount of carbohydrate (6–8 percent of the molecule’s R. B ELLVÉ , C ELL 23:434, 1981 © WITH PERMISSION FROM weight). Band 3 protein serves as a channel for the passive ex- ELSEVIER .) change of anions across the membrane. As blood circulates through the tissues, carbon dioxide becomes dissolved in the fluid of the bloodstream (the plasma) and undergoes the fol- The Red Blood Cell: An Example lowing reaction: S S Ϫ ϩ of Plasma Membrane Structure H2O ϩ CO 2 H2CO 3 HCO 3 ϩ H Ϫ Of all the diverse types of membranes, the plasma membrane The bicarbonate ions (HCO 3 ) enter the erythrocyte in ex- of the human erythrocyte (red blood cell) is the most studied change for chloride ions, which leave the cell. In the lungs, and best understood (Figure 4.32). There are several reasons where carbon dioxide is released, the reaction is reversed and for the popularity of this membrane. The cells are inexpensive bicarbonate ions leave the erythrocyte in exchange for chlo- Ϫ Ϫ to obtain and readily available in huge numbers from whole ride ions. The reciprocal movement of HCO 3 and Cl blood. They are already present as single cells and need not be occurs through a channel in the center of each band 3 dimer. dissociated from a complex tissue. The cells are simple by Glycophorin A was the first membrane protein to have its 4.6 comparison with other cell types, lacking nuclear and cyto- amino acid sequence determined. The arrangement of the MembranePlasma the of Nature Dynamic The plasmic membranes that inevitably contaminate plasma mem- polypeptide chain of glycophorin A within the plasma membrane preparations from other cells. In addition, purified, brane is shown in Figure 4.18. (Other related glycophorins, B, intact erythrocyte plasma membranes can be obtained simply C, D, and E, are also present in the membrane at much lower by placing the cells in a dilute (hypotonic) salt solution. The concentrations.) Like band 3, glycophorin A is also present in cells respond to this osmotic shock by taking up water and the membrane as a dimer. Unlike band 3, each glycophorin A swelling, a phenomenon termed hemolysis . As the surface area subunit spans the membrane only once, and it contains a of each cell increases, the cell becomes leaky, and the con- bushy carbohydrate cover consisting of 16 oligosaccharide tents, composed almost totally of dissolved hemoglobin, flow chains that together make up about 60 percent of the mole- out of the cell leaving behind a plasma membrane “ghost” cule’s weight. It is thought that the primary function of gly- (Figure 4.32 b). cophorin derives from the large number of negative charges Once erythrocyte plasma membranes are isolated, the borne on sialic acid, the sugar residue at the end of each car- proteins can be solubilized and separated from one another bohydrate chain. Because of these charges, red blood cells re- (fractionated), providing a better idea of the diversity of pel each other, which prevents the cells from clumping as they proteins within the membrane. Fractionation of membrane circulate through the body’s tiny vessels. It is noteworthy that proteins can be accomplished using polyacrylamide gel persons who lack both glycophorin A and B in their red blood electrophoresis (PAGE) in the presence of the ionic detergent cells show no ill-effects from their absence. At the same time, Chapter 4 The Structure and Function of the Plasma Membrane 146 (e) (a) (d) Tropomyosin Actin (b) B V arrangement of the proteins of the inner membrane s membrane inner the of proteins the of arrangement Figure 4.32 Figure A S HF. M M. M. irgaho ua rtrcts ( erythrocytes. human of micrograph . rti tblzsatnseti opee.( complexes. actin–spectrin stabilizes protein 4.1 dime 3 band The skeleton. internal membrane’s the up arra the and bilayer lipid the in embedded proteins the from viewed as membrane plasma erythrocyte the were isolated by allowing erythrocytes to swell and swell to erythrocytes allowing by isolated were teins of the erythrocyte membrane, which are identifi are which membrane, erythrocyte the of teins ( electrophoresis gel SDS–polyacrylamide of results HIH , IOCHEM OET Band 3 E : OFFMAN ERDCDWT EMSINOF PERMISSION WITH REPRODUCED -C B, HUN IOCHEMISTRY OREL . VOL ; L The plasma membrane of the human erythrocyte. human the of membrane plasma The C R, IU . 45; © 1976 1976 © 45; . : FROM L, ICHARD AURA 2, .T M T. V. EDD .DH. .BF. BY .; AKER Glycophorin A ARCHESI ERICK A COPYRIGHT NNUAL Ankyrin b , irgahsoigpam ebaegot,whichghosts, membrane plasma showing Micrograph ) AND , AND .F H. , R H T EVIEWS J HE AROLD IRI 95 J1995, © Band 4.1 URTHMAYR e Spectrin R P ) Electron micrograph showing the the showing micrograph Electron ) ALEK OCKEFELLER hmlz sdsrbdi h et (text. the in described as hemolyze ngement of peripheral proteins that make that proteins peripheral of ngement α SDS–PAGE) used to fractionate the pro- the fractionate to used SDS–PAGE) Band 4.1 G3PD Actin Band 4.2 Band 4.1 Anykrin INTEGRAL Spectrin PERIPHERAL β I eeo.( keleton. da h ie ftegl (gel. the of sides the at ed W NC internal surface, showing the integral the showing surface, internal r shown here is simplified. The band The simplified. is here shown r OHN AYLAND .C J. , .; D , : AND W AFTER ELL A ILEY C: ; U .TM. B B ( a NIVERSITY : OURTESY IOL (c) ) Scanning electron Scanning ) OREYOFCOURTESY .VD. & S & OMITA 0:2,1987; 104:527, . ONS E ANDOET Band 3 Minor Major Band 7 Glycophorin A I, F A, P RANÇOIS NC d RESS NNU ) A model of model A ) .; J .G. J. OSEPH E .) R. : c AChe FROM ATPase ) ) The EV . 147 the band 3 proteins in these individuals are more heavily gly- sible for causing muscular dystrophy, a devastating disease cosylated, which apparently compensates for the otherwise that cripples and kills children. As in the case of cystic fibrosis missing negative charges required to prevent cell–cell interac- (page 163), the most debilitating mutations are ones that lead tion. Glycophorin also happens to be the receptor utilized by to a complete absence of the protein in the cell. The plasma the protozoan that causes malaria, providing a path for entry membranes of muscle cells lacking dystrophin are apparently into the blood cell. Consequently, individuals whose erythro- destroyed as a consequence of the mechanical stress exerted cytes lack glycophorin A and B are thought to be protected on them as the muscle contracts. As a result, the muscle cells from acquiring malaria. Differences in glycophorin amino die and eventually are no longer replaced. acid sequence determine whether a person has an MM, MN, or NN blood type. R E V I E W 1. Describe two techniques to measure the rates of diffu- The Erythrocyte Membrane Skeleton The peripheral sion of a specific membrane protein. proteins of the erythrocyte plasma membrane are located on its internal surface and constitute a fibrillar membrane skele- 2. Compare and contrast the types of protein mobility ton (Figure 4.32 d,e) that plays a major role in determining the depicted in Figure 4.28. biconcave shape of the erythrocyte. As discussed on page 143, 3. Discuss two major functions of the integral and periph- the membrane skeleton can establish domains within the eral proteins of the erythrocyte membrane. membrane that enclose particular groups of membrane pro- 4. Compare the rate of lateral diffusion of a lipid with that teins and may greatly restrict the movement of these proteins. of flip-flop. What is the reason for the difference? The major component of the skeleton is an elongated fibrous protein, called spectrin . Spectrin is a heterodimer approximately 100 nm long, consisting of an ␣ and ␤ subunit that curl around one another. Two such dimeric molecules are linked at 4.7 | The Movement of Substances their head ends to form a 200-nm-long filament that is both flexible and elastic. Spectrin is attached to the internal surface Across Cell Membranes of the membrane by means of noncovalent bonds to another Because the contents of a cell are com- peripheral protein, ankyrin (the green spheres of Figure pletely surrounded by its plasma mem- 4.32d), which in turn is linked noncovalently to the cytoplas- brane, all communication between the cell mic domain of a band 3 molecule. As is evident in Figures and the extracellular medium must be mediated by this struc- 4.32d and e, spectrin filaments are organized into hexagonal or ture. In a sense, the plasma membrane has a dual function. On pentagonal arrays. This two-dimensional network is con- one hand, it must retain the dissolved materials of the cell so structed by linking both ends of each spectrin filament to a that they do not simply leak out into the environment, while on cluster of proteins that include a short filament of actin and the other hand, it must allow the necessary exchange of materi- tropomyosin , proteins typically involved in contractile activi- als into and out of the cell. The lipid bilayer of the membrane is ties. A number of genetic diseases ( hemolytic anemias ) charac- ideally suited to prevent the loss of charged and polar solutes terized by fragile, abnormally shaped erythrocytes have been from a cell. Consequently, some special provision must be made traced to mutations in ankyrin or spectrin. to allow the movement of nutrients, ions, waste products, and 4.7 If the peripheral proteins are removed from erythrocyte other compounds, in and out of the cell. There are basically two ghosts, the membrane becomes fragmented into small vesi- means for the movement of substances through a membrane: MembranesCell Across Substances of Movement The cles, indicating that the inner protein network is required to passively by diffusion or actively by an energy-coupled trans- maintain the integrity of the membrane. Erythrocytes are cir- port process. Both types of movements lead to the net flux of a culating cells that are squeezed under pressure through micro- particular ion or compound.The term net flux indicates that the scopic capillaries whose diameter is considerably less than that movement of the substance into the cell ( influx ) and out of the of the erythrocytes themselves. To traverse these narrow pas- cell ( efflux ) is not balanced, but that one exceeds the other. sageways, and to do so day after day, the red blood cell must be Several different processes are known by which sub- highly deformable, durable, and capable of withstanding stances move across membranes: simple diffusion through the shearing forces that tend to pull it apart. The spectrin–actin lipid bilayer; simple diffusion through an aqueous, protein- network gives the cell the strength, elasticity, and pliability lined channel; diffusion that is facilitated by a protein trans- necessary to carry out its demanding function. porter; and active transport, which requires an energy-driven When first discovered, the membrane skeleton of the ery- protein “pump” capable of moving substances against a concen- throcyte was thought to be a unique structure suited to the tration gradient (Figure 4.33). We will consider each in turn, unique shape and mechanical needs of this cell type. However, but first we will discuss the energetics of solute movement. as other cells were examined, similar types of membrane skeletons containing members of the spectrin and ankyrin The Energetics of Solute Movement families have been revealed, indicating that inner membrane skeletons are widespread. Dystrophin, for example, is a mem- Diffusion is a spontaneous process in which a substance ber of the spectrin family that is found in the membrane moves from a region of high concentration to a region of low skeleton of muscle cells. Mutations in dystrophin are respon- concentration, eventually eliminating the concentration Chapter 4 The Structure and Function of the Plasma Membrane 148 lasfo iht o ocnrto.( from high to lowalways concentration. constant, rdet (gradient. Movement occurs against a co as ATPsuch hydrolysis. released by an with energy driven change in affinity with a specific binding sit of a protein transporter rti are afclttv rnpre) As in (a facilitative transporter). protein carrier solute moleculesdiffusion bind in which specificall prot of such protein membrane integral or a cluster oeeti lasdw ocnrto rdet ( downmovement a concentration gradient. is always where into lowing relationship describes the movement of a non concentrationence the in membrane.theeachsideofon is, that gradient, concentration the of magnitude depends membrane a across diffuses nonelectrolyte) stances across membranes. diffus to discussion following the restrict We will i increase an by driven process exergonic an is and s of motion thermal random the on depends diffusion on discussed As regions. two the between difference of an erythrocyte.membrane (directions of the concentration gradients. acrossmove membranes. Figure 4.33 ( proceeds from high to always low which the bilayer, b ) Simple diffusion through forme an aqueous channel ) Simple h fe-nry hne hn n nhre slt (a solute uncharged an when change free-energy The the cell: (e) ⌬ G e ) Examples of each type of mechanism as it occurs i occurs it as mechanism of type each of Examples ) O T C B A is the free-energy change (Section 3.1), 2 Nonmediated Four solute molecules which by basic mechanisms a b c (d) (c) (b) (a) O is the absolute temperature, and [C and temperature, absolute the is C B A 2 ¢ ¢ G G Na Na Passive ϭ ϭ + + The relative sizes of the letters indicate the of the letters indicate The relative sizes RT 2.303 ln Glucose [C RT [C Glucose Transporter mediated o i ] log ] d ) Active transport by means) Active transport a ) Simple diffusion through) Simple 10 [C [C a ATP ATP and o e that undergoes a i Na ] ] y to a membrane is As in eins. Na exergonic process, Active concentration. c + + b ) Facilitated D D movement is, ncentration ncentration d within an K i + ]/[C K ADP ADP+P R ion of sub-of ion + P + n entropy.n electrolyte i is the gas page 88,page The fol-The o a n n the differ- ] is theis ] , n theon i olutes tration ratios becomes ratios tration [C f term the movementfor cell, the ofsolute out a of rdet erae,te trd nry s dissipated, is energy stored the decreases, gradient conc the cell, themoves intosolute As st kcal/mol. 1.4 a represents gradient concentration tenfold a of decreases until, at equilibrium, at until, decreases usd o ufcso h ebae At 25 of the membrane. outside (o) surfaces ins the on solute the of concentration the of ratio If the ratio of [Cof ratio the If about i cell a of membrane the across voltage the Because c the in than cell the outside concentration higher conditions would be movement of a mole of Naof mole a of movement fteeetoyehvn n oeo hre,and charge), of mole one having electrolyte the of (23.06 kcal/V (23.06 kcal/V where isthe cell The free-energy change for the diffusion of an elec differences are combined to form an Togeth charge. in difference the by determined dient, sub the and between the of the electric two poten compartments, difference concentration the by termined a chemical gradi partments depends on two gradients: t between diffuse to electrolyte an of tendency the the greater the difference in free ene compartments, betwee voltage) or difference potential (the charge differ the greater The favored. thermodynamically is the moving, is it which into compartment the to sign is electrolyte the of charge the if Conversely, sign. the of charge net a having compartment another into compartm one from membrane a across move to trolyte fo unfavorable thermodynamically is it charges, like o repulsion mutual the of result a As considered. be all charge difference between the two compartments xenl ocnrto o slt i 1 tms h in the times 10 is concentration, solute of concentration exampl external for If, (exergonic). favored thermodynamically ratio ratio is negative, gradient consisted of Na generates a generates concentration of a nonelectrolyte across a membrane a across nonelectrolyte a of concentration indifference tenfold a that example previous the in saw We potential difference (in volts) between the two com

¢ ¢ G G

If the solute is an electrolyte (a charged species) Ϫϭ Ϫϭ Ϫϭ Ϫ z is the charge of the solute,the of charge the is 0 V pg 15,te reeeg cag fr the for change free-energy the 165), (page mV 70 1.4 3.1 kcal/mol 1.4 ⌬

kcal/mol kcal/mol G ؒ ⌬ ¢ qiaet where an equivalent is the amountequivalent, of ¢ G G ⌬ i G ]/[C G Ϫ Ϫϭ ϭ ϭ 1.4 kcal/mol. Suppose the concentrationthe Suppose kcal/mol. 1.4 sngtv,and the net inﬂux of solute isis negative, 1.4 kcal/mol o ϩ ϩ RT ] is less than 1.0, then the log of the of log the then 1.0, than less is ] . clml.Tu,the maintenance Thus, 1.4 kcal/mole. ϩ o (1)(23.06 kcal/V(1)(23.06 zF ]/[C ions, which which were present at tenfoldions, ln ϩ ¢ [C [C E i ions into the cell under theseunder cell the into ions ]). ⌬ m o i ] ] G ϩ # electrochemical gradient F szr.(To calculate zero. is log zF is the Faradayconstantthe is 10 ¢

[C [C E # m Њ mol) ( mol) o C, i ] ] opposite inopposite trolyte trolyte into ⌬ or concen-or partments. ide (i) and(i) ide Ϫ the over-, s typicallys n the twothe n g.Thus,rgy. ytoplasm. r an elec-an r must also E o com-wo entration orage of of orage f ions ofions f at 25at 0.07 V) and and process tial gra- er theseer n,de-ent, m ne inence stance same ternal ternal ,thee, is theis ⌬ ⌬ ent Њ C G G . 149

Thus under these conditions, the concentration difference and 10 - 3 the electric potential make similar contributions to the storage

- 4 of free energy across the membrane. 10 ~ * Z The interplay between concentration and potential dif- Y ϩ W X ferences is seen in the diffusion of potassium ions (K ) out of - 5 ϩ 10 T U V a cell. The efﬂux of the ion is favored by the K concentration S ϩ R gradient, which has a higher K concentration inside the cell, P Q O 10 - 6 N but hindered by the electrical gradient that its diffusion cre- K M L I J ates, which leaves a higher negative charge inside the cell. We H G C E F will discuss this subject further when we consider the topic of 10 -7 D membrane potentials and nerve impulses in Section 4.8. permeability (cm / sec) Cerebrovascular A B

10 - 8 - 1 10 - 4 10 - 3 10 - 2 10 1 10 10 0 10 00 Diffusion of Substances through Membranes Octanol - water partition coefficient Two qualifications must be met before a nonelectrolyte can A. Sucrose J. Vinblastine S. Misonidazole B. Epipodophyllotoxin K. Curare T. Propylene glycol diffuse passively across a plasma membrane. The substance C. Mannitol L. Thiourea U. Metronidazole D. Arabinose M. Dianhydrogalacticol V. Spirohydantoin mustard must be present at higher concentration on one side of the E. N-methyl nicotinamide N. Glycerol W. Procarbazine membrane than the other, and the membrane must be perme- F. Methotrexate O. 5-FU X. PCNU G. Vincristine P. Ethylene glycol Y. Antipyrine able to the substance. A membrane may be permeable to a H. Urea Q. Acetamide Z. Caffeine I. Formamide R. Ftorafur ~. BCNU given solute either (1) because that solute can pass directly *. CCNU through the lipid bilayer, or (2) because that solute can traverse an aqueous pore that spans the membrane. Let us begin Figure 4.34 The relationship between partition coefficient and by considering the former route in which a substance must membrane permeability. In this case, measurements were made of dissolve in the lipid bilayer on its way through the membrane. the penetration of a variety of chemicals and drugs across the plasma Discussion of simple diffusion leads us to consider the membranes of the cells that line the capillaries of the brain. Substances penetrate by passage through the lipid bilayer of these cells. The polarity of a solute. One simple measure of the polarity (or partition coefficient is expressed as the ratio of solubility of a solute in nonpolarity) of a substance is its partition coefficient , which octanol to its solubility in water. Permeability is expressed as is the ratio of its solubility in a nonpolar solvent, such as oc- penetrance (P) in cm/sec. For all but a few compounds, such as tanol or a vegetable oil, to that in water under conditions vinblastine and vincristine, penetrance is directly proportional to lipid where the nonpolar solvent and water are mixed together. Fig- solubility. (F ROM N. J. A BBOTT AND I. A. R OMERO , M OLEC . M ED . ure 4.34 shows the relationship between partition coefficient TODAY , 2:110, 1996; C OPYRIGHT 1996, WITH PERMISSION FROM and membrane permeability of a variety of chemicals and ELSEVIER SCIENCE .) drugs. It is evident that the greater the lipid solubility, the faster the penetration. Another factor determining the rate of penetration of a compound through a membrane is its size. If two molecules have approximately equivalent partition coefficients, the than do dissolved ions or small polar organic solutes, which 4.7 smaller molecule tends to penetrate the lipid bilayer of a are essentially nonpenetrating. Because of this difference in membrane more rapidly than the larger one. Very small, un- the penetrability of water versus solutes, membranes are said MembranesCell Across Substances of Movement The charged molecules penetrate very rapidly through cellular to be semipermeable . Water moves readily through a semi- membranes. Consequently, membranes are highly permeable permeable membrane from a region of lower solute concentra- to small inorganic molecules, such as O 2, CO 2, NO, and tion to a region of higher solute concentration. This process is H2O, which are thought to slip between adjacent phospho- called osmosis , and it is readily demonstrated by placing a cell lipids. In contrast, larger polar molecules, such as sugars, into a solution containing a nonpenetrating solute at a con- amino acids, and phosphorylated intermediates, exhibit poor centration different than that present within the cell itself. membrane penetrability. As a result, the lipid bilayer of the When two compartments of different solute concentra- plasma membrane provides an effective barrier that keeps tion are separated by a semipermeable membrane, the com- these essential metabolites from diffusing out of the cell. partment of higher solute concentration is said to be Some of these molecules (e.g., sugars and amino acids) must hypertonic (or hyperosmotic ) relative to the compartment of enter cells from the bloodstream, but they cannot do so by lower solute concentration, which is described as being hypo- simple diffusion. Instead, special mechanisms must be avail- tonic (or hypoosmotic ). When a cell is placed into a hypo- able to mediate their penetration through the plasma mem- tonic solution, the cell rapidly gains water by osmosis and brane. The use of such mechanisms allows a cell to regulate swells (Figure 4.35 a). Conversely, a cell placed into a hyper- the movement of substances across its surface barrier. We will tonic solution rapidly loses water by osmosis and shrinks (Fig- return to this feature of membranes later. ure 4.35 b). These simple observations show that a cell’s volume is controlled by the difference between the solute con- The Diffusion of Water through Membranes Water centration inside the cell and that in the extracellular medium. molecules move much more rapidly through a cell membrane The swelling and shrinking of cells in slightly hypotonic and Chapter 4 The Structure and Function of the Plasma Membrane 150 urudn wl (iue 4.36 (Figure wall surrounding to develop an internal ( internal an develop to there is a tendency for water to enter the c result, environmen fluid their to compared hypertonic erally is generally are which with the medium in plant which cell cells, they are bathed, animal Unlike ways. ent face would you Plants utilize osmosi prospect of rapid diarrhea, dehydration. extreme of cases in happens osmoticall If cells this that fluid reabsorbed line weren’tyour intestine. is which daily, fluid secretes of liters example, for tract, digestive Your functions. into or out of the cells occurs (Figure 4.35 occursinto (Figure or out of the cells their plasma membranes and then placed the oocytes oocytes the placed then and membranes plasma their pr discovered newly the incorporate to oocytes frog eng they protein Tohypothesis, their test a membrane. throcyte identified they t of channel water long-sought the be might pursuit, thought this During re of cells. surface the on antigen Rh the for responsible membrane the purify and isolate to attempting were th In Peter bilayer. Agre and colleagues 1990s, at Johns Hopkins lipid Un the through diffusion simple by cells are much more permeable to water than can be and wilt. plants to lose their support causes ysis ul aa fo te urudn cl wl,a process a plasmolysis wall, cell surrounding the from membr away plasma pulls the as shrinks volume its medium, tonic placed is cell plant a If leaves. the as such trees, pa nonwoody the for and plants nonwoody for support wlsbcueo e ano ae yomss ( of a net gain of water by osmosis. because swells (one having a lower solute concentration than the c membrane. plasma the of sides opposite on solutes of tration flux of water is equal flux. to the outward tion maintains a constant the inward volume because are exte and internal the concentration, solute external includes a high concentration of dissolved ( proteins concentration solute internal the Once medium. the i gain cells the as occurs recovery medium, pertonic thereby reducing their internal osmotic ions, pressu ce the as occurs recovery medium, hypotonic a In ume. recover the cells and return to their o minutes, few hypertonic media are events. usually only temporary Figure 4.35 fwtrb soi.(of water by osmosis. of a because solution shrinks cell in a hypertonic isotonic Osmosis is an important factor in a multitude of bo of multitude a in factor important an is Osmosis o l el r qal emal owtr In fac Not all cells are permeable to water. equally ( a The effects of differences in the concen-The ) A cell placed) in A cell a hypotonic solution (Figure (Figure 4.36 (or isosmotic c ) A cell placed in an isotonic solu- turgor b .The loss of water due to plasmol-). ), and no net movement of waterof movement net no and ), ) pressure) itsagainst pushes that a .Tro pesr provides pressure Turgor ). net loss c b ). ) A ell) rasre,as reabsorbed, into a hyper-a into ell, causing causing itell, ) equals the riginal vol-riginal e In a hy-re. s in differ- rnal fluids rnal otein intootein explained s are gen- Within Within a n fromons H b the by y (a) H proteins bloodd several 2 ,manyt, ineered he ery-he iversity 2 early e t. As a As t. called O lls lose lls Hypotonicsolution otonic O which Net watergain rts of rts they the n a in Cell swells i ydil ane H H 2 2 O O H H 2 raigtro rsue (creating turgor pressure. Water therefore tends to flow hypotonic environment. ouin uha ewtr h ellsswtr and loses water, the cell as seawater, such solution, ebaeplsaa rmtecl al (E away from pulls wall. membrane the cell 2 O O ( Figure 4.36 a ) Aquatic plants living in freshwater are surrounde (b) (a) (b) H H 2 2 H Hypertonic solution Hypertonic O O The effects of osmosis on a plant cell. a plant on osmosis of effects The 2 Net waterloss O Cell shrinks b H ) If the plant is placed in a hypertonic ) If the plant is placed in a hypertonic 2 O H H 2 2 O O D R E SCHK E pressure Normal turgor Hypotonic: pressure No turgor Hypertonic: H H the plasma (c) No netlossorgain 2 2 O O Plasmolysis Isotonicsolution into the cells, .) d by a H 2 0 H H 2 2 O O 151 Figure 4.37 Passage of water molecules through an aquaporin channel. (a) Snapshot from a molecular dynamics simulation of a stream of water molecules (red and white spheres) passing in single file through the channel in one of the subunits of an aquaporin molecule residing within a membrane. (b) A model describing the mechanism by which water molecules pass through an aquaporin channel with the simulta- neous exclusion of protons. Nine water molecules are shown to be lined up in single file along the wall of the channel. Each water molecule is depicted as a red circular O atom with two associated Hs. In this model, the four water molecules at the top and bottom of the channel are oriented, as the result of their interaction with the carbonyl (C PO) groups of the protein backbone (page 51), with their H atoms pointed away from the center (a) of the channel. These water molecules are able to form hydrogen bonds (dashed lines) with their neighbors. In contrast, the single water molecule in the center of the channel is oriented in a animations.html (A: F ROM (b) position that prevents it from forming hydrogen bonds with other water BENOIT ROUX AND KLAUS molecules, which has the effect of interrupting the flow of protons SCHULTEN , S TRUCTURE 12:1344, 2004, WITH PERMISSION FROM through the channel. Animations of aquaporin channels can be found ELSEVIER ; B: F ROM R. M. S TROUD ET AL ., C URR . O PIN . S TRUCT . at www.nobelprize.org/nobel_prizes/chemistry/laureates/2003/ BIOL . 13:428, © 2003, WITH PERMISSION FROM ELSEVIER .)

hypotonic medium. Just as predicted, the oocytes swelled due Aquaporins are particularly prominent in cells, such as to the influx of water and eventually burst. The team had dis- those of a kidney tubule or plant root, where the passage of covered a family of small integral proteins, called aquaporins , water plays a crucial role in the tissue’s physiologic activities. that allow the passive movement of water from one side of the The hormone vasopressin, which stimulates water retention plasma membrane to the other. Each aquaporin subunit (in by the collecting ducts of the kidney, acts by way of one of 4.7 the four-subunit protein) contains a central channel that is these proteins (AQP2). Some cases of the inherited disorder lined primarily by hydrophobic amino acid residues and is congenital nephrogenic diabetes insipidus arise from mutations in MembranesCell Across Substances of Movement The highly specific for water molecules. A billion or so water mol- this aquaporin channel. Persons suffering from this disease ecules can pass—in single file—through each channel every excrete large quantities of urine because their kidneys do not ϩ second. At the same time, H ions, which normally hop along respond to vasopressin. a chain of water molecules, are not able to penetrate these open pores.The apparent mechanism by which these channels The Diffusion of Ions through Membranes The lipid are able to exclude protons has been suggested by a combina- bilayer that constitutes the core of biological membranes is tion of X-ray crystallographic studies, which has revealed the highly impermeable to charged substances, including small ϩ ϩ ϩ Ϫ structure of the protein, and molecular dynamics simulations ions such as Na , K , Ca 2 , and Cl . Yet the rapid move- (page 60), which has put this protein structure into operation. ment ( conductance ) of these ions across membranes plays a A model based on such simulations is shown in Figure 4.37 a. critical role in a multitude of cellular activities, including for- Very near its narrowest point, the wall of an aquaporin chan- mation and propagation of a nerve impulse, secretion of sub- nel contains a pair of precisely positioned positive charges stances into the extracellular space, muscle contraction, (residues N203 and N68 in Figure 4.37 b) that attract the oxy- regulation of cell volume, and the opening of stomatal pores gen atom of each water molecule as it speeds through the con- on plant leaves. striction in the protein. This interaction reorients the central In 1955, Alan Hodgkin and Richard Keynes of Cam- water molecule in a position that prevents it from maintaining bridge University first proposed that cell membranes contain the hydrogen bonds that normally link it to its neighboring ion channels , that is, openings in the membrane that are per- water molecules. This removes the bridge that would normally meable to specific ions. During the late 1960s and 1970s, allow protons to move from one water molecule to the next. Bertil Hille of the University of Washington and Clay Chapter 4 The Structure and Function of the Plasma Membrane 152 atedg such channelsan open are or a closed conformation; of the ion channels that have been identified can ex from a stateis, of higher energy to a state of lowe the diffusion of ions through a channel is always d across solutes of types other of diffusion passive one particular type of ion to pass through the pore 2. 1. categories of gated channels are distinguished: Thre channel. particular the on depending factors of complex physiologic regulation and can be induced b page 162). le can (see diseases serious many Table channels PerHuman the of 1 ion encoding genes the in pre mutations be might As prote pore. aqueous membrane central a integral enclose that by formed varie each bewildering channels, a ion identified have biologists Today, vestigations into the activities of individual prot (Fi succes first the marked studies landmark measured These be 4.38). can pipette the by surrounded brane patc small the in currentoriginating the and value, odcac tdfeetapidvlae.(conductance at different applied voltages. its as well as channel, gated a single of ing lows investigators to monitor the opening and clos- al- which containingmembrane a single ion channel, o a patch enclose the micropipette can in the figure, As indicated be measured. can channels membrane and the responding flow of ions through thepipette, by theacross enclosed of membrane the patch electrode (a the pipette is wired Because as anplasma membrane. of the pipette against theapplied to seal the rim and suction of a cell, of the outer surface portion polished glass micropipette is placed agains highly ebae a b mitie ( maintained be can acros voltage membrane The suction. by membrane the to su sealed cell outer the on placed are that glass polished accomplished using fine very micropipette-electrodes the ionic current passing through a single ion chan m to techniques developed who 1980s early and 1970s the Erwin in Germany and in Institute Sakmann Max-Planck the Bert at Neher of work the through emerged fina The channels. such of existence the for evidence t Pennsylvaniabegan of University the of Armstrong recording.by patch-clamp Figure 4.38 right) is sealed against a small patch of the is plasm sealed patch against a small right) while a second micropipette micropipette by suction, in is drawn of the cell One portion of a salamander. made from a single photoreceptor of the retina cell recordingsmicrograph shows being patch-clamp pends on the binding of a specific molecule (the lig channelsLigand-gated of the membrane. pends on the difference in on ionic the two charge Voltage-gated channels ot o canl ae ihy eetv i allowing in selective highly are channels ion Most h oeig n coig f h gts r sbet t subject are gates the of closing and opening The . microelectrode Measuring ion channel conductance channel ion Measuring ), a voltage can be applied a voltage can ), ( a nti ehiu,a) In this technique, whose conformational state de- whose conformational state de- clamped b ) The a ay particular any at ) a membrane ona membrane is t a to a glass f -electrode (lower nry Mostr energy. ein molecules. (a) As with the. whl,thatownhill, membranes, ist in either e.This isnel. h of mem-of h y a variety fc and rface said to be pof”l “proof made of spective, o obtaino majore sful in-sful sides and), dicted, onitor d to ad ty of of ty thes late gure onl y ins ins o peared over the following years. billion one or two refineme many although evolution, in early relatively we channel ion an operating in challenges basic the Evi operating in the much larger mammalian channels. ident virtually are channel bacterial this in tance and selectivity ion of mechanisms the that see also image of an ion channel protein, in this case, a bact a case, this in protein, channel ion an of image atomic-res first the provided University Rockefeller be channels these are the best understood. ion potassium voltage-gated of function We will focus in the following discussion on the st M nte oto ftecl.( of the cell. another portion rapid conductance of K esadn o te ehns b wih hs remarka these overwhelmingl select to able are machines molecular which by mechanism the of derstanding the formulation of this structure led direc shortly, 3. ERDCDWT EMSINOF PERMISSION WITH REPRODUCED ions over Naover ions Kthe th example better a find to difficult be would it but biologica the in everywhere evident is function and st between relationship The KcsA. called channel ion ATTHEWS inner surface of certain calcium ion channels. calcium of certain inner surface act on t as cAMP, such nucleotides, while cyclic nels, cation of certain act on the outer surface choline, as acety such neurotransmitters, For example, channel. lowing the binding of a ligand to the inner surface others are opened (or closed of the channel; surface following the binding of a molecule to the closed) are ligand-gated channels opened (or Some channel. not the solute that passes through is usually which the inner ear in response to sound or motions of th ments of stereocilia 9.54) on the hair (see Figure are opened by the move- for example, channels, cation o Members of one family are applied to the membrane. stretch tension) forcesdepends on mechanical (e.g., channelsMechano-gated n 98 oeik aKno ad i clege at colleagues his and MacKinnon Roderick 1998, In ϩ ion channel depicted in Figure 4.39. As we will see will we As 4.39. Figure in depicted channel ion , AND ϩ ions, yet at the same time allow an incrediblyan allow time same the at yet ions, .TV. ORRE (b) ϩ B ostruhtemmrn.Weions through the willmembrane. .P J. , F: ROM HYSIOLOGY whose conformational state .D L D. T. J OHN W AMB 7:1,18.©1986, 1986. 372:319, ILEY .R. H. , & S ONS tly tly to an un- ical to thoseto ical ructure ructure and an that ofthat an conduc- cells ofcells re solvedre .) chan- y for Kfor y ra K erial world,l of the the outer e head. olution t ap-nts ructure ) fol- that dently, cause he 35 l- ble f ϩ ϩ 153 light brown in Figure 4.39) that forms the lining of a narrow G O O G + selectivity filter , so named because of its role in allowing only K ϩ Y O O Y the passage of K ions. G O O G The lining of the selectivity filter contains a highly con- K+ V O O V served pentapeptide—Gly-Tyr-Gly-Val-Thr (or GYGVT in single-letter nomenclature). The X-ray crystal structure of the T O O T KcsA channel shows that the backbone carbonyl (C PO) K+ groups from the conserved pentapeptide (see the backbone structure on page 51) create five successive rings of oxygen Selectivity atoms (four rings are made up of carbonyl oxygens from the Filter polypeptide backbone, and one ring consists of oxygen atoms from the threonine side chain). Each ring contains four oxygen atoms (one from each subunit) and has a diameter of ap- P proximately 3 Å, which is slightly larger than the 2.7 Å diameter of a K ϩ ion that has lost its normal shell of hydration. Consequently, the electronegative O atoms that line the selectivity filter can substitute for the shell of water molecules ϩ K+ that are displaced as each K ion enters the pore. In this ϩ ions model, the selectivity filter contains four potential K ion binding sites. As indicated in the top inset of Figure 4.39, a Kϩ ion bound at any of these four sites would occupy the center of a “box” having four O atoms in a plane above the ion and four O atoms in a plane below the atom. As a result, each K ϩ ion in one of these sites could coordinate with eight O atoms of the selectivity filter. Whereas the selectivity filter is a precise fit for a dehydrated K ϩ ion, it is much larger than the diame- M2 M1 ter of a dehydrated Na ϩ ion (1.9 Å). Consequently, a Na ϩ ion helix helix cannot interact optimally with the eight oxygen atoms necessary to stabilize it in the pore. As a result, the smaller Na ϩ ions cannot overcome the higher energy barrier required to penetrate the pore. ϩ Figure 4.39 Three-dimensional structure of the bacterial KcsA Although there are four potential K ion binding sites, ؉ channel and the selection of K ions. This K ϩ ion channel consists of only two are occupied at any given time. Potassium ions are four subunits, two of which are shown here. Each subunit is comprised thought to move, two at a time—from sites 1 and 3 to sites 2 of M1 and M2 helices joined by a P (pore) segment consisting of a and 4—as indicated in the top inset of Figure 4.39. The entry short helix and a nonhelical portion that lines the channel through of a third K ϩ ion into the selectivity filter creates an electro- which the ions pass. A portion of each P segment contains a conserved static repulsion that ejects the ion bound at the opposite end pentapeptide (GYGVT) whose residues line the selectivity filter that 4.7 ϩ of the line. Studies indicate that there is virtually no energy screens for K ions. The oxygen atoms of the carbonyl groups of these residues project into the channel where they can interact selectively barrier for an ion to move from one binding site to the next, MembranesCell Across Substances of Movement The ϩ which accounts for the extremely rapid flow of ions across the with K ions (indicated by the red mesh objects) within the filter. As ϩ indicated in the top inset, the selectivity filter contains four rings of membrane. Taken together, these conclusions concerning K carbonyl O atoms and one ring of threonyl O atoms; each of these ion selectivity and conductance provide a superb example of five rings contains four O atoms, one donated by each subunit. The how much can be learned about biological function through diameter of the rings is just large enough so that eight O atoms can an understanding of molecular structure. coordinate a single K ϩ ion, replacing its normal water of hydration. The KcsA channel depicted in Figure 4.39 has a gate, just ϩ Although four K binding sites are shown, only two are occupied at like eukaryotic channels. The opening of the gate of the KcsA one time. (F ROM RODERICK MAC KINNON , N ATURE MED . 5:1108, channel in response to very low pH was illustrated in Figure © 1999, REPRINTED BY PERMISSION FROM MACMILLAN 4.22. The structure of KcsA shown in Figure 4.39 is actually PUBLISHERS LTD .) the closed conformation of the protein (despite the fact that it contains ions in its channel). It has not been possible to crystallize the KcsA channel in its open conformation, but the structure of a homologous prokaryotic K ϩ channel (called The KcsA channel consists of four subunits, two of which MthK) in the open conformation has been crystallized and its are shown in Figure 4.39. Each subunit of Figure 4.39 is seen structure determined. Comparison of the open structure of to contain two membrane-spanning helices (M1 and M2) and MthK and the closed structure of the homologous protein a pore region (P) at the extracellular end of the channel. P KcsA strongly suggested that gating of these molecules is ac- consists of a short pore helix that extends approximately one- complished by conformational changes of the cytoplasmic third the width of the channel and a nonhelical loop (colored ends of the inner (M2) helices. In the closed conformation, as Chapter 4 The Structure and Function of the Plasma Membrane 154 (R two-dimensional portrait of a K charged S4 helix of the sequence of amino acids of the positively The partinset of theshows channel’s the wall. helix or P) that dips into the protein to form a portion of the polypeptide (called the pore unit showing its six transmembrane helices and distinct domai be grouped functionally intocan two wh are These shown si two-dimensionally in Figure 4.41. S1–S6, named helices, membrane-associated six tain subuni channel Kv Eukaryotic chapter. the of end the ex is which function, nerve and muscle in role their kno best are animals of channels Kv The volume. cell reg in and balance water and salt in role important plant of channels Kv The scrutinized. proteins their Kv) channels have been isolated and the molecular a Shaker vigorously when Theanesthesized with ether. certain mutations in the protein shake is located. glyci a where point hinge specific a at bend helices pore. the the of channel opens when t model shown face in Figure 4.40, cytoplasmic the seals that bundle” fo to another one over cross and straight are lices th drawing, left seen in Figure 4.39 and Figure 4.40, 2003; FROM identified and cloned in 1987. is called a This member of thedrophobic Kv family helix. thirdat every residue along the otherwise hy- The positively charged side chains are situated which asserves a voltage sensor.ion channel, voltage-gated Ka eukaryotic, Figure 4.41 1. in,wih r togt o efr i a iia man similar a voltage-gat in distinct of variety a perform encode that Genes to thought are which sions, eukaryot complex more underst the to of function position and better structure a in are we operate, nels which opens the gate at the intracellular end of th opens the gate at the which intracellular helices from subunit at a specific bend outward each PIT YPRISO FROM PERMISSION BY EPRINTD a of the entire bacterial channel illustrated in Figu Now that we have seen how these prokaryotic K prokaryotic these how seen have we that Now pore domain Figure 4.40 model for the opening of the bacterial KcsA channel model for the opening of the bacterial COPYRIGHT .L K L. B. channel was the first K Shaker The structure of one subunit of subunit one of structure The LYANDELLY channel because flies with which has the , same basic architecture as that 2003.) Schematic illustration of the hinge-bending illustration Schematic lsdOpen Closed Drosophila .GA. ϩ ؉ ROSS Hinge helix ϩ channel to be channel. M2 channel sub- K N, M ϩ Shaker ACMILLAN ATURE A S TRUCT e channel to Ke channel P Inside cell Outside cell UBLISHER glycine residue, B. IOL re4.39 and rm a “helixa rm ne residuene ed Ked natomy of ulation ofulation . ns: e M2 he- plored atplored 10:280,. s play anplay s The M2 x helices ϩ n the and L ts con-ts wn forwn he M2 c ver-ic n the In TD ϩ N chan- ϩ ions. 1S 3S S5 S4 S3 S2 S1 ner. : ich (or Voltage-sensing domain 2. 6 (see here the selectivit described and another involving of the opening and closing one involving mechanisms: The voltage-sensing domain is seen to be connected be to seen is domain voltage-sensing The volt the of element key the as acts 4.41), Figure of amino acid residues spaced alongch the polypeptide 4.42 represents the open state of the channel.in depicted protein The 4.40). Figure in (shown nel ma a in close and open roughly similar to to that of the M2 helices of thought the ba is and helices S6 en inner the by formed is channel Kv a into leading The proteins. Kv eukaryotic and KcsA prokaryotic the sumed sumed mechanism of Kthus and filter, selectivity The pore. ion-conducting mologous subunits around arranged the symmetrically of consists channel Kv eukaryotic single a channel, Like moting its function as a voltage-gated channel. taining the native structure of the membrane protei in important be to thought is phospholipids charged of presence The process. crystallization and cation throughout lipid and detergent mixtureof a of use possi made was structure this of Determination 4.42. karyotic Kv channel purified from rat brain is shown Recent studies strongly suggest Recentthat K studies strongly a cussed below). senses the voltage across the plasma membrane (as dis- to the channel is open or closed.to the channel and their whether configuration determines the pore, the four S6 helices line much of theM2 helices of KcsA, eukaryotic channel illustrated Like in Figure 4.41. helices S5 and S6 and the P segment of the voltage- 4.39 areof of the Figure KcsA channel homologous t passage of K the filter that permits selcontains the selectivity PNAS + + + + The S4 helix, which contains several positively char positively several contains which helix, S4 The The three-dimensional structure crystal of a comple voltage-sensing domain 0:63 2010 and 107:7623, Pore domain

P ϩ os Helices M1ions. segmentand M2 and the P S6 ϩ Nature ion selection, is virtually is identical virtually inion selection, C consisting of helices S1–S4 that 6:0,2010). 466:203, ϩ channels are distinct gated by two y filter, which is not discussed which y filter, + + the inner helix bundle as the inner helix bundle S4 Segment K R F V F L 6 + S V R cterial cterial chan- V K age sensor.age n and pro- ective the purifi-the negatively the KcsA in Figure + + ain (inset ds of the of ds I or ho-four the four the pre-the R S I central main- G Figure gated l byble o theto te eu- R H gate o I nner gate L + ged L L R + 155

(a) (b) (c)

Figure 4.42 Three-dimensional structure of a voltage-gated and a separate ␤ polypeptide. ( c) Ribbon drawing of a single subunit ؉ mammalian K channel. (a) The crystal structure of the entire showing the spatial orientation of the six membrane-spanning helices tetrameric Kv1.2 channel, a member of the Shaker family of K ϩ ion (S1–S6) and also the presence of the S4–S5 linker helix, which channels found in nerve cells of the brain. The transmembrane portion connects the voltage-sensing and pore domains. This linker transmits is shown in red, and the cytoplasmic portion in blue. The potassium the signal from the S4 voltage sensor that opens the channel. The inner ion binding sites are indicated in green. ( b) Ribbon drawing of the same surface of the channel below the pore domain is lined by the S6 helix channel shown in a, with the four subunits that make up the channel (roughly similar to the M2 helix of the bacterial channel shown in shown in different colors. If you focus on the red subunit, you can see Figure 4.39). The channel shown here is present in the open conﬁgura- (1) the spatial separation between the voltage-sensing and pore domains tion with the S6 helices curved outward (compare to Figure 4.40) at of the subunit and (2) the manner in which the voltage-sensing domain the site marked PVP (standing for Pro-Val-Pro, which is likely the from each subunit is present on the outer edge of the pore domain of a amino acid sequence of the “hinge”). (F ROM STEPHEN B. L ONG ET neighboring subunit. The cytoplasmic portion of this particular channel AL ., S CIENCE 309:867, 899, 2005, COURTESY OF RODERICK consists of a T1 domain, which is part of the channel polypeptide itself, MAC KINNON ; © 2005, REPRINTED WITH PERMISSION FROM AAAS.)

pore domain by a short linker helix denoted as S4–S5 in the onds, the movement of K ϩ ions is “automatically” stopped by model in Figure 4.42. Under resting conditions, the negative a process known as inactivation. To understand channel inac- 4.7 potential across the membrane (page 165) keeps the gate tivation, we have to consider an additional portion of a Kv closed. A change in the potential to a more positive value (a channel besides the two transmembrane domains discussed MembranesCell Across Substances of Movement The depolarization, page 166) exerts an electric force on the S4 he- above. lix. This force is thought to cause the transmembrane S4 helix Eukaryotic Kv channels typically contain a large cytoplas- to move in such a way that its positively charged residues shift mic structure whose composition varies among different from a position where they were exposed to the cytoplasm to channels. As indicated in Figure 4.43 a inactivation of the a new position where they are exposed to the outside of the channel is accomplished by movement of a small inactivation cell. Voltage sensing is a dynamic process whose mechanism peptide that dangles from the cytoplasmic portion of the pro- cannot be resolved by a single static view of the protein such tein. The inactivation peptide is thought to gain access to the as that shown in Figure 4.42. In fact, several competing mod- cytoplasmic mouth of the pore by snaking its way through one els describing the mechanism of action of the voltage sensor of four “side windows” indicated in the figure. When one of are currently debated. However it occurs, the movement of the these dangling peptides moves up into the mouth of the pore S4 helix in response to membrane depolarization initiates a (Figure 4.43 a), the passage of ions is blocked, and the channel series of conformational changes within the protein that is inactivated. At a subsequent stage of the cycle, the inactiva- opens the gate at the cytoplasmic end of the channel. tion peptide is released, and the gate to the channel is closed. Once opened, more than ten million potassium ions can It follows from this discussion that the potassium channel can pass through the channel per second, which is nearly the rate exist in three different states—open, inactivated, and closed— that would occur by free diffusion in solution. Because of the which are illustrated schematically in Figure 4.43 b. large ion flux, the opening of a relatively small number of K ϩ Potassium channels come in many different varieties. It is channels has significant impact on the electrical properties of remarkable that C. elegans , a nematode worm whose body the membrane. After the channel is open for a few millisec- consists of only about 1000 cells, contains approximately 80 Chapter 4 The Structure and Function of the Plasma Membrane 156 many ways to an enzyme-catalyzed reaction. Like enzy Like reaction. enzyme-catalyzed an to ways many voltage required to open or close a particular K particular addia close or open requiredto voltage In voltages. different to response in close and this chapter. the subject of the is Experimental Pathways section at receptor, acetylcholine nicotinic ligand-gated type the different channel, very a of function and structure The regulat of set complex and diverse a of control the It is apparent that ion channel funct other factors. hormon protei by regulated is turn channel in which the phosphorylated, not or whether on depending vary the two sides of the membrane.the two flux depends on the relative concentration of the directisu The directions. both in well equally solutes mov the mediate can transporters facilitated system, without being coupled that to is, an passively, energ the Because gradien 4.44. Figure in illustrated is mechanism concentration its down diffuse can it where membran membr the of surface other the to solute the posing the of side one thought to trigger a conformational change in the p on transporter facilitative solu the of binding The process. diffusion the tates Facilitated Diffusion spanning protein, called a called spanningprotein, di a to selectively binds first substance diffusing always the not do they but In m through the lipid bilayer or through a channel. side, other the on tration higher concentration on one side to a region of low Substances always diffuse across a membrane from a ige elwehr n nmtd,hmn r plant—is or human, Kdifferent of variety a possess to likely nematode, a in cell—whether single different genes that encode Kencode that genes different domain Cytoplasmic Inside cell Outside cell (a) Facilitated diffusion, facilitative transporter,facilitative sti rcs scle,is as similar this inprocess is called, ϩ channels. It is evident that athat evident is It channels. ϩ Pore Central cavity channels that open that channels Inactivation peptide Lateral window ϩ membrane- channel can channel ion is under ory agents. ory y-releasing bstance on er concen- thatfacili- the end of oen ex-rotein, any cases, on of netof on n,fromane, y operatey ement ofement region of te to theto te in thetion, f ion of .Thist. membrane Cell s andes ffuse mes, is e is n ( transport, discriminating, for example, between between example, for discriminating, molec the transport, for specific are transporters facilitative 1998, C VOL Figure 4.43 EMSINFROMPERMISSION (Rof the membrane. that exposes the glucose binding site to eithe rier tated diffusion of glucose depicts the alternating Figure 4.44 an enzymes both addition, In 44). (page stereoisomers channel. t notectpamcoeigo h hne.( opening of the channel. fits into the cytoplasmic portion dangle from which the cytoplasmic peptides, occurs as one of t Inactivation activity of channel sentation of a view into a Ksentation of a view L rsig,oe,adiatvtdsae ( and inactivated state. open, (resting), showing the channel side, frombrane the cytoplasmic (b) IE NHAR D ANL ANDHANNELS 0 .M A M. C. 20, . IHPRISO FROM PERMISSION WITH Recovery etOe Inactivated Open Rest ( a ) KThree-dimensional model of a eukaryotic T, Conformational states of a voltage-gated K states of a voltage-gated Conformational Facilitated diffusion. R E NDS MTOGANDRMSTRONG E LECTRICAL E PITDFROMEPRINTED B LSEVIER IOCHEM ϩ ion channel, perpendicular to the mem- perpendicular ion channel, L, Dissociation E S. Glucose E TD Binding SCITABILITY LSEVIER CI .HB. .) A schematic model for the facili-A schematic :1,18.U 1981. 6:210, . B .A B A. S. R: ILLE PITDFROMEPRINTED S CIE NCE V, , LWNANDALDWIN P OL T AGE 377;. r the inside or outside conformation of a car-conformation he inactivation b ) Schematic repre-) Schematic E WITHSED .) of the complex, in the closed COPYRIGHT Transport -G ϩ ؉ ion channel. ATED ls they ules ion D N .E.G. EURON and and I ON L d , 157

V Active Transport max

) Life cannot exist under equilibrium conditions (page 93). v Protein-mediated transport Nowhere is this more apparent than in the imbalance of ions (facilitated diffusion) across the plasma membrane. The differences in concentration of the major ions between the outside and inside of a typical mammalian cell are shown in Table 4.3. The ability of a cell to generate such steep concentration gradients across its plasma membrane cannot occur by either simple or facilitated diffusion. Rather, these gradients must be generated by active transport . Rate of solute movement ( Simple diffusion Like facilitated diffusion, active transport depends on integral membrane proteins that selectively bind a particular solute and move it across the membrane in a process driven by changes in the protein’s conformation. Unlike facilitated dif- Solute concentration fusion, however, movement of a solute against a gradient requires the coupled input of energy. Consequently, the Figure 4.45 The kinetics of facilitated diffusion as compared to that endergonic movement of ions or other solutes across the of simple physical diffusion. membrane against a concentration gradient is coupled to an exergonic process, such as the hydrolysis of ATP, the ab- sorbance of light, the transport of electrons, or the flow of other substances down their gradients. Proteins that carry out transporters exhibit saturation-type kinetics (Figure 4.45). active transport are often referred to as “pumps.” Unlike ion channels, which can conduct millions of ions per second, most facilitative transporters can move only hundreds Primary Active Transport: Coupling Transport to ATP to thousands of solute molecules per second across the mem- Hydrolysis In 1957, Jens Skou, a Danish physiologist, dis- brane. Another important feature of facilitative transporters is covered an ATP-hydrolyzing enzyme in the nerve cells of a that, like enzymes and ion channels, their activity can be reg- crab that was only active in the presence of both Na ϩ and K ϩ ulated. Facilitated diffusion is particularly important in medi- ions. Skou proposed, and correctly so, that this enzyme, which ating the entry and exit of polar solutes, such as sugars and was responsible for ATP hydrolysis, was the same protein that amino acids, that do not penetrate the lipid bilayer. This is was active in transporting the two ions; the enzyme was called illustrated in the following section. the Na ϩ/K ϩ-ATPase, or the sodium–potassium pump . Unlike the protein-mediated movement of a facilitated The Glucose Transporter: An Example of Facilitated diffusion system, which will carry the substance equally well Diffusion Glucose is the body’s primary source of direct en- in either direction, active transport drives the movement of ergy, and most mammalian cells contain a membrane protein ions in only one direction. It is the Na ϩ/K ϩ-ATPase that is that facilitates the diffusion of glucose from the bloodstream responsible for the large excess of Na ϩ ions outside of the cell into the cell (as depicted in Figures 4.44 and 4.49). A gradient and the large excess of K ϩ ions inside the cell. The positive favoring the continued diffusion of glucose into the cell is charges carried by these two cations are balanced by negative 4.7 maintained by phosphorylating the sugar after it enters the charges carried by various anions so that the extracellular and cytoplasm, thus lowering the intracellular glucose concentra- MembranesCell Across Substances of Movement The intracellular compartments are, for the most part, electrically tion. Humans have at least five related proteins (isoforms) that neutral. Cl Ϫ ions are present at greater concentration outside act as facilitative glucose transporters. These isoforms, termed of cells, where they balance the extracellular Na ϩ ions. The GLUT1 to GLUT5, are distinguished by the tissues in which abundance of intracellular K ϩ ions is balanced primarily by they are located, as well as their kinetic and regulatory charac- excess negative charges carried by proteins and nucleic acids. teristics. Insulin is a hormone produced by endocrine cells of the pancreas and plays a key role in maintaining proper blood sugar levels. An increase in blood glucose levels triggers the Table 4.3 Ion Concentrations Inside and Outside secretion of insulin, which stimulates the uptake of glucose of a Typical Mammalian Cell into various target cells, most notably skeletal muscle and fat Extracellular Intracellular Ionic cells (adipocytes). Insulin-responsive cells share a common concentration concentration gradient isoform of the facilitative glucose transporter, specifically ϩ GLUT4. When insulin levels are low, these cells contain rela- Na 150 mM 10 mM 15 ϫ Kϩ 5 mM 140 mM 28 ϫ tively few glucose transporters on their plasma membrane. In- – stead, the transporters are present within the membranes of Cl 120 mM 10 mM 12 ϫ 2ϩ –3 –7 ϫ cytoplasmic vesicles. Rising insulin levels act on target cells to Ca 10 M 10 M 10,000 Hϩ 10 –7.4 M 10–7.2 M Nearly 2 ϫ stimulate the fusion of the cytoplasmic vesicles to the plasma (pH of 7.4) (pH of 7.2) membrane, which moves transporters to the cell surface where they can bring glucose into the cell (see Figure 15.26). The ion concentrations for the squid axon are given on page 177. 158

Extracellular

Plasma K+ Na + membrane E1 conformation Occluded + Na K+

+ + Na K ATP ATP ADP P ADP P P P P ATP ATP

Open Occluded E2 Conformation 1 234 5 6 78

Cytosol

(a)

؉ ؉ Figure 4.46 The Na /K -ATPase . ( a) Sim- pliﬁed schematic model of the transport cycle as described in the text. Note that the actual Na ϩ/K ϩ-ATPase is composed of at least two different membrane-spanning subunits: a larger ␣ subunit, which carries out the transport activity, and a smaller ␤ subunit, which functions primarily in the maturation and assembly of the pump within the membrane. A third ( ␥) subunit may also be present. (b) A model of the E 2 conformation of the protein based on an X-ray crystallographic study. The cation binding sites are located deep within the transmembrane domain, which consists of 10 membrane- spanning helices. The two rubidium ions are located where the potassium ions would normally be bound. ( B: F ROM AYAKO TAKEUCHI , COURTESY OF DAVID C. G ADSBY , N ATURE 450:958, Cytosol 2007, © 2007 REPRINTED BY PERMISSION FROM MACMILLAN PUBLISHERS LTD .) (b) ?

The ratio of Na ϩϺKϩ pumped by the Na ϩ/K ϩ-ATPase is ions. Then the protein must release the ions on the other side of not 1 Ϻ1, but 3 Ϻ2 (see Figure 4.46). In other words, for each the membrane into a much greater concentration of each ion.To ATP hydrolyzed, three sodium ions are pumped out as two do this, the affinity of the protein for that ion must decrease. potassium ions are pumped in. Because of this pumping ratio, Thus, the affinity for each ion on the two sides of the membrane the Na ϩ/K ϩ-ATPase is electrogenic , which means that it con- must be different. This is achieved by ATP hydrolysis and the tributes directly to the separation of charge across the mem- subsequent release of ADP, which induces a large conforma- brane. The Na ϩ/K ϩ-ATPase is an example of a P-type ion tional change within the protein molecule. The change in pro- pump. The “P” stands for phosphorylation, indicating that, tein structure—between the E 1 and E 2 conformations—also during the pumping cycle, the hydrolysis of ATP leads to the serves to alternately expose the ion binding sites to the opposite transfer of the released phosphate group to an aspartic acid side of the membrane, as discussed in the following paragraph. residue of the transport protein. As denoted by the name of A proposed scheme for the pumping cycle of the Na ϩ/K ϩ- The The Structure and Function of the Plasma Membrane the protein, this reaction is catalyzed by the pump itself. ATPase is shown in Figure 4.46 a, which is based on recent X-ray Consider the activity of the protein. It must pick up sodium crystallographic structures of P-type ion pumps. In step 1, Fig- or potassium ions from a region of low concentration, which ure 4.46 a, the pump is in the E 1 conformation, and the ion bind- means that the protein must have a relatively high affinity for the ing sites are accessible to the inside of the cell. In this step, the Chapter Chapter 4 159 protein has bound three Na ϩ ions and an ATP.In step 2, a gate from the foxglove plant that has been used for 200 years as a within the protein has closed, shifting the pump into an oc- treatment for congestive heart disease, binds to the Na ϩ/K ϩ- ϩ cluded E 1 state in which the Na ions can no longer flow back ATPase. Digitalis strengthens the heart’s contraction by inhibit- into the cytosol. Hydrolysis of the bound ATP (step 2 S3) and ing the Na ϩ/K ϩ pump, which leads to a chain of events that S 2ϩ release of ADP (step 3 4) cause the change from the E 1 to the increases Ca availability inside the muscle cells of the heart. E2 conformation. In doing so, the binding site becomes accessible to the extracellular compartment, and the protein loses its Other Primary Ion Transport Systems The best studied affinity for Na ϩ ions, which are then released outside the cell. P-type pump is the Ca 2ϩ-ATPase whose three-dimensional Once the three Na ϩ ions have been released, the protein picks up structure has been determined at several stages of the pump- two K ϩions (step 5).The closing of another gate within the pro- ing cycle. The calcium pump is present in the membranes of tein shifts the pump into an occluded state (step 6), preventing the endoplasmic reticulum, where it actively transports cal- back flow of the K ϩ ions into the extracellular space. Occlusion cium ions out of the cytosol into the lumen of this organelle. is followed by dephosphorylation (step 6 S7) and ATP binding Plant cells have a H ϩ-transporting, P-type, plasma mem- (step 8), which induces the return of the protein to the original brane pump. In plants, this proton pump plays a key role in E1 conformation. In this state, the binding site is open to the in- the secondary transport of solutes (discussed later), in the con- ternal surface of the membrane and has lost its affinity for K ϩ trol of cytosolic pH, and possibly in control of cell growth by ions, leading to the release of these ions into the cell.The cycle is means of acidification of the plant cell wall. then repeated. A model of the crystal structure of the Na ϩ/K ϩ- The epithelial lining of the stomach also contains a ATPase structure is shown in Figure 4.46 b. Because they require P-type pump, the H ϩ/K ϩ-ATPase, which secretes a solution complex conformational changes, these active transport pumps of concentrated acid (up to 0.16 N HCl) into the stomach move ions across membranes at rates that are several orders of chamber. In the resting state, these pump molecules are situ- magnitude lower than their flow through ion channels. ated in cytoplasmic membranes of the parietal cells of the The sodium–potassium pump is found only in animal cells. stomach lining and are nonfunctional (Figure 4.47). When This protein is thought to have evolved in primitive animals as food enters the stomach, a hormonal message is transmitted the primary means to maintain cell volume and as the mecha- to the parietal cells that causes the pump-containing mem- nism to generate the steep Na ϩ and K ϩ gradients that play such branes to move to the apical cell surface, where they fuse with a key role in the formation of impulses in nerve and muscle cells. the plasma membrane and begin secreting acid (Figure 4.47). These same ionic gradients are used in nonexcitable cells to In addition to functioning in digestion, stomach acid can power the movement of other solutes as discussed below. The also lead to heartburn. Prilosec and related drugs are widely importance of the sodium–potassium pump becomes evident used to prevent heartburn by inhibiting the stomach’s when one considers that it consumes approximately one-third of Hϩ/K ϩ-ATPase. Other acid-blocking heartburn medications the energy produced by most animal cells and two-thirds of the (e.g., Zantac, Pepcid, and Tagamet) do not inhibit the energy produced by nerve cells. Digitalis, a steroid obtained Hϩ/K ϩ-ATPase directly, but block a receptor on the surface

Gastric + + + + pit of Inactive H /K -ATPase Active H /K -ATPase Pump-inhibiting stomach drug 4.7 The Movement of Substances Across Cell MembranesCell Across Substances of Movement The

Parietal cell ATP + + H H

Food + K K+

ADP+Pi

Cytosol

Acid-neutralizing agents (OH–) Histamine Acid-blocking drug Histamine Figure 4.47 Control of acid secretion in the stomach. In the resting at the surface, the transport protein is activated and pumps protons state, the H ϩ/K ϩ-ATPase molecules are present in the walls of into the stomach cavity against a concentration gradient (indicated cytoplasmic vesicles. Food entering the stomach triggers a cascade of by the size of the letters). The heartburn drugs Prilosec, Nexium, hormone-stimulated reactions in the stomach wall leading to the and Prevacid block acid secretion by directly inhibiting the H ϩ/K ϩ- release of histamine, which binds to a receptor on the surface of the ATPase, whereas several other acid-blocking medications interfere with acid-secreting parietal cells. Binding of histamine to its receptor stimu- activation of the parietal cells. Acid-neutralizing medications provide lates a response that causes the H ϩ/K ϩ-ATPase-containing vesicles to basic anions that combine with the secreted protons. fuse to the plasma membrane, forming deep folds, or canaliculi. Once Chapter 4 The Structure and Function of the Plasma Membrane 160 oae i te aa ad aea pam mmrn,wh membrane, plasma lateral and basal the in located Transport to Existing Ion GradientsIon Existing Transportto Secondary Active Transport (or Cotransport): Coupli f ocnrto gains uh s hs o Na of those as such gradients, concentration of in the next chapter.as described used by subsequ an ATP-synthesizing to is enzyme phosphorylate gradient This membrane. plasma the across primary the by cells the within low very kept is centration H ternal environment, thereby generating a steep Hsteep a generating thereby environment, ternal cytoplasm the from protons of translocation the in proce this effect, In cytoplasm. the from protein the trans proton another by ph replaced the is by retinal donated excited proton The 4.48). (Figure exterior through a in channel the protein, the retinal group, tional changes in the protein that cause a proton t light byenergy the retinal group induces a series light-abs ab The the retina. vertebrate the rodsof the protein of rhodopsin, in present group prosthetic t retinal, contains bacteriorhodopsin 4.48, Figure in orhodopsin, oo de o h peec o oe atclr protein particular one of presence the to due color a on take prokaryotes these of membranes conditio plasma the anaerobic under grown When Lake. Salt Great lives in such extremelyas thatsalty environments, multi-subunit complexes (shown in Figure 4.4 Figure in (shown complexes multi-subunit are pumps V-type urine. forming the into protons ing kidn of tubules helps maintain the membranes body’s acid–base balance plasma the in pump V-type a ample, cells of variety a of membranes plasma the in found V-typetain the pumps lowhave a pH of the contents. th where vacuoles cell plant and granules, secretory lysos line that membranes the in occur They V-type). of organelles cytoplasmic and vacuoles (hence the d V-type pumps actively transport hydrogen ions acros ATP without forming a protein phosphorylated interm ions is the is ions in structure to that of the ATP synthase shown in F activated by the hormone. thereby stopping the cells fr of the cells, parietal ihsdu os silsrtdi iue44.The as illustrated in Figure 4.49. with sodium ions, by occurs gradient, concentration a against cells, ep the of membrane plasma apical the across glucose by the The epithelial cells that line the intestine. which areweight polysaccharides into simple sugars, intestine. Within its enzymes lumen, hydrolyze high-m acti physiologic the Consider solutes. other of port including by a cell in various ways to perform work, gradients ionic in stored energy potential The cell. terium salinariumterium Using Light Energy to Actively Transport Ions porter is described in the accompanying Human Persp in the accompanying is described porter trans ABC studied best The domain. ATP-binding ogous because all of the members of this shar superfamily ϩ provides be store can a means by freewhich energy , Unlike P-type pumps,Unlike P-type Another diverse group of proteins that actively tra actively that proteins of group diverse Another cie rnpr sse (h Na (the system transport active which acts a light-driven proton pump. As shown As pump. proton light-driven a acts which ATP-binding cassette ATP-binding (or .halobiumH. V-type ( ) is an archaebacterium thatarchaebacterium an is ) ABC pumps utilize the energy of the energy pumps utilize ) transporters The establishmentThe ϩ /K d movement of om becoming of conforma- ) and similarand ) o move from cotransport found in the ϩ ϩ action of aof action sorption of sorption e a homol- ϩ igure 5.23. -ATPase), is utilizedis the trans- vity of theof vity esignation , so called so , K, by secret- s the walls to the cell to the ex-the to Na absorbed ey main- ey erd to ferred ss results ss , Halobac- Forex- . olecular- e samehe gradient lso been bacteri- purple ective. ithelial ϩ ϩ orbing ediate. nsport ADP, omes, large d in a and, ently con- oto- ich ng ns, ey - tpe”b te pteil el t die h cotran the drive cell the into to molecules glucose cells epithelial the by “tapped” apical plasma membrane down their concentration gra The tendency for sodium ions to dient. diffuse back pumps sodium ions out of the cell against a concent Na transport. active by driven be to said are molecules glucose The ent. rti.(Fprotein. through exterior a c to the cell from the cytoplasm As a result of these even in the next pumping cycle. Asp85 is deprotonated to receiving (step 5) prior a proton fr EMSINFROMPERMISSION Asp96 is then reprotonated by a H side o residue near the cytoplasmic (Asp96) located (step 3) when it accepts a proton from an undissoci The deprotonated retinal is returned to its pathway. hydrogen-bonded water molecules that help shuttle p these residues The spaces between are ﬁ and Glu194). amino acid residrelay consisting of several system then released side of the memb to the extracellular acid residue aspartic (#85) (ste charged negatively Figure 4.48 transfer of a protontransfer from the —NH in the electronic a change of causes structure reti of a pho Absorption element (chromophore). absorbing serve which retinallocated group (shown in purple), helices an membrane-spanning protein contains seven L membrane by facilitated diffusionmembrane (page 157). across moved are and cell the through diffuse cules the gluco Once inside, cose molecule with each cycle. ANYI ϩ guoecotransporter/glucose S, CIE NCE ROM Bacteriorhodopsin: a light-driven proton pump. a light-driven Bacteriorhodopsin: H 8:5,1999; 286:255, ARTMUT n hs ae h tasot rti,cle a called protein, transport the case, this In AAAS.) moves two sodium ions and one , glu- L EK TAL ET UECKE © 1999, ϩ ϩ against from the cytoplasm (step 4).from the cytoplasm group to a closely associated,group to a closely ERNE WITHREPRINTED ., a concentration gradi-concentration a OREYOFCOURTESY a,leading to thenal, ) The proton isp 1). e Ap2 Glu204,ues (Asp82, entral channel in the channel entral rane (step 2) by a rane ated aspartic acidated aspartic original state original f the membrane. s protons movets, s as the light- rotons along the lled with d a centrally d a centrally ton of light ration ration gra- om retinal across the the basalthe secondary J se mole- pr of sport ANOS dient is The K. 161 ϩ This calculation indicates that the Na /glucose cotransporter is capable of transporting glucose into a cell against a concentration gradient greater than 20,000-fold. Lumen Plant cells rely on secondary active transport systems to take up a variety of nutrients, including sucrose, amino acids, and nitrate. In plants, uptake of these compounds is coupled ϩ to the downhill, inward movement of H ions rather than ϩ + GL Na ions. The secondary active transport of glucose into the 2Na epithelial cells of the intestine and the transport of sucrose into a plant cell are examples of symport , in which the two ϩ ϩ transported species (Na and glucose or H and sucrose)

Na +/glucose move in the same direction. Numerous cotransporters have cotransporter been isolated that engage in antiport , in which the two trans- Na + GL ported species move in opposite directions. For example, cells Glucose transporter often maintain a proper cytoplasmic pH by coupling the in- + + ϩ Na Na GL (GLUT2). ward, downhill movement of Na with the outward move- + + Facilitated ϩ K K diffusion of ment of H . Cotransporters that mediate antiport are usually glucose. called exchangers . The three-dimensional structures of a num- + + Blood Na /K -ATPase ber of secondary transporters have been solved in recent years, ϩ ϩ GL and, like the Na րK -ATPase, they exhibit a transport cycle in which the protein’s binding sites gain alternating access to Figure 4.49 Secondary transport: the use of energy stored in the cytoplasm and the extracellular space. A proposed model ϩ ϩ an ionic gradient. The Na /K -ATPase residing in the plasma of the transport cycle of one of the major families of second- membrane of the lateral surface maintains a very low cytosolic concen- ϩ ϩ ary transporters is shown in Figure 4.50. tration of Na . The Na gradient across the plasma membrane represents a storage of energy that can be tapped to accomplish work, such ϩ as the transport of glucose by a Na /glucose cotransporter located in Extracellular the apical plasma membrane. Once transported across the apical 1 2 surface into the cell, the glucose molecules diffuse to the basal surface Outer gate where they are carried by a glucose facilitative transporter out of the + cell and into the bloodstream. The relative size of the letters indicates Leu Na ϩ the directions of the respective concentration gradients. Two Na ions ϩ are transported for each glucose molecule; the 2 Ϻ1 Na /glucose provides a much greater driving force for moving glucose into the cell than a 1 Ϻ1 ratio. Inner gate

To appreciate the power of an ion gradient in accumulat- ing other types of solutes in cells, we can brieﬂy consider the 4.7 ϩ

energetics of the Na /glucose cotransporter. Recall from MembranesCell Across Substances of Movement The page 148 that the free-energy change for the movement of a ϩ mole of Na ions into the cell is equal to Ϫ3.1 kcal/mol, and ϩ thus 6.2 kcal for two moles of Na ions, which would be 4 3 available to transport one mole of glucose uphill into the cell. Cytoplasm Recall also from page 148 that the equation for the movement of a nonelectrolyte, such as glucose, across the membrane is Figure 4.50 A schematic model of the transport cycle of a secondary transporter. Four different conformational states during the transport [C ] ¢G ϭ RT ln i cycle of a bacterial symporter of the LeuT family are shown. The pro- [C ] tein actively transports the amino acid leucine into the cell using the o ϩ established Na ion gradient as its source of energy. In step 1, the outer [C ] ϩ ¢ ϭ i gate in the protein is open, whch allows both Na and leucine to reach G 2.303 RT log 10 [C o] their binding sites from the extracellular space. In step, 2, the outer gate closes, occluding the substrates within the protein. In step 3, a second Using this equation, we can calculate how steep a concentra- leucine molecule binds to another site just outside the outer gate. In tion gradient of glucose ( X ) that this cotransporter can gener- step 4, the inner gate opens and the substrates are released into the Њ ate. At 25 C, cytoplasm. The protein returns to its original state when the inner gate Ϫ ϭ # is closed and the outer gate is opened. (R EPRINTED BY PERMISSION 6.2 kcal/mol 1.4 kcal/mol log 10 X FROM MACMILLAN PUBLISHERS LTD : N ATURE , 465:171, 2010 BY ϭ Ϫ log 10 X 4.43 N. K. K ARPOWICH AND DA-N ENG WANG (NYU S CHOOL OF 1 MEDICINE ) X ϭ 23,000 Chapter 4 The Structure and Function of the Plasma Membrane 162 See See conse Clinical Gene Type of channel Ca Familial (FHM) hemiplegic migraine Inherited disorder Table 1 ytcfirssCl Na Cl Cl Na Na K Cardiac arrhythmias fibrosis Cystic Bartter’s IV type syndrome K Myotonia congenita Dent’s disease K Myasthenia gravis Syndrome Liddle K Ca Ca QT syndrome Long Nonsyndromic dominant deafness Benign familial neonatal convulsions Episodic ataxia type-1 paralysis periodic Hypokalemic (EA-2) Episodic ataxia type-2 yeklmcproi aayi Na paralysis periodic Hyperkalemic ion channels of epithelialion channels cells. stud best the from results disorder, fibrosis, channel ion inherited common cystic contrast, In 167). (page t or develop to cells these of ability the reducing and nerve, muscle, (i.e., cells excitable of membranes in listed ions Tableders of movement the affect 1 genes Mo that encode ion channel proteins (Table 1). traced been have disorders inherited severe, Several de nmru cmlctos o h soy s t has it as story s the subsequent to complications But numerous added transporter. a not channel, chloride into artificial lipid andbilayers, shown to act as purifie a was protein the after answered be to thought that researchers weren’t sure of its precise functi ductance regulator The protein superfamily. was named apparent that the polypeptide was a member of the A was correspondingthepolypeptide of sequence acid amino the and determined was gene CF the of sequence the Once sively destroy pulmonary function. destroysively pulmonary wh inflammation, and infections lung chronic from fer Afflicted individu hard to propel out of the airways. sticky mu of Victims CF produce a thickened, effects. the respiratory tract usually exhi productive g tract, sweat pancreas, intestine, the including organs, ous fibros cystic Although (CF). fibrosis cystic with born tion (1/25 tion Co carriers. are 2500 infants in approximatelythis C 1 out of every they that unaware are heterozygotes mutant the of symptoms no show they Because fibrosis. ca that gene mutant the of copy one carries descent E V I T C E P S R E P N A M U H E H T Nature Cell Biol On average, 1 out of every 25 persons of Northern Eu Northern of persons 25 every of out 1 average, On The gene responsible for cystic fibrosis was isolate was fibrosis cystic for responsible gene The ϫ Defects in Ion Channels and Defects in Ion Channels Transporters Disease as a Cause of Inherited 1/25 :00 04 or 2004, 6:1040, . ( CFTR ϫ 1/4) is homozygous recessive at this locus andlocus this at recessive homozygous is 1/4) ,an ambiguous term that reflected the fact), Nature cystic fibrosis transmembrane con- 4:4,20,for a more complete list. 2006, 440:444, cyclic AMP-regulated n The question wason. bits the most severe across the plasmathe across asi impulses ransmit K Na Ca aucasian popula-aucasian Cl to mutations inmutations to als typically suf-als typically ,incorporatedd, deduced, it was it deduced, ϩ ϩ ϩ ϩ ϩ BC transporter st of the disor- sensory cells), sensory cus that is very Ϫ Ϫ Ϫ Ϫ a defect in thein defect a is affects vari- affects is n cause cystic cystic cause n 2 2 2 2 lands, and re- and lands, ϩ ϩ ϩ ϩ ϩ e ad most and ied en shown been ϩ ϩ ϩ ϩ ich progres- ich uis have tudies gn,mostgene, nsequently, d in 1989.in d ropean aydfeetIrregular heartbeat or rapid many different CFTR CLC-Kb CLC-1 CLCN5 nAChR B-ENaC SCN4A orKCNQ1, HERG KCNQ4 KCNQ2 KCNA1 CACNL1A3 CACNL1A4 CACNL1A4 genes SCN5A GS-9411) with the potential to reduce Na conducts bicarbonate (HCO bicarbonate conducts in addition to functioning as that, a chloride chann leig h mvmns f os n n ot f h ep the Na include compounds These of 1). out (Figure and in ions of movements the s altering of volume the increase to potential the have that Clinical trials are also of being the carried mucus. reduc and airways the into water more draw to helps hypertoni of mist a inhaling by helped are patients respiratory the of out bacteria and mucus push that funct the impair mucus, secreted the of viscosity in r a and liquid, surface the of volume reductionin A the of cells epithelial the bathes that fluid the in Cl potentia the with Moli1901) (e.g., CFTR) than (other an epithelial Na HCO sis follows the movement of salts, abnormalities in abnormalities salts, of movement the follows sis iwy ud no h eihlu ad ciaos f C of activators and epithelium the into fluid airway mains of the CFTR polypeptide. Subsequent research has revealedhas research Subsequent polypeptide. cyt CFTR the the of mains of one within 508, position at phenylalanine many researchers would agree with the following sta there cons is While chronicof infections. ment lung le protein this in defect a how precisely establish become has it complex, more become has CFTR of role chloride/bicarbonate epithelial of family a of ity ntd tts oti te ae eei atrto ( alteration genetic same ⌬ the contain States United c for responsible alleles the of percent 70 imately different mutations that give rise to cystic fibrosi F508)—they F508)—they are all missing three base pairs of DNA Ϫ ion conductance from the epithelium into the airway Because the Because movement of water out of epithelial cel In the past decade, researchers have identified more than 1000than more identified have researchers decade, past the In Ϫ 3 and/or Na, ϩ o hne Ea) and (3) stimulates the activ- (ENaC), ion channel ϩ Lung congestion andLung infections deafnessKidney dysfunction, Periodic myotonia Kidney stones weaknessMuscle (high blood pressure)Hypertension Periodic myotonia and paralysis sudden death from ventricularDizziness, Deafness Epileptic convulsions Ataxia Periodic stiffness) and paralysis myotonia (muscle of balanceAtaxia and coordination) (lack headachesMigraine caused by CFTR deficiency leads to deficiency a decrease by caused CFTR fibrillation 3 Ϫ ) ions, (2) suppresses the activity of activity the suppresses (2) ions, ) quences ϩ ϩ -channel inhibitors (e.g., inhibitors -channel ion absorption from the .Hwvr approx- However, s. airways (Figure 1). (Figure airways ads to the develop-the to ads out on compounds exchangers. As the As exchangers. ystic fibrosis in thein fibrosis ystic el, CFTR also CFTR (1)el, esulting increaseesulting tract. Many CF Many tract. the flux of Clof flux the iderable debate, iderable tements. ion of the ciliathe of ion aie whichsaline, c rae ud by fluid urface e the viscosity viscosity the e pamc do-oplasmic that encode a ithelial cells cells ithelial l l to increaseto l Ϫ ls by osmo- designated difficult todifficult fluid. channels Ϫ , 163

Hydrated Suspended mucous layer CFTR bacteria

Layer of pathogenic Flow Dehydrated mucous layer bacteria (biofilm) Deficient. flow Figure 1 An explanation for the debilitating effects on lung function from the absence of the CFTR protein. In the Cilia airway epithelium of a normal individual, water flows out of the epithelial cells in response to the outward movement of ions, Epithelial thus hydrating the surface mucous layer. Na + Cl – H O Na + H O Cl – 2 cell 2 The hydrated mucous layer, with its trapped bacteria, is readily moved out of the airways. Airway epithelium from normal individual Airway epithelium from CF patient In the airway epithelium of a person with cystic fibrosis, the abnormal movement of ions causes water to flow in trapped bacteria cannot be moved out of the airways, which allows the opposite direction, thus dehydrating the mucous layer. As a result, them to proliferate as a biofilm (page 13) and cause chronic infections. that CFTR polypeptides lacking this particular amino acid fail to be Ever since the isolation of the gene responsible for CF, the devel- processed normally within the membranes of the endoplasmic retic- opment of a cure by gene therapy—that is, by replacement of the de- ulum and, in fact, never reach the surface of epithelial cells. As a fective gene with a normal version—has been a major goal of CF result, CF patients who are homozygous for the ⌬F508 allele com- researchers. Cystic fibrosis is a good candidate for gene therapy pletely lack the CFTR channel in their plasma membranes and have because the worst symptoms of the disease result from the defective a severe form of the disease. When cells from these patients are activities of epithelial cells that line the airways and, therefore, are ac- grown in culture at lower temperature, the mutant protein is trans- cessible to agents that can be delivered by inhalation of an aerosol. ported to the plasma membrane where it functions quite well. This Clinical trials have been conducted using several different types of de- finding has prompted a number of drug companies to screen for livery systems. In one group of trials, the normal CFTR gene was in- small molecules that can bind to these mutant CFTR molecules, corporated into the DNA of a defective adenovirus, a type of virus that preventing their destruction in the cytoplasm and allowing them to normally causes upper respiratory tract infections. The recombinant reach the cell surface. One of these candidate drugs, VX-809, is in virus particles were then allowed to infect the cells of the airway, deliv- phase II clinical trials. VX-809 is given in combination with ering the normal gene to the genetically deficient cells. The primary another drug, Kalydeco, which can keep the ion channel open if it disadvantage in using adenovirus is that the viral DNA (along with the arrives at the cell surface. Kalydeco has recently been approved as a normal CFTR gene) does not become integrated into the chromo- solo treatment for the approximately 4 percent of CF patients whose somes of the infected host cell so that the virus must be readministered CFTR protein carries a single amino acid substitution, G551D. frequently. As a result, the procedure often induces an immune re- While this mutation does not interfere with the protein reaching the sponse within the patient that eliminates the virus and leads to lung cell surface, it does prevent the ion channel from opening properly. inflammation. Researchers are hesitant to employ viruses that inte-

According to one estimate, the ⌬F508 mutation had to have grate their genomes for fear of initiating the formation of cancers. In 4.7 originated more than 50,000 years ago to have reached such a high other trials, the DNA encoding the normal CFTR gene has been frequency in the population. The fact that the CF gene has reached linked to positively charged liposomes (page 128) that can fuse with the MembranesCell Across Substances of Movement The this frequency suggests that heterozygotes may receive some selec- plasma membranes of the airway cells, delivering their DNA contents tive advantage over those lacking a copy of the defective gene. It has into the cytoplasm. Lipid-based delivery has an advantage over viruses been proposed that CF heterozygotes may be protected from the ef- in being less likely to stimulate a destructive immune response follow- fects of cholera, a disease that is characterized by excessive fluid se- ing repeated treatments, but has the disadvantage of being less effec- cretion by the wall of the intestine. One difficulty with this proposal tive in achieving genetic modification of target cells. To date, none of is that there is no record of cholera epidemics in Europe until the the clinical trials of gene therapy has resulted in significant improve- 1820s. An alternate proposal suggests that heterozygotes are pro- ment of either physiologic processes or disease symptoms. The devel- tected from typhoid fever because the bacterium responsible for this opment of more effective DNA delivery systems, which are capable of disease adheres poorly to the wall of an intestine having a reduced genetically altering a greater percentage of airway cells, will be re- number of CFTR molecules. quired if a treatment for CF based on gene therapy is to be achieved.

R E V I E W 1. Compare and contrast the four basically different ways 3. Describe the relationship between partition coefficient that a substance can move across the plasma mem- and molecular size with regard to membrane brane (as indicated in Figure 4.33). permeability. 2. Contrast the energetic difference between the diffusion 4. Explain the effects of putting a cell into a hypotonic, of an electrolyte versus a nonelectrolyte across the hypertonic, or isotonic medium. membrane. Chapter 4 The Structure and Function of the Plasma Membrane 164 muss which is the impulses, subject of the remainder propertie of t basic same the membranes on that dependslead to the ameba formation and an propagatio in ity and rounding moving up, offdia, in another direction with a fine respondsglass needle, by withdrawing its as to ferred pro a stimulation, external to respondorganisms All 4.8 Figure 4.51 and Nerve Impulses within the central nervous system are called oligod are system called nervous within the central ( nodes of are Ranvier. called wrapping The sites where the axon around the axon. themselves that have cells individual Schwann sheath comprises As the inset s simple neuron with a myelinated axon. Nucleus .How does the Na6. .Describe two ways in which energy is utilized to 5. .What isthestructuralrelationbetweenpartsof 8. .What is the role of phosphorylation in the mechan 7. .Because of its smaller size, one9. would expect Na Cell body Dendrites be able to penetrate any pore large enough for a K regulated K How does the K ions and solutes against a concentration gradient. of theplasmamembrane? action of the Na prokaryotic KcsA K tion) occur? of these processes (ion selectivity, gating, and in and which part in channel inactivation? How does ea involved in ion selectivity, which part in channel gating, (a) | Membrane Potentials The structure of a nerve cell. nerve a of structure The Myelin sheath irritability Axon ϩ channel? Which part of the channel is ϩ ϩ ϩ /K channel select for this specific ion? vn snl-eld mb,i poked if single-celled ameba, a Even . /K ϩ ϩ channel and the eukaryotic voltage- ϩ -ATPase? -ATPase illustrate the asymmetry Node ofRanvier Note: Layers of Terminal knob myelin ( Myelin-forming cellsMyelin-forming a ) Schematic drawing of a drawing ) Schematic endrocytes ratherendrocytes Schwann cell Nucleus of wrapped os the myelinhows, lacks myelin lacks he chapter. pseudopo- ϩ n of nerve Irritabil-. activa- move perty re-perty ϩ ions to ism of ion. ch Axon ofs (b) ie hslnt.( times this length. in larger ma cells Motor nerve 1 cm in length (red). neuron (pu hippocampal body with and dendrites cell than Schwann cells.) ( cells.) than Schwann B cess Voltages of negative ions at the othera point. when there is an excess of positive ions at one poi such as the inside and outside of the plasma membra A voltage (or electric potential difference) betwee The Resting Potential tension, the the tension, promin more single, a is body cell the from emerging other typically neu formation from external sources, called extensions, fine neuronsaremost of a bodies cell fromthetending manufacture are contents material its of most where smaller processes, each ending in a in ending e each their near processes, smaller split axons Most whale. or giraffe a as extend for many meters in the body of a large verte Al length, in micrometers few a only be cell(s). may axons some target the toward and body cell the from ern i te etbae oy r wapd n lip a sheathmyelin in wrapped are body vertebrate 167 the page in on neurons discussed As targets. potential of sands wit communicate to cells brain these allowing knobs, of thousands in end brain the in neurons Many cell. ized site where impulses are transmitted from neuro cell bodycell regionexpanded an within located is neuron the of 4.51. Figure in areneuronillustrated typical a of The in the form of fast-moving electrical impulses. is which information, of transmission and conduction, IOL Nerve cells Nerve 750 07 ©2007, 2007, 17:520, . , which is the metabolic center of the cell and the and cell the of center metabolic the is which , axon , whose function is described below. whose function is described , (or B F: , which conducts conducts which , b neurons ) A composite micrograph of a single rat ROM dendrites C IHPRISO FROM PERMISSION WITH ) are specialized for the collection,the for specialized are ) ARLOS , which receive which , .IF. BÁÑEZ terminal knobterminal outgoing T, mmals can be 100mmals can R E NDS rple) and an axonrple) muss away impulses nt and an ex- E n two points, incoming The nucleusThe cross plasma —a special-—a LSEVIER rt,suchbrate, C number ofnumber basic parts n to target os Alsorons. e resultsne, called the called ELL d intonds terminal h thou-h though ent ex-ent id-rich .Ex-d. coded most , others .) site in- 165 membranes can be measured by inserting one fine glass elec- Those that are open are selective for K ϩ; they are often re- trode (or microelectrode ) into the cytoplasm of a cell, placing an- ferred to as Kϩ leak channels . K ϩ leak channels are members of other electrode in the extracellular fluid outside the cell, and the K2P family of K ϩ channels, which lack the S4 voltage connecting the electrodes to a voltmeter, an instrument that sensor (page 155) and fail to respond to changes in voltage. measures a difference in charge between two points (Figure Because K ϩ ions are the only charged species with signif- 4.52). When this experiment was first carried out on a giant icant permeability in a resting nerve cell, their outflow axon of the squid, a potential difference of approximately 70 through the membrane leaves an excess of negative charges on millivolts (mV) was recorded, the inside being negative with the cytoplasmic side of the membrane. Although the concen- respect to the outside (indicated with a minus sign, Ϫ70 mV). tration gradient across the membrane favors continued efflux The presence of a membrane potential is not unique to nerve of K ϩ, the electrical gradient resulting from the excess nega- cells; such potentials are present in all types of cells, the mag- tive charge on the inside of the membrane favors the retention nitude varying between about Ϫ15 and Ϫ100 mV. When a of K ϩ ions inside the cell. When these two opposing forces are nerve or muscle cell is in an unexcited state, the membrane po- balanced, the system is at equilibrium, and there is no further tential is referred to as the resting potential because it is sub- net movement of K ϩ ions across the membrane. Using the ject to dramatic change, as discussed in the following section. following equation, which is called the Nernst equation, one The magnitude and direction of the voltage across the can calculate the membrane potential ( Vm) that would be plasma membrane are determined by the differences in con- measured at equilibrium if the plasma membrane of a nerve ϩ 7 centrations of ions on either side of the membrane and their cell were permeable only to K ions. In this case, Vm would relative permeabilities. As described earlier in the chapter, the be equal to the potassium equilibrium potential ( E ): ϩ ϩ ϩ ϩ K Na /K -ATPase pumps Na out of the cell and K into the ϩ cell, thereby establishing steep gradients of these two ions RT # [Ko ] EK ϭ 2.303 log 10 ϩ across the plasma membrane. Because of these gradients, you zF [Ki ] might expect that potassium ions would leak out of the cell ϩ For a squid giant axon, the internal [K i ] is approximately and sodium ions would leak inward through their respective ϩ 350 mM, while the external [K o ] is approximately 10 mM; ion channels. However, the vast majority of the ion channels thus at 25 ЊC (298 K) and z ϭ ϩ 1 (for the univalent K ϩ ion), in the plasma membrane of a resting nerve cell are closed. EK ϭ 59 log 10 0.028 ϭ Ϫ 91 mV ϩ A similar calculation of the Na equilibrium potential ( ENa ) +80 +80 Sodium equilibrium potential +60 +60 would produce a value of approximately ϩ55 mV. Because +40 +40 measurements of the voltage across the resting nerve mem- +20 +20 brane are similar in sign and magnitude ( Ϫ70 mV) to the 0 0 Electrode –20 –20 enters cell potassium equilibrium potential just calculated, the move- Voltage (mV) Voltage –40 –40 ment of potassium ions across the membrane is considered the –60 –60 –80 –80 most important factor in determining the resting potential. ϩ –100 –100 The difference between the calculated K equilibrium poten- Potassium equilibrium potential tial ( Ϫ91 mV) and the measured resting potential ( Ϫ70 mV,

Time Time Figure 4.52) is due to a slight permeability of the membrane to Na ϩ through a recently described Na ϩ leak channel. Voltmeter The Action Potential Recording Reference electrode electrode Our present understanding of membrane potentials and nerve 4.8 impulses rests on a body of research carried out on the giant ax- Membrane Potentials and Nerve ImpulsesNerve and Potentials Membrane ++ + + + + + + + + + + + + + + + ++ + + + + + + + + + + + + + + + ons of the squid in the late 1940s and early 1950s by a group of – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – British physiologists, most notably Alan Hodgkin, Andrew Axon Axon Huxley, and Bernard Katz. These axons, which are approximately 1 mm in diameter, carry impulses at high speeds, en- (a) (b) abling the squid to escape rapidly from predators. If the membrane of a resting squid axon is stimulated by poking it Figure 4.52 Measuring a membrane’s resting potential. A with a fine needle or jolting it with a very small electric current, potential is measured when a difference in charge is detected some of its sodium channels open, allowing a limited number of between the reference and recording electrodes. In (a), both electrodes sodium ions to diffuse into the cell. This opportunity for posi- are on the outside of the cell, and no potential difference (voltage) is measured. As one electrode penetrates the plasma membrane of the tively charged ions to move into the cell reduces the membrane axon in ( b), the potential immediately drops to Ϫ70 mV (inside negative), which approaches the equilibrium potential for potassium ions, 7The Nernst equation is derived from the equation provided on page 148, by that is, the potential that would result if the membrane were imperme- setting ⌬G at zero, which is the case when the movement of the ions is at equi- able to all ions except potassium. librium. Walther Nernst was a German physical chemist who won the 1920 Nobel Prize. Chapter 4 The Structure and Function of the Plasma Membrane 166 4.53 membrane potential are called an an called are potential membrane ch these Collectively, state. resting its to membrane sium channels to close (see Figure 4.43Figure (see close to channels sium voltage-gate the causes potentialmembrane negative membrane voltage is approximatelymembrane Kto permeabilitycreased ae,leading to an influx of Nagates, opening the voltage-regu beyond the threshold value, has The membrane phase: shows the depolarization box, n esalsigtersigptnil (and reestablishing the resting potential. path of ion movement across channels as the primary leaving the potassium gates close, open, soon as they that location (that location more neg and establish an even of the drawing) part allowing potassium ions to diffuse across the open, the sodium gates are inactivated and th of a second, With shows phase: the repolarization box, upper right potentialof membrane that constitutes the action p proximately reac reverse itself, voltage to temporarily membrane hnei h oe rp) The increased Nachange in the lower graph). fuse freely into the cell 4.53(Figure io sodium result, a As open. to channels sodium gated of events Theis launched. change in voltage causes th in membraneavoltage causesin Becausepositthe negative. less makingit potential, hne htocrdrn nato oeta,as de an action potential, changes that occur during Figure 4.53 ceased (Figure 4.53 returns restingitsto potentialth soonas as idly larizes the membrane beyond a certain point, called called point, certain a threshold beyond membrane the larizes meability of the membrane to Nato membrane the of meability incr The gradients. electric and concentration their e ilvls say from a few millivolts, the two sides it is of called the membrane, a swing back to a negative value of about ig oeet f oiie hre no h cl caus cell 4.53 (Figure briefly potential reverse to membrane the into charge positive of movement ding ing positive at about at positive ing rium potential for Narium brane potential caused by Naby caused potential brane K des that to similarmanner a in porechannel the of th into peptideinactivation an of diffusionrandom results inactivation view, prevailing the to cording eul iatvt,bokn frhr nu o Na of influx further blocking inactivate, neously the voltage-gated potassium channels (Figure 4.53 decreased permeability of the membrane to Nato membrane the of permeabilitydecreased grad concentration steep their down cell the of out discussed As potassiumon a page ions result, 154. di resting potential, in which only the K only in which resting potential, c in this region The membrane of the nerve box: left that of the Kthe of that ϩ channels on page 155. Meanwhile, the change in mem-in change the Meanwhile, 155. page on channels If the stimulus causes the membrane to depolarize b fe prxmtl sc thesodium channels spon Afterapproximately 1 msec, b .Theentire series ofchanges during anaction pot ). , which occurs at about at occurs which , ϩ Formation potential. of an action Ϫ 0m ntesudgatao tm ) It is this r 40 mV in the squid giant axon (time 2). ϩ 0m)ta hto h etn oeta.Almost as than that of the resting potential. 80 mV) equilibrium potential (Figure 4.52). The large The 4.52). (Figurepotentialequilibrium a etbx.I,hwvr the stimulus depo- however, If, left box). , ϩ ϩ 40 mV, which approaches the equilib-the approaches which mV, 40 (Figure 4.52).(Figure Ϫ ϩ 70 to ϩ cause the membrane potential topotentialmembrane the cause ions (indicated in the permeabilityions (indicated ϩ decrease Ϫ influx triggers the opening ofopening the triggers influx ϩ Ϫ 0m.Tm ,upper middle 2, Time 70 mV. leak channels areleak channels open and the Ϫ ϩ 0m,the 60 membrane mV, rap- b a action potential action ) A summary of the voltage) A summary 50 mV, then a new series new a then mV, 50 , middle box) middle down both, ions and the correspon-the and ions in the polarity betweenpolaritythe in ϩ Ϫ b permeability causes the causes permeability ), which returns thereturns which ), 0m,approaching80 mV, ( a depolarization ie1 upper ) 1, Time membrane (lowermembrane scribed in part in part scribed otential. Time 3, Time otential. hing a value of ap- e potassium gates ative potential at ell exhibits theell the potassium leak the membrane lated sodium in a tiny fraction e stimuluse has ϩ depolarized ϩ a and the in-the and right box), b ffuse freely ive changeive eased per-eased rm the from e openinge cribed forcribed ,becom-), e voltage- os Ac-ions. et Theient. ne inanges d potas-d (Figure ns dif-ns s the es eversal y only ential a . the . ta- 4.53 The strikingtial. changes in membrane potential seen sidestheofmembrane areinvolved anyingiven act o ionspercentageminutethe ofa only dramatically, inactivating peptide has swung out of the opening o occur only conformationcan closed the to activated the transformationFigure 4.43, of the ion channeldepi f As stimulus. another to response in reopened be be potentialactionclosemustthe ofinitialstage inactivatedwerechannelssodiumthatthebecaused peri refractory The restimulated. be cannot it which eta,te ebae nes brief a enters membrane the tential, in a myelinated Followingmammalian nerve cell. an a takes only about 5 msec in the squid axon and less leak channel Potassium sodium gatesclosed + K b actionAlthoughpotentialthechangesmembrane volt voltage =–70mV Resting potential + + arenot caused by changes in Na – – + Sodium closed – – gate – Na Outside + + + – + – Potassium + – closed (a) (b) Membrane potential (mV) Ion permeability gate + –70 K + 40 + 0 + 2 1 0 0 + – + + – – + + – – Time + + – – 1 + + Depolarization phase – – sodium gatesopen – + + voltage =+40mV + + – – + + + – – Sodium – – open gate + – – Time (ms) Time (ms) + + Time – – – + + 2 – – Na Outside Na + + – – 1 – + Na + Potassium – – closed + + + – – + gate permeability + + + – – + + + + – – Time + + – – + 3 ϩ + + – – ercoy periodrefractory – + K + + – – + andK + + – – permeability + + – – potassium gatesopen Repolarization phase 2 + + – – + voltage =–80mV – + + Sodium closed ϩ – – gate – + ion fore they cantheyfore Outside – than 1 msec Potassium + – rom the in- – ionpoten- open + gate f the pore. n the twothe n + concentra- od occursod in Figure – – after theafter uring theuring ction po- + td incted during K K + + + age 167 tions on the two sides of the membrane (such changes are in- significant). Rather, they are caused by the movements of +++++++++ – – – – – – – –++++++++++++++++++++++ charge in one direction or the other that result from the fleeting –– – – – – – – – ++++++++– – – – – – – – – – – – – – – –– – – – – – changes in permeability to these ions. Those Na ϩ and K ϩ ions that do change places across the membrane during an action Axon Direction of propagation

ϩ ϩ

– – – – – – – – ++++++++– – – – – – – – – – – – – – – –– – – – – –

potential are eventually pumped back by the Na /K -ATPase. –

++++++++ – – – – – – – –++++++++++++++++++++++ Even if the Na ϩ/K ϩ-ATPase is inhibited, a neuron can often + continue to fire thousands of impulses before the ionic gradients established by the pump’s activity are dissipated. Region in Region of Region where depolarization Once the membrane of a neuron is depolarized to the refractory action will trigger threshold value, a full-blown action potential is triggered with- period potential action potential out further stimulation. This feature of nerve cell function is known as the all-or-none law. There is no in-between; sub- Figure 4.54 Propagation of an impulse results from the local flow threshold depolarization is incapable of triggering an action po- of ions. An action potential at one site on the membrane depolarizes tential, whereas threshold depolarization automatically elicits a an adjacent region of the membrane, triggering an action potential at maximum response. It is also noteworthy that an action poten- the second site. The action potential can only flow in the forward tial is not an energy-requiring process, but one that results from direction because the portion of the membrane that has just the flow of ions down their respective electrochemical gradients. experienced an action potential remains in a refractory period. Energy is required by the Na ϩ/K ϩ-ATPase to generate the steep ionic gradients across the plasma membrane, but once that is accomplished, the various ions are poised to flow through the pulses than weaker stimuli. Yet, we are clearly capable of detect- membrane as soon as their ion channels are opened. ing differences in the strength of a stimulus. The ability to make The movements of ions across the plasma membrane of sensory discriminations depends on several factors. For example, nerve cells form the basis for neural communication. Certain a stronger stimulus, such as scalding water, activates more nerve local anesthetics, such as procaine and novocaine, act by clos- cells than does a weaker stimulus, such as warm water. It also ac- ing ion channels in the membranes of sensory cells and neu- tivates “high-threshold” neurons that would remain at rest if the rons. As long as these ion channels remain closed, the affected stimulus were weaker. Stimulus strength is also encoded in the cells are unable to generate action potentials and thus unable pattern and frequency by which action potentials are launched to inform the brain of events occurring at the skin or teeth. down a particular neuron. In most cases, the stronger the stimulus, the greater the number of impulses generated. Propagation of Action Potentials as an Impulse Speed Is of the Essence The greater the diameter of an Up to this point, we have restricted the discussion to events axon, the less the resistance to local current flow and the more occurring at a particular site on the nerve cell membrane rapidly an action potential at one site can activate adjacent re- where experimental depolarization has triggered an action po- gions of the membrane. Some invertebrates, such as squid and tential. Once an action potential has been initiated, it does not tube worms, have evolved giant axons that facilitate the ani- remain localized at a particular site but is propagated as a nerve mal’s escape from danger. There is, however, a limit to this impulse down the length of the cell to the nerve terminals. evolutionary approach. Because the speed of conduction in- Nerve impulses are propagated along a membrane be- creases with the square root of the increase in diameter, an cause an action potential at one site has an effect on the adja- axon that is 480 ␮m in diameter can conduct an action poten- cent site. The large depolarization that accompanies an action tial only four times faster than one that is 30 ␮m in diameter.

potential creates a difference in charge along the inner and During the evolution of vertebrates, an increase in con- 4.8 outer surfaces of the plasma membrane (Figure 4.54). As a re- duction velocity was achieved when the axon became wrapped sult, positive ions move toward the site of depolarization on in a myelin sheath (see Figures 4.5 and 4.51). Because it is ImpulsesNerve and Potentials Membrane the outer surface of the membrane and away from that site on composed of many layers of lipid-containing membranes, the the inner surface (Figure 4.54). This local flow of current myelin sheath is ideally suited to prevent the passage of ions causes the membrane in the region just ahead of the action across the plasma membrane. In addition, nearly all of the potential to become depolarized. Because the depolarization Na ϩ ion channels of a myelinated neuron reside in the un- accompanying the action potential is very large, the mem- wrapped gaps, or nodes of Ranvier , between adjacent Schwann brane in the adjacent region is readily depolarized to a level cells or oligodendrocytes that make up the sheath (see Figure greater than the threshold value, which opens the sodium 4.51). Consequently, the nodes of Ranvier are the only sites channels in this adjacent region, generating another action where action potentials can be generated. An action potential potential. Thus, once triggered, a succession of action poten- at one node triggers an action potential at the next node (Fig- tials passes down the entire length of the neuron without any ure 4.55), causing the impulse to jump from node to node loss of intensity, arriving at its target cell with the same without having to activate the intervening membrane. Propa- strength it had at its point of origin. gation of an impulse by this mechanism is called saltatory Because all impulses traveling along a neuron exhibit the conduction . Impulses are conducted along a myelinated axon same strength, stronger stimuli cannot produce “bigger” im- at speeds up to 120 meters per second, which is more than Chapter 4 The Structure and Function of the Plasma Membrane 168 next resting node along the axon. Figure 4.55 accomplished as current flows directly from an activ an action potent of forming and capable depolarized the in membrane the nodal regiononly of the axon b Axon Na Na + + Saltatory conduction.Saltatory Node of Ranvier + + – – Current flowdepolarizes next nodeofRanvier sheath Myelin During saltatory conduction, saltatory During Next node Synaptic cleft Synaptic vesicles Membrane ofpostsynaptictarget cell Terminal knobofpresynaptic neuron ated node to the a.This is ial. ecomes + + – – of impulse Direction Muscle fiber Axon ofnervecell a eetr el r nuo) odcs muss tow impulses conducts neuron) a or cell receptor (a ad,dfclyi akn,and difficulty problems in walking, withhands, their patientsexperienceweakness adulthood; youngin gin of Manifestationsthe nervous system. of the disease tionofthe myelin sheath that surrounds axons inv disease a bymultipleassociated sclerosiswit (MS), myelinated neuron of the same diameter. un-an in travel impulses that speed the than faster times 20 This gap is called the the called is gap This contact separated direct make from not each other do by cells a narrow two the gap of that about veals called tions speci at cells target their with linked are Neurons Neurotransmission: Jumping the Synaptic Cleft The importance of myelination is dramaticallyillusmyelinationis importance of The synapses . Careful examination of a synapse re- synapse a of examination Careful . NE FROMINSET h ufc ftemsl el(le.(L (blue). cell of the muscle the surface neuron are binding to receptors (yellow) on of the presynaptic vesicles from synaptic Neurotransmitter molecules (red) released plasma membrane. cell against the muscle shows knob pressed the terminal closely inset The right target cell. postsynaptic knob and the the terminal between cleft knob of the axon and the narrow synaptic residing within the terminal vesicles synaptic inset shows the The left skeletal muscle. fibers of a with the muscle synapses form is the site where from branches a motor axon Figure 4.56 R EESE yatc cleft synaptic /P HOTO D The neuromuscular junctionThe ON R ESEARCHERS .FW. . A A . AWCETT rsnpi cell presynaptic 20 to 50 nm. alized junc- alized h deteriora-h I, ariousparts usually be- /T . NC vision. u are but in theirin .) trated r a ard EFT 169 synapse, and a postsynaptic cell (a neuron, muscle, or gland The sequence of events during synaptic transmission can be cell) always lies on the receiving side of a synapse. Figure 4.56 summarized as follows (Figure 4.57). When an impulse reaches shows a number of synapses between the terminal branches of a terminal knob (step 1, Figure 4.57), the accompanying depo- an axon and a skeletal muscle cell; synapses of this type are larization induces the opening of a number of voltage-gated called neuromuscular junctions . Ca 2ϩ channels in the plasma membrane of this part of the presy- How does an impulse in a presynaptic neuron jump across naptic nerve cell (step 2, Figure 4.57). Calcium ions are normally the synaptic cleft and affect the postsynaptic cell? Studies car- present at very low concentration within the neuron (about 100 ried out decades ago indicated that a chemical substance is in- nM), as in all cells. When the gates open, calcium ions diffuse volved in the transmission of an impulse from one cell to from the extracellular fluid into the terminal knob of the neuron, another (page 171). The very tips (terminal knobs) of the causing the [Ca 2ϩ] to rise more than a thousandfold within lo- branches of an axon appear in the electron microscope to con- calized microdomains near the channels. The elevated [Ca 2ϩ] tain large numbers of synaptic vesicles (Figure 4.56, left in- triggers the rapid fusion of one or a few nearby synaptic vesicles set) that serve as storage sites for the chemical transmitters with the plasma membrane, causing the release of neurotrans- that act on postsynaptic cells. Two of the best studied neuro- mitter molecules into the synaptic cleft (step 3, Figure 4.57). transmitters are acetylcholine and norepinephrine, Once released from the synaptic vesicles, the neurotransmitter molecules diffuse across the narrow gap and bind selec- O tively to receptor molecules that are concentrated directly + across the cleft in the postsynaptic plasma membrane (step 4, CH 3 C O CH 2 CH 2 N(CH3)3 Figure 4.57). A neurotransmitter molecule can have one of Acetylcholine (ACh) two opposite effects depending on the type of receptor on the target cell membrane to which it binds: 8 OH H 1. + The bound transmitter can trigger the opening of cation- HO C CH 2 NH 3 selective channels in the membrane, leading primarily to an

OH 8It is important to note that this discussion ignores an important class of neuro- Norepinephrine transmitter receptors that are not ion channels and thus do not directly affect membrane voltage. This other group of receptors are members of a class of proteins called GPCRs, which are discussed at length in Section 15.3. When a neurotrans- which transmit impulses to the body’s skeletal and cardiac mitter binds to one of these receptors, it can initiate a variety of responses, which muscles. often includes the opening of ion channels by an indirect mechanism.

1 Nerve impulse Synaptic vesicle Acetylcholine

Presynaptic membrane

2 4.8 3 4 5a 5b 6 2+ Synaptic cleft Ca Ca 2+ 0 0 gates Release Binding of Nerve impulse ImpulsesNerve and Potentials Membrane open of AcCh AcCh by receptor mV –70 or mV –70

Na + gates open Cl -- gates open Postsynaptic membrane

Figure 4.57 The sequence of events during synaptic transmis- pulse may be generated there (6). If, however, the binding of sion with acetylcholine as the neurotransmitter. During neurotransmitter causes a hyperpolarization of the postsynaptic steps 1–4, a nerve impulse reaches the terminal knob of the axon, membrane (5b), the target cell is inhibited, making it more difficult for calcium gates open leading to an influx of Ca 2ϩ, and acetylcholine is an impulse to be generated in the target cell by other excitatory stimu- released from synaptic vesicles and binds to receptors on the postsynap- lation. The breakdown of the neurotransmitter by acetylcholinesterase tic membrane. If the binding of the neurotransmitter molecules causes is not shown. a depolarization of the postsynaptic membrane (as in 5a), a nerve im- Chapter 4 The Structure and Function of the Plasma Membrane 170 h rutk o srtnn nuornmte implic neurotransmitter a serotonin, of reuptake the prescribed antidepressants, including Prozac and Zol neurotransmittersout Aof numbe the synaptic cleft. each impulse milliseconds. lasts no moreeach than a few the molecules, neurotransmitter of reuptake or tion ot ertasitr oeue bc t te presyna the to called process back terminals—a molecules neurotransmitter port proteins by and cleft synaptic the in molecules ter destroyneurotrthat enzymes by ways: two in synapse eliminated is neurotransmitter A recover. not would postsynaptic the and extended, be would transmitter t of effect the otherwise neuron; presynaptic a from neurotransmitter has only a short half-life followi ized by the loss of acetylcholine-releasing neurons by the loss of acetylcholine-releasing ized treat the which symptoms is of Alzheimer’s disease, a acetylcholinesterase of inhibitors Milder choline. of concentrations high of presence continued the to contra violent undergo body the of muscles skeletal exam for DFP, gas nerve the to exposure by inhibited thi If acetylcholine. hydrolyzes that cleft synaptic w localized enzyme an is Acetylcholinesterase fects. have physiologicdramatic and can transmitters beha Synapses on Drugs of Actions tic neuron. whether or not an impulse will be generated in the dete that influences opposing these of summation the signals hibitory from many different presynaptic ne excitato both receive brain the in cells nerve Most insomnia, and schizophrenia, exert exert their action at th and schizophrenia, de insomnia, anxiety, as such disorders, affecting drugs ferent section, following the in evident be will As prima brain’s switch. the of activity the well enhancing and as tor anesthetics, general Valium of act by and binding its to derivatives, the GA number A transmitter. inhibitory glut primary the as (GABA) acid aminobutyric brain, the Within serves as the primary excitatory neurotransmitter a muscle. skeletal of tractility inhibits contractility of the heart but sti example, Acetylcho another. on effect inhibitory an and brane have a effect stimulatory on one particular postsyn neurotransmitte given a However, neurotransmitter(s). 2. tion potential of its steps own 5a (Figureand 4.57, respond to this or subsequent stimuli by generating tic membrane membrane This depolarization potential. of the posts influx of sodium ions and a less negative (more posi orahtemmrn’ hehl Fgr .7 step to reach the membrane’s threshold 4.57, (Figure greater r tial because sodium influx is subsequently an acti generate will the cell makes it less likely membr of the postsynaptic Hyperpolarization tential. membrane and a more negative (hyperpolarized) ions, to an influx of ch leading mainly selective channels, the opening of an trigger The bound transmitter can Many drugs act by inhibitingtransportersthebythat act drugs Many neu of reuptake or destruction the with Interfering l tria kos f gvn ern ees te sa the release neuron given a of knobs terminal All excites h el making the cellthe morecell, likely to reuptake . Because of the destruc-the of Because . t s motn ta a that important is It mulates con- ng its release s enzyme is enzyme s nd gamma- aptic mem- . that trans-that on poten- postsynap- rn.It isurons. f,inhibitoft, rof widely ry and in-and ry BA recep- character- re used toused re many dif-many e synapse. he neuro-he ction duection vioral ef-vioral 6). pression, ithin theithin from thefrom effect of effect an ac- y“f ”“off ry tive) ie forline, ansmit- equired acetyl- l,theple, td inated neuron loride neuro- ynap- amate rmine mayr s w e e p ion- 5b). ane ptic po- as ro- me and block their stimulation by dopamine. tain subtypes of the dopamine receptor on postsynap the A number synaptic of cleft. antipsychotic drugs neurotransmitterreuptakethetheferewithof mole releasedopamineof frompresynaptic terminals and Amphetaminesareneurons. thought stimulate to e the dopamine-res postsynaptic dopamine-releasing or tic lackingthe or amphetamines has no additional behavioral effect been given cocaine or amphetamines. Administration o take—showt normalmicebehaviorofthat similar to transporter d (DAT)—the the lack protein to engineeredresponsible genetically been for havedopam that strongdesirerepeataa totheaswell as euphoria, limbictheofcleftssystemtic produces short-li a centers.The ward” sustained presence of dopamine in Thelimbic system. system contains the brain’s “plea certain nerve cells in a portion of the brain known neurotransmitterthereuptake ofr dopamineis that on the interferesmood Cocaine, other disorders. hand, cannabinoid cannabinoid (CB1) receptors located on the I mechanism. different totally a by acts cannabinol) achieves it mature configuration.achieves when the neuronalof circuitry and fancy childhood, importa particularly is plasticity Synaptic ticity.” display a remarkable dynamic t quality known as “syna structures, unchanging fixed, as perceived often are While pieces back or rerouting them in another direction. holding while another, to neuron one from pass inform of pieces some allowing pathways, various the These synapses act like gates sta trillion synapses. one least at contain to thought is brain human The nervou the through impulses of routing the in nants are key they neurons; adjacent between sites necting ftm,the synapsesof time, that connect these neurons to pocampal neurons are repeatedly stimulated over a s tally important in learning and short-term W memory. neurons from the a hippocampus, portion of the brain body called called body produced compounds with interact normally receptors neurotransmitt release will neurons these that hood reduces which brain, the of neurons certain of nals yatc Plasticity Synaptic fromhas been pulled the market of side eff because Acomplia, called drug weight-loss CB1-blocking a of d the reasoningto of led line This creaseappetite. receptors these blocking that follows it receptors, bindi by appetite increases marijuana If spectively. appetite and coordination, motor memory, on marijuana effe the explains which hypothalamus, and cerebellum, hipp the including brain, the of areas many in cated CB1 receptor suppressing transmission. synaptic tors, where bindthey to CB1 to the presynaptic membrane, depolarizat following across the syna These neurons substances diffuse “backwards” postsynaptic by duced h atv cmon i mrjaa ( marijuana in compound active The Synaptic plasticity is most readily observed in stu in observedreadily most isplasticity Synaptic DAT endocannabinoids ee Numerousother gene. drugs act on presynap- yass r mr ta sml con- simply than more are Synapses noanbnis r pro- are Endocannabinoids . presynaptic ⌬ 9 ved feelingvedof nt during in- during nt as the limbic -tetrahydro- tioned along tvt.Micectivity. s on animals their neigh- evelopment ue or “re-sure” bind to cer- hort period ng to CB1to ng tic neurons ih de-might cules from ects. the synap- eleased by alsointer- the likeli-the t binds tobinds t that is vi- ocampus, determi- ptic plas- synapses the brain s system.s r.CB1ers. ptic cleft ine reup- with the opamine f cocaine hundred hen hip- a havehat ponding s are lo- ation toation hey can hey xcessive is ondies in thein termi- recep- which cts ofcts other re-, ion. 171 bors become “strengthened” by a process known as long-term R E V I E W potentiation (LTP), which may last for days, weeks, or even 1. What is a resting potential? How is it established as a longer. Research into LTP has focused on the NMDA receptor, result of ion flow? which is one of several receptor types that bind the excitatory 2. What is an action potential? What are the steps that neurotransmitter glutamate. When glutamate binds to a post- lead to its various phases? synaptic NMDA receptor, it opens an internal cation channel within the receptor that allows the influx of Ca 2ϩ ions into the 3. How is an action potential propagated along an axon? postsynaptic neuron, triggering a cascade of biochemical What is saltatory conduction, and how does such a changes that lead to synaptic strengthening. Synapses that have process occur? undergone LTP are able to transmit weaker stimuli and evoke 4. What is the role of the myelin sheath in conduction of stronger responses in postsynaptic cells. These changes are an impulse? thought to play a major role as newly learned information or 5. Describe the steps between the time an impulse memories are encoded in the neural circuits of the brain. When reaches the terminal knob of a presynaptic neuron and laboratory animals are treated with drugs that inhibit LTP,such an action potential is initiated in a postsynaptic cell. as those that interfere with the activity of the NMDA receptor, 6. Contrast the roles of ion pumps and channels in estab- their ability to learn new information is greatly reduced. lishing and using ion gradients, particularly as it applies There are numerous other reasons the study of synapses is to nerve cells. so important. For example, a number of diseases of the nervous system, including myasthenia gravis, Parkinson’s disease, schizophrenia, and even depression, are thought to have their roots in synaptic dysfunction.

EXPERIMENTAL PATHWAYS The Acetylcholine Receptor In 1843, at the age of 30, Claude Bernard moved from a small French rate of the second heart slowed dramatically, as though its own in- town, where he had been a pharmacist and an aspiring playwright, to hibitory nerve had been activated. 2 Loewi called the substance re- Paris, where he planned to pursue his literary career. Instead, Bernard sponsible for inhibiting the frog’s heart “Vagusstoff.” Within a few enrolled in medical school and went on to become the foremost phys- years, Loewi had shown that the chemical and physiologic properties iologist of the nineteenth century. Among his many interests was the of Vagusstoff were identical to acetylcholine, and he concluded that mechanism by which nerves stimulate the contraction of skeletal mus- acetylcholine (ACh) was the substance released by the tips of the cles. His studies included the use of curare, a highly toxic drug isolated nerve cells that made up the vagus nerve. from tropical plants and utilized for centuries by native South Ameri- In 1937, David Nachmansohn, a neurophysiologist at the can hunters to make poisonous darts. Bernard found that curare para- Sorbonne, was visiting the World’s Fair in Paris where he observed lyzed a skeletal muscle without interfering with either the ability of several living electric fish of the species Torpedo marmarota that nerves to carry impulses to that muscle or the ability of the muscle to were on display. These rays have electric organs that deliver strong contract on direct stimulation. Bernard concluded that curare somehow shocks (40–60 volts) capable of killing potential prey. At the time, acted on the region of contact between the nerve and muscle. Nachmansohn was studying the enzyme acetylcholinesterase, which This conclusion was confirmed and extended by John Langley, a acts to destroy ACh after its release from the tips of motor nerves. physiologist at Cambridge University. Langley was studying the ability Nachmansohn was aware that the electric organs of these fish were 4.8 of nicotine, another substance derived from plants, to stimulate the con- derived from modified skeletal muscle tissue (Figure 1), and he asked traction of isolated frog skeletal muscles and the effect of curare in in- if he could have a couple of the fish for study once the fair had ended. ImpulsesNerve and Potentials Membrane hibiting nicotine action. In 1906, Langley concluded that “the nervous The results of the first test showed the electric organ was an extraor- impulse should not pass from nerve to muscle by an electric discharge, dinarily rich source of acetylcholinesterase. 3 It was also a very rich but by the secretion of a special substance on the end of the nerve.” 1 source of the nicotinic acetylcholine receptor (nAChR),* the recep- Langley proposed that this “chemical transmitter” was binding to a “re- tor present on the postsynaptic membranes of skeletal muscle cells ceptive substance” on the surface of the muscle cells, the same site that bound nicotine and curare. These proved to be farsighted proposals. Langley’s suggestion that the stimulus from nerve to muscle was *The receptor is described as nicotinic because it can be activated by nicotine as transmitted by a chemical substance was confirmed in 1921 in an in- well as by acetylcholine. This contrasts with muscarinic acetylcholine receptors genious experiment by the Austrian-born physiologist, Otto Loewi, of the parasympathetic nerve synapses, which can be activated by muscarine, but the design of which came to Loewi during a dream. The heart rate not nicotine, and are inhibited by atropine, but not curare. Smokers’ bodies become accustomed to high levels of nicotine, and they experience symptoms of of a vertebrate is regulated by input from two opposing (antagonis- withdrawal when they stop smoking because the postsynaptic neurons that possess tic) nerves. Loewi isolated a frog’s heart with both nerves intact. nAChRs are no longer stimulated at their usual level. The drug Chantix, which is When he stimulated the inhibitory ( vagus ) nerve, a chemical was re- marketed as an aid to stop smoking, acts by binding to the most common version leased from the heart preparation into a salt solution, which was al- of brain nAChR (one with ␣4␤2 subunits). Once bound, the Chantix molecule lowed to drain into the medium bathing a second isolated heart. The partially stimulates the receptor while preventing binding of nicotine.