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Home , Cotransporter, Facilitated diffusion, Passive transport

MEMBRANE TRANSPORT

Bob Mercer [email protected] Transport Defects

Cardiac arrhythmias Renal transport defects Epilepsy Deafness Parkinson disease Autism Ataxia Hypertension Mental Retardation CYSTIC FIBROSIS

1/2000 births in white Americans

30,000 children and adults in the US

Median age for survival late 30s

Autosomal recessive inheritance Hartnup Disease With an estimated overall prevalence of 1 case per 24,000 population Hartnup disease ranks among the most common disorder in humans.

Wide clinical spectrum: Most patients remain asymptomatic, but, in a minority of patients, skin photosensitivity and neurologic and psychiatric symptoms may have a considerable influence on quality of life. Rarely, severe CNS involvement may lead to death, mental retardation and short stature. Malnutrition and a low- diet are the primary factors that contribute to morbidity.

Caused by mutations in the SLC6A19 (B0 AT1) neutral amino acid transporter. Homozygotes fail to transport neutral amino acids in the and the renal tubules. The B0 AT1 transporter is a Na-dependent that transports all neutral amino acids. B0 AT1 expression appears mainly restricted to the kidneys and intestine. Although tryptophan is transported inefficiently, it is thought to be one of the key substrates in the development of the nonrenal symptoms of Hartnup disorder. Tryptophan converted in the to niacin, and approximately half of the NADPH synthesis in humans is generated through tryptophan. As a result, tryptophan and niacin deficiencies generate similar symptoms. In addition, symptoms in persons with Hartnup disorder quickly respond to nicotinic acid supplementation. WILSON’S DISEASE

1 in 100 individuals carry mutation in ATP7B gene (Cu- ATPase)

1-4 per 100,000 people

Autosomal recessive inheritance

Neurological or psychiatric symptoms

Liver disease

Kaysar-Fleischer (KF) ring THE RELATIVE PERMEABILITY OF A SYNTHETIC BILAYER TO DIFFERENT

O2 HYDROPHOBIC CO2 MOLECULES N2 benzene

SMALL H2 O UNCHARGED urea POLAR glycerol MOLECULES

LARGE UNCHARGED POLAR sucrose MOLECULES

H +, Na+ + HCO¯3 , K Ca2+ , Cl¯ Mg2+

synthetic COMPARISON OF CONCENTRATIONS INSIDE AND OUTSIDE A TYPICAL MAMMALIAN

Intracellular Extracellular Concentration Concentration Component (mM) (mM)

Cations

Na 5-15 145 K 140 5 Mg 0.5 1-2 Ca 10-4 1-2 H 8 x 10-5 (pH 7.1) 4 x 10-5 (pH 7.4)

Anions

Cl 5-15 110

Because the cell is electrically neutral the large deficit in intracellular anions reflects the fact that most cellular constituents are negatively charged. The concentrations for Mg and Ca are given for free ions. THE PROBLEM: - + A CELL - - H O - 2 - + + - + + + + - + ------+ + + - - + + + +

- + - +

SOLUTIONS: IONS - + + - + - - + - - + + - + + - - - + - H 2 O - + - - H 2 O + + - H O + - - 2 - + + - - - - + + - + + - + - + - + - - + + + + - + + + + - + - - + + + - - + + - + - + - 500 Normal Died Asymptomatic Lethargic

450

400

350 Brainwater (g/100 gdrywt) 300 139 baseline 119 in 2 h 122 in 3.5 d 99 in 16 d Plasma Na concentration (mEq/l) Woman drinks so much water she dies January 13, 2007

SACRAMENTO, California (AP) -- A woman who competed in a radio station's contest to see how much water she could drink without going to the bathroom died of water intoxication, the coroner's office said Saturday.

Jennifer Strange, 28, was found dead Friday in her suburban Rancho Cordova home hours after taking part in the "Hold Your Wee for a Wii" contest in which KDND 107.9 promised a Nintendo Wii video game system for the winner.

"She said to one of our supervisors that she was on her way home and her head was hurting her real bad," said Laura Rios, one of Strange's co-workers at Radiological Associates of Sacramento. "She was crying, and that was the last that anyone had heard from her."

Copyright 2007 The Associated Press. All rights reserved.This material may not be published, broadcast, rewritten, or redistributed.

Simple

• Flux is proportional to external concentration • Flux never saturates Flux

[S]o PROTEIN MEDIATED

• • PRIMARY • SECONDARY ACTIVE • / Membrane Flux (moles of solute/sec)

• Simple Diffusion

• Carrier • Facilitated Diffusion • Primary Active Transport • Secondary Active Transport

• Ion Channels .

TRANSPORT OF MOLECULES THROUGH

transported

lipid electrochemical bilayer gradient

ENERGY simple channel- carrier- diffusion mediated mediated diffusion diffusion

PASSIVE TRANSPORT ACTIVE TRANSPORT (FACILITATED DIFFUSION) CARRIER MEDIATED TRANSPORT

lipid bilayer

UNIPORT SYMPORT OR ANTIPORT OR COTRANSPORT COUNTERTRANSPORT

COUPLED TRANSPORT Review • The lipid bilayer is impermeable to ions and acts like an electrical capacitor. • Cells express ion channels, as well as pumps and exchangers, to equalize internal and external osmolarity. • Cells are permeable to K and Cl but nearly impermeable to Na. • Ions that are permeable will flow toward electrochemical equilibrium as given by the Nernst Equation.

Eion = (60 mV / z) * log ([ion]out / [ion]in) @ 30°C • The Goldman-Hodgkin-Katz equation is used to calculate the steady-state in cells with significant relative permeability to sodium.

& P ∗[K] + P ∗[Na] + P ∗[Cl] # V 60mV log$ K out Na out Cl in ! m = ∗ $ ! % PK ∗[K]in + PNa ∗[Na]in + PCl ∗[Cl]out " Structure of a Channel

Doyle et al., 1998 Carrier-Mediated Transport

• Higher flux than predicted by solute permeability

Mmax • Flux saturates • Binding is selective (D- versus L-forms) 0.5 • Competition Flux • Kinetics:

[S]o << Km M α [S]

[S]o = Km M = Mmax / 2

Km [S] >> K M = M [S]o o m max Transport Kinetics

+ k

So + Co - SCo Si S = Solute C = Carrier k

+ - dSCo/dt = k [S]o [C]o – k [SC]o = 0 at equilibrium

+ - ⇒ k [S]o [C]o = k [SC]o

- + k / k = ([S]o [C]o)/[SC]o = Km ⇒ [SC]o = ([S]o [C]o)/Km

Fractional Rate = M / Mmax = [SC]o / ([C]o + [SC]o)

M = Mmax / (1 + [C]o/[SC]o) = Mmax / (1 + Km/[S]o) Reversible Transport

Co Ci

So Si

SCo SCi

Mnet = Min – Mout =

1 1 Mmax - ( 1 + Km / [S]o 1 + Km / [S]i ) Facilitated Diffusion

• Uses bidirectional, symmetric carrier

• Flux is always in the directions you expect for simple diffusion

• Binding is equivalent on each side of the membrane

Examples include: Glucose Transporters (GLUT); Anion Exchanger; Organic Anion Transporters; Urea Transporters; Monocarboxylate (lactate) Transporters (MCTs); Amino Acid Transporters; Zn Transporters (ZIP) (GLUT), Solute Carrier (SLC) family 2

13 members of the GLUT/SLC2 identified

Facilitated Diffusion: Band 3/AE1 4 Facilitated Diffusion: Band 3/AE1 Cytoskeletal/AE1 Interactions Aquaporins

In mammals at least 13 isoforms present Primary Active Transport: Driven by ATP • Class P – all have a phosphorylated intermediate • Na,K-ATPase H,K-ATPase • Ca-ATPase Cu-ATPase • H-ATPase bacterial K-ATPase • Flippase • Class V • H+ transport for intracellular organelles • Class F • Synthesize ATP in mitochondria • ABC ATPases • ATP Binding Cassette 48 known members- Multiple Drug Resistance; Sulfonylurea receptor; CFTR Primary Active Transport: Na,K-ATPase

3 Na

ATP ADP + Pi

2 K

• 3 Na outward / 2 K inward / 1 ATP

• Km values: Nain ≈ 20 mM Kout ≈ 2 mM • Inhibited by digitalis and ouabain • “opens” • 2 subunits, beta and alpha (the pump) • Two major conformations E1 & E2 • Turnover = 300 Na+ / sec / pump site @ 37 °C Na,K-ATPase Reaction Scheme

Extracellular 2K 3Na . . E2P K2 E2P E1P E1P Na3

Pi . E2 (K2) ATP . E1P (Na3)

. ADP E2 (K2)ATP

...... E2 ATP K2 E2 ATP E1 ATP Na3 E2P ADP Na3

2K 3Na Intracellular

Membrane Transport and Cellular Functions that Depend on the Na,K-ATPase Amino Acid Homology Among the Na,K-ATPase Subunit Isoforms

Y K Y L Q P L L A V Q F T N L T L D T E I R I E C K A G E N I G Y S E D R F Q G R F D V K I E V K S COOH K 300 P Q L G GY F G G M E E C N L K G Y Y P Y Y Q L P F Y F G F Y I N G V K D K D D R K G T Q V P L V P 250 N Y E G K C I L K N E S K Extracellular G Y K K P I I K N R V L G F K P P P K L E T Y P L T M Y 200 S E D N L G S C N G L W D L K F R C V K R E G R E H N F E G R E K P E S P M S G C D E F I M D D T W D D R W I N D V E K R E D S 150 I Y Q A G G Q I Q K T E I S F R P N D P K S Y E A Y V L N I I R F L E K Y K D S L Q P 100 L Q 900 I H W Q E E T L G P P A V R D Q Y T E P F T α T P P Y P A N L E K F G N E I K R Q L S D P A V E Y T W L L L R M R D E E P G L E L Y S I L A I T 800 I A P I G I L V I F N V F V F T C G A L K L T A Y Y L G L I A L Y G M L L S L I G I I T I F T H T A F C P P T W L M V S L Q L C F V L S F F I I V A N I F C I D G G F V S L S Y W F C I V 300 L L F F 52 I F I A I V G V P E G T D Q A A A P G T I G A V L V P F G I I V V V L A F G A M1 100 M2 G M3 F T M4 M5 M6 M7 M M8 A M9 M10 M1 L L L W I T A V L L A E I T M V P I G W A E E T S L C G I V C P Q Q Y Y V S M T G V T V N I I S Y G 850 L F I F I F G F C F I S A A D L V F G L F Y G H L T L T M L L L F S F I L A Y T L T L A Y L I S I I L I 950 V Y D I Q Y H K A E R C K E K R Y E R I Q E K N V F C Q I M S A N T K R 1000 W F E E A K E V R M K S K A E R K S L R G L G L V K A K D K N Q I G 3/3 IDENTICAL NH N I D S Q I 2 W S I N 350 V F T 2/3 IDENTICAL G D E S 150 P C M T R R R F K NONE P K T L K R G I R D T I Q V P R L K L Q N T M G K P F R P R Y P E G N G β E G E P S E L 750 G K P E P F L E E V G T V I S A F N D D W K L A T K G A V E A L L N S V E S V M I R M A E V G S D V S K Q A A D K N S Q V L T I E W E P P T L G T I H G Q E M Y G S A G A V G I D A K K L A P S D N V G D G T V A V I F G D Q T E I S A Y COOH K D R A L P R T G K K 700 A L S Q G S Q W K H T E I V F A R T S P Q Q K L I I V E G C Q R L V S T M T Y S S I I N R V I R G K E R D S T C L E K 650 A N V P R S I E A D D L E E S T M D K L D S G H V V C A K A D R P N V Q N V P I N L R A A I D E P G K D D D V K K R E C F G K T A K E A K G T T T K E N P M N N Y G 600 G R G T S A E V T A V P D A V G K C R S A G I K V I M V T G D H P I T A K A I A K G V G I I S E R D A L I S N L A M V R G N I P I T M D P NH R A I 200 L G Q 2 E P S E R E R 250 N L D 550 L D L T A R I K M S I L G V F C L N D V P F N V E D T D F Q F G E P F Q E D P L L L H C F G L V R E D D R M K V T G T A N T L E V G G V P I E V 500 G V G Y 50 G I A A L L V M K G A P E R I L D R C S S I L L H G K E Q P L D E E L K D A F Q N A Y L E L S V K H K D R F H M C P R L D F N M D S T E H G D V W S 450 L E G F A H V K N P N K H I S L Q Y K N T S N F P I E V I K T Y K E R M E M V S G C C V E I C K L L A S E S K E D 4/4 IDENTICAL L S L D A N D 3/4 IDENTICAL Q G 2/4 IDENTICAL I 400 A H E A D T T E N Q S G V S F D K T S A T W F A L S R I A G L C N R A V F Q A N Q E N L P I L K R A V NONE Cytoplasmic The Na,K-ATPase As a Receptor For Signal Transduction SR Ca-ATPase Predicted topology of Flippase-ATPase

Tanaka K et al. J Biochem 2011;149:131-143 F-type, V-type ATPases V-type ATPase H-ATPase

Nat Commun. 2012 February 21; 3: 687 Experimental Evidence for Rotation Multidrug Resistance Protein 1 (MDR1)/P-glycoprotein 1 ATP-binding cassette sub-family B member 1 (ABCB1) Secondary Active Transport

• stored in the Na+ (H+ or K+) gradient is used to power the transport of a variety of solutes glucose, amino acids, ions and other molecules are pumped in (cotransport) Ca2+ or H+ are pumped out 2 or 3 Na+ / 1 Ca2+ ; 1 Na+ / 1 H+ (countertransport)

• These transport proteins do not hydrolyze ATP directly; but they work at the expense of the ion gradient which must be maintained by an ATPase Secondary Active Transport

• In humans over 40 families of Na coupled transporters

Examples include: Na+/H+ exchanger; Na+/Ca2+ exchanger; Na+/aspartate cotransporter; Na+/amino acid cotransporter; Na+/glucose cotransporter; Na+/urea + + + 2+ cotransporter; Na /PO4; cotransporter; (H /Na )/Zn exchanger (ZnT) Energy available from ATP

H2O ATP ADP + Pi

ΔG = Gproducts – G reactants

Chemical Energy (G) = RT ln [C]

ΔG = ΔG° + 2.3 RT (log ([ADP] [Pi]) – log [ATP])

2.3 RT = 5.6 kiloJoules / mole @ 20° C

ΔG° = -30 kiloJoules /mole @ 20°C, pH 7.0 and 1M [reactants] and [products] “Standard Conditions” Energy Depends on Substrate Concentrations

ΔG = -30 – 5.6 log [ATP] kJ / mole [ADP] [Pi]

The energy available per molecule of ATP depends on: [ATP] ≅ 4mM, [ADP] ≅ 400 µM, [Pi] ≅ 2 mM

⇒ per mole of ATP hydrolyzed:

Δ G = -30 kJ – 5.6 kJ * log 4 x 10-3 2 x 10-3 * 4 x 10-4

= -30 kJ - 21 kJ = -51 kiloJoules per mole of ATP

Converting to approximately -530 meV/molecule of ATP Energy in the Sodium Gradient

Consider Na+ movement from outside to inside:

ΔG = Gproducts – Greactants = Ginside – Goutside

ΔGtotal = ΔGelectrical + ΔGchemical

Conditions for our sample calculation:

+ + Vm = -60 mV [Na ]out = 140 mM [Na ]in = 14 mM

and 2.3 RT = 60 meV / molecule

Energy in the Na Gradient: Electrical Term

ΔGelectrical = e * mVin – e * mVout

= +1e * -60 mV – (+1e) * 0 mV

= -60 meV

• negative sign means energy is released moving from outside to inside

• 60 MeV is the energy required to move a charged ion (z=1) up a voltage gradient of 60 mV (assuming zero concentration gradient) Energy in the Na Gradient: Chemical Term

+ + ΔGchemical = 2.3 RT (log [Na ]in – log [Na ]out)

= 60 meV * (-1)

= -60 meV

• negative sign means energy is released moving from outside to inside

• 60 meV is the energy required to move a molecule up a 10 fold concentration gradient (true for an uncharged molecule or for a charged molecule when there is no voltage gradient) Energy in the Na Gradient: Total

ΔGtotal = ΔGelectrical + ΔGchemical = -120 meV

• 120 milli-electron-Volts of energy would be required to pump a single Na+ ion out of the cell up a 10 fold concentration gradient and a 60 mV voltage gradient.

• Hydrolysis of a single ATP molecule can provide at least 500 + meV of energy – enough to pump 4 Na ions.

• A single Na+ ion moving from outside to inside would be able to provide 120 meV of energy, which could be used to pump some other molecule, such as glucose, an amino acid, Ca2+ or H+ up a concentration gradient Cotransport Proteins

Phlorizin Bumetanide Thiazide Furosemide Cotransport Proteins Countertransport /Exchanger Proteins

SITS Amiloride DIDS Na Glucose Cotransporter- Solute carrier family 5 12 members of the human SLC5 gene family, including for sugars, anions, vitamins, and short-chain fatty acids

Gene Substrate K0.5, mM Distribution SGLT1 (SLC5A1) Glucose 0.5 Intestine, trachea, kidney, heart, Cotransporter Galactose 0.5 brain testis, prostate

SGLT2 (SLC5A2) Glucose 6 Kidney, brain, liver, , Cotransporter muscle, heart

SGLT3 (SLC5A4) Glucose 20 Intestine, testis, uterus, , Cotransporter brain, thyroid

SGLT4 (SLC5A9) Glucose 2 Intestine, kidney, liver, brain, Cotransporter Mannose 0.15 lung, trachea, uterus,

SGLT5 (SLC5A10) Glucose ND Kidney cortex Cotransporter Galactose ND Na Glucose Cotransporter- Solute carrier family 5

SGLT1 consists of the 664 residues arranged in 14 transmembrane domains with both the N- and C- termini facing the extracellular surface. A single N-glycosylation site occurs at Asn (N) 248. Na Glucose Cotransporter- Solute carrier family 5 Na Glucose Cotransporter- Solute carrier family 5 Na Glucose Cotransporter- Solute carrier family 5 Na Glucose Cotransporter- Solute carrier family 5 Na Glucose Cotransporter- Solute carrier family 5 Na Glucose Cotransporter- Solute carrier family 5 12 members of the human SLC5 gene family, including cotransporters for sugars, anions, vitamins, and short-chain fatty acids

Gene Substrate K0.5, mM Distribution SGLT1 (SLC5A1) Glucose 0.5 Intestine, trachea, kidney, heart, Cotransporter Galactose 0.5 brain testis, prostate

SGLT2 (SLC5A2) Glucose 6 Kidney, brain, liver, thyroid, Cotransporter muscle, heart

SGLT3 (SLC5A4) Glucose 20 Intestine, testis, uterus, lung, Cotransporter brain, thyroid

SGLT4 (SLC5A9) Glucose 2 Intestine, kidney, liver, brain, Cotransporter Mannose 0.15 lung, trachea, uterus, pancreas

SGLT5 (SLC5A10) Glucose ND Kidney cortex Cotransporter Galactose ND http://www.fda.gov/Drugs/DrugSafety/ucm446845.htm Na/H Exchanger- Solute carrier family 9, subfamily A NHE1-9

Exchanges 1 Na+ for 1 H+ Signal Transduction Complex of NHE-3 REGULATION OF NA/H EXCHANGER TRAFFICKING Na/Ca Exchangers- Solute carrier family 8 & 24

3 Mammalian isoforms 5 Mammalian isoforms

The crystal structure of bacterial Na/Ca Exchanger from Methanococcus jannaschii has been reported (1.9-Å resolution), describing ten transmembrane helices ( Liao et al., 2012). Sequence similarity suggests the eukaryotic exchanger may share the same ten-TMS topology rather than the suggested nine-TMS topology. Example: Na+/Ca2+ exchange

Compare the internal [Ca2+] for exchange ratios of

2 Na+ : 1 Ca2+ vs. 3 Na+ : 1 Ca2+

2+ 2+ Vm = -60 mV, [Ca ]out = 1.5 mM [Ca ]in = ?

Ca2+ moves from inside to outside

ΔG = Gproducts – Greactants = Goutside – Ginside

ΔGelectrical = (+2e) * (0 mV) – (+2e) * (-60 mV)

= +120 meV

ΔGchemical = 60 meV (log 1.5 – log ?) Na+/Ca2+ exchange

ΔGtotal = ΔGE + ΔGC = 120 meV + 60 meV log (1.5 / ?) 2 Na+ ⇒ 240 meV 240 = 120 + 60 log (1.5 / ?)

2+ 120 / 60 = log (1.5 / ?) Internal [Ca ] 2 can be reduced 10 = 1.5 / ? 100 fold lower ? = 15 µM for 3 Na : 1 Ca vs 2 Na : 1 Ca 3 Na+ ⇒ 360 meV 360 = 120 + 60 log (1.5 / ?) 240 / 60 = log (1.5 / ?) 104 = 1.5 / ? ? = 0.15 µM Structure of the Na/Ca Exchanger

Summary: Energetics

Transport Energetics • A molecule of ATP donates about 500 meV • It takes 60 meV to transport up a 60 mV electrical gradient • It takes 60 meV to transport up a 10 fold concentration gradient • A single sodium ion donates approximately 120 meV Summary: Membrane Flux (moles of solute/sec)

Simple Diffusion • Flux is directly proportional to external concentration • Flux never saturates Carrier-Mediated Transport • Higher flux than predicted by solute permeability • Flux saturates • Binding is selective D- versus L-forms • Competition • Kinetics Facilitated Diffusion • Uses bidirectional, symmetric carrier proteins • Flux is in the direction expected for simple diffusion • Binding is equivalent on each side of the membrane Primary Active Transport – driven by ATP hydrolysis Secondary Active Transport – driven by ion gradients Ion Channels CELLULAR MECHANISMS FOR INFLUENCING TRANSPORT ACTIVITY

• Expression of Specific Isoforms

• Assembly of Different Isoform Subunits

• /Endocytosis

• Specific Regulation by Protein Kinases

• Modification Through Inhibitors

•Assembly with Accessory/Regulatory Proteins

•Changes in Rate of Synthesis

•Changes in Rate of Degradation Transporters Regulated by Signaling Cascades

Na/H Exchangers Na/Phosphate Cotransporter Na/K/2Cl Cotransporter Na/Cl Cotransporter K/Cl Cotransporter Na/Ca Exchanger Na Channels K Channels Na,K-ATPase H,K-ATPase Unidirectional Transport Assays

1. Cells washed in isotonic buffered solution 2. Required transport inhibitor(s) added 3. Flux medium containing radioactive isotope added 4. At required times flux medium rapidly removed and Cells growing cells washed (3-4 x) in ice-cold isotonic saline in multi-well plates 5. Final wash removed, cells lysed and radioactivity and protein content of samples determined Unidirectional Transport Assays Calculations:

Specific Activity of medium:

Measure radioactivity in known volume of flux medium.

For example: For unidirectional uptake of Na into cells in medium containing:

50 mM Na 100 mM Cl 25 mM K-Hepes, pH 7.4 22Na (≈ 1 µCu/ml)

Measure radioactivity in 5 µl flux medium

cpm (22Na) 1 L 1 mole cpm (22Na) X X = 5 x 10-6 L 0.050 moles Na 109 nmoles nmoles Na

Measure radioactivity and protein content in sample.

Determine Na influx using specific activity of medium Determine transport rate/protein content (Na uptake nmoles/µg protein/min) THICK ASCENDING LIMB CELL

Na+

K+ Na+ Na+ K+ Cl 2Cl K+ Na+

K+ K+ Na+ K+

Lumen Blood GASTRIC PARIETAL CELL

K+ + BLOOD + Na K "alkaline + tide" H + H HCO3 HCO3 HCO3 CA Cl K+ H2O Na+ + Cl + CO2 H

CO2

Lumen SMALL INTESTINAL CELL

Na+ K+ + H2O Na

Na+ K+ Cl 2Cl

cAMP K+

Lumen