Nobel Prize in Chemistry 2003 the Gateway for Perfect Health

GENERAL I ARTICLE Nobel Prize in Chemistry 2003 The Gateway for Perfect Health

S M Srideshikan and S K Srivatsa

Nature has provided a specific protein channel for each molecule to achieve rapid transport across the cell mem brane. The number of molecules exchanged in this process is as large as 109 per second. This article discusses this year's Chemistry Nobel Prize which was awarded for the discovery of the water channel-aquaporins and the eluci dation of the structure of potassium ion channels. S M Srideshikan is a research scholar at the Existence of some kind of openings for the transport of water Department of Biochemistry, Indian Institute of Science. was known for almost a century. Yet, it was left for Peter Agre, in His interests are in the the 1990s, to discover the water channel. He named it aquaporin, field of 'secondary metabo which means water pore. lites'. He also takes interest in science popularisation "I became so shaky I couldn't hit the keys, and I had no one to through various activities. tell". This was how Roderick MacKinnon recalls his moment of eureka at midnight on Jan 1st, 1998 when the image of the potassium channel became clear on his computer. It was exactly the same structure as predicted by other investigators earlier.

Agre's and MacKinnon's discoveries are relevant in medicine and drug development. A number of diseases can be attributed S K Srivatsa with research interest in to poor functioning of the water and ion channels in the human biophysics, is also a visiting body. The knowledge of their structure and how they work will faculty at the St. Paul's enable scientists and doctors in developing new and more effec School, Bangalore. He takes tive pharmaceuticals. interest in developing new methodologies to present Water Channels science at various levels.

A separate channel for water transport was proposed as early as the mid nineteenth century. Yet, the observed high permeation rates (-3 xl09 molecules per second) in RBC and renal tubules Keywords Nobel Prize in Chemistry 2003, could not be explained by the concept of simple diffusion. aquaporins, potassium ion Instead, a specialized, highly selective water pore was thought channels.

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Peter Courtland Agre: Born in 1949, Minnesota, USA. Did his MD at the Johns Hopkins University School of Medicine, Baltimore, USA. Currently a professor of Biological Chemistry, and of Medicine there. He is a recipient of numerous awards including Kekule Prize, Clinical Investigator Award of National Heart, Lung and Blood Institute, and Basil O'Connor Award of March of Dimes Birth 'Defects Foundation' .

Roderick MacKinnon: Born in 1956 at Burlington, USA. Earned MD from Tufts University, in 1982. Presently he is Howard Hughes Medical Institute investigator at the Rockefeller Univer sity. He has been a recipient of numerous awards foremost of them being the Lasker A ward.

necessary to achieve this. All efforts to purify and identify the water-transporting protein proved unsuccessful till 1987.

The Discovery

Eventually, Peter Agre discovered the water channel. He was studying the Rh antigen of RBC and in 1988 isolated a 28 KDa membrane protein of unknown function from RBC and renal tubules. Researchers had neglected this protein as it did not take the usual histological stains. To determine the function of this protein, Agre ran its amino acid sequence in the software called Agre found that the BLAST (Basic Local Alignment Search Tool) which looks for genetic cousins of genetic similarities. The data he obtained was very intriguing. 28 KDs membrane He found that the genetic cousins of this protein were found in protein were found in human kidneys, fly brains, plant roots and cow eyes. His friend human kidneys, fly and mentor John Parker realized what was common in this brains, plant roots entire bizarre list that Agre gave him: all were highly permeable and cow eyes. His to water. So Parker suggested that this unknown protein may be friend and mentor the long-sought-after water channel. John Parker realized what was common in Agre's approach to test this hypothesis was to introduce the this entire bizarre list mRNA encoding this mystery protein and express it in a cell, that Agre gave him: which is not permeable to water. The cell he chose for testing all were highly was Xenopus oocytes. His postdoc, Gregory Preston, prepared permeable to water. two sets of egg cells: in one set he caused the expression of

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0.5 min 1.5 min 2.5 min 3.5 min

Cell with aquaporin

Cell without aquaporin

mRN A encoding this mystery protein and another control set Figure 1. Peter Agre's ex without it. The moment of discovery came on 9th October 1991, perimentwith cells contain when Preston dropped the genetically modified oocytes in dis ing or lacking aquaporin. tilled water. The cells swelled and burst before he could adjust Theaquaporin is necessary for making the cell absorb the instrument for recording. When he dropped the control set, water and swell. the egg cells did not swell and burst (see Figure 1).

Mark Zeidel, a co-investigator of Agre, conducted an experi ment, which silenced all the skeptics. They wanted to see the protein being purified in physiological conditions and put to work as water channel. Zeidel introduced this purified protein in liposomes, an artificial membrane, and measured the perme ability of water by Stopped-flow Fluorescence. In this technique, a steady flow is abruptly stopped and the retreating front of the flow measured optically. He found that the protein acts as water channel and conducts water 10-100 folds faster than a channel free membrane. Also it was known that Hg+ reversibly inhibited water diffusion. The inhibition of swelling of oocytes and lipo some by Hg+ was also confirmed. Zeidel found that Structure the protein acts as The prevalent strategy in determining the function of a protein water channel and is by elucidating ana analyzing its structure. A continuous conducts water 10- column of water joined by hydrogen bond would allow the 100 folds faster leakage of protons (Grotthus Effect or proton hopping). The proton than a channel gradient across the membrane, essential for ATP (adenosine free membrane.

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The extreme triphosphate) synthesis, is destroyed by their leakage. The ex specificity of treme specificity of aquaporin to water alone and not even to aquaporin to water protons (Le., oxonium ions, H30+) was not understood until the alone and not even to crystal structure was elucidated. The high-resolution 3D pic protons (i.e., oxonium tures of the structure of the channel revealed that water molecule ions, H30+) was not reorients its dipole to break this continuous column, effectively understood until the preventing the proton leakage. Further, in different physiologi crystal structure was cal conditions water is either taken up or given out by the cell. elucidated. The hourglass model explains this hi-directionality of water flow in the cell.

The Hourglass Model of AQP

AQP is found to be a tetramer with intracellular Nand C termini, a characteristic feature of channel proteins. Each mono mer is made up of six tilted spanning a-helices enclosing the pore formed by two NPA (Asparagine, Proline, and Alanine) containing loops that enter from the opposite side of the bilayer and are juxtaposed in the center. Many helix-helix interactions stabilize the monomer. Loops LB and LE, each containing an NPA motif and a short helix HB and HE, bend into the six-helix bundle to form the channel (see Figure 2).

The 'hourglass' AQP structure has a cone-shaped intra and inter cellular vestibule. Separating the two vestibules is a 20A narrow channel through which water has to flow in a single file without hydrogen bonding. About 8A from the centre is the 2A Ar/R (aromatic amino acid/arginine195) constriction. The partial posi The water molecule tive charge on the arginine and histidine repels positively charged is found to reorient molecules and the aromatic bulky groups provide size restric its dipole, such that tion to molecules larger than water. The water molecule is found oxygen in the water to interact with the amino groups of the asparagine (N) in the 2 molecule facing the NPA motif, which are opposite and juxtaposed. The water mol pore center while ecule is found to reorient its dipole, such that oxygen in the entering the channel water molecule facing the pore center while entering the chan will still face the pore nel will still face the pore center while leaving the pore. The center while leaving carbonyl groups of a few amino acids in the pore wall will aid in the pore. hydrogen bonding of the water molecule during transport. As

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Figure 2. Left: Ribbon model of AQP1 monomer showing six tilted bilayers spanning domains and two pore-forming loops with short transmembrane K-helices entering the membrane from the extracellular surface (golden yellow) and intracellular surface (light blue). Right: Schematic diagram representing channel pore in same orientation. Flow of water from extracellular to intracellular chambers occurs through the narrow column. Proton conduction (hydronium ion, H,O+) is prevented at the narrowest segment 8 AT above the channel center by size restriction and electrostatic repulsion (H180 and R195). A second barrier to proton conduction exists at the center of the channel where partial positive dipoles contributed by the short pore-lining K-helices and the two highly conserved asparagines (N76 and N192) in the signature NPA motif cause a transient dipole reorientation of an isolated water molecule. (Reproduced with permission from PAgre et ai, JI. Clin.lnvest. 109, 1395, 1992) the number of interacting residues that stabilize the molecule by hydrogen bonding is small, the rate of transfer is high.

AQP plays a crucial role in fluid balance as they transport water, the vital component of life. Eleven mammalian aquaporins, named AQPO, AQPI, and so on, have been identified to date. Their distribution is different in various tissues. With the ex ception of AQP2, their regulatory mechanisms are poorly un derstood. AQP2 is regulated by anti-diuretic hormone (ADH) also called vasopressin. Caffeine and alcohol (diuretic agents) can regulate AQP by inhibiting the ADH secretion.

Analysis of several disease states has confirmed that aquaporins

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Analysis of several are involved in different illness including disorder of kidney, disease states has onset of brain edema, and loss of vision. AQPI and AQP2, confirmed that abundantly present in kidney cells, help reabsorb much of the aquaporins are primary urine (glomerular filtrate). AQPI renders re-absorption involved in different to about 70% and the rest by vasopressin regulated AQP2. In illness including normal individuals, release of vasopressin causes AQP2 proteins disorder of kidney, to become localized to the apical plasma membrane permitting onset of brain edema, water re-absorption from filtrate. If too much AQP2 is expressed and loss of vision. then water re-absorption may become excessive, and this is believed to contribute to common fluid overload states found in patients with congestive heart failure or pregnancy.

In a diseased condition called nephrogenic diabetes insipidus (NDI), the hormone is deficient and thus re-absorption of water by AQP2 is affected. This state results in the excretion of about 10-20 liters of urine everyday. AQP4 is implicated in the onset of brain edema and AQPO in inherited cataract. The diseased states may either occur from a mutation in the gene encoding for the protein (cataract) or improper localization of the protein (dia betic insipidus). Therefore AQPs can be considered as potential drug targets in the treatment of various diseases.

Potassium Channel

Wilhelm Ostwald, as early as 1890, suggested that electrical currents in living tissues might be caused by ions moving across cellular membranes. The ions acted as signaling molecules in these cases. Soon, the notion of narrow channels for conduction of these ions was proposed. Alan Hodgkin and Andrew Huxley Wilhelm Ostwald, (Nobel Prize in Physiology or Medicine, 1963), at the Univer as early as 1890, sity of Cambridge, UK, showed in 1952 that sodium ions are suggested that rapidly and transiently transported into the nerve cell during electrical currents the transmission of the signal. This causes the membrane to in living tissues depolarize (+0.04 V). Further, the membrane becomes perme might be caused able to potassium ions, which flow out to re-polarize (-0.07 V). by ions moving In 1965 Hodgkin and Keynes demonstrated that K+ ions are across cellular selectively transported in a single file. Though ion selectivity, membranes. voltage sensing, rapid transport, channel gating and channel

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By 1970 it was shown that ion channels are selective because they are equipped with some kind of ion filter. The difficult task was to identify ion channels that allowed only K+ and not Na+ ions. It was proposed that selectivity was achieved by the proper placement of oxygen atoms in the selectivity filter in a way that ions of correct size would be preferentially solvated during the transport through the narrow filter. But to support this hypoth esis high resolution X-ray pictures were needed. The crystalliza tion of membrane channels was difficult and ion channels were no exception. Ion channels of plants and animals are compli cated and hence bacterial KcsA K+ channels were studied, as it resembled a human ion channel.

The major breakthrough was obtained in 1998 when Roderick MacKinnon solved the crystal structure of KcsA. MacKinnon's life is an inspiring one. He was a biochemist who later qualified as a doctor. He pursued his career as a physician for some time before he decided to dwell on the mysteries of potassium chan Ion channels of nels. Realizing the need for high-resolution crystal structure he plants and animals ventured into a subject that was totally alien to him - X-ray are complicated and crystallography. Despite criticisms from his colleagues and hence bacterial friends he pursued his work. He learnt the fundamentals of X KcsA K+ channels ray crystallography and only in a few years astonished the whole were studied, as it research community by presenting the structure of an ion chan resembled a human nel in April 1998. ion channel.

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Structure and Mechanism

MacKinnon determined the first high-resolution structure of an ion channel. The original picture, though a bit fuzzy revealed the molecular design of these pores. This gave hope to under stand the perplexing functional hallmark of K+ channels: total obstruction to Na+ movement but transport of K+ ions at high rates as if the protein presented no barrier at all.

This structure enabled him to predict why potassium (K+) ions are preferred over sodium (Na+) ions, and the high permeation rate. K+ channel resembles a funnel with a central water cavity, and a narrow selectivity filter in the shape of oxygen lined electro-negative tunnel in which dehydrated K+ (and not Na+) fits precisely. The oxygen atoms are spatially placed such that they mimic the hydration shell ofK+ ions in the outside aqueous environment (see Figure 3). Hence, K+ ions willingly leave the aqueous solution and enter the selectivity filter in a largely dehydrated form. Na+ ions (atomic radius - 0.95 A), though

Figure 3. The ion channel smaller than K+ ions (1.33 A), do not fit precisely in the oxygen permits passage of potas lined electronegative tunnel and thus are not permitted. sium ions but not sodium ions. The oxygen atoms of Inside the filter the ion filter form an envi ronment very similar to the water environment outside 0, 0 the filter. Outside the cell o 0 the ions are solvated by water molecules. The sol vation shell depends on the atomic radius. The distance to the oxygen atoms in the ! ! ion filter is the same as in water for potassium ions. Theso!Jium ions, which are smaller, do not fit in be tween the oxygen atoms in the filter. This prevents them from entering the Outside the li.Jter channel.

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The crystal structure also shows K + ions in seven distinct sites along the pore axis. Four of these are in the selectivity filter, one in the central cavity and the other two at the external end of the pore. At any time, two of the sites in the filter are occupied by K+ ions separated by a single water molecule distance (see Figure 4). The presence of more number of ions in the cavity causes electrostatic repulsion between them and pushes the ions across the filter. This helps achieve high permeation rate.

Figure 4(1). There are seven main sites for ions along the pore axis: one in the pore cavity, four in the selectivity filter and two just beyond the external end ofthe pore. The cavity site is fully occupied, but (as indicated in 2) only half of the remaining six is occupied at any other time. (2) The two main ion configurations, known as outer and inner, that are postulated to exist within the pore. Black arrows indicate ion shifts that are linked directly to concerted ion entry into and exit from the pore. Pink Arrows represents shifts within the pore without ion entry and exit. As shown here, then, ion passage through the selectivity filter and extracellular sites occurs in bucket-brigade fashion.

ExtrflceUulu

'. Itlletior of

1 water molecule distance I 3 o

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