Fifty Years of Progress in Ion Channel Research

IEEE TRANSACTIONS ON NANOBIOSCIENCE, VOL. 4, NO. 1, MARCH 2005 3

Fifty Years of Progress in Ion Channel ResearchPeter C. Jordan

Abstract—Fifty years ago, ion channels were but a reason-able hypothesis. I outline some major steps in transformingthis idea from a plausible description of the biological assem-blies responsible for controlling passive ion transport acrossmembranes to established fact. Important electrophysiological,biochemical, molecular biological, structural, and theoretical toolsare discussed in the context of the transition from studying wholecell preparations, containing many channels, to investigatingsingle channel behavior. Six channel families are exemplified:the model peptide, gramicidin, the acetylcholine receptor, andthe sodium, potassium, calcium, and chloride channels. Somequestions of current interest are posed.

Index Terms—Biological nanodevices, electrophysiology, gating,ion channels, selectivity.

I. INTRODUCTION

B IOLOGISTS have long recognized that the transport ofions and of neutral species across cell membranes is cen-

tral to physiological function. Cells rely on their biomembranes,which separate cytoplasm from the extracellular medium, tomaintain the two electrolytes at very different composition.Specialized molecules, essentially biological nanodevices, haveevolved to selectively control the movement of all the majorphysiological species. As should be clear, there have to be atleast two distinct modes of transport. In order to maintain thedisequilibrium, there must be molecular assemblies that driveions and other permeable species against their electrochem-ical potential gradients. Such devices require energy input,typically coupling a vectorial pump with a chemical reaction,the dephosphorylation of adenosine triphosphate (ATP). Theseenzymes (biochemical catalysts) control highly concerted, andrelatively slow, process, with turnovers of 100 s .

Another class of enzymes, the focus of this issue, controlsthe transmembrane flux of ions and other permeant speciesdown their electrochemical potential gradients. Two types ofmolecules are immediate candidates for this purpose. Naturecould have designed specialized carrier molecules, whichbind ions or other lipophobic species at the water–membraneinterface and then diffuse across the membrane. Alternatively,transport could be effected by channel forming molecules,whose water filled interiors form electrical shunts that provideessentially barrierless pathways for the transport of chargedand polar species. Both types exist, and have similar designfeatures: lipophilic exteriors, stabilizing their interaction withmembranes, and polar interiors, stabilizing their interactionwith charged and polar species. But it is ion channels, which

Manuscript received November 4, 2004. This work was supported by the Na-tional Institutes of Health under Grant GM28643.

The author is with the Department of Chemistry, Brandeis University,Waltham, MA 02454-9110 USA (e-mail: jordan@ brandeis.edu).

Digital Object Identifier 10.1109/TNB.2004.842467

can support fluxes as high as 10 s , that control biologicalelectrical signaling. All share three crucial properties: they arehighly permeable; they are highly selective; and their openingand closing is exquisitely controlled. Understanding theirbehavior hinges on determining structure and relating it tofunction.

Hodgkin and Huxley’s 1952 [1] analysis of electrical activityin squid giant axon established that both Na and K con-tributed to the ionic current and that the fluxes were opposed.It suggested channels as the ionic pathways, but it took 20 moreyears until Hladky and Haydon [2] definitively demonstrated theexistence of ion channels, and then only in studies of the smallantibiotic gramicidin. When incorporated in lipid bilayer mem-branes bathed by electrolytes, it exhibited what has come to bethe characteristic electrical signature of an ion channel: quan-tized bursts of current of variable duration arising in response toapplication of a transmembrane electric field.

From these beginnings, ever more detailed pictures of ionchannels have evolved. The links between structure and func-tion have relied on great technical strides in electrophysiology,biochemistry, molecular biology, structure determination, com-puter power, theoretical chemistry, and bioinformatics. ThreeNobel prizes, in 1963, 1991, and 2003, specifically cite studiesdevoted to ion channels. The remainder of this overview de-scribes important results of the past half century, beginning withone Nobel bookend, the Hodgkin–Huxley model [1], that postu-lated independent sodium and potassium pathways, and endingwith another, MacKinnon’s [3] atomic-level determination of apotassium channel’s structure.

II. CLASSICAL BIOPHYSICS

When electrically stimulated, a polarization wave propagatesalong the length of an axon. As squid giant axon is over 1 mlong, Hodgkin et al. [4], using 1950s technology, could perfectthe “voltage clamp” method in which an electrode is insertedin the axon interior, permitting local perturbation of the mem-brane potential from its resting value of 60 mV (a Nernstpotential reflecting the fact that the axon interior concentratespotassium to which the membrane is selectively permeable)and the transmembrane current measured. They studied howvarying both membrane potential and extracellular electrolytecomposition altered electrical response, distinguished and sep-arately measured the currents carried by Na and K [5] anddetermined their voltage-dependent kinetics. The ionic path-ways were separate; each flux was activated and deactivated inresponse to voltage; each was electrodiffusive, controlled byits own resting potential. Properly parameterized, their kineticmodel for axon behavior [1] accounted for all salient featuresof the action potential. After a local partial depolarization,there is a large inward flow of Na and the interior potential

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4 IEEE TRANSACTIONS ON NANOBIOSCIENCE, VOL. 4, NO. 1, MARCH 2005

rises sharply, inverting membrane polarity; the sodium pathwaythen shuts down and the potassium pathway opens, restoringthe resting potential, which causes it to shut down. The initiallarge depolarization provides the stimulus that propagates thepolarization wave further along the axon. Further studies of thepotassium pathway provided more detailed insight, indicatingit was multiply occupied in its conducting state [6].

Hodgkin and Huxley demonstrated the existence of indepen-dent pathways for K and Na flow. They showed that the ionicfluxes were Nernst-like, due to electrodiffusive potential differ-ences. Further studies showed membranes were selectively per-meable to other ions as well, suggesting individual pathways forother physiologically important ions, i.e., Ca , H , Cl , andHCO . By varying the composition of the external electrolyte, apathway’s relative permeability to different ions could be estab-lished, thus determining a selectivity sequence. For the axon’spotassium pathway, it is

K Rb Cs Na Li (1)

If electrodiffusion governs permeation, such relationships mustreflect the underlying thermodynamics. What is the free energychange in removing an ion from electrolyte and inserting it intothe transmembrane pathway? Are there general molecular levelprinciples governing selectivity? Electrostatic design obviouslypermits discrimination based on ionic polarity. Eisenman [7]noted that for the five alkali cations, only 11 of 120 possibleselectivity sequences were commonly found. Why might this bethe case? Could electrostatic influences dominate here as well?His answer developed from the study of glass electrodes and thethermodynamics of ion exchange. For an electrode that selects

over , the relative free energy for binding to glass mustbe more favorable than the relative free energy for hydration,i.e.,

glass glass aqueous aqueous (2)

Hydration-free energy differences for isovalent ions are known.He modeled ion-electrode interaction as purely electrostatic,governed by the contact distance between the bound cation andthe anionic site in the glass. For (univalent) alkali cations, theinteraction energy is

(3)

where is the anionic valence, the electronic charge,Avogadro’s number, the dielectric constant, the vacuumpermittivity, the cation radius and the radius of theanionic binding site. For large anions, ionic hydration [theright-hand side of (2)] governs the equilibrium; the electrode(or peptide) would select for Cs . As the anion becomessmaller, the left-hand side of (2) becomes ever more important,ultimately leading to selection for the smallest cation Li . Asthe radius of the anionic site decreases, this simple electrostatictheory generates the 11 observed selectivity sequences. Nothingabout the Eisenman argument is limited to glass electrodes.Similar qualitative considerations apply to binding sites alongtransmembrane ionic pathways and Eisenman’s sequence III or

IV [7] describes the selectivity of the potassium pathway (1).Recent work suggests that slightly modified, these electrostaticconsiderations may quantitatively account for selectivity in thepotassium channel [8].

As transmembrane ionic pathways are narrow water-filledshunts surrounded by protein and lipid, electrostatics is centralto understanding the influence of these surroundings. Parsegian[9] was the first to provide quantitative estimates of how theassociated permittivity differences could affect ionic trans-port. The energy barrier associated with ionic motion fromaqueous electrolyte directly into a membraneis prohibitive. It is much reduced, but not eliminated, forwater-filled transmembrane conduits. Even if water in the pathis dielectrically equivalent to bulk water, an ion induces chargesalong the water–lipid interface, which impede its transloca-tion. Were trapped water electrically inequivalent to ambientwater, its ability to reorganize and shield an ion from the low

surroundings would be reduced, thus increasing the barrier.In addition a charging energy would be associated with ionictransfer to lower surroundings, also increasing the barrier.These dielectrically induced energy barriers, impeding elec-trodiffusion, are reduced due to interaction with Eisenman-likebinding sites along the path.

Classical electrodiffusion views ion flow as movement downan electrochemical potential gradient, modulated by travelover a sequence of wells and barriers, reflecting a series of ionbinding sites. This is naturally treated by biochemical kinetics,which, at its simplest, invokes a two-step translocation process:ion transfer from water to the binding well from one side of themembrane, followed by dissociation to the other side. This ismechanistically expressed as

Ch Ch (4a)

Ch Ch (4b)

where and represent ions to the left and right of the mem-brane, Ch is the channel, and Ch the ion at the binding site.The rate of entry from the left is Ch and the rateof back reaction is Ch ; and are rate constantsand square brackets signify species concentrations. Similar ex-pressions describe processes occurring on the right. Analysis ofelectrophysiological data can establish the rate constants. Mi-croscopic interpretation involves using these parameters to de-duce the associated energetics. The classic work of Arrhenius[10] showed that rate constants take the form

(5)

where is an “activation energy,” the absolute temperature,and the gas constant. The topological picture identifiesas the energy required to surmount an activation barrier alongthe electrodiffusive pathway. The biochemical problem requiresdeconvoluting and . In ordinary chemical kinetics, this isdone by determining how varies with . It is more difficultbiochemically: wide temperature ranges cannot be accessed;proteins denature; membranes undergo phase transitions. Fromquantum statistics Eyring [11] developed absolute reaction rate

JORDAN: FIFTY YEARS OF PROGRESS IN ION CHANNEL RESEARCH 5

theory where, given an energy profile, rate constants may becomputed. His expression takes the form

(6)

where is the standard free energy change in forming the“activated complex” from reactants and and are thecorresponding standard enthalpy and entropy changes. The ac-tivated complex is a saddle point on a multidimensional poten-tial surface describing interacting species and is the frequencywith which molecules in this activated state proceed to products.While the correspondence between (5) and (6) is superficiallyseductive, in the absence of temperature variation studies ex-tracting activation enthalpies or free energies from (6) is fraughtwith difficulty, since neither the structure reorganizational term

nor the frequency factor is easy to estimate [12].

III. PHARMACOLOGY AND SINGLE CHANNELS

Hodgkin and Huxley’s work [1], [4], [5] was done onwhole cell preparations. Distinguishing potassium and sodiumcurrents and eliminating perturbations from other membranecomponents required both clever technique and a system rel-atively rich in sodium and potassium pathways. More generalstudy entailed suppressing the competing currents or devel-oping techniques for pathway isolation.

Neurotoxins, which selectively block the pathways, phar-macologically separate sodium and potassium contributions tothe action potential. Tetrodotoxin (TTX), which makes fugusuch a risky delicacy, eliminates the sodium current [13], [14],thus isolating the potassium pathway. The quaternary amine,tetraethyl ammonium (TEA), has a complementary effect,blocking the potassium pathway [15], [16]. The discovery thateach ionic pathway could be individually suppressed providedstrong inferential evidence for the notion that these were, in fact,transmembrane channels. Even more importantly, it impliedthese channels were molecular receptors, with toxins actingat specific sites. Viewed this way, channels are enzymes thatfacilitate ion flux and toxins are reversible inhibitors, describedby the physical chemist’s Langmuir adsorption isotherm orthe biochemist’s Michaelis–Menten expression. The 1972Hladky–Haydon [2] studies on gramicidin provided final andcompelling evidence for the existence of an ion channel, albeita simple one.

The acceptance of the channel paradigm radically alteredthe conceptual framework, focusing interest on identifyingand characterizing specific structural features, and developingputative models, “cartoons,” which often bore an uncanny re-semblance to the actual structures found years later. Studies byHille [17], [18], with a set of organic cations, estimated the di-mensions of the sodium and potassium channel pores, showingthat in neither case could occlusion account for selectivity.A chemically attractive hypothesis, similar to Eisenman’sselectivity theory [7], came immediately to mind, that theorifices were surrounded by rings of carbonyl oxygen atoms,regions sufficiently negatively charged to compensate for thefree energy of dehydration [18], [19]. Thirty years later, thisidea was confirmed by MacKinnon’s X-ray structure [3] of a

potassium channel. While no sodium channel structure has yetbeen determined, subsequent work has demonstrated that herecharged residues, not carbonyl groups, control selectivity [20],[21].

Sodium channel kinetics is complex. Whole cell studiesshowed that, in addition to open and closed states, there is afunctionally distinct “inactivated” state. When the channel isopen, Na streams down the electrochemical potential gradiententering the cell, toward a locally negative region. But thechannel does not long remain conducting. It shuts down andrequires potential reversal to slowly undo inactivation. Thesekinetics are consistent with reversible block of the pore’s innermouth by a positively charged group, which Armstrong intuitedacted like a “ball and chain” [22], a large tethered group thatswings into the pore’s inner mouth when it becomes negativelycharged. Work of the last 30 years has confirmed this hypoth-esis: large organic cations, injected into the cell, immobilizerecovery from inactivation [23]; excision of the “ball and chain”domain, eliminates inactivation [24], [25]. Channel openingand closing modify structure somewhere along a permeationpathway; in inactivation a bulky charged group occludes achannel entrance.

Inspired pharmacology clearly provides insight at the molec-ular level. But once gramicidin was proved to be an ion channel[2], the race was on to isolate individual channel proteins, re-constitute them in bilayer membranes, and make single-channelmeasurements free from electrical interference by other chan-nels. Two groups reported success in 1976 [26], [27], totallychanging the experimental landscape. Even though whole cellmeasurements made accurately enough can in principle pro-vide even more information than single channel studies, thecomplexity of a membrane mosaic makes teasing this out un-feasible. Thus, if a single channel’s conductance is sufficientlylarge, the pharmacological techniques developed to study wholecell preparations are immediately transferable, permitting studywith greater accuracy and in more detail.

The discovery of ClC chloride channels provides striking ev-idence of the value of single channel studies. Unitary cationconductance measurements showed that there is a unique ionpathway associated with each channel protein. Chloride channelcurrent records were strikingly different. They could not be ra-tionalized in terms of a single ionic pathway [28]. The dataimplied each protein had two identical pathways opening inseparate fast processes and an additional slow step activatingthe whole dimer. The system behaved like a double-barreledshotgun. This remarkable cartoon was confirmed 20 years later,by X-ray structure determination [29].

IV. PATCH CLAMP, SEQUENCING, AND MUTAGENESIS

Isolation of single-channel proteins by biochemical separa-tory methods is difficult and laborious. Electrophysiology onwhole cell preparations is severely limited in the systems thatcan be studied. In 1976, Neher and Sakmann, in work hon-ored by the 1991 Nobel Prize, reported a way to observe single-channel currents from tiny patches of living cellular material[30], a technique that was refined to permit fusing cell mem-branes with the tip of a micropipette [31]. The contents of thepipette bathing the membrane surface could be adjusted at will;


the patching protocol could expose either membrane surface tothis electrolyte. Thus, single-channel recording became simple,accurate, and reliable, and there was no limit to the cells thatcould be studied.

Patch clamp recording methods gave unprecedented freedomin assessing how channel function was altered by differentstimuli. But mechanistic understanding was still limited tocartoon models. Channel proteins are linear arrays comprisingup to a few thousand amino acid residues. They fold and formchannels, but what groups give rise to ionic pathways, whichto the assemblies that respond to gating stimuli, and which areresponsible for channel selectivity? New tools were needed torelate structure and function. These were soon forthcoming.Numa and coworkers, applying recombinant DNA techniquesto the acetylcholine receptor (AChR) channel of the electriceel, developed efficient methods to determine its primary aminoacid sequence [32]. This assembly, formed from five similarsubunits [33], plays a crucial role in neuromuscular transmis-sion. Located at synaptic junctions, it governs nerve–musclecommunication. The subunit sequences exhibited considerablehomology, i.e., substantial residue similarities [34]. Couplingthese results with rules for identifying secondary structuralelements [35] and regions of hydrophilicity [36] provided thefirst solid hypothesis for channel protein architecture [34].

The AChR sequences from both calf and electric eel aresubstantially homologous, suggesting that the similar regionsare functionally important. Using cloning techniques, Numa’sgroup was able to create a hybrid AChR, mixing subunits fromthe two organisms [37]. In a collaboration with Sakmann, itselectrical properties were measured; the hybrid and its parentshad the same unitary conductance, strongly suggesting thatchannel conductance was controlled by a string of 22 aminoacids, highly homologous in each of the five subunits [38].By selectively mutating specific residues in the protein theyconfirmed this speculation [39]. The era of site-directed muta-genesis had arrived.

These tools form the everyday arsenal of modern electro-physiology. The patch clamp permits near total control of theenvironment in studying ion channels. Site directed mutationpermits near complete freedom in protein design. Their appli-cations showed that the cation channels—sodium, potassium,and calcium—form a superfamily. Na and Ca channelproteins are single stranded, linking four similar peptides; Kchannel proteins are homologous, with sequences similar toindividual Na or Ca peptide subunits [40]. Clever thermo-dynamics connected these observations, showing K channelsto be tetramers [41]. But what makes them unique? Primary se-quence comparisons identify “signature” domains that controlselectivity; these regions are residue stretches common to, e.g.,all K channels, etc. In K channels, the conserved feature isa five-peptide sequence [42]. In Ca channels, the signatureis a set of four negatively charged residues, one from eachsubunit. This filter is remarkable, favoring passage of Caover Na , even though the latter is 100 times more prevalentphysiologically. Na channels have residues with a netcharge in place of the four negative signature residues of Cachannels. Mutational signature interconversion makes a Nachannel selective for Ca and vice versa [20], [21].

Selectivity gives channels chemical individuality. Gating,which can be coupled with permeation [43], provides func-

tional control. A branch of the cation superfamily is voltageregulated, with the sensor a highly charged domain. Unlikeselectivity, which is basically understood, molecular detailsof gating remain murky. Like the tale of the blind men andthe elephant, different experiments suggest different structuralinterpretations. In one picture, the sensor snuggles up to coun-tercharged regions of the protein [44]–[46]. Another suggeststhe sensor is on the outer side of the assembly, attracting waterto stabilize its charges [47]. The jury is out.

Patch clamp, sequencing, and mutagenesis provided muchfunctional information. However, the structures inferred werestill cartoons. The 15-residue, channel-forming peptide grami-cidin is small, amenable to nuclear magnetic resonance (NMR)structure determination [48]. Long before X-ray structures ofchannel proteins were available, designed chemical mutationof this cation selective channel provided detailed insights con-necting structural modification to changes in channel behavior[49]. Slight sequence variations can have major consequences,making the channel voltage sensitive [50] or totally altering itsfold [51].

V. STRUCTURE

With the exception of gramicidin, atomic resolution struc-tures were unavailable until 1998. Channel portraits were eithercartoons or silhouettes. The histories of gramicidin and AChRare illustrative. As gramicidin has only 15 residues, conforma-tional analysis combined with secondary structure deductiontools [35], [36] would appear adequate for reliable struc-ture determination. The original hypothesis, a head-to-headleft-handed -helix was close to the mark [52]. The channeldoes form by hydrogen bonding carbonyl and amino groupssix residues apart, but Arseniev’s 1985 NMR study showed thescrew was right-handed [48]. Cation selectivity arises from in-teraction with channel lining carbonyls, Eisenman’s selectivitysequence II [7].

Oriented two-dimensional preparations of AChR have beenstudied for 20 years by electron microscopy (EM). From theoutset they portrayed gross channel architecture [53], [54], butresolution is not yet at atomic level, the best being 4 [55]. Thevalue of an extra angstrom is clearly demonstrated in studies ofwater channels, the aquaporins. There are significant, resolutionrelated, differences between cryo-EM results, at 3.8 [56] andthe X-ray determinations, at 2.2 [57]. Cryo-EM’s advantage isthe small samples needed; the corollary is a limit to the numberof images that can be acquired.

In 1998 MacKinnon solved two daunting problems: isolatingand purifying channel proteins “in bulk” and crystallizing them.X-ray methods, with their superior signal-to-noise ratios, werethen applied, determining the structure of an ion occupied bac-terial potassium channel KcsA [3]. It completely substantiated40 years of cartooning. The selectivity filter, formed of residuesidentified a decade earlier [42], is a narrow cylinder near thechannel’s external mouth, 15 long, just adequate to accom-modate two potassium ions easily; further in is a water-filledpool, with a third ion, all substantiating the prediction of mul-tiple occupancy [6]. Ions are stabilized by interaction with car-bonyl oxygens of the filter residues. This lining, reminiscent ofgramicidin, is only possible because the two glycines of the filter


have more folding options than any other residues. Both the foldand the aqueous cavity were utterly unexpected.

KcsA was crystallized in a nonconducting state, its inte-rior end too narrow to permit ion passage. Another bacterialpotassium channel, caught with its interior mouth open [58],provided a different view of K-channels, again consistentwith electrophysiological canon; it suggested strongly that aglycine residue was the gating hinge [59]. A year later, thereappeared another tour de force, the structure of a voltage-gatedbacterial K-channel [60]. As noted above (Section IV), it wasnot only surprising, but apparently totally at odds with yearsof experimental inference. Why the differences? Possiblyeukaryotic and prokaryotic organelles (cells with and withoutnuclei) differ fundamentally. Possibly there are alternate datainterpretations. Possibly crystallization conditions have greatlydistorted the channel. Possibly the accepted view of gating iswrong. New single-molecule techniques simultaneously usingelectrophysiological and optical probes may provide the neededinsights [61].

A bacterial ClC chloride assembly suggests there are im-portant differences between prokaryotes and eukaryotes. Thecrystal structure of a bacterial ClC [29] confirmed Miller’s car-toon with its two identical parallel pathways [28]. Another struc-ture indicates how chloride ions get into the path, but not howthey exit [62]. The bacterial protein exhibits signature regionscharacteristic of chloride selectivity, but its conductance is toosmall for single channel analysis. In fact, even though its pro-tein is significantly homologous with that of eukaryotes, it isnot a channel but a pump with exceptionally high turnover. Forevery two Cl that pass in one direction, a proton goes the otherway [63]. The turnover is 1000 times faster than a typical pumpand 100 times slower than a channel. Might it be a missing link,which could provide insight into subtle differences that inter-convert pumps and channels?

VI. THEORY

As experimental tools developed, so did theoretical ways torelate structure an function. Early studies, based on cartoonstructures, treated channel–water–membrane ensembles asproblems in electrostatics. They focused on discriminatingamong these pictures and gave qualitative insights [9], [64].The aqueous shunt was presumed electrically equivalent tobulk water. Studies of narrow, selective channels were limitedto treating reaction field effects induced by the low permittivitymembrane surrounding the channel protein. Läuger presentedthe chemical kinetic, Eyring-like view, conductance as passageover a series of structurally induced barriers [65], while Levittconsidered conductance as an electrodiffusional process [66]where kinetic barriers created potential fields. Such treatmentswere most useful as correlational tools [67], [68].

Once a reasonable structure for gramicidin was available,its behavior became the focus for applying two powerfulmolecular-level methods, Brownian dynamics (BD) [69], andmolecular dynamics (MD) [70], and developing a more generalelectrodiffusional approach, Poisson–Nernst–Planck (PNP)theory [71], [72]. PNP views ions as diffuse charge clouds,an adequate model for wide channels but problematical fornarrow, selective ones. However, it is a powerful, flexible tool,

generating current-voltage – profiles for direct compar-ison with data. BD treats ions as discrete entities; it trackstheir motion through a pore, also generating – profiles.It treats the aqueous pore as a viscous, dielectric continuumand describes ionic motion in the potential field of the proteinas stochastic, frictionally retarded by pore water. Both PNPand BD impose severe dielectric assumptions, similar to theearlier electrodiffusional approaches [66], ones which must beused cautiously [73]. Their special strength is their ability toefficiently establish the electrophysiological consequences ofalterations in protein structure and charge distribution. MD isless constrained. It describes atomic level motion, governed byempirical force fields, but even now limits to computationalpower preclude directly determining – profiles. Wilson’spioneering study [70] focused on ion–water–peptide correla-tions in gramicidin, concluding that water, in these confinedsurroundings, formed an oriented, hydrogen-bonded chain evenin the ion-free channel.

Two BD studies are of special interest. Open and closed statestructures demonstrate that K-channels’ inner mouths are veryflexible [3], [58]. BD shows that small changes in the size ofthe inner mouth can easily account for the observed hundredfoldspread in K-channel conductances [74]. While the bacterial ClCchloride assembly is not a channel [63], its pore may nonethe-less be a template for true channels. Combining this assumptionwith bioinformatics methods yields models of eukaryotic ClCchannels, which BD studies demonstrate account for observedconductance behavior [75].

Although attempts have been made [76], MD has not yetdirectly reproduced – profiles; however, there is an indirectpathway—computing a potential of mean force (PMF), inessence the permeation free energy for ion transfer from bulkelectrolyte to the channel interior. It has provided surprisinginsights, predicting a K-channel ion binding site [77] beforeexperimental confirmation [78] and generating a PMF thatreproduces conductance measurements [79]. The origin ofgating in AChR poses a challenge. Its closed pore is stillquite wide, 3.1 in radius [55]. What is the exclusionarymechanism? Possibly a hydrophobic one [80], [81]. But thereremain problems. Gramicidin, which is structurally superblycharacterized [82] and for which the electrophysiological dataset is essentially unlimited [49], is the critical testing ground.Here the picture is mixed. PMF computations based on stan-dard force fields yield energy barriers implying conductances

10 -fold too small [83]. What has gone wrong? Possiblymethodological artifacts or possibly the force fields [84]. Thequestion remains open.

VII. WHAT IS NEXT?

Predicting scientific advances is a fool’s errand, but irre-sistible. A bacterial sodium channel has been crystallized and,with good fortune, its structure will soon appear and put specu-lation to rest. The structure of AChR seems tantalizingly closeto resolution. The next few years should resolve the mechanismof voltage gating. The functional basis for differences betweenprokaryotic and eukaryotic ClCs should be clarified, providinginsight into the structural features distinguishing channels from


pumps. New theoretical tools, applicable to millisecond pro-cesses, will provide insight into gating, the slow conformationalchanges that control channel opening; attempts have alreadybeen made [85]. Channels will be engineered in novel ways,creating practical nanodevices, which has already been donewith gramicidin; it is only a matter of time until similar modifi-cations are grafted onto biological channels. Detailed structuralknowledge will lead to hosts of pharmaceutical products,specifically targeted at biological ion channels. Theory will bemore prominent, providing quantitative physical explanationsof the mechanisms of permeation, selectivity, and gating.

ACKNOWLEDGMENT

The author would like to thank G. Miloshevsky andM. Partensky for helpful comments and S.-H. Chung forhis encouragement and his critique.

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Peter C. Jordan received the B.S. degree fromthe California Institute of Technology, Pasadena,in 1957 and the Ph.D. degree from Yale University,New Haven, CT, in 1960.

He has held postdoctoral positions at CambridgeUniversity and the University of California, SanDiego. He has been a Visiting Scientist at Univer-sité Paris Sud, Universität Konstanz, Universityof Houston, Rijks Universiteit Groningen, andthe Australian National University. He has heldGuggenheim and Whiting Fellowships, is the author

of one book and of nearly 100 research papers. He is currently Professor andChair of the Chemistry Department at Brandeis University, Waltham, MA. Hiscurrent research focuses on theoretical modeling of transmembrane biologicalchannels.

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Fifty Years of Progress in Ion Channel Research