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Evolution of the Ion Channel Concept: The Historical Perspective Jan C. Behrends* Laboratory for Membrane Physiology and -Technology, Department of Physiology, University of Freiburg, Hermann-Herder-Str. 7, 79104 Freiburg, Germany Freiburg Centre for Materials Research (FMF), Stefan-Meier-Str. 21, 79104 Freiburg, Germany limited overview is, necessarily, strongly informed by Hille’s account and can only serve the purpose of a reminder of, or placeholder for, the historical perspective without which a special volume on “Ion Channels and Disease” might seem incomplete. The ion channel or ionic channel was developed as a concept in General Physiology and Biophysics to explain the following facts: (1) Biological membranes have electrical conductance due to permeability for ions. (2) This permeability can show a high degree of ion selectivity. (3) This permeability is regulated and can be changed locally CONTENTS and very rapidly by electrical or chemical stimuli. 1. Introduction A By thus explaining both steady-state behavior and rapidly 2. Ion Channels As Pores B time-variant flux of ions at the interface beween the interior of 2.1. The Membrane Theory of Bioelectric Phecells and their surroundings, the ion channel concept became nomena B the cornerstone of rational explanation of the excitability of 2.2. The Membrane Problem D tissues, notably muscle and nerve, and of the conduction of the 2.3. Ionic Pathways for Excitability E excited state or impulse, which we call the action potential. 2.4. The Individuation of Ion Channels F However, ion channels were never actually discovered as 3. Ion Channels and Disease G responsible for these phenomena, in the sense of a sudden Author Information H insight. In retrospect, it looks as if their existence was Corresponding Author H postulated on excellent theoretical and experimental grounds Notes H and with growing confidence for over a century. The final proof Biography H of their function as independent molecular entities came when References H the evidence had been quite ineluctable for many years. The concept evolved so gradually probably because other structural and functional concepts, on which it in turn 1. INTRODUCTION dependschiefly that of the cell membrane (itself a highly plastic term), which ion channels allow ions to crosshad to “I see no present, definite and certain indications of the be developed in parallel. nature of the mechanism by which the permeabilities of a Undoubtedly one of the greatest advances in membrane membrane are controlled. There is no dearth of proposals.” biophysics was the realization that rapidly changing ionic K. S. Cole, Membranes, Ions and Impulses (1968), p 521 An acquaintance with the historical foundations of a field of permeabilities underlying both nervous conduction and study is useful especially when they lie in many different chemical synaptic transmission are a consequence of the disciplines. Following the threads of reasoning from their opening and closing of individual membrane pore structures origins through points of branching and fusion to their present with a “stable identity”. Another was the discovery that these position in the closely woven fabric of science helps to identify pore structures are transmembrane proteins creating a and separate the contribution they make to current ideas, which hydrophilic pathway through a lipid membrane’s hydrocarbon thereby become clearer. core. By far the most complete overview on the historical There are, in fact, two intertwined but separate stories, one in development of the ion channel concept can be gained from the realm of physiology and biophysics and the other one in the authoritative, standard textbook on Ion Channels of Excitable biochemistry and structural biology. The present review can Membranes by Bertil Hille.1 Hille’s historical passages, which only address the first. often serve to introduce new concepts, not only contribute essentially to the didactic quality of his representation but, by Special Issue: 2012 Ion Channels and Disease relating directly to a detailed and exhaustive treatment of the subject, are also more precise and complete than would be Received: August 19, 2012 possible in a purely historical narrative. The present short and © XXXX American Chemical Society
A
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Figure 1. Emil du Bois-Reymond (1818−1896, left) and Ernst (Ritter von) Brücke (1819−1892, right). With Ludwig and Helmholtz, du BoisReymond and Brücke formed the physicalist movement of mid-19th century physiology. In a kind of rationalist Sturm and Drang, they felt united in the struggle to overcome traditional concepts of Naturphilosophie such as “animal spirit” and “vital force” and to apply the laws of physics to biological problems. Their spirit of youthful belligerence is conjured up in the Ossianic passage that an aged du Bois-Reymond used in dedicating the 1886 edition of his famous collected speeches7 to his bosom friend Brücke (center): “Tell, Oscar, to Inis-thona’s King, that Fingal remembers his youth; when we strove in the combat together, in the days of Agandecca”. Reprinted with permission from ref 7b. Copyright 1886 Veit & Comp., Leipzig.
Figure 2. Left: Moritz Traube (1826−1894), a native of Ratibor (Racibórz) in Upper Silesia had studied chemistry in Berlin and Giessen, obtained his doctorate, and continued to study medicine for two years before returning home to succeed his father as a wine merchant. Quite successful in his family business, he used his private means to continue in biochemical and biophysical research. In his wide-ranging interests, which often ventured into physiology, there was, in his own view, one center of gravity: “It is this wonderful constituent of the atmosphere: oxygen, supporting all life on this planet, the chemical actions of which I sought to unravel” (writer’s translation).8 Indeed, his view of the cell membrane, which remained highly influential well into the 20th century, was that it formed in a kind of precipitation reaction of the colloidal protoplasmic substance with oxygen. His model systems, ranging from drops of glue in tannic acid to the famous copper ferrocyanide membrane, were designed to reproduce this phenomenon. Despite his “amateur” status, Traube’s contribution was highly acknowledged by contemporary professional scientists. In 1867 he received a honorary doctorate from the Medical Faculty of the University of Halle-Wittenberg and in 1886 was unanimously elected a corresponding member of the Berlin Academy of Sciences (proposed by eight members, among them du Bois-Reymond). Right: All but forgotten today, Rudolf Höber (1873−1953) was an influential and remarkably independent figure. As a medical doctor who had written his thesis on wound shock (1897), he published a widely read monograph on Physical Chemistry of Cells and Tissues (1902) that went through six German editions until 1926, as well as a textbook of Human Physiology (first ed. 1919−eighth ed. 1938). Removed from his position as Chair of Physiology in Kiel because of Jewish ancestry (1933), he emigrated via London to the United States where he eventually became Professor of Physiology in Philadelphia.9 His contribution to the Membrane Theory of Biolelectricity was perceived as very strong by his contemporaries, who sometimes referred to it as the Bernstein−Höber theory (ref 10, p 175).
2. ION CHANNELS AS PORES
are pores through which ions move much as they do in water, i.e., by electrodiffusion, which is a superimposition of random walk and drift in a force field. Electrodiffusion of ions was truly introduced to physiology when in 1902 Julius Bernstein not only proposed it as a general explanation for bioelectricty but
2.1. The Membrane Theory of Bioelectric Phenomena
The most important distinguishing property of ion channels as pathways for charge movement across membranes is that they B
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Figure 3. (A) Bernstein’s apparatus. The muscle, M (horizontal line pattern), is kept under oil in a glass jar (G) with a cut end held toward the left of two elecrodes (E) with a ligature while its intact lateral surface is touched by the other. The injury current flowing between the two electrodes is thus driven by the resting potential of the muscle fibers. A thermometer, T, is used to monitor temperature. Electrodes and thermometer are fixed in a cork lid, which can be rapidly transferred between bowls containing oil brought to different temperatures.2 (B) Measurement of the driving force of the injury current by compensation through a battery (D)-driven circuit. The position of the contact (r) on the wire (N-S) is varied to null the force on the indicator of the galvanometric bussole (M) through a Pohlian gyrotrope or commutator (G), which allows the current to be inverted. The contact with the muscle, μ, is here shown only schematically.21
also provided the first evidence as to the validity of such a model for the basic electrical phenomena that were then measurable in muscle and nerve.2 These were (a) the so-called injury current (or the potential driving it) between the transversally cut end of a muscle or nerve and its intact surface, first shown in muscle by Carlo Matteucci in 1840,3 and (b) the reduction (or “negative variation”) in this resting current upon electrical stimulation of muscle or nerve that Bernstein’s teacher Emil du Bois-Reymond (Figure 1) had measured.4 These phenomena are, as we now know, directly related to the resting membrane potential and the action potential, respectively. Bernstein ascribed the electromotive force of the resting current to a Nernstian diffusion potential. This he supposed to be created by a selective permeability for potassium ions, relatively concentrated inside nerve or muscle fibers with respect to the exterior space, of the boundary of their cytoplasm. Bernstein was inspired by pioneering studies on semipermeable membranes such as the copper ferrocyanide precipitation membranes that Moritz Traube (Figure 2) had presented in 1867.5 In a study on “Electric properties of semipermeable partitions”, Wilhelm Ostwald in 18906 showed that the apparent impermeability of Traube’s membranes to a given salt can be explained by assuming that it is, in fact, impermeable to only one of its ions, say the anion, and permeable to the cation, or vice versa, because in that situation diffusion will create an electric field that will also prevent the permeant ions from equilibrating over the partition. Bernstein cites Ostwald verbatim with the prediction that “not only the currents in muscle and nerve but in particular the mysterious actions of electric fish will find their explanations in the properties of semipermeable membranes”. Considerable background was necessary to make this conjecture: Svante Arrhenius in 188711 had established that cations and anions of salts dissociate spontaneously in water. Friedrich Kohlrausch in 187612 had shown that they are independently mobile in aqueous solution, and Walter Nernst in 1888 and 188913 and Max Planck in 189014 had explained
how an electrical potential or potential difference (PD) is generated by diffusion at the interface of two electrolytic compartments, provided that cations and anions have different mobilities in the border zone. Concerning such a border zone or membrane, Ludimar Hermann’s core-conductor theory explaining electrotonus,15 i.e., the polarization of fibers by steady applied current, provided an idea that individual nerve or muscle fibers were bounded with a polarizable interface that provided for larger transversal than longitudinal resistance to electric current and could possibly assume the role of a selectively permeable membrane. Diffusion through pores with sizes admitting only a few water molecules had been discussed by Carl Ludwig (1858)16 and Adolf Fick (1855)17 following earlier work by Ernst Brücke (Figure 1), who in 1843 had published a German language excerpt from his M.D. thesis “De diffusione humorum per septa mortua et viva”.18 Involving considerably less intricate apparatus than his equally famous earlier differential rheotome recordings of the negative variation, i.e., the action current, in nerve, the measurements that were to form the basis of Bernstein's Membrane Theory were quite simple. Using the apparatus shown in Figure 3A and measuring the driving force with a compensation circuit devised by du Bois-Reymond (Figure 3B), he measured the electromotive force of the resting current and showed that its changes with temperature were predicted by the Nernst equation. His conclusion is that there is “somewhere in the substance of the muscle fiber (fibril, sarkoplasm) a lack of permeability for one ion, e.g., for the anion of the electrolyte” leading to the stablishment of an “electrical double layer”. Because of the importance of the concept of a semipermable membrane, Bernstein calls his theory “the Membrane Theory”. The basic phenomenon of semipermeability of the “protoplasmic skin” had been shown in plant cells by Hugo de Vries (1871)19 and Wilhelm Pfeffer (1877)20the latter of whom Bernstein mentions. Bernstein’s original (1902) statement of the Membrane Theory, however, contained no reference to a mechanism for a C
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Figure 4. Fricke’s 1925 high-frequency Wheatstone bridge for measuring the capacitance of red blood cell suspensions. Left, layout; right, photograph of the apparatus. Reprinted with permission from ref 36. Copyright 1925 Rockefeller University Press.
selective permeability of ions. This question was first explicitly raised in 1905 by the physiologist Rudolf Höber (Figure 2) when he reported on his tests of Bernstein’s hypothesis by measuring the effects of various electrolytes on the resting current of muscle or nerve and establishing, for instance, that high concentrations of extracellular potassium, as expected, decrease the resting potential.22 In that paper's introduction, Höber starts with Ernest Overton’s well-known observation that the efficiency of uptake of a substance (e.g., a dye) by cells (diosmosis) depends on the degree of its lipid solubility23 and from which Overton developed his theory that protoplasmic membranes are composed of lipoids (mainly cholesterol). Höber, in turn, argues that those substances that are chiefly important for cellular life (sugars, salts, amino acids, etc.) are hardly lipid-soluble at all but still apparently must be able to enter cells. He concludes that in addition to what he calls “physical-diosmotic permeability” there must a different, second kind that depends more on the cell’s physiological state: “physiological permeability”. Höber remarks, with what du Bois-Reymond would have called “manly resignation”,7 that “'physiological' here means hardly more than 'unexplained'”. Here we have then, in a nutshell, the problem to which the ion channel concept provides one solution and “the” solution as far as electrical signaling by rapid changes in membrane conductance is concerned. The membrane theory gained a lot of credibility through further work on model membranes published in 1925 by Leonor Michaelis (of Michaelis−Menten fame), who used dried collodium (nitrocellulose) membranes to show that they supported diffusion potentials due to a selective permeability for cations brought about by negative charges in the membrane.24 Cation-selectivity had already been shown (1912) in a biological preparation (apple skin) by Jaques Loeb and Reinhardt Beutner.25 Using a membrane made of gelatin, the charge of which can be titrated using different pH values, Michaelis’s co-worker Akiji Fujita26 showed the absence of a potential difference at gelatin’s isoleelectric pH of 4.7, while at acidic pH the potential shows decreased mobility of cations and reversed sign at alkaline pH to approach that of the negatively charged collodium membrane. In addition, Michaelis and his co-workers found that more strongly hydrated cations have lower permeability than less hydrated ones, in accordance with their mobility in free solution.24 Michaelis explained these findings by the presence in the membrane of very narrow and charged “capillary canals”, i.e., pores, which promote electrostatic and frictional interaction
of the permeant ion and its hydration shell, respectively, with the substrate material. A few years later, Rudolf Mond and Friedrich Hoffmann were even able to create a positive charge in the collodium pores using the base Rhodamin B, leading to anion-selective behavior.27 The permeability series of anions, interestingly, departed from the mobility sequence in free solution, instead following the lyotropic Hofmeister series.28 2.2. The Membrane Problem
These and similar model experiments using porous membranes (e.g., ref 29) might, in fact, have quickly brought about a rechristening of the “Membrane Theory” as the “Membrane Pore Theory” of bioelectricity. But it was not to be. Reading the literature of the time, one is left with the impression that the membrane theory lacked one important prerequisite for progress toward a pore theory: that, ironically, was the membrane itself or, more precisely, a concept of the biological cell membrane that would allow pores to exist in it. As it was, there seems to have been an almost general consensus that the membrane is not a structure apart from the protoplasm or cytosol; still very much in the vein of Traube’s conception,5 it was thought to consist of the same constitutents as the protoplasm in a form that was more condensed and gel-like, somewhat like the skin that forms on a pudding. Consequently, it was also widely believed that the protoplasmic membrane might be fundamentally different in composition and structure from one cell to another and also change composition according to the physiological state of the cell or even disappear and reform easily. In fact, in biological textbooks of the 1920s, the cytoplasmic membrane was not even considered a necessary attribute of a cell (e.g., ref 30). Ernst Gellhorn, in his exhaustive monograph on The Permeability Problem,10 for instance, is quite critical of the lipoid theory of Overton, instead proposing an amalgamated colloid−molecular-sieve model for a “protoplasmic skin” that can change its permeability in response to stimuli. He clearly does not believe in a protoplasmic membrane as a structure separate from the cytoplasm. This view is likewise expressed in Williams Bayliss’s textbook on General Physiology31 and even a decade later (1937) in Herbert Gasser’s chapter on the “Sequence of Potential Changes” in his famous book with Joseph Erlanger.32 The fundamental idea of a canonical membrane structure based on a bimolecular phospholipid layer33 was not yet born. Meanwhile, in a series of experiments on the frequencydependent resistivity of red blood cell suspensions (1910− D
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Figure 5. (A) Transverse impedance measurement by Cole and Curtis of a squid axon undergoing excitation. The impedance band is superimposed with the extracellularly recorded action potential, showing that the impedance decrease follows the depolarization with a delay (time marks 1 ms). This proved correct the original idea of Hermann that the initial depolarization of the membrane comes about by axial, not transmembrane, current flow. Reprinted with permission from ref 47. Copyright 1939 Rockefeller University Press. (B) Family of current responses to step changes of membrane voltage from resting membrane potential. Reprinted with permission from ref 50b. Copyright 1949 Centre National de la Recherche Scientifique (CNRS).
these peripheral membrane proteins was to account for the low surface tension of cell membranes, first measured in sea urchin eggs by Cole.43 Integral membrane (or transmembrane) proteins in a lipid bilayer finally appeared with the fluid mosaic model of S. J. Singer and Garth L. Nicolson,44 which was the first to be strongly influenced by the concept of hydrophobic interactions.45 By depicting the membrane as a barrier consisting chiefly of a hydrocarbon, dielectric core, these models gave rise to theoretical considerations presaging important facts regarding the possibility of passage of ions, in particular the energy required for (partial) shedding of the hydration shell in narrow pores.46
1913), Höber had shown that their cytosol had a conductance similar to an electrolyte solution so that their membrane had to be a poor conductor, behaving like a dielectric.34 Kenneth S. Cole, in his 1968 monograph,35 actually uses the values published by Hö ber to estimate a specific membrane capacitance of 3.6 μF/cm2, showing that one might have concluded on a membrane of molecular dimensions from Höber’s data already. In fact, the first direct measurements of membrane capacitance in cell suspensions were made by Hugo Fricke (Figure 4).36 He found a specific value of 0.81 μF/cm2 and quite deliberatelybut probably following Overton’s lipoid theoryassuming an oily membrane with a dielectric constant of 3, he arrived at membrane thickness of 3.3 nm, in line with today’s ideas about lipid bilayer thickness. None of the texts mentioned makes reference to the work on thin lipid layers by Agnes Pockels (1891), Lord Rayleigh (John Strutt, third Baron Rayleigh, 1892), and Irving Langmuir (1917).37 Langmuir’s work on monomolecular layers, however, provided the inspiration for Evert Gorter and François Grendel who (in 1925) spread lipid extracts of red blood cells on water and, from the area of the resulting lipid film, concluded that the membrane must have been a bimolecular layer.38 Apparently, the lipid extraction was incomplete, but as they simultaneously underestimated the erythrocyte surface, the result came out right.39 In 1935, James F. Danielli actually makes the permeability problem the starting point in arguing for his famous membrane model.40 He sets out to show by relatively simple arguments that in a thin film of lipid “it is possible for a pore structure of molecular dimensions to have a relatively permanent existence”. The double layer of lipids is not yet the clearly favored option but an allowed one “between unimolecular and trimolecular”, and a drawing just illustrates two peripheral leaves with lots of space between the two layers for further lipid. A later publication,41 however, was widely interpreted as indicating a preference for a bilayer.42,39 This view of the plasma membrane dominated for three decades. It was largely on the strength of electron microscopic observations using a particular stain (KMO4) that in 1966 J. David Robertson proposed a “unit membrane”33 with the general structural layout of exactly two leaflets of amphiphilic lipids, forming the boundary of all cells and of organelles inside them. A common feature of both the Davson−Danielli and the unit membrane (Davson−Danielli− Robertson) model was that proteins were adsorbed, as globules or as sheets, to the lipid headgroups. Originally, the role of
2.3. Ionic Pathways for Excitability
Using impedance measurements similar to those of Fricke on cell suspensions, Cole with Howard J. Curtis tested Bernstein’s membrane hypothesis by looking for the predicted breakdown of membrane resistance during an impulse in a nerve fiber. They succeeded (1939)47 and thereby produced one of the iconic figures of electrophysiology (Figure 5A). The preparation used in these experiments, which were conducted at the marine Biological Laboratory at Woods Hole, was the giant nerve fiber or axon of the squid Loligo, that had been anatomically described in 1910 by Leonard W. Williams48 and recently discovered for physiological experiments by J. Zachary Young.49 It was noted on both sides of the Atlantic that this axon, with a diameter of about half a millimeter, permitted insertion of an electrode to measure, for the first time, the full extent of the membrane potential and of its changes during excitation. Just after the steep decline in membrane resistance during the action potential had seemed to finally corroborate Bernstein’s hypothesis, the first results came as a surprise. Alan L. Hodgkin and Andrew F. Huxley, working in the Plymouth Laboratory of the Marine Biological Association, found the expected negative membrane potential at rest, but instead of approaching 0 mV during excitation (the result expected for loss of membrane selectivity), the membrane potential went positive (overshot the zero line) by about 30−40 mV.51 Curtis and Cole did the same experiment and published an even larger overshoot (+110 mV),52 which is likely to have been due to overcompensation of capacitances35 and created some confusion.53 A greater diversion, however, was due to the second World War, and it was only afterward that Hodgkin and Bernard Katz were able to test the new hypothesis that the action potential was not due to a general breakdown of the membrane selectivity for potassium E
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Figure 6. Two drawings by Bertil Hille illustrating the development of the ion channel concept from rather featureless ion-specific defects in what seems like a Danielli-membrane with a central oily sheet and protein adsorbed periperally to individual protein macromolecule spanning the membrane with a selectivity filter located toward the extracellular space and a voltage sensor operating a cytoplasmically located gate. Left: Reprinted with permission from ref 68. Copyright 1999 Nature Publishing Group. Right: Reprinted with permission from ref 69. Copyright 1977 Elsevier.
that leads to a model where relatively sparse, single entities provide a pathway for several ions at a time. Discussing possible explanations for the steep voltage dependence of the ionic conductance changes observed, Hodgkin and Huxley write: “Details of the mechanism will probably not be settled for some time, but it seems difficult to escape the conclusion that the changes in ionic permeability depend on the movement of some component of the membrane which behaves as though it had a large charge or dipole moment. If such components exist it is necessary to suppose that their density is relatively low and that a number of sodium ions cross the membrane at a single active patch.” This insightful reasoning is not mentioned again, it seems, for almost a decadepossibly because there was no way apparent in which such a hypothesis might be tested.
but rather to the opening of a second, selective pathway for another ion, one with a Nernst equilibrium potential on the positive side. From what was known about the ionic concentration relationships between the squid axoplasm and seawater, an obvious candidate was sodium, and also Overton’s ancient observation on the requirement of sodium ions for muscle contraction54 might have given a hint. By replacing half or two-thirds of the sodium chloride in seawater by choline chloride or sugars, Hodgkin and Katz found that, indeed, the action potential became slower in its rising phase and the overshoot was gone at low extracellular Na+ concentrations.55 The transatlantic exchange of ideas between Cole and coworkers and the group of Hodgkin, Huxley, and Katz then brought fourth a revolutionary technique: the “voltage clamp”. Electronic feedback circuitry in conjunction with long, specially treated axial Ag/AgCl-chloride electrodes was used to set and hold the voltage across the membrane to a certain command value and to record the current that had to be delivered in order to keep it there (Figure 5B). The command voltage could be changed rapidly in stepwise fashion, with the long axial wire and extracellular guard electrodes providing spatial uniformity (“space clamp”) so that, by Ohm’s law, the current flowing in response to such voltage steps became a direct measure of the membrane conductance.50 This technique became known as the voltage clamp and, particularly in combination with the possibility to exchange solutions, even on the inside of the axon,56 has remained the basis of all quantitive work done on membrane ion conductances since (ref 1). In 1952 Hodgkin and Huxley published results of voltageclamp experiments on squid axon to test a carrier hypothesis for sodium influx developed earlier57 in order to explain the overshooting action potential. This series of papers58 can be said to be the culmination of an era. They disproved the carrier hypothesis and led to a quantititativebut mechanistically neutraldescription of the action potential mechanism with a voltage-dependent, transient sodium inward current followed by a delayed and more slowly rising potassium outward component that is still valid, even if discrepancies sometimes arise (for a recent interesting case, see ref 59). The Discussion of the last paper in this series, while largely devoted to discounting the carrier hypothesis, contains the first reasoning
2.4. The Individuation of Ion Channels
The term ion channel (or ionic channel), when it first came up, was not usually used to denote a specific mechanism of conductance. As did its meaning, so did the term develop in stages. With few exceptions (e.g., ref 60), the expressions “sodium channel”61 and “potassium channel”,62 in the singular, designate the relative contribution of the respective ion to the voltage-dependent membrane conductance underlying excitability, impulse conduction, and chemical sensitivity of membrane potential. Bernard Katz, in a review on synaptic transmission,63 is probably the first to use the general term “ionic channel”, but in a manner that makes it very clear that he is not yet speaking about a molecular entitity: “How do transmitter substances alter the membrane potential? Only a very incomplete answer to this question can be given. In general, the primary action leads to the opening of some ionic permeability channel in the membrane. Depending upon the size or specificity of this ionic channel, the membrane potential either tends to fall toward a low level, well beyond the firing threshold of an impulse, or it may become stabilized in the vicinity of the resting level or even tend to rise somewhat (hyperpolarize).” Thinking about channels for ions in terms of multiple separate entities, i.e., molecules, really began with the earnest study of the effects of substances that blocked the voltage-dependent potassium and sodium conductances in nerve.64 Sometimes, authors are still careful to stress that “By F
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Bert Sakmann in Göttingen to electrically isolate micrometersized patches of membrane of muscle fibers, which had been denervated to make acetylcholine receptors appear all over their membrane. The small size of the membrane under the tip of a fire-polished pipet filled with ACh-containing electrolyte solution reduced the background conductance, and therefore electrical noise, to the point that it was indeed possible to resolve rectangular current steps corresponding to the stochastic opening and closing of ACh-gated cation channels.77 Such single ion channel measurements with the extracellular patch clamp, as it was called, enhanced in clarity by drawing the small patch of membrane into the pipet to increase the isolating resistance beyond a GOhm and thereby further reduce noise,78 and repeated in all the classical preparations (for an overview, see ref 1), put the final seal of truth on the idea that membrane ionic conductance is controlled by the stochastic opening and closing of macromolecular ionic pores. There were many and impressive further developments, which, however, did not fundamentally change the firmly established concept of the ion channel as it has been known since the early 1980s. Molecular cloning confirmed the basic idea of a transmembrane protein and added hugely important facts such as multimeric structure as well as a vast array of new channel types. It also enabled the establishment of structure− function relationships by amino-acid exchange and reporter labeling as well as investigations of the atomic structure of some bacterial channels by X-ray crystallography, which in turn provided the structural basis of ion selectivity79 to the utter satisfaction of the pioneers, who saw that they had been on the right track.68,80 Still on the agenda is the quest to fully understand the dynamics of channel protein conformation in relation to gating, i.e., the change in the equilibrium between the open and closed state of the ion-conductive path in response to electrical, mechanical, and chemical stimuli.
channel, we do not imply any particular mechanism by which ions traverse the membrane but use this term simply as an equivalent of 'pathway'”.65 Others, in particular Bertil Hille66 and Clay M. Armstrong,67 were less reticent, even if their ideas on ionic pores were viewed as premature, for instance, by Cole (see ref 68). Their careful studies of the block of sodium and potassium conductances by tetrodotoxin and tetraethylammonium (TEA) and its derivatives, as well as of the differential conduction by the same mechanism of various ionic species (i.e., of the ionic selectivity of the conductive path), revealed properties strongly indicating that ionic conductances are indeed mediated by pores. Even more fundamentally, the effects of toxins and drugs, by obeying the law of mass action for a bimolecular reaction, showed that the membrane conductance to an ion behaves as a number of binding sites or receptors, so as to change the conceptual status of the ion channel from “principle” to “(molecular) entity” (see ref 1, p 66). The development of the concept in the crucial decade 1967−1977 is well-illustrated by a comparison of two cartoons made by Hille (Figure 6). In the early 1970s, Armstrong and co-workers wrote:70 “The ionic channels of nerve membrane and the gates that control ion movement through them are widely supposed to be composed of protein, but there is surprisingly little evidence on the question.” Apart from the effects of proteases on ionic conductances, suggestive pieces of evidence came from studies on free-standing artificial lipid bilayers in which a poorly characterized protein called excitability-inducing material (EIM)71 and the peptide Gramicidin A72 had been shown to induce ionic conductances. Remarkably, these peptide-doped membranes showed discrete conductance changes as would be expected from the opening and closing of microscopic channels. The reconstitution from purified proteins in such pure lipid bilayers of the conductance mechanism underlying voltage-dependent membrane currents finally confirmed that ion channels are membrane proteins (for an overview, see ref 73). Meanwhile, Bernhard Katz with Ricardo Miledi analyzed the minute stochastic fluctuations in membrane potential that accompanied the depolarization of muscle cells by neuromuscular transmitter acetylcholine (ACh).74 Similar “noise analysis” had been used on frog nerve fibers by Hans A. Derksen and Alettus A. Verveen in 1965 and 1966.75 Katz and Miledi concluded from a statistical analysis of the ACh-induced voltage noise that the ACh response is a superposition of stochastic pulses (“shots”) that occur at a mean frequency that depends on acetylcholine concentration. The way in which such observations influenced thinking on ion channel function is well expressed in this passage form the last paper: “The opening and closing of an individual ionic channel is more probably a sudden on/off event, but it might be argued that if the duration of individual 'on' states varies in random fashion, the final result might approximate to that for an exponential average shape.” Would it ever be possible to directly observe the opening and closing of biological ion channels, as it had been possible with peptide-doped synthetic lipid membranes? That story has been told many times. Briefly, Erwin Neher, as a graduate student with Hans Dieter Lux in Munich, had recorded ionic currents pressing an extracellular glass pipet against the membrane of a nerve cell soma to achieve perfect isopotentiality in a small patch of membrane (pipet spot clamp).76 A miniaturized and simplified version of this arrangement was used by Neher and
3. ION CHANNELS AND DISEASE The clinical importance of the pathways for membrane ionic conductances became clear immediately, at least when it had been finally shown that they are the basis of tissue excitability. For example, the Hodgkin−Huxley−Model of excitability was used to predict and interpret changes in, e.g., cardiac or cerebral excitability associated with arrhythmias or epileptiform discharges as a consequence of enhanced or suppressed function of voltage-dependent ion conductances (overview in refs 81 and 82), often with a view to understanding the underlying diseases in terms of primary ion channel malfunction. Many drugs that had been in use for such conditions for a long time were subsequently foundusing the techniques of single-cell electrophysiologyto more or less specifically interact with ion channels (e.g., ref 83). Since then, a number of hereditaryand fairly rarediseases that are caused monogenetically by mutations in ion channel genes have been identified and fashionablyif unclassicallycalled “channelopathies” (overview in ref 84). These discoveries are instructional showcases of pathophysiological principles and provide important information as to possible underlying causes of other diseases. Not least important is the fact that the identification of such ion channel pathologies has led to the discovery of new roles for ion channels in unexpected places (e.g., ref 85). However, the true clinical importance of ion channels relates to their role as therapeutic drug targets for more widespread medical conditions. This role is accorded to them by their strategic location in the web of cellular signaling G
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and their eminent accessibility for drugs. The pharmacological challenge, therefore, is to discover new and better, more specific, and less toxic substances to modulate ion channel function. Novel technologies have been and continue to be developed, allowing automated and high-throughput electrophysiological analysis of ion channel modulation.86 Any arrival in the clinic of a novel, specific, and safe ion-channel active drug developed in a rationally directed fashion using biophysical tools and structural information would deserve to be celebrated as a fulfillment of the long story outlined above.
AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare the following competing financial interest(s): The author is shareholder and Chairman of the Board of Nanion Technologies GmbH, Munich, a producer of automated patch-clamp devices. Biography
Dr. Jan C. Behrends directs the Laboratory for Membrane Physiology and -Technology at the Department of Physiology in the University of Freiburg, Germany. He received his M.D. in Cellular Neurophysiology from the University of Munich and was Postdoctoral Fellow at the Institut Pasteur in Paris and at the Max-Planck-Institute for Psychiatry and later Assistant Professor at the University of Munich, where he obtained his Habilitation before moving to Freiburg as Professor of Physiology in 2003. His research interests are centered on membrane physiology and synaptic transmission with a penchant towards developing electrophysiological techniques. In 2002, he cofounded Nanion Technologies, a provider of chip-based and automated patchclamp devices.
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