Neuron

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Ion Channels: From Conductance to Structure
Francisco Bezanilla1,*
1Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637, USA *Correspondence: fbezanilla@uchicago.edu DOI 10.1016/j.neuron.2008.10.035

In this perspective I tell the story (albeit a clearly abridged version) of how our knowledge of ion conduction through ion channels has evolved from a purely electrical concept to a structural dynamics view of ions interacting with a membrane protein. Our progress in this eld has shown steady growth over the years but has also been interspersed with sudden jumps of discovery. These leaps have normally been associated with the introduction of a new technical advance or the development of a new biological preparation; therefore, it is quite certain that we have not seen them all.
Introduction The ion channels eld is a longstanding and distinguished one that has steadily grown and matured, often as a direct reection of our ability to address questions in new and unique ways. In telling this brief history of the ion channels eld, Ive therefore organized this story around the experimental jumps that have expanded our understanding and opened up new avenues for investigation. As you read on, you will see that the early description of conductances was simply a study of voltage and current at the level of a black box because there was no indication of the physical basis of the conductances. It is remarkable, however, that the concept of a conducting pore as part of an ionic channel with properties of permeation, selectivity, and gating was introduced quite early, even before there were any data on ion channel molecular structure. In fact, many of these concepts were pretty well established in the 1970s from biophysical experiments when the channels were drawn as cartoons as an attempt to describe what was in the black box. This era was followed by an expansion in the use of unique biological preparations that allowed us to address new and interesting questions, along with leaps in insight gained from our understanding of the operation of single molecules by the introduction of the patch-clamp technique. The subsequent era of cloning initiated our understanding of ion channels at the molecular level, an understanding that has been further expanded by the crystal structures and spectroscopic methods that then followed. And, while the future is always hard (one might even say impossible) to predict, its clear that there will be many more exciting advances to come. I trust you will indulge me as I also attempt to give you some of my ideas of what we might expect in the near future, given the present advances. You as the reader may decide to go quickly over the rst two sections, spending more time in the last three sections because those are directly relevant to the research done today in ion channels. Yet, please remember that we can often learn much from our past. Exposing the experiments and concepts developed during those rst eras gives an appropriate historical perspective and, in my view, helps one to gain a deeper understanding of the rich and unique nature of the ion channels eld. In the Beginning Was the Squid Giant Axon The lipid bilayer that makes up the cell membrane is an essentially insurmountable barrier for the ow of ions; therefore, ion transport is carried out by membrane-embedded specialized proteins in the form of transporters and ion channels. Although this statement is obvious to everyone today, it was not clear at all just over half a century ago. In the early 1900s Bernstein explained correctly the resting potential of a cell as a diffusion potential (Bernstein, 1902), but most of the attention in the eld was given to the changes in membrane potential. This explains why the history of ion channel research is intimately connected with the study of the physical basis of the initiation and propagation of the nerve impulse. Bernstein also proposed a mechanism for the action potential, attributing the sudden potential change to a simple breakdown of the normal K+ selectivity of the membrane. This concept had to be revised when Hodgkin and Huxley and Curtis and Cole measured the actual time course of the action potential in the squid giant axon, revealing an actual reversal of the membrane potential at the peak of the impulse (Hodgkin and Huxley, 1939; Curtis and Cole, 1940). This experiment was possible because the large size of the squid axon allowed the introduction of an internal electrode and thus permitted the measurement of the real transmembrane potential difference. The measurement of the action potential is one of the multiple examples of the critical role that the squid giant axon has played in our understanding of membrane transport. In fact, its importance can hardly be overemphasized. Electrical Models and the Voltage Clamp The concept of the membrane behaving electrically as a capacitor in parallel with a resistance (or conductance) with an electromotive force was well accepted in the 1930s (see Cole, 1972). However, the origin of the sudden change in membrane potential that occurs during an action potential was not well dened. It was the landmark paper of Cole and Curtis (Cole and Curtis, 1939) using impedance analysis of the squid axon membrane during the action potential that established that the transient large decrease in conductance (and not a change in capacitance) was the main event underlying the impulse. Yet, a picture of the events underlying the generation of the impulse remained elusive. The main problem was that the all-or-none nature of the impulse made it impossible to gradually control the voltage when

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elicited by current pulses. It took the development of a new technique, the ability to control the membrane potential (the voltage clamp), that initiated the revolution that has led to our understanding of membrane conductance changes (Cole, 1949). But, while illuminating some aspects of the initiation and propagation of the nerve impulse, the voltage clamp was not enough: a more complete understanding also required the sodium hypothesis (Hodgkin and Katz, 1949). The Hodgkin and Huxley Axon The beauty and simplicity of voltage-dependent conductances in the Hodgkin and Huxley (HH) equations goes way beyond explaining the generation and propagation of the action potential. In fact, it would not be a stretch to say that the concept of the ion channel was established at that point. Hodgkin and Huxley, referring to the dipoles or charges responsible for the voltage dependence (that predicted the later-found gating currents) stated: 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 (Hodgkin and Huxley, 1952). This statement implies that the ionic conductance is not pictured as a diffuse property of the membrane, but rather as the result of specic, discrete sites. For example, in their concept of the potassium conductance, there was a conduction term (gK ) and a voltage-dependent gating process that can turn the conductance on or off (n4). This is precisely the concept accepted today, where we say gK = Ng, where N is the total number of channels and g is the conductance of one channel, or site. It should be emphasized that the work of Hodgkin and Huxley also established the basic pulse protocols, such I-V curves, instantaneous I-V curves, and double and triple conditioning pulse protocols, that are the basis for most electrical recordings in use today. After the monumental work of Hodgkin and Huxley, many advances were made on the properties of the Na+ and K+ conductances still using the squid axon preparation under voltage clamp. The development of internal perfusion (Baker et al., 1961; Oikawa et al., 1961) and dialysis (Brinley and Mullins, 1967) techniques that introduced tubing or semipermeable capillaries into the axon to exchange the internal medium allowed a detailed study of reversal potentials and conductances (Chandler and Meves, 1965). The introduction of the Vaseline gap to the voltage-clamp technique provided a way to measure the ionic currents in myelinated nerve where it was found that the conductances in the node followed rules similar to their counterparts in the squid axon (Dodge and Frankenhaeuser, 1958). But, most importantly, what followed Hodgkin and Huxley was the development of the channel concept as we understand it today. Separate Pathways of Conduction through Pores The identication of tetrodotoxin (TTX) (Narahashi et al., 1964) as a specic blocker of the Na+, and not K+, currents was an important conrmation that there were separate paths for Na+ and K+ in the membrane. The concept that the paths of conduction were through discrete pores was not new at that time and had been presented much earlier (see Hille, 2001). Mullins put forward the idea that the pore is a cage where ions lose, at least partially, their hydration shell to t snugly, and could thus differentiate between Na+ and K+ (Mullins, 1959), a concept that is not too far away from what we know today (see below). The subsequent discovery that tetraethylammonium ion (TEA) blocked K+ currents without changing the Na+ currents further conrmed the idea of separate pathways (Armstrong and Binstock, 1965; Tasaki and Hagiwara, 1957). The pioneering work by Clay Armstrong using TEA derivatives as blockers of the K+ conductance from the internal side of the squid axon gave us the rst insights into the basic architecture of the channel. He found that by replacing one of the ethyl groups of the TEA molecule with a longer hydrocarbon chain, the block of the K+ current occurred after the channel opened, introducing the concept of a discrete gate. Then he went further in demonstrating that the blocker was displaced by an inward ow of K+ ions, giving us the rst indication that the blocker and the ions occupied the same region in the channel (Armstrong, 1971). The blocker of the K+ conductance could also be a less permeable ion such as Na+ or Cs (Bezanilla and Armstrong, 1972) that could be dislodged of its blocking site when large electric elds were applied (French and Wells, 1977). The TEA results were also obtained from the inside of the node of Ranvier (Armstrong and Hille, 1972) and allowed the general proposal that K+ ions crossed the channel in single-le fashion, as was previously inferred by Hodgkin and Keynes (1955), and implied that a gate controlling the ions ow was located in the intracellular region of the channel. Therefore, in the early 70 s the concept of an ion conducting pore that had an intracellular gate and an extracellular region that selected the type of ions (selectivity lter) was denitely taking shape (Hille, 1975). The Molecular Basis of Conductances The type of molecules that formed the conductance pathway was a subject of intense debate because it was thought that ions could go either through the lipids or through specialized proteins embedded in the membrane. One of the rst pieces of evidence that proteins were involved in this process was provided by the work of Rojas and Luxoro in Montemar, Chile, who showed the effects of proteases on excitability of the squid giant axon (Rojas and Luxoro, 1963). This work was followed by the study of the effect of other proteases on the ionic currents under voltage clamp. Pronase is a protease that was found to selectively destroy Na+ inactivation, leaving the potassium conductance unaltered, and this result prompted the hypothesis that pronase was clipping part of the protein that normally moved into the mouth of Na+ channel pores and thereby blocking the current (Armstrong et al., 1973). This nding is the origin of the ball and chain hypothesis of inactivation that is still discussed today. It took another biological preparation and advances in biochemical techniques to nally show that the Na+ channel was a protein. The electric eel contains specialized cells (electrocytes) with a large density of sodium channels that are stacked in such a way that when they re an action potential, the voltage can go as high as 600 Volts along the whole animal. This arrangement makes the eel a swimming killing battery that can easily stun and capture its prey, and it also makes it the richest natural source of Na+ channels. Taking advantage of the eel and the high afnity of the Na+ channel to TTX, Agnew and colleagues were able to extract and purify a 230 kDa protein (Agnew et al., 1978), demonstrating the protein nature of the Na+ channel, which was later found to transport Na+ in liposomes and, even later, demonstrated to induce Na+ channel currents in liposomes and bilayers (Correa et al., 1990; Rosenberg et al., 1984).

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The Voltage Dependence of the Conductances The voltage dependence of Na+ and K+ conductances requires that the channels have a sensor, such as a charge or a dipole that responds to voltage changes (Hodgkin and Huxley, 1952). The movement of these putative charged components is expected to produce a transient current that is about 100 to 200 times smaller than the ionic currents, making their detection difcult. Toward the end of the summer of 1972, while we scribbled possible diagrams on the sand in Woods Hole with Clay Armstrong, we came up with a simple system that allowed the subtraction of the large linear component of the capacitive current to extract the nonlinear gating current. We also had to minimize the ionic currents using nonpermeant ions and TTX as a blocker to successfully measure the gating current of the Na+ channel in the squid axon (Armstrong and Bezanilla, 1973; Keynes and Rojas, 1974). The detailed analysis of the gating currents revealed that the charge movement correlated with the opening and closing of the Na+ channel (its activation process) but that the inactivation process did not have its own voltage sensor. Rather, it was an almost voltage-independent event that affected the voltage dependence and kinetics of the activation charge. It is after these ndings that the ball and chain hypothesis of inactivation was put forward. An important implication of this work was the idea that channel inactivation inherited most of its voltage dependence from the intrinsically voltage-dependent activation process (Armstrong and Bezanilla, 1977), a concept that had been put forward before by Goldman and Schauf (1972) and that departed from the HH hypothesis of independent activation and inactivation processes. The Unitary Currents However, if channels were discrete units of conduction, it should be possible to measure the (albeit minute) current through a single pore. The idea of measuring the unitary currents, also called the single-channel currents, was mentioned by biophysicists in the 1960s. The rst breakthrough occurred when Bean et al. (1969) recorded discrete miniature current jumps in articial lipid bilayers doped with a contaminant called the excitability inducing material (EIM) (Mueller et al., 1962). These single-channel currents were analyzed in detail by Ehrenstein and coworkers (Ehrenstein et al., 1970, 1974) and constituted the rst correlations done between stochastic single-channel uctuations and macroscopic currents. Although EIM currents were not the classical Na+ or K+ channels, they stimulated research into achieving the same with classical channels. The rst success in this direction was the detection of excess noise produced by an ensemble of channels due to the stochastic nature of the single-molecule gating (Fishman, 1973; Katz and Miledi, 1970; Wanke et al., 1974), but the recording of the single-channel currents had to wait for the development of a technique that isolated a small patch of membrane with minimal leak: the patch clamp. Expanding the Horizon of Channel Research: Introduction of the Patch Clamp The rst single-channel recordings of Ach receptor channels were done by Neher and Sakmann (1976) using small pipettes pressed against denervated, enzymatically treated muscle bers from the frog. This landmark achievement was a nal conrmation of the discrete nature of channels. After these recordings, it was remarkable that these authors immediately recognized that, upon application of negative pressure to a clean pipette attached to the cell, the decrease in the background noise meant they must have obtained a seal between the pipette and the membrane that exceeded a gigaohm: in that precise instant the patch-clamp technique was invented (Hamill et al., 1981). It is not possible to summarize here the tremendous impact the patch-clamp technique has had or will have in the future, but it is undeniable that it radically changed the scope and the way we do electrophysiology. The patch-clamp technique was not only useful for recording single channels, but it also allowed voltage clamping of the whole cell, and thus gave us the ability to record macroscopic currents with much higher accuracy than found when using microelectrodes. Most importantly, this technique opened up the use of the voltage-clamp technique to essentially any cell that has its membrane available to be touched by the patch pipette to obtain a gigaohm seal, thus nishing the hegemony of the squid axon and other large, cylindrical cells types in electrophysiology. Initially, the patch clamp was used in a few tissue culture cell lines that presented an easy surface to achieve the gigaseals. But it did not take long before the technique was used in many other cell lines and also in cells dissociated from tissues. This extended the voltage-clamp technique to tissues to which we had no access before, and new conductances were found that had different selectivity, permeability, and voltage dependencies. The simplicity of the squid axon, which essentially has a Na+ conductance, a K+ conductance, a minor Ca2+ conductance, and a poorly dened leak conductance, was rarely found in other cells such as neurons or cardiac myocites. These cells showed a myriad of different types of conductances that were found to play roles in frequency generation, fast repolarization, and plateau generation. The patch-clamp technique was also used to directly record from almost intact tissue (Edwards et al., 1989), opening a new era that allowed voltage clamping of neurons in situ to study synaptic transmission and connections in brain slices. The dream of describing the elementary properties of neurons in situ was nally a reality. Permeation and Gating Measuring single-channel events gave us, for the rst time, the actual value of the conductance of a single pore. It was found that the single-channel conductance varied widely among different types of channels, and it was also found that there were several types of channels that, although showing the same selectivity, had different single-channel conductances. These results also stimulated the development of theories of permeation based on the idea that ions had to overcome energy barriers separated by energy wells, or binding sites, within the pore (see Hille, 2001). At the time, these theories were phenomenological in the sense that they required the construction of mathematical models of permeation to describe the observed reality without knowledge about the chemical groups that were actually lining the pore or the 3D structure of the pore itself. Proof of such theories would await future technological advances. The single-channel recording also made important advances in our understanding of channel gating because it was now possible to develop stochastic models of gating that showed the individual opening and closing of the channel. These were kinetic

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models that still had no molecular basis. It became clear, however, that single-channel recordings have very high sensitivity to events that are kinetically near to the open state, but they are pretty insensitive to deeper closed states. On the other hand, the gating currents were quite sensitive to any transition that has some charge movement. Thus, the ideal combination would be to measure single channels, macroscopic currents, and gating currents in the same preparation. This was difcult to achieve in the cells normally used to record single-channels because, as they have limited channel densities, gating currents had too low signal-to-noise ratio. The squid axon still offered the best recordings of gating currents, but no single-channel recordings were possible because they are surrounded by connective tissue and a Schwann cell layer. This changed when Conti and Neher were able to use a bent pipette inside of the axon to record the rst single potassium channel currents from the squid axon (Conti and Neher, 1980). The development of the cut-open axon technique allowed the routine recording of potassium channel from the axon (Llano et al., 1988) where it was found that even the squid axon contains several types of K+ channels. When the technique was extended to record single Na+ channels (Bezanilla, 1987), it was nally possible to record the three main electrical manifestations of channel operation in the same preparation. Thus, using gating currents, macroscopic currents, and single-channel recordings in the squid giant axon, a much more constrained full kinetic model of the Na+ channel was developed (Correa and Bezanilla, 1994; Vandenberg and Bezanilla, 1991). Molecular Cloning Opens the Black Box and Expands the Realm of Channel Families Though electrophysiological methods can give us very precise accounts of the relation between voltage and currents, they essentially describe a black box. Models can be built based on these measurements, but they do not contain physical elements because the content of the black box is unknown. Finding the components inside the box amounted to unraveling the structure of the channel protein, which required at least knowing what amino acids make up the protein. Molecular cloning was the tool that nally gave us the amino acid sequence of channels, and the rst ones were the sequences of the subunits forming the Ach receptor (Noda et al., 1983). Only a year later the Na+ channel of the eel was cloned (Noda et al., 1984) and the Shaker K+ channel primary sequence was obtained by more than one group three years after (Kamb et al., 1988; Pongs et al., 1988; Tempel et al., 1987). The primary structures are not only useful in locating the amino acids in the sequence but, using hydropathy plots, they provided information about transmembrane topology. A pattern emerged for Na+, K+, and Ca2+ channels in that there was a basic structure of six putative transmembrane segments, repeated four times in the same polypeptide for Na+ and Ca2+ channels. Biochemical and biophysical experiments conrmed that the basic six transmembrane segments plus a reentrant loop were common in all these voltage-gated channels. In contrast, the subunits in the ACh channel had only four transmembrane segments. The stoichiometry of ve subunits for the ACh channel had been determined before (see Karlin, 1991), but to nd the stoichiometry of four subunits for the Shaker K+ channel required several biophysical tricks (Christie et al., 1990; Isacoff et al., 1990; MacKinnon, 1991). For K+ channels, four subunits were more or less expected because it would make the nal assembled K+ channel very similar to the Na+ and Ca2+ channels with a total of 24 transmembrane segments. The cloning of the Ach receptor from Torpedo, the Na+ channel from the eel, and a Shaker K+ channel from Drosophila produced an explosion of new sequences from other similar proteins using homology cloning. In this way it has been possible to classify channels into families and superfamilies, each containing a large number of members. New Families of Channels with a Large Variety of Functions Functional analysis, homology cloning and the sequencing of genomes of several species have revealed a large number of channel types that have been classied into families and larger superfamilies. The number of channels known today is overwhelming. For example, The Transport Classication Database (TCDB) from the University of California in San Diego has a classication that divides all ion channels in seven categories based on whether the channels are mainly a-helical, beta barrels, pore forming toxins, etc. Within the a-based category, there are 69 subcategories called families or superfamilies. Examples within these 69 categories are the voltage-gated channels superfamily (which contains 26 members with some of its members with as many as 11 different types; see for example Hille, 2001), the inward rectier family, the epithelial Na+ channels family, the ATP-gated channel family, the ryanodine receptor channels, the cysteine-loop ligand gated channels, etc. (see http://www. tcdb.org). Such a wealth of information makes it impossible for me to go into any detail on the different types of channels, but at least two examples of the new superfamilies of channels should be mentioned briey, given that they have had a large impact. The transient receptor potential (TRP) family is made of six transmembrane segments and a reentrant loop, similar to the voltage-gated channels, although its members differ signicantly in their function from the classical voltage-gated K+ channels or cyclic-nucleotide gated channels (Nilius et al., 2007). This group originated from a defect in phototransduction in Drosophila that was traced to a channel that instead of giving a sustained response, gave a more transient response. Within the superfamily there are four families, such as the Vanilloid family that contains the TRPV1 channel. TRPV1 opens in response to high temperatures or to a chemical stimulus such as capsaicin, the main ingredient in chili peppers, with a larger Ca2+ than Na+ selectivity. Another family of the TRP superfamily, TRPM, has (as an example) TRPM8 that opens with cold temperatures or with chemicals such as menthol and is selective to both Na+ and Ca2+. The discovery and characterization of TRP channels have pushed forward somatosensory and pain research. A different family is the glutamate-gated ion channel group of neurotransmitter receptors that includes NMDA, AMPA, and kainite receptors (see Mayer and Armstrong, 2004). They are of great importance in neuroscience because L-Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system (that also acts on metabotropic receptors). Their pore region is made of two (in prokaryote) or three (in eukaryote) transmembrane segments and a reentrant loop that is

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reminiscent of an inverted K+ channel. These receptors are involved in synaptic plasticity such as long-term potentiation (LTP) and long-term depression (LTD). They are also possible targets for therapies for disorders of the central nervous system such as Alzheimers diseases or epilepsy. Heterologous Expression Molecular cloning expanded our view of the type and variety of channels even further than patch clamp, and the new task that emerged was the identication of these cloned channels with the recorded counterparts in the native cells. This required the recording of the currents from these new cloned channels in an appropriate expression system. The development of the Xenopus laevis oocytes as a heterologous expression system has been crucial in describing the function of the cloned channels and, as we will see below, in understanding the relation between structure and function (Barnard et al., 1982; Sumikawa et al., 1981). The Xenopus laevis oocyte normally has an intrinsically low expression of channels in its surface; therefore, when it is injected with the mRNA that codes for the channel protein in study, it can synthesize the protein and properly trafc it to the surface membrane where it can be detected by electrophysiological methods such as two microelectrode clamp, patch clamp (Hamill et al., 1981), or cut-open oocyte clamp (Stefani and Bezanilla, 1998). With this expression method many newly cloned channels, initially identied only by sequence, could be correlated with channels recorded from cells by measuring their selectivity, gating kinetics, voltage dependence, and pharmacology. Other expression systems have since been very successfully added to our repertoire, including transfected mammalian cells or insect cells where ion channel currents can be studied with patch-clamp techniques. The Search for Structural Motifs that Underlie Function The long sequence of amino acids that make up the channel protein can present a daunting obstacle for unraveling the specic role of each amino acid in the function of the protein. Sitedirected mutagenesis is a direct approach whereby a single amino acid or a group of amino acids are changed at the DNA level, and the resulting mutant can then be injected as mRNA into the oocyte to test the effect of the mutation on the function. Unless one is lucky, this method would take an impossibly long time to be productive because many amino acids have only a structural role and do not participate in the active sites such as the conduction pore or the binding site of the transmitter. Therefore an important guide in site-directed mutagenesis was the search for conserved or slightly modied sequence patterns using sequence homology among members of a superfamily. An excellent example of a very well-conserved pattern is the string of basic residues (arginine or lysine), each separated from the next by two hydrophobic residues in the fourth transmembrane segment (the S4 segment) of Na+, K+ and Ca2+ channels. In the paper where the rst sequence of a voltage-gated channel appeared, Noda et al. (1984) pointed out that the S4 pattern is a likely candidate to be the voltage sensor because it has charged residues that may move within the membrane electric eld. Studies were immediately initiated to modify the S4 pattern by site-directed mutations to see if the voltage dependence was affected (Papazian et al., 1991; Stuhmer et al., 1989). The result was that neutralization of basic residues did change the voltage dependence of the conductance, as expected, but it was also found that mutations of noncharged residues had similar effects on the voltage dependence of the conductance. The resolution of this problem required a different approach: if the charge in question is part of the voltage sensor, the total gating charge per channel should be decreased upon its neutralization. The rst important step was done by Schoppa et al. (1992) when they measured the total gating charge of Shaker K+ channel by integrating the gating currents in the same patch where they determined the number of channels by noise analysis. The total charge was large: 13 e0. Notice that this number is actually the product of the charge and the fraction of the eld it traverses; therefore, it cannot be directly related to the number of physical charges moving unless it is known how far into the eld they move. Using the limiting slope method (Almers, 1978; Sigg and Bezanilla, 1997) a similar number was obtained for the muscle Na+ channel (Hirschberg et al., 1995) and Ca2+ channel (Noceti et al., 1996). The second step, done by two groups (Aggarwal and MacKinnon, 1996; Seoh et al., 1996), was to measure how much the total charge per channel was affected when each of the charged amino acids was removed. It was found that only the rst four most extracellular charges of S4 and one acidic residue near the intracellular end of S2 were part of the gating currents. These results gave the long-sought correspondence between amino acids and gating currents but still did not tell us how much and how those residues moved when the voltage was changed. Another example of a conserved pattern was the signature sequence of the K+ channel GYGD, which was traced to the reentrant loop between S5 and S6 (Heginbotham et al., 1992, 1994) and was found to be the basis of conductance and selectivity. The reentrant loop was studied in great detail using mutagenesis, scorpion toxins, and TEA blocking, giving a rough picture of the pore region of the channel. However, un covering how ions may interact with this part of the protein had to wait for the unveiling of the atomic structure (see below). Similar studies were also done in Na+ and Ca2+ channels (Noda et al., 1989; Yang et al., 1993) and in CNG channels (Goulding et al., 1993). Kinetic Models and the First Correlations to Structure Heterologous expression of large numbers of channels allowed the recording of single channels, macroscopic ionic currents, and gating currents from a well-dened cloned channel such as Shaker in a common preparation. This was ideally suited to the development of kinetic models that reproduced the data quite accurately and that could be further constrained using the stoichiometry of the channel. As it was shown with the squid axon (see above), the three simultaneous electrophysiological measurements signicantly improve the accuracy of kinetic models. Thus, several groups were able to propose improved kinetic models of Shaker K+ channels (Bezanilla et al., 1994; Schoppa and Sigworth, 1998; Zagotta et al., 1994). The excellent expression of cloned channels permitted experiments not possible before and allowed researcher to ask questions such as how the sensor moves at the single-molecule level. Two possible mechanisms were proposed: ion charge moves in a diffusional regime or charge moves by making sudden jumps. The difference between these two views has important molecular implications. If it occurs in jumps, the detection of such shots

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of charge is below the resolution of present techniques, but the analysis of the noise of the gating currents can provide the answer. Conti and Stuhmer were able to record the uctuations in the gating currents of a large number of Na+ channels and found that they were consistent with shots that carry a charge of 2.4 e0 (Conti and Stuhmer, 1989), while Sigg et al. found the same value for the Shaker K+ channel (Sigg et al., 1994). It was later found that the gating current is preceded by a very fast transient that carries little charge but does not show uctuations (Sigg et al., 2003), implying that the charge movement occurs in many steps and that those steps are of differing natures depending on the landscape of energy encountered by the gating charge as it progresses from a deep closed state to the active state. These results have important implications in the dynamics of the structural changes occurring during voltage sensing. While advances continue to be made in assessing channel structure (see below), the structural counterparts of these physiological ndings have yet to be demonstrated. One of the rst structural breakthroughs followed from the nding that different Shaker K+ channel subtypes show different degrees of inactivation. Such results suggested the classical experiments that subsequently demonstrated the molecular basis of the ball and chain hypothesis of inactivation. After deleting residues 646 of the N-terminal part of the protein, it was found that fast inactivation was removed. Remarkably, in this truncated channel, inactivation was restored after injection of the ball peptide made with the deleted residues (Hoshi et al., 1990; Zagotta et al., 1990). Knowing that the residues 646 were responsible for inactivation, it was nally possible to demonstrate the molecular basis of gating charge immobilization by the inactivating particle by comparing the gating currents in oocytes expressing the full Shaker B channel or the truncated version. The kinetics of the gating current tails was drastically slowed down by the inactivating particle, as well as by internal TEA, providing support for the origin of the charge immobilization seen in the squid axon Na+ channel (Bezanilla et al., 1991). Chemistry and Spectroscopy Give Hints on the Structure and the Function Changing one amino acid at a time in the cloned channel allowed the replacement of a given residue with the easily reacting amino acid cysteine. This is a particularly useful residue because it is generally not very abundant and it reacts quite specically with several thiol-modifying agents. The technique of scanning residues with cysteine and testing its accessibility with thiol reactive agent (Akabas et al., 1992) has played a fundamental role in assessing whether a region of the protein is exposed or buried and even deciding whether exposure changes during the normal operation of a channel. The pattern of accessibility in the M2 segment of the Ach receptor a subunit was consistent with a long a-helical structure as a pore lining (Akabas et al., 1994). In the case of the Shaker K+ channel, the classical result proposed by Armstrong, that TEA derivatives can only access the channel when the gate was open, was conrmed at the molecular level when Yellens group found activation-dependent accessibility changes using cysteine scanning along the S6 segment (Choi et al., 1993). The actual location of the gate along the S6 segment was found as the point in the scanning that switched from a large difference to a small difference in accessibility when the channel closed relative to when it was open (Liu et al., 1997). The voltage sensors of the Na+ and Shaker K+ channels were also subjected to cysteine scanning and the accessibility was found to depend on the membrane potential, giving direct evidence of the movement of the S4 segment in response to changes in the electric eld (Larsson et al., 1996; Yang and Horn (1995)). The same charges could be found to be accessible on the inside upon hyperpolarization and on the outside upon depolarization. Another scanning perturbation method was histidine scanning. Our idea in this case was not a chemical modication but a protonation change depending on the pH where the histidine was exposed. This technique was born as a way to replace at will the charge of the basic residues of the S4 sensor. The results showed that the second and third charges of the S4 segment in Shaker were exposed alternatively to the inside or the outside, depending on whether the potential was negative or positive, thus making a voltage-dependent proton transporter (Starace et al., 1997). This result was in agreement with the cysteine scanning results, but the replacement of the rst and fourth charges by histidine gave a new clue about the extent of channel movement because proton pores were found in the closed and open states, respectively (Starace and Bezanilla, 2001, 2004). The simplest interpretation was that there was a very short separation between the internal and external solutions, possibly created by deep water crevices penetrating the protein. This was also an indication that the electric eld traversed by the gating charges was concentrated and that the S4 did not have to make a large transmembrane movement to carry the rather large amount of 13 e0. When the group of Isacoff (Tombola et al., 2005) replaced the rst charge with a cysteine, they found that in the closed state, there was a cation current through the voltage sensor (the omega current). Therefore, there was nothing special about histidine; instead, these results pointed to a property of the structure of the sensor that in the closed state put in very close proximity the internal with the external solution in the region where the rst charge is located. Inserted Probes Can Track Local Structural Changes As cysteine can be conjugated to many different molecules, one could insert molecules such as electron paramagnetic resonance (EPR) or uorescent probes in specic sites in the protein. A uorophore can sense the environment in which it is located by the hydrophobicity of the surrounding environment or by being quenched by a local group. This technique therefore allowed the measurement of local changes during the function of the protein. Cysteine-reactive rhodamine was inserted in engineered cysteines in or near the S4 segment of the Shaker K+ channel, and the time course of its uorescence was studied under voltage clamp with simultaneous recording of the ionic or gating current (Mannuzzu et al., 1996; Cha and Bezanilla, 1997, 1998) The changes in uorescence intensity by differential quenching gave the rst direct evidence of local movement of a channels sensor as a result of changing the membrane potential. In the case of the Na+ channel, where each domain can be labeled separately, gave direct evidence that the different domains of the Na+ channel have different functions in the operation of the channel. Thus, domains III and IV were involved in inactivation while domains I and II were not (Cha et al., 1999a), and domains I, II, and III generated the fast component of the gating currents that correlate

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with the opening of the channel, while domain IV generated the slow component of the gating currents that was a prelude to the inactivation process (Chanda and Bezanilla, 2002). Introducing two uorophores in the protein, via cysteine attachments, further allowed the measurement of intramolecular distances with uorescence resonance energy transfer (FRET). Both regular FRET and lanthanide-based transfer (LRET) were applied to the voltage sensor of the K+ channel, and it was possible to demonstrate for the rst time that there were changes in molecular distances that correlated with the voltage dependence of the charge movement (Cha et al., 1999b; Glauner et al., 1999). These studies have further expanded with improved techniques and preparations (Chanda et al., 2005; Posson et al., 2005; Posson and Selvin, 2008; Richardson et al., 2006; Sandtner et al., 2007). The ndings with these techniques indicate that during voltage sensing the S4 segment moves a limited amount ) across the membrane plane. FRET techniques are of (about 7 A course not limited to intramolecular distances and have now also been used extensively to measure proximity between molecules and even to assess membrane potential. Structural Techniques: X-Ray, Spectroscopy, and Computational Chemistry The evidence accumulated on the structure of ion channels until 1998 was all based on indirect methods that, at best, gave a rough picture as to where important groups were located inside the protein. What was really needed to make further progress was the 3D structure of the channel protein. It was the remarkable achievement of MacKinnon and collaborators in 1998 that gave us the rst crystal structure of a K+ channel (Doyle et al., 1998). This is again another turning point in the research of ion channels that initiated a new era in the study of structurefunction relationship. By using a bacterial K+ channel homolog, KcsA, that only had two transmembrane segments and a pore loop, the MacKinnon group presented the location of almost every atom in the ion channel. It was nally possible to see where the critical groups discovered in the previous decade were located in 3D space. The structure has a beauty of its own, with the four predicted subunits (MacKinnon, 1991) intertwined via the inner transmembrane segment (the S6 in voltage-gated channels) making a closed gate in the predicted intracellular side (Armstrong, 1971). A wide aqueous cavity resides almost at the center of the membrane, a feature never seen in any previous protein structure. The pore loop forms the selectivity lter with the GYGD motif, with their carbonyl groups lining the pore and pore helices pointing their dipoles into a central lake enclosed by the TM2 segments. The series of predictions that have been made from biophysical experiments for several decades suddenly became real when one could actually see that, in the selectivity lter, there were several K+ ions in a row (Armstrong, 1971; Hodgkin and Keynes, 1955) coordinated by the carbonyl groups (Eisenman and Dani, 1987). The Physical Basis of Permeation and Selectivity The answer to how the channel easily conducts K+ appeared to be very much in line with what Mullins had envisioned so many years ago. In the crystal structure carbonyls replaced the hydration water of the K+ ion (Bezanilla and Armstrong, 1972; Mullins, 1959)of course, Na+ would not t as well in the cage because it is smaller and it would not permeate as well as K+. When we were discussing this issue with Benoit Roux, he pointed out to me a paradox in this reasoning. The problem, he said, is that the size of the cage is expected to easily change due to thermal vibration of the backbone chain, and in that case one would expect that it would have no trouble accommodating Na+ as well as K+, which would negate selectivity! When Roux went back to carry out molecular dynamic free energy computations, he discovered that the repulsion between the carbonyls that are coordinating the ion provides a hidden energy term that makes it unfavorable to coordinate a smaller ion like Na+, because they then have to get closer to each other (Noskov et al., 2004). Therefore, besides the steric factor, there were local dynamic and electrostatic effects that were important. Also, the concept of eld strength predicted many years earlier than the publication of the crystal structure was playing a role (Eisenman, 1962). The contribution of computational chemistry illustrated above is just one example of what it can do after a structure is available. Once the coordinates of the atoms are known, the tools of molecular dynamics can be used to relate structure to function at the atomic level. The availability of a channel in puried form opened many new possibilities in experimentation besides crystallization. Thus, the group of Eduardo Perozo was able to reconstitute a functional KcsA in the lipid bilayer and scan its sequence with an EPR probe attached to engineered cysteines. EPR methods also allowed one to determine whether a region was exposed to lipid or aqueous solutions, to determine whether amino acid groups were free or restrained in their movements, and even to assess whether/ how two groups were moving relative to each other. By lowering the pH, Perozo showed that he could open the channel and detect the changes in the position of TM2 that opened the bundle crossing (Perozo et al., 1999). Thus, the combination of crystal structure and EPR spectroscopy illuminated how and where a channel changed from the closed to the open states, with answers in agreement with Armstrong and Yellens previous work using more indirect methods. Molecular Basis of Slow Inactivation The ultimate situation would be to combine crystal structure with spectroscopy, electrophysiology, and molecular dynamics simulations, allowing the identication of the states of the molecule (normally thought of as kinetic states) with the physical states of the structure. We are not quite there yet, but the groups of Perozo and Roux have gotten close. The electrical activity of the KcsA channel when recorded in bilayers or liposomes has a very low maximum open probability, of about 0.1. CorderoMorales et al. found that this low open probability is due to an inactivation process that takes a few seconds to develop (Cordero-Morales et al., 2006). When the channel opening was tested by estimating the separation of the bundle crossing using EPR (Cordero-Morales et al., 2006) or uorescence spectroscopy (Blunck et al., 2006), it was found that the gate is actually open at the time when there is no ion conduction, as tested with electrophysiology. This result pointed to the existence of another gate in series that would be responsible for the inactivation. Cordero-Morales et al. traced this inactivation to an amino acid that is behind the selectivity lter that can be mutated to accelerate or abolish inactivation (Cordero-Morales et al., 2006).

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What is really interesting is that they found that the crystal structure of the mutant shows a change in the ne structure of the selectivity lter. Other Structures and the Voltage-Gated Channel Crystal structures of other ion channels have continued to be published following the landmark publication by MacKinnon and colleagues of the rst KcsA crystal. Such ndings have expanded our knowledge of the structural details of a Ca-activated channel in the open conformation (Jiang et al., 2002) and showed the inactivation particle inside the channel, giving a structural view of inactivation (Zhou et al., 2001). In addition structures of other inward rectiers were obtained (Kuo et al., 2003). Electron microscopy showed us the Ach receptor structure down to 4 A resolution (Unwin, 2005) where all the critical parts of the channel could be resolved, giving us a rst glimpse of the possible correlation between the structure and function. The details of the binding pocket have been provided by the crystal structure of the ACh binding protein (Brejc et al., 2001), and a recent crystal structure of a bacterial homolog, ELIC (Hilf and Dutzler, 2008), has provided a higher-resolution map of the whole receptor including details of the pore and possible gate regions that are now waiting to be correlated with the function. There were no structures of voltage-gated channels until the MacKinnon group published a crystal structure of KvaP, an archea voltage-gated potassium channel (Jiang et al., 2003a). Although this was a long-awaited structure, it produced quite a controversy among researchers because the structure t only a few of the predictions that had come out of biophysical methods used over the prior two decades. Although the pore region (S5, S6, and pore loop) was exactly as expected and similar to KcsA except that it was in the open state, the voltage sensing region (S1 through S4) had the S3 and S4 segments in the intracellular solution, and the S2 segment was not crossing the membrane. In an accompanying paper, Jiang et al. modied the structure and created the paddle model that accounted for their bilayer results of KvaP tagged with biotin that interacted with avidin in the baths (Jiang et al., 2003b). In that model segments S3 and S4 move together across the bilayer in a total distance of , and the arginine side chains are directly exabout 20 to 25 A posed to the hydrocarbon region of the lipid bilayer. Yet Cuello et al. did an EPR study of KvaP in the lipid bilayer (Cuello et al., 2004) and found that the inferred structure was not compatible with the crystal structure of Jiang et al. (2003a). This publication was only one of a number of papers that addressed, with a variety of methods, the plausibility of the paddle model. Most of them found that such a large movement, as well as locating the arginines in the bilayer, were not supported (Cohen et al., 2003; Ahern and Horn, 2004; Bezanilla, 2005). These discrepancies were resolved to a large extent by a new structure. In 2005, the MacKinnon group presented the crystal structure of Kv1.2, the mammalian homolog of Shaker (Long et al., 2005), that had the voltage sensor in a conformation that was in agreement with most of the biophysical data (Laine et al., 2003). This study suggested that the previously published KvaP structure was not in its native conformation. In the Kv1.2 structure there were arginines pointing toward a region wherein, when the channel was embedded in the bilayer, the side chains would be in the hydrophobic part of the lipids. When that structure was put into a lipid bilayer and water and ions were added, it was found that after 1 nanosecond of molecular dynamics computation all the arginines were in water crevices enclosed by the transmembrane segments of the voltage sensor or in contact with the polar heads, and none were embedded in the nonpolar core of the bilayer (Jogini and Roux, 2007). The previously proposed concentrated electric eld near the sensor (Chanda et al., 2005), based on biophysical experiments, was also clearly conrmed by these molecular dynamics computations based on the Kv1.2 structure. The Operation of the Voltage Sensor The crystal structure of Kv1.2 and of the chimera of Kv1.2 and Kv2.1 (Long et al., 2007) gave the location of the charged groups that compose the voltage sensor, but it should be remembered that such ndings represented only a snapshot of what seems to be the open state. The large number of biophysical experiments done before the structures and the new constraints found using state-dependent cysteines bridges between S4 and S1 and S2 (Campos et al., 2007) and uorescence scanning (Pathak et al., 2007) allowed the proposal of better-constrained models of the closed state. Using these two states, a model of voltage sensing emerges whereby the change in voltage moves the S4 segment in a focused electric eld translating perpendicular to , with a simultaneous rotation the membrane no more than 7 A of as much as 180 and a change of its tilt by about 30 . Recent distance measurements with LRET by Posson and Selvin (2008) and cysteine bridges between S4 and S3 (Broomand and Elinder, 2008) are consistent with this hypothesis. There is one problem, however. The structures of Kv1.2 and the chimera have been obtained in absence of membrane potential. At zero membrane potential the channel is expected to be open but also inactivated by the well-known slow inactivation process. Since the work in squid axon, it has been known that the voltage dependence of the gating charge is changed by prolonged depolarization (Bezanilla et al., 1982) and that the hysteresis of the Q-V curve is common to all S4-based voltage sensors, including a voltage-dependent phosphatase that has no pore (Villalba-Galea et al., 2008). We recently showed that the voltage sensor itself, and not only the pore, undergoes an inactivationlike process that changes its conformation. This new state has been called the relaxed state, and it is likely to be the state represented by the crystal structures because these structures have been obtained under prolonged depolarization. Biophysical experiments suggest that the movement between the closed and open states is mediated by an S4 segment in 310 helical conformation, while the S4 in the crystal structure is mainly an a-helical conformation, and would correspond to the relaxed state (Villalba-Galea et al., 2008). If this is correct, because the lifetime of the open state lasts only tens of milliseconds, it will be difcult to observe the open state in a crystal structure. This leaves the details of the structural basis of voltage sensor operation open to speculation until time-resolved structural experiments become feasible. What Is Next? The progress in the ion channel eld has been a series of jumps, with each jump followed by a steady growth. The brief history just told shows that the jumps have been the result of the

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introduction of new techniques. Today we are witnessing the outcome of a myriad of techniques that have enlarged our knowledge on the variety of ion channels, their distribution, and their cell biology, as well as our knowledge of some specic aspects of the relation between structure and function for some of them. It is clear that the impact of ion channel research on our understanding of the nervous system is only starting. The classication of channel type and their distribution have provided new insights on the workings of nuclei and connections, but there are many areas that have not been explored yet; the brain is too complex and channels are involved in multiple functions from synaptic transmission to cell homeostasis. We have started seeing the results of advanced techniques for detecting how several types of channels interact in systems such as the excitation-contraction coupling machinery, the storeoperated CRAC channels where ion channels are involved in a concerted fashion in different membranes to perform complex functions. We have a few examples of the details of synthesis and maturation of ion channels before they get inserted in the membrane and also some of the coding signals that retain them in the endoplasmic reticulum or direct them to specic regions of the cell. We expect to see many more channels and systems of channels that play roles in other cell functions being studied as the above examples are, where it has been possible to make progress by taking advantage of combining electrophysiology, pharmacological agents, cloning, labeling, and imaging. Crystal structures are appearing more frequently. For example, Jasti et al. (2007) recently reported the crystal structure for an acid-activated channel that belongs to the family of ENaC, a Na+-selective non-voltage-gated channel that is critical in epithelial function. This particular channel is made as a trimer. This structure expands the accepted pattern that puts tetramers for high selectivity (Na+, K+, Ca2+), pentamers for moderate selectivity (Ach receptor), and hexamers and above for no selectivity (gap junctions, MscS) and opens the possibility that other ion channels may be assembled according to this new pattern. In addition, new structures showing the channel in different states are expected to be obtained by nding mutations that stabilize a channel in a particular state that allows crystallization, opening the possibility of direct correlation of structure with kinetic states of the channel. The future in the understanding of structure-function relation looks very bright. There are now multiple structural techniques that can be applied to channels in situ and after reconstitution. It is clear that crystal structures are critical in locating side chains and key atoms in the structure, but because they are snapshots, they must be complemented with more dynamic techniques. In addition, because crystals are obtained in conditions that are not a good emulation of the lipidic membrane environment, it is imperative to have complementary techniques, spectroscopy and computational, that can study the protein embedded in the bilayer and the intracellular and extracellular aqueous environment. Many detailed studies require labeling that must be incorporated as part of the backbone of the protein so that they can follow the movement closer to where the action takes place. Genetic methods to incorporate nonnatural amino acids are expected to play an important part in these studies, as is the chemical synthesis of sections of the protein incorporating nonnatural amino acids that can be joined to other parts of the protein to form a functional channel. I have mentioned the example of the voltage sensor that seems to undergo changes in secondary structure on the scale of tens of millisecond. This will be a perfect candidate to which to apply fast methods, such as optical or infrared, that can detect these structural changes, but the challenge will be to carry out these measurements with simultaneous detection of the channel function. There are also techniques that only recently have been applied to ion channel research such as nuclear magnetic resonance (Etzkorn et al., 2007) and single molecule uorescence (Sonnleitner et al., 2002). In the latter, a channel is labeled in specic sites and the uorescence is followed as the channel is functioning. It should be remembered that when single-channel recording was nally possible after the introduction of the patch clamp, we learned the stochastic behavior and steady-state and kinetic properties of the channels, which was not possible by simply recording macroscopic currents. The single molecule uorescence goes one step further because it allows us to infer the properties of the single molecule at the structural level. One example is the detection of the movement of single subunits when the main gate of KcsA opens and closes using single molecule uorescence. These experiments have shown that the subunits do not move independently (Mc Guire et al., 2008, Biophys. J., abstract). In the future, it is expected that the individual movements of the voltage sensor could be followed by single molecule uorescence techniques and thus record the movement that generates the single shots that were inferred from noise analysis of the gating currents (see above). The correlation of the structure with function has been done with a limited number of channels, mainly because not all the techniques can be applied to all channel types or there are channels that currently have no known homologs that are easily crystallized. Although many basic principles will be applicable to most channels, it will be necessary to study in detail multiple types of channels because the diversity in structure and function forewarns that they may operate with different mechanisms. There are obvious candidates that require a more detailed study such as the Na+ and Ca2+ channels: our understanding of permeation, selectivity, and gating of these important channels is not at the level we have for some voltage-gated K+ channels. Permeation and selectivity of K+ channels could be explained at the atomic level only after the structure was obtained, because before the structure, there was no indication that the carbonyls were pointing to the conduction pathway. The structures of the Na+ and Ca2+ pores are not available, and even the bacterial homolog NaChBac (Ren et al., 2001), which is not a perfect homolog, has not yielded a high-resolution structure. Ion channels are not only crucial in healthy individuals, but several of them have been implicated in disease, either genetic or acute. The possible treatments to channel-associated disease will be accelerated if we understand in detail how channels are implicated in the physiology of the cell, and if we could design modications that restore normal function. The latter will be aided by a description of the molecular basis of channel function. The ingenuity in the use of homology mapping and chimera construction may prove fruitful in our progress to expand

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our knowledge of structure-function relation to many other ion channels. The real understanding of an ion channel at the molecular level will only happen when the kinetics and steady state of the channel function can be predicted from the dynamics of the structure. We are far from that objective, but there is no question that to achieve that goal, we will require a synergy between structural, functional, and spectroscopic techniques that will be merged with computational analysis.
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