voltage-gated ion channels - electrophysiology and the molecular

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

Voltage-Gated Ion ChannelsFrancisco Bezanilla

Abstract—Voltage-dependent ion channels are membrane pro-teins that conduct ions at high rates regulated by the voltage acrossthe membrane. They play a fundamental role in the generationand propagation of the nerve impulse and in cell homeostasis. Thevoltage sensor is a region of the protein bearing charged aminoacids that relocate upon changes in the membrane electric field.The movement of the sensor initiates a conformational change inthe gate of the conducting pathway thus controlling the flow of ions.Major advances in molecular biology, spectroscopy, and structuraltechniques are delineating the main features and possible struc-tural changes that account for the function of voltage-dependentchannels.

Index Terms—Gating currents, ionic currents, S4 segment,voltage sensor.

I. INTRODUCTION

THE BIT OF information in nerves is the action potential, afast electrical transient in the transmembrane voltage that

propagates along the nerve fiber. In the resting state, the mem-brane potential of the nerve fiber is about 60 mV (negativeinside with respect to the extracellular solution). When the ac-tion potential is initiated the membrane potential becomes lessnegative and even reverses sign (overshoot) within 1 ms and thengoes back to the resting value in about 2 ms, frequently after be-coming even more negative than the resting potential. In a land-mark series of papers, Hodgkin and Huxley studied the ionicevents underlying the action potential and were able to describethe conductances and currents quantitatively with their classicalequations [1]. The generation of the rising phase of the actionpotential was explained by a conductance to Na ions that in-creases as the membrane potential is made more positive. Thisis because, as the driving force for the permeating ions (Na) wasin the inward direction, more Na ions come into the nerve andmakes the membrane more positive initiating a positive feed-back that depolarizes the membrane even more. This positivefeedback gets interrupted by the delayed opening of anothervoltage dependent conductance that is K-selective. The drivingforce for K ions is in the opposite direction of Na ions; thus,K outward flow repolarizes the membrane to its initial value.The identification and characterization of the voltage-dependentNa and K conductances was one of the major contributionsof Hodgkin and Huxley. In their final paper of the series, theyeven proposed that the conductance was the result of increasedpermeability in discrete areas under the control of charges or

Manuscript received November 4, 2004; revised November 15, 2004. Thiswork was supported in part by the U.S. Public Health Service National Institutesof Health under Grant GM30376.

The author is with the Departments of Physiology and Anesthesiology, D.Geffen School of Medicine and the Biomedical Engineering InterdepartmentalProgram, University of California, Los Angeles, CA 90095 USA (e-mail:[email protected]).

Digital Object Identifier 10.1109/TNB.2004.842463

Fig. 1. General architecture of voltage-gated channels. Top part shows thebasic subunit (or domain in the case of Na and Ca channels). The graybackground represents the lipid bilayers. The cylinders are transmembranesegments. The region between S5 and S6 forms the pore. Segments 1 through4 are called the voltage sensor part of the channel. The + or � signs in whiteindicate charges that have been implicated in voltage sensing. In the bottompart, a schematic view of the channel from the outside showing the assembledfour subunits or domains.

dipoles that respond to the membrane electric field. This was aninsightful prediction of ion channels and gating currents.

Many years of electrical characterization, effects of toxinson the conductances, molecular biological techniques, andimprovement of recording techniques led to the identificationof separate conducting entities for Na and K conductances.These conductances were finally traced to single-membraneproteins, called ion channels, that can gate open and closed anion conducting pathway in response to changes in membranepotential (see introductory paper by P. Jordan in this issue).

II. VOLTAGE-DEPENDENT ION CHANNELS ARE

MEMBRANE PROTEINS

The first voltage-dependent ion channel that was isolated andpurified was extracted from the eel electroplax where there is alarge concentration of Na channels [2]. Several years later, thesequence of the eel Na channel was deduced from its mRNA[3]. The first K channel sequence was deduced from the Shakermutant of Drosophila melanogaster [4]. These initial sequenceswere the basis to subsequent cloning of a large number of Na ,

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BEZANILLA: VOLTAGE-GATED ION CHANNELS 35

Fig. 2. Comparison between a field-effect transistor (FET) and a voltage-gated ion channel. The FET transistor is represented as a p-channel device to make acloser analogy to a cation selective voltage-gated channel. (a) and (c) are the closed states; (b) and (d) are the open states. Notice that, in contrast with the FET, thegate in the voltage-gated channel indicates the actual point of flow interruption. BLM is the lipid bilayer. In the FET, D is the drain, S is the source, and G is thegate. For details, see text.

K and Ca channels in many different species. Hydropathyplots were helpful in deciding which parts of the sequence wastransmembrane or intra or extracellular. A basic pattern emergedfrom all these sequences: the functional channels are made upof four subunits (K channels) or one protein with four homol-ogous domains (Na and Ca channels). Each one of the do-mains or subunits has six transmembrane segments and a poreloop. (see Fig. 1). The fifth and sixth transmembrane segments(S5 and S6) and the pore loop were found to be responsible forion conduction. The fourth transmembrane segment (S4) con-tains several basic residues, arginines or lysines and was ini-tially postulated to be the voltage sensor [3] In addition S2 andS3 contain acidic residues such as aspartate and glutamate. Mostof the channels have additional subunits that modify the basicfunction but they are not necessary for voltage sensing and ionconduction.

III. THE PARTS OF THE VOLTAGE DEPENDENT CHANNEL

We think of voltage-dependent channels as made of threebasic parts: the voltage sensor, the pore or conducting pathwayand the gate. These three parts can be roughly mapped in theputative secondary structure (Fig. 1). The pore and the gate arein the S5-loop-S6 region and the voltage sensor in the S1–S4region. As the conduction is dependent on the voltage acrossthe membrane, a useful analogy is a field effect transistor (see

Fig. 1). If we take a typical V-dependent K channel, its voltagesensor corresponds to the gate of a p-channel FET transistor,the conducting pathway of the ion channel corresponds to thep-channel (Fig. 2) and the gate of the channel is the space chargein the p-channel. As we will see below, this analogy is useful todiscuss the parts of the channel but it cannot be pushed very farbecause, although the functions are similar, the actual mecha-nisms are quite different.

A. The Conducting Pathway

Living cells, and in particular nerve fibers, are surrounded bya thin membrane made of a bimolecular layer of lipids. The per-meability of ions through the lipid bilayer is extremely low be-cause it takes a large amount of energy to put a charged ionspecies inside the low dielectric constant lipid bilayer [5]. Theconducting pathway of ion channels lowers that energy barrierby providing a favorable local environment, thus allowing largeflows under an appropriate driving force. Details of the ion con-duction pore structure, conductance, and selectivity are coveredin other papers in this issue [6]. What is important to emphasizehere is that the ion flow is proportional to the driving force forthe selected ion. The driving force corresponds to the differencebetween the voltage applied and the voltage at which there isno flow, or reversal potential . If the channel is perfectly selec-tive to one type of ion, say, K , then is the Nernst potential;


Fig. 3. Time course of single-channel and macroscopic ionic currents. The applied voltage is in the top trace and the current recorded through one channel isshown for four different trials. The mean current is the result of thousands of trials. (a) Small depolarization to�30 mV opens the channel infrequently. (b) A largedepolarization (to +30 mV) opens the channel most of the time. c is the closed state and o is the open state.

otherwise, is predicted by the Goldman–Hodgkin and Katzequation that considers concentrations and relative permeabili-ties. Knowing the conductance of the conducting pathway ,wecan compute the current flow through the open conducting poreas

(1)

The – curve of an open channel may be nonlinear becausein general is voltage dependent.

B. The Gate

The ion conduction through the pore may be interrupted byclosing a gate (see Fig. 2). Thermal fluctuations will close andopen the gate randomly and the current will have interruptions.In voltage-dependent channels, the probability that the gate isopen depends on the membrane potential. In the majorityof -dependent Na , K , and Ca channels from nerve andmuscle, the increases with membrane depolarization (i.e.,decrease in the resting potential). There are a few cases, such asKat1 channel, where increases on hyperpolarization.

The operation of the gate can be seen by recording the currentflowing through a single ion channel. This is possible with thepatch clamp technique [7], which records currents from a verysmall patch of membrane with a small glass pipette and a low-noise system that can resolve currents in the order of 1 pA.

An example of the operation of one K channel is shownin a simulation in Fig. 3. As the internal concentration of Kis more than 10 times higher in the cell as compared to theextracellular space, the reversal potential for K channels isaround 80 mV. Starting with a negative membrane potential

100 mV , the channel is closed most of the time. A depolar-izing voltage pulse to 30 mV increases the open probabilityand the channel spends some time in the open state [Fig. 3(a)].As we are dealing with one molecule, thermal fluctuations willgenerate different responses for each repetition of the samepulse (four of such are shown in the figure). A larger depolar-ization 30 mV increases the even more by increasing

the open times and decreasing the closed times, as seen inFig. 3(b). Notice also that the time elapsed between the start ofthe pulse and the first opening (first latency) is decreased forthe larger depolarization Apart from increasing the open times,the magnitude of the current through the pore was increasedby the larger depolarization. This is because the appliedis now further away from , increasing the driving force forion movement. Thus, this increase in current is not a result ofincreasing but is just a passive property of the open pore. Anaverage of several thousands of repetitions gives us the macro-scopic ionic currents. Provided the channels do not interact,the average of thousands of repetitions is the same as havingthousands of channels operating simultaneously. The bottomtrace (labeled mean) in Fig. 3(a) and (b) shows the macroscopiccurrents for and 30 mV, respectively. Notice that theturn-on kinetics is faster for a more positive potential and thatthe current magnitude is also increased. The kinetics change isthe result of an increased while the magnitude increase isthe result of both increased and driving force. We can nowwrite the expression for the macroscopic current as

(2)

where is the channel density and is the voltage- andtime-dependent open probability.

C. The Voltage Sensor

How does become voltage dependent? It is clear that to de-tect changes in membrane potential, a voltage sensor is needed.The electric field in the bilayer could be detected by electricor magnetic charges or dipoles that change their position ac-cording to changes in the field. As there is no evidence of mag-netic charges, electric charges or dipoles remain as the primecandidates. We will see below that the actual charges involvedin voltage sensing have been identified and a schematic repre-sentation of their relocation is shown in Fig. 2(b). In the resting(hyperpolarized) condition, the membrane is negative inside andthe positive charges are located in contact with the interior of


the cell. Upon depolarization, the positive charges are driventoward the outside. This movement in the electric field has twoconsequences: it is coupled to the gate resulting in pore opening[Fig. 2(b)] and the charge translocation produces another mem-brane current that is transient in nature, called gating current. Itis called gating current because it ultimately gates the channelopen and closed, and it is transient because the charge locationsare bound to limiting positions as they are tethered to the protein.

IV. GATING CHARGE AND THE VOLTAGE SENSOR

An understanding of the voltage sensor requires a character-ization of the gating charge movement and a correlation of thatmovement to structural changes in the protein. In this section,we will address two functional questions. The first question iswhat are the kinetics and steady state properties of the gatingcharge movement and how does this charge movement relate tochannel activation. The second is how many elementary chargesmove in one channel to fully activate the conductance and howdoes this movement occur in one channel.

A. The Gating Currents and the Channel Open Probability

The movement of charge or dipole reorientation is thebasic mechanism of the voltage sensor and was predicted byHodgkin and Huxley [1]. Gating currents are transient and theyonly occur in the potential range where the sensor respondsto the electric field; therefore, they behave like a nonlinearcapacitance. In addition, as gating currents are small, to recordthem it is necessary to decrease or eliminate the ionic currentsthrough the pore and eliminate the normal capacitive currentrequired to charge or discharge the membrane. This is normallyaccomplished by applying a pulse in the voltage range thatactivates the current and then subtracting the current elicitedby another pulse or pulses in the voltage range that does notactivate the currents to eliminate the linear components [8], [9].Using these subtraction techniques, the kinetics of Na gatingcurrents were studied in detail in squid giant axon and otherpreparations where a high channel density was found. Thecombination of gating currents, macroscopic ionic currents andsingle-channel recordings were used to propose detailed kineticmodels of channel operation (see [10]). When voltage-de-pendent channels were cloned and expressed in oocytes orcell lines, it was possible to achieve large channel densitieson the surface membrane and study those channels in virtualabsence of currents from other channels. In comparison to thecurrents in natural tissues such as the squid giant axon, theexpression systems gating currents were much larger and madethe recording easier and cleaner. In addition, the study of thepore region gave us the possibility of mutating the channelprotein to eliminate ionic conduction but maintaining the op-eration of the gating currents [11]. We can illustrate the basicfeatures of gating currents and their relation to ionic currentsin recordings from Shaker K channels with fast inactivationremoved (Shaker-IR) as shown in Fig. 4(a).

In this figure, two separate experiments are shown. The toptraces are the time course of the ionic currents recorded duringpulses that range from 120 to 0 mV, starting and returning to

90 mV. The bottom traces are gating currents recorded for the

Fig. 4. Ionic and gating currents in Shaker-IR K channel. (a) Top traces:time course of ionic currents for pulses to the indicated potentials starting andreturning to �90 mV. Bottom traces: time course of the gating currents for thepulses indicated. Notice the difference in the amplitude calibration for ionic ascompared to gating currents. (b) The voltage dependence of the open probabilityP (V ) and the charge moved per channel Q(V ). For details, see text.

same set of pulses from Shaker-IR with a mutation that changesa tryptophan into a phenylalanine in position 434 (W434F) thatrenders the pore nonconducting [11]. Several features that arecharacteristic of most voltage-dependent channels can be ob-served. First, the ionic currents do not show significant activa-tion for potential more negative than 40 mV while the gatingcurrents are visible for all the pulses applied, implying that thereis charge displacement in a region of potentials where most ofthe channels are closed. Second, the time course of activationof the ionic current is similar to the time course of decay ofthe gating current. Third, the time course of the return of thecharge (gating current “tail”) changes its kinetics drasticallywhen returning from a pulse more positive than 40 mV, whichis precisely the potential at which ionic currents become clearlyvisible. The gating tails are superimposable for potentials morepositive than 20 mV, showing that most of the charge hasmoved at 20 mV. The total charge moved at each potentialmay be computed as the time integral of the gating current foreach pulse. As we will see below, it is possible to estimate thetotal charge moved per channel molecule; therefore, the voltagedependence of the charge moved can be plotted as shown in


Fig. 4(b). Knowing the number of channels present (see below),using (2) it is possible to estimate the voltage dependence of

, as shown in the same figure. Fig. 4(b) establishes the rela-tionship between the charge movement and channel opening andclearly shows that the opening of the channel is not superimpos-able with charge movement as expected from a simple two-statemodel. A striking feature of these plots is that the relationis displaced to the left of the curve so that there is quitea large charge movement in a region where the is essentiallyzero. This is an expected feature of a channel that requires sev-eral processes to occur to go from closed to open, such as theclassical Hodgkin and Huxley model where four independentparticles are needed to be simultaneously in the active positionfor the channel to be open.

Current recordings of the type shown in Fig. 4(a) can be usedto formulate kinetic models of channel gating. These models arenormally written as a collection of closed and open states in-terconnected with rate constants that, in general, are a functionof the membrane potential. Initially, kinetic models were devel-oped from the macroscopic ionic currents only [1]. The additionof single-channel recordings and gating currents recordings im-poses several constraints in the possible models and in the pa-rameters fitted, thus obtaining a more robust representation ofthe kinetic parameters that characterize the channel.

The common assumption in kinetic models is that the move-ment of the charge or dipole in the field has a finite numberof low-energy positions and energy barriers in between them.According to kinetic theory, the transition rates across the en-ergy barrier are exponentially related to the negative of the freeenergy amplitude of the barrier. This free energy contains non-electrical terms, and an electrical term such that the membranevoltage can either increase or decrease the total energy bar-rier, resulting in changes in the forward and backward rates ofcrossing the barriers. There are multiple examples in the lit-erature with varied levels of complexity that describe well thekinetic and steady state properties of several types of voltage-de-pendent channels (see, for example, [10], [12]–[14]).

A more general approach to modeling is based on a represen-tation of a landscape of energy using the charge moved as thereaction coordinate. In this type of modeling, the above-men-tioned discrete kinetic models are also represented when thelandscape of energy has surges that exceed 4 kT [15].

When gating currents are recorded with high bandwidth, newcomponents are observed [16]. Fig. 5(a) shows a typical gatingcurrent trace recorded with a bandwidth of 10 kHz. Notice thatbefore the initial plateau and decay of the gating current, thereis a brief surge of current. When the bandwidth is increased to200 kHz, the first surge of current is the predominant amplitudeas shown in the fast timescale recording of Fig. 5(b). The largespike of current is followed by a long plateau of current thatcorresponds to the dip observed at 10 kHz in Fig. 5(a). However,the area spanned by the large spike is a very small fractionof the total charge moved during the entire time course of thegating current. The interpretation of this early component isbest understood using the representation of the gating chargemoving in a landscape of energy that undergoes a change intilt when the membrane potential is changed such that thecharge advances in its initial energy well before making the

Fig. 5. Early component of the gating current. (a) Gating current recorded at10-kHz bandwidth. (b) Gating current recorded at 200-kHz bandwidth. Noticethe differences in the amplitude and time scales. (c) The time course of thecharging of the membrane capacitance for the experiment in (b). Recordingsdone in collaboration with Dr. E. Stefani.

jump across the first energy barrier. Using this approach, Sigget al. [16] computed the viscosity encountered by the gatingcharge in its well of energy.

What have we learned with kinetic modeling? In fact, a greatdeal. It is now clear that voltage-dependent channels have mul-tiple closed states and, in some cases, several open states.

In general, the opening of the gate requires all four subunitsto be activated, although there are cases where intermediatestates have been observed. Each subunit undergoes severaltransitions before reaching the active state, and in the case ofK channels, they do not seem to interact until the final stepthat opens the channel [17], [18]. However, in the muscle Nachannel site-directed fluorescence studies show that the inter-domain interactions are manifested prior to channel opening[19]. Kinetic modeling has given us a picture of the channel interms of channel physical states with transitions between themthat are regulated by voltage. Kinetic modeling is a critical stepin developing a physical model of channel operation becauseall the predicted features of channel function should be repro-duced by the structure of the protein and its voltage-inducedconformational changes.

B. Gating Charge Per Channel

When the gating charge moves within the electric field, wedetect a current in the external circuit. The time integral of that


current represents the charge moved times the fraction of thefield it traverses; therefore, our measurement of gating chargedoes not represent the exact number of charges displaced be-cause it includes the arrangement of the electric field. We mustkeep this in mind when we represent the reaction coordinate ofthe activation of the channel in the variable . A channel evolvesfrom to traversing many closed and/or open states.Activation of the channel corresponds to the opening of the poreand, analogous to the chemical potential, we define the activa-tion potential as

(3)

Then the activation charge displacement corresponds to thenegative gradient of the activation potential

(4)

The equilibrium probabilities in each physical state of thechannel can be explicitly written using the Boltzmann distri-bution knowing the potential of mean force for each state. Then, by assigning open or closed (or intermediate states)

conductances to every state, we can wite an expression forthat includes the voltage dependence of . The final result ofthe derivation [20] gives a relation between , , andthe charge moving between open states

(5)

Note that is the – curve shown in Fig. 5. This result isgeneral and includes cases with any number of open and closedstates connected in any arbitrary manner. If there is no chargemovement between open states , then the – curvesuperimposes on . In addition, it is possible to estimate ,the total charge per channel, by taking the limiting value ofthat makes go to zero. In the typical case of a channel thatcloses at negative potentials, we obtain

(6)

a result that was first obtained by [21] for the special case of asequential series of closed states ending in an open state. Thismethod has been applied to several types of voltage-dependentchannels, and the charge per channel obtained ranges between9 and 14 [22]–[24].

Another way to estimate the charge per channel is to mea-sure the maximum charge from the – curve and divide bythe number of channels present ( method). The number ofchannels can be estimated by noise analysis [25] or by toxinbinding [26]. The value of charge per channel estimated bythe method was 12–13 for the Shaker K channel, avalue that was not different from the value obtained by the lim-iting slope method [24]. As the limiting slope measures only thecharge involved in opening the channel, the agreement betweenthe two methods implies that in the case of the Shaker channelthere is no peripheral charge.

The large value of 12 to 13 per channel explains the verysteep voltage dependence of the superfamily of voltage gated

ion channels. At very negative , has a linear dependence on, so that is exponential in , such that it increases by in

only 2 mV

(7)

This very steep voltage dependence explains why the –curve of Fig. 4(b) shows no visible at potentials more neg-ative than 40 mV; in fact, there is a finite value of of lessthan 10 at potentials as negative as 100 mV.

Voltage-dependent channels that have several open stateswith charge moving between them , show plots of

– that may not be used to compute . One example is themaxi K channel, activated by voltage and Ca, that shows a

– curve with a slope that decreases as the potential is mademore negative. This result is consistent with the multiple-stateallosteric model proposed for this channel [27].

Gating current of one channel. The previous paragraph showsthat we can estimate the total charge that moves in one channelbut does not give any ideas of how that charge movement oc-curs at the single-channel level. Two limiting cases can be pro-posed. In one case, the time course of the gating current is justa scaled-down version of the macroscopic gating current shownin Fig. 4. The other case assumes that the charge movement oc-curs in large elementary jumps and that the macroscopic gatingcurrent is the sum of those charge shots. In principle, if thecharge is moving in discrete packets, it should be possible todetect those elementary events as it has been possible to detectthe elementary events of pore conduction. The problem is thatthose events are predicted to be very fast and extremely smalland not detectable above background noise with present tech-niques. However, if those events do exist, they should producedetectable fluctuations in the gating currents recorded from arelatively small number of channels. The fluctuation analysis isdone by repeating the same voltage pulse and recording a largenumber of gating current traces. From these traces, an ensemblemean value and an ensemble variance are obtained that allowsthe estimation of the elementary event (Fig. 6)

Gating current noise analysis was first done in Na channelsexpressed in oocytes [28] where indeed it was possible to detectfluctuations that were used to estimate an elementary charge of2.2 . A similar analysis was done in Shaker-IR K channel[29], and the elementary charge was found to be 2.4 . Thisvalue corresponds to the maximum shot size, and if it were persubunit it would account for only 9.6 or 3.6 less than the13 measured for the whole channel. This means that thereis a fraction of the total charge that produces less noise andthen it may correspond to smaller shots or even a continuousprocess. Fig. 6 shows the mean and variance for a large pulse(to 30 mV) and a smaller depolarization (to 40 mV). For thesmall depolarization [Fig. 6(b)], gating current noise increasesby the time that more than half of the charge has moved. This in-dicates that at early times the elementary event is smaller than atlonger times. The gating current decreases with two exponentialcomponents, which are more separated in time at small depo-larizations. Therefore, it is possible to attribute the small shotsto the fast component and the large shots to the slow compo-nent. This would indicate that the early transitions of the gating


Fig. 6. Current fluctuations in gating currents. (a) Top trace is the time courseof a pulse to+10 mV; middle trace is the variance and bottom trace is the meancomputed from several hundred traces. (b) Top trace is the time course of asmaller pulse to �40 mV; middle trace is the variance; lower trace is the meancomputed from hundreds of traces. Modified from [28].

current are made up of several steps, each carrying a small el-ementary charge. The total movement of charge in the earlytransitions would have to account for the 3.6 needed to makethe total of 13 per channel. We conclude from these experi-ments that the gating current of a single channel is made up ofsmall shots at early times followed big shots of currents that inthe average show two exponential decays.

V. STRUCTURAL BASIS OF THE GATING CHARGES

In this section, we will address the relation between the func-tion of the voltage sensor and its structural basis. We will firstask where are the charges or dipoles in the structure of thechannel and then how those charges or dipoles move in responseto changes in membrane potential.

A. The Structures Responsible for the GatingCharge Movement

There are many ways one could envision how the chargemovement is produced by the channel protein. The putative

-helical transmembrane segments have an intrinsic dipolemoment that upon tilting in the field would produce an equiv-alent charge movement. Also, induced dipoles of amino acidsside chains could accomplish the same. However, 13 perchannel is very large, and charged amino acids become the most

likely possibility. Since the first channel was cloned, it wasrecognized that the S4 segment with its basic residues wouldbe the prime candidate for the voltage sensor. By introducingmutations that neutralize the charges in S4, several studiesfound that there were clear changes in the voltage dependenceof the conductance. However, shifts in the voltage dependenceof the – curve or even apparent changes in slope in thevoltage range of detectable conductance do not prove thatcharge neutralization is decreasing the gating charge. The proofrequires the measurement of the charge per channel foreach one of the neutralizations. If, after neutralization of acharged residue, the charge per channel decreases, one mayassume such charge is part of the gating current. Using themethods to measure total charge per channel discussed abovein Shaker, two groups found that the four most extracellularpositive charges in S4 and that one negative charge in the S2segment were part of the gating charge [24], [26] (see whitesymbols in Fig. 1). It is interesting to note that in severalinstances a neutralization of one particular residue decreasedthe total gating charge by more than 4 . This indicates thatsomehow the charges interact with the electric field where theyare located such that the elimination of one charge can affectthe field seen by the remaining charges. If most of the gatingcharge is carried by the S4 segment (4 per subunit), it givesa total of 16 . Therefore, to account for the 13 for thetotal channel obtained from charge/channel measurements,they all must move at least 81% of the membrane electric field

.

B. Movement of the Charges in the Field

Knowing the residues that make up the gating charge is a bigadvance because it makes it possible to test their positions as afunction of the voltage and, thus, infer the possible conforma-tional changes.

As we will see below, the literature contains a large number ofpapers (for a review, see [30] and [31]) with many types of bio-physical experiments testing the accessibility, movement, andintramolecular distance changes. With all these data, modelsof the voltage sensor movement were proposed that could ac-count for all the experimental observations but in total absenceof solid information on the three-dimensional (3-D) structureof the channel. In Section VI, we will discuss many of the bio-physical measurements of the voltage sensor and the models thathave emerged from them and the recently solved crystal struc-ture of KvAP [32].

VI. STRUCTURAL BASIS OF THE VOLTAGE SENSOR

A 3-D structure of the channel, even in only one conforma-tion, would be invaluable as a guideline in locating the chargesinside the protein and in proposing the other conformations thataccount for the charge movement consistent with all the bio-physical measurements. We will see below that the only crystalstructure of a voltage-dependent channel available is not in anative conformation. Therefore, we are still relying on data thatcan only be used to propose models that are still uncertain asthey lack information on the 3-D structure of the channel.


Fig. 7. Three models of the voltage sensor. (a) Helical screw model. (b) Transporter model. (c) Paddle model. Following the chemical convention, positivelycharged residues are in blue. For details, see text.

A. The Crystal Structure of KvAP

Recently, the long-awaited first crystal structure of a voltage-dependent channel, KvAP from archea Aeropirum pernix, hasbeen published [32]. The structure was a surprise because itshowed the transmembrane segments in unexpected positionswith respect to the inferred bilayer. For example, the N-terminalwas buried in the bilayer whereas it has been known to be intra-cellular; the S1–S2 linker is also buried although it has been pre-viously shown to be extracellular. The S4 segment, along withthe second part of S3 (S3B) formed the paddle structure thatwas intracellular and lying parallel to the bilayer, a location thatwould be interpreted as the closed position of the voltage sensor.However, the pore gates in the same crystal structure clearlycorrespond to an open state. Thus, the crystal structure is in aconformation that was never observed functionally, raising thequestion whether that crystal structure of KvAP is indeed repre-sentative of the native conformation of the channel in the bilayer.The authors mention that the channel is inherently floppy; there-fore, in obtaining the structure, it was necessary to cocrystallizeit with Fab fragments attached to the S3–S4 loop of the channel.This raises the possibility that the fragments may have distortedthe channel structure that was in the final crystal. The authorsfunctionally tested the structure by incorporating the channelin bilayers and recording the currents through the channel afterFab fragments were added to the inside or to the outside of thechannel. The Fab fragment did not attach from the inside butonly from the outside, showing that the position of the S3–S4shown in the crystal structure (obtained in detergent) did notrepresent a native conformation of the channel in the bilayer[33].

Along with the crystal structure of the full KvAP channel,Jiang et al. [32] solved the crystal structure of just the S1 throughS4 region of KvAP. This crystal showed similarities but was notidentical to the S1–S4 region of the full KvAP crystal structure[34]. This second crystal was docked to the pore region of thefull crystal maintaining the S2 segment within and parallel tothe bilayer to obtain a new structure that was proposed in theopen and closed conformations as the paddle model of channelactivation. It is important to note here that the structures shownin the second paper [33] do not correspond to the original KvAPstructure. In fact the proposed structures of the model depart somuch from the crystal structure that we should treat them justlike any other model of activation proposed before.

B. Models of Sensor Movement

A compact way of reviewing the large body of biophysicalinformation on structural changes of the voltage sensor is todescribe the models that are currently proposed to explain theoperation of the voltage sensor.

Fig. 7 shows schematically three classes of model that havebeen proposed to explain the charge movement in voltage-de-pendent channels. In all cases, only two of the four sensors areshown and the coupling from the sensor to the gate is not shownexplicitly. In addition, the charge that moves resides completelyin the first four charges of the S4 segment. The common featureof all three models is that the charge is translocated from theinside to the outside upon depolarization. However, there areimportant differences as of how those charges relocate in theprotein structure.

Fig. 7(a) shows the conventional helical screw model [35],[36]. Although there are variations on this model, the general


idea is that upon depolarization the S4 segment rotates alongits axis and at the same time translates as a unit perpendicularto the membrane, thus changing the exposure of the chargesfrom the intracellular to the extracellular solution, effectivelytranslocating 4 per subunit [see Fig. 7(a)]. In the originalversion of the model, the change of exposure required a large16- translocation of the S4 segment, and the positively chargedarginines were making saline bridges with aspartate or gluta-mates residues that had to be broken to initiate the movement. Inmore recent versions [37]–[39], the charges are in water crevicesin both the closed and open position, decreasing the amount oftranslation required of the S4 segment.

Fig. 7(b) shows the transporter model [40]–[42]. In the closedposition, the charges are in a water crevice connected to theintracellular solution, and in the open position they are in an-other water crevice connected to the extracellular solution. Thetranslocation of the charges is achieved by a tilt and rotation ofthe S4 segment with little or no translation. In this case, the fieldis concentrated in a very small region that changes from aroundthe first charge in the closed state to the fourth charge in the openstate.

Fig. 7(c) shows the paddle model introduced by theMacKinnon group [33] where the S4 segment is locatedin the periphery of the channel and the charges are embeddedin the bilayer. The S4 segment makes a large translation suchthat the most extracellular charge goes from exposed to theextracellular medium in the open state to completely buried inthe bilayer in the closed state.

The helical screw and transporter models are similar but theydiffer dramatically from the paddle model in that the gatingcharges in the paddle model are embedded in the bilayer, whilein the helical screw and transporter models, the charges aresurrounded by water, anions, or making salt bridges. In con-trast with the transporter model, the helical screw model hasin common with paddle models the large translation of the S4segment.

In Sections VI-C–VI-E, we will review biophysical experi-ments that were designed to test the topology of the channel andthe extent of the conformational changes of the gating charge. Inthe absence of a native crystal structure, these experiments arethe only data that we can use to support or reject the availablemodels of voltage sensor operation.

C. The Topology of the Channel and Gating Charge Location

Alanine and tryptophan scanning have been used to infer therelative positions of the transmembrane segments [43], [44],and the results indicate that the S1 segment is in the peripheryof the channel. This result is at odds with the recent report byCuello et al. [45] where using electron paramagnetic resonance(EPR) scanning, they found that in KvAP the S1 segment isnot exposed to the bilayer but rather surrounded by the rest ofthe protein. This difference could be an inherent limitation inthe ala or tryp scanning techniques that test the function of thechannel, or it could be a genuine difference between eukaryoticand prokaryotic channels. On the other hand, their report showsthat the S4 segment seems to be exposed to the bilayer, in agree-ment with the paddle model although the charged residues seem

to be protected, contrary to the location proposed in the paddlemodel.

Laine et al. [46] found that the extracellular part of the S4segment gets within a few Ångstroms of the pore region in theopen state of Shaker-IR channel. This result is consistent withthe helical screw and the transporter model but is inconsistentwith the paddle model presented in [33] because in their modelthe extracellular portion of S4 is well separated from the restof the channel protein, giving a distance of about 98 be-tween segments across the pore region. In experiments usingresonance energy transfer with lanthanides [47], [48] or withorganic fluorophores [49], that distance was found to be around50 A, also in agreement with measurements done with tetheredTEA derivatives [50] These results indicate that the S4 segmentis not extended into the bilayer but is against the bulk of thechannel protein. If the paddle model were modified, so that theS4 segments would be almost perpendicular to the plane of thebilayer in the open state and almost parallel to the bilayer in theclosed state but still flush against the rest of the protein, it wouldbe consistent with the distance constraints just mentioned. In amore recent report, the MacKinnon group made such modifica-tion at least for the open-inactivated state [51]. However, evenwith these modifications the paddle model locates the charges inthe bilayer, a proposal that is inconsistent with the EPR results[45] and several other biophysical experiments [52], [54]–[63].Locating the charges in the bilayer has been a subject of inten-sive debate because the energy required in moving a charge intothe low dielectric constant bilayer is extremely high [5]. If pos-itively charged gating charges are neutralized by making saltbridges with acidic residues, the energy decreases [5] but therewould not be any gating charge movement.

Evidence obtained by charge measurements in the squid axonsodium channel has shown that the gating charges do not movein the bilayer [52] In these experiments, addition of chloroformincreased the kinetics of translocation of the hydrophobic iondipycrilamine while it did not change the kinetics of the sodiumgating currents. The conclusion was that the gating charge, un-like hydrophobic ions, does not move in the bilayer. Ahern andHorn [38] have explored this subject in more detail. As in thepaddle model the S4 segment is in the bilayer, they reasoned thaton addition of more charges in the S4 segment, the net gatingcharge should increase. Their results show that the charge ad-dition at several positions did not increase the gating charge,indicating that only the charges in aqueous crevices are respon-sible for gating and can sense the changing electric field. Boththese experimental results are hard to reconcile with the paddlemodel where the voltage sensor is immersed in the hydrophobiccore of the lipid bilayer.

The S1–S2 loop has been clearly located in the extracellularregion by several criteria. One is that a glycosylation site inShaker occurs in this loop [53]. In addition, fluorescence signalsfrom fluorophores attached in this loop are consistent withthis region being extracellular [54], as well as the recent EPRscanning of KvAP [45]. These results are in agreement with thehelical screw and transporter models but are again inconsistentwith the location proposed in the paddle model. In the KvAPcrystal structure the S1–S2 linker is buried in the bilayer withan S2 segment almost parallel and also buried in the bilayer.


In fact, this orientation of the S2 segment was the basis todock the S1–S4 crystal to the rest of the crystal to proposethe paddle model.

D. Voltage-Induced Exposure Changes of the S4 Segment andIts Gating Charges

Testing the exposure of the gating charges in the intra- or ex-tracellular medium would give us an idea whether their voltage-induced movement takes them out of the protein core or fromthe lipid bilayer. There have been three different approaches totest exposure. The first method, cysteine scanning mutagenesis,consists of replacing the residue in question by a cysteine andthen test whether a cysteine reagent can react from the extra orintracellular solution and whether that reaction is affected byvoltage [55]–[59]. The second method, histidine scanning mu-tagenesis, consists on titrating with protons the charge in theresidues. In this case, as the pKa of the arginine is very high,histidine was used as a replacement allowing the titration in apH range tolerated by the cell expressing the channel [41], [60],[61]. The third method consists of replacing the residue in ques-tion with a cysteine, followed by tagging it with a biotin groupand then testing whether streptavidin can react from the insideor outside depending on the membrane potential [33]. The out-come of all these experiments is that indeed the charged residuesin S4 undergo changes in exposure when the membrane poten-tial is changed. However, it is important to look at each of theseprocedures with their limitations and compare their results be-cause that may reveal many details of the actual movement ofthe charge.

Cysteine scanning mutagenesis. The experiments by Yangand Horn [55] were the first to test the accessibility of chargedgroups to the extra and intracellular solutions and its depen-dence on membrane potential. The idea of these experimentsis to test whether a cysteine reacting moiety can attach to an en-gineered cysteine in the channel depending where the moiety isand what the electric field is. This method works provided theattachment induces a detectable change in the channel currents.In addition, the attachment of the moiety will depend on thelocal pH and state of ionization of the cysteine residue. Yangand Horn [55] showed that the reaction rate of MTSET to thecysteine replaced most extracellular charge of the S4 segmentof the fourth domain of a sodium channel depended on mem-brane potential. To conclude that a particular site changes itsexposure, the reaction rate must differ by more than an orderof magnitude between the two conditions. This is important be-cause if one just measures whether the reaction occurred or not,one may be sampling a rarely occurring conformational state.Their result showed for the first time that the accessibility ofthis group changed with voltage or alternatively that the ioniza-tion state was changed by voltage. This work was expanded tothe deeper charges in the S4 segment of domain IV of the Nachannel [56]. The conclusion was that upon depolarization themost extracellular charge was exposed and that at hyperpolar-ized potential it became buried. In addition, the two followingcharges could be accessed from the inside at hyperpolarized po-tential and from outside at depolarized potential. The next twodeeper charges were always accessible from the inside regard-less of the membrane potential. These results strongly suggest

that the three outermost charges change exposure with voltageand are consistent with the idea of a conformational change thattranslocates charges from inside to outside upon depolarizationgiving a physical basis to the gating currents.

These studies were also done in the Shaker-IR K channel[57]–[59] and showed the same trend: the outermost chargeswere exposed to the outside upon depolarization and the inner-most to the inside on hyperpolarization. While some residueswere inaccessible from one side to the cysteine-reacting com-pound on changing the voltage, there were a few that reactedwhen the reactive agent was added to the other side.

The conclusion from the cysteine scanning experiments isthat the residues bearing the gating charges change their expo-sure with the voltage-dependent state of the channel. Anothervery important conclusion is that the charges are exposed to thesolutions and not to the lipid bilayer, since the SH group in thecysteine has to be ionized to react with the cysteine modifyingreagent. This would be energetically unfavorable in the low di-electric medium of the bilayer. It is important to note that thereactivity of thiol groups on some the sites tested was compa-rable to their reactivity in free solution.

Histidine scanning mutagenesis. Testing the titration of thecharged residues is a direct way to address exposure of thecharges to the solutions. In this approach, each arginine orlysine is exchanged to a histidine residue that can be titratedin a pH range that is tolerated by the expression system. Thetitration of the histidine with a proton can be easily detectedas a change in the gating current, provided the ionic currentis blocked. For this reason, most of these experiments werecarried out in the nonconducting Shaker-IR K channel thatbears the W434F mutation.

The logic of this procedure can be understood by taking somelimiting cases.

1) If the histidine is not exposed to the solutions and/or ifit does not move in the field, it would not be possible totitrate it from either side or under any membrane potential;therefore, no change in the gating currents are observed.

2) The histidine can be exposed to the inside or to the out-side depending on the membrane potential and thus onthe conformation of the voltage sensor. In this case, ifa pH gradient is established, every translocation of thevoltage sensor would also translocate a proton, thus pro-ducing a proton current. This proton current would bemaximum at potentials where the sensor is making mostexcursions which occurs around the midpoint of the –curve. At extreme potentials, the gating current would beaffected but no steady proton current would be observed.Therefore, the – curve of such proton current would bebell-shaped.

3) One particular conformation of the sensor locates the his-tidine as a bridge between the internal and external so-lutions forming a proton selective channel. In this casea steady current would be observed that would have analmost linear – curve in the range of potentials wherethat conformation is visited and would be zero otherwise.

The analysis of histidine scanning was done in Shaker-IR Kchannels and six charges were tested starting from the most


Fig. 8. Results of histidine scanning mutagenesis. (a) Each mutant is listedwith the type of current observed and their accessibility to the internal andexternal solutions. In the case of K374H and R377H, the histidine may notbe accessible and/or they do not move in the field. (b) Interpretation of thehistidine scanning experiments with the transporter model based on a molecularmodel built with KvAP and KcsA crystal structures [42], [80]. A change intilt of the S4 segment exposes the first four charges to the outside in thedepolarized condition and to the inside in the hyperpolarized condition. In thehyperpolarized condition, there is a very narrow region bridged by histidine inposition 362 and in the depolarized condition a bridge is formed by histidine inposition 371. For details, see text.

extracellularly located [41], [60], [61]. Fig. 8(a) shows the re-sults where the accessibility of each of the mutant is shown. Thefirst four charges are accessible from both sides depending onthe membrane potential, while the next two are not titratable.

These results are consistent with what we know about the roleof each charge. Only the first four charges, which are responsiblefor most of the gating charge, seem to translocate, while the nexttwo either do not move in the field or are never accessible fromthe solution. The results also show that some of the charges thatappeared buried to the cysteine scanning mutagenesis methodare accessible to protons indicating that they are pointing intodeep crevices that are too narrow for the cysteine-modifyingreagent but large enough for protons to reach.

Fig. 8(b) shows how the results could be explained in terms ofan actual structure of the S4 segment with its associated mem-brane segments and bilayer, based on a transporter-like molec-ular model [42], [80] built with the crystal structures of KvAPand KcSA. The alpha-helical S4 segment is represented by acylinder showing the first four charged arginines, as labeled. The

next two arginines are on the back side of the S4 helix and arenot visible. The hydrophobic region, which includes other seg-ments and the bilayer, is simply shown as a gray area. In theclosed state, the first four charges are in contact with the in-ternal solution by way of a water crevice (up arrow); however,the most extracellular charge (R362) is in the boundary betweenthe intracellularly connected water crevice and the extracellularsolution. If this arginine were replaced by a histidine, it wouldform a proton pore by making a bridge between both solutions.Upon depolarization, the S4 segment changes its tilt, and all fourcharges become exposed to a water crevice (down arrow) con-nected to the extracellular solution. Now the fourth charge isin the boundary between the extra and intracellular solutions sothat a histidine in this position would form a proton pore. No-tice that R365 (and also R368) change exposure in such a waythat if each one were replaced by a histidine in the presence ofa proton gradient, every transition would be able to shuttle oneproton from one side to the other. It was found that R371H is atransporter but it can also form a proton pore at large depolar-ized potentials [61].

The consequence of these results is that in the two extremespositions the electric field is concentrated [62] in a very narrowregion of the protein: near 362 in the closed position and near371 in the open position. This means that there is no need for alarge movement of the voltage sensor to transport a large amountof charge. Asamoah et al. [63] have shown that indeed the fieldis concentrated in this region by using a cysteine reactive elec-trochromic fluorophore. By attaching this fluorophore in dif-ferent regions of the channel and comparing the fluorescencesignal induced by voltage changes with the same electrochromicgroup in the bilayer, the field in the S4 region was found to beat least three times more intense.

The results of histidine and cysteine scanning experimentssuggest that the charged arginine residues are stabilized by waterand possible anions residing in the water filled crevices. It hasbeen shown by Papazian and collaborators [64], [65] that theacidic residues in S2 and S3 segments play a stabilizing role inthe structure of Shaker-IR channel but they could also lower theenergy of the ariginines in the crevices and possibly contributeto the gating charge by focusing the field in that region.

Biotin and streptavidin scanning. Biotin has a length of about17 and its cysteine modifying derivative can react with cys-teines engineered in the channel protein. In addition, the biotingroup binds avidin, a large soluble protein molecule that is notexpected to cross the membrane. Jiang et al. [33] studied sev-eral positions by mutating residues to cysteine in S3b and S4segments of KvAP and attached to it a biotin molecule. Afterincorporating these channels into bilayers, they tested whetherthe currents were affected by avidin in the external or internalsolutions. They found that, depending on the site of biotin at-tachment, the currents were decreased by externally or inter-nally applied avidin. There were two sites, (121 and 122) lyingin between the second and third charges of KvAP, where theysaw inhibition by avidin from both sides. They indicated that inthe conventional model (helical screw or transporter models) itwould be extremely difficult to relocate the linker of the biotinalong with the charges moving within the protein core. There-fore, they proposed that those sites must move a large distance


within the bilayer to reach for the avidin present in the solu-tion and gave them the basic restrictions on the extent that thepaddle must move. Although this result is consistent with thepaddle model, it is not inconsistent with the transporter modelbecause those residues may in fact be facing the bilayer [42] andthus allowing the biotin to reach to either side of the bilayer toattach to the avidin. It should be noted that no reaction rate wasmeasured in those experiments [33], so that it is also possiblethat their inferred conformations were rarely visited.

E. Fluorescence Spectroscopy Reveals ConformationalChanges of the Voltage Sensor

Site-directed fluorescence is a powerful technique to followconformational changes in a protein. In the case of voltage de-pendent channels, the idea is to label specific sites of the channelprotein with a fluorophore and measure changes in fluorescenceinduced by changes in the field. The salient feature of this tech-nique is that the changes measured reflect local changes in ornear the site where the fluorophore is located in the protein asopposed to electrical measurements that reflect overall confor-mations of the protein. Two main types of measurements havebeen done with site-directed fluorescence. One is the detec-tion of fluorescence intensity changes of one fluorophore in thelabeled site, and the other is the estimation of distance and dis-tance changes between two fluorophores using fluorescence res-onance energy transfer (FRET).

Site-directed fluorescence changes. In these experiments, thesite of interest is mutated to a cysteine and then is reacted toa fluorophore that has a cysteine reactive group and the timecourse of fluorescence is monitored during pulses applied toopen or close the channel [66], [67]. To detect a fluorescencechange, the conformational change must change the environ-ment around the fluorophore so that the intensity changes be-cause of spectral shifts, quenching, or dye reorientation. In mostcases, the changes in fluorescence have been traced to changesin the quenching environment around the fluorophore [67], [68],and in a few cases it is produced by spectral shifts of the fluo-rophore as the hydrophobicity of the environment changes withthe change in conformation [54]. In some cases, the presence ofquenching groups in the protein is crucial in obtaining a signal.For example, in the bacterial channel NaChBac, the signals arevery small or in some sites not detectable [69], although thereare four charges in the S4 segment following the classical pat-tern and the total gating charge was recently measured to beabout 14 [70]. This result has been traced to the lack ofquenching groups in the structure of this channel [69]. To obtainsignals from sites near the S4 segment upon changes of mem-brane potential, a requirement seems to be the presence of gluta-mate residues in the nearby region that act as quenching groups[69].

Fluorescence changes in site-directed fluorescent labelinghave provided information of local conformational changesaround the S4 segment, the S3–S4 linker, the S1–S2 linker, andthe pore region as a result of changes in membrane potential[66], [67], [71]. In the absence of 3-D structure, the interpre-tation of these fluorescence changes is not straightforwardbecause the exact location of the fluorophore is unknown.

However, several important qualitative results have been ob-tained. For example, it has been found that the kinetics of thefluorescence changes in S4 are slower than around the S1–S2linker, suggesting that there might be earlier conformationalchanges that precede the main conformational change normallyattributed to the S4 segment [67]. Also, the time course offluorescence changes near the pore region are much slower thanchannel activation and their kinetics can be traced to anothergating process called slow inactivation in Shaker K channel[67], [71].

Probably one of the most informative results have been ob-tained in the sodium channel because, as this technique detectslocal changes, it has been possible to distinguish specific func-tions for each one of the four domains of the Na channel. Fastinactivation is another gate that operates by blocking the channelpore [1], [72], [73]. By labeling the S4 segment of each domainof the Na channel, it was found that the gating charge immobi-lization produced by inactivation only occurred in domains IIIand IV, thus locating the regions of the channel that interact withthe inactivating particle [73]. The kinetics of the fluorescence ofsites in S4 was found to be very fast in domains I–III but slowerin domain IV, indicating that domain IV followed the other threedomains [75]. Finally, by comparing the effect of a perturbationin one domain to the fluorescence signal in another domain, itwas found that all four domains of the Na channel move incooperative fashion. This result is important in explaining whysodium channels are faster than potassium channels, an abso-lute requirement in eliciting an action potential [19]. For moredetails on the voltage-dependent sodium channel, see the paperby French and Zamponi in this issue.

Resonance energy transfer. By labeling the protein with adonor and an acceptor fluorophore, it is possible to estimate thedistance between them using the Förster theory of dipole–dipoleinteraction [76]. Depending on the fluorophore pair used, thosedistances can be from a few to about 100 , thus enablingthe measurements of intramolecular distances and changes indistances.

This technique was used to estimate distances and distancechanges between subunits in Shaker-IR channel by Cha et al.[47] and Glauner et al. [49]. Cha et al. [47] used a variant ofFRET called LRET that uses a lanthanide (terbium) as a donorand has the advantage that the estimation of the distances aremore accurate mainly because the orientation factor is boundbetween tight limits giving an uncertainty of 10 [77]).

Both studies [47], [49] showed that the distances betweenS4 segments were around 50 and that it did not change verymuch from the closed to the open states. The maximum distancechange measured by Cha et al. [47] was about 3 while Glauneret al. [49] measured about 5 . These measurements were alldone between subunits; therefore, they do not completely ruleout a translation across the bilayer, as proposed in the paddlemodel.

In Shaker K channel, the linker between S3 and S4 is madeof about 30 residues. At least six of the residues close to theextracellular part of S4 seem to be in alpha-helical conformation[78] suggesting that S4 is extended extracellularly as an alphahelix. This would explain why Cha et al. [47] measured changesin distance in the S3–S4 linker as a result of membrane potentialchanges. One of those changes is a rotation in the linker and


the other is a decrease in the tilt of the S4 with its extensionupon depolarization, providing a possible mechanism for chargetranslocation (see Fig. 8).

The question of how much translation the S4 undergoesacross the bilayer with depolarization has been approachedwith two other variants of FRET. In the first series of experi-ments [79], green fluorescent protein (eGFP) was inserted afterthe S6 of Shaker K channel and was used as the donor to anacceptor attached in the extracellular regions of the channel.In this case, the acceptor was a sulforhodamine with an MTSreactive group that can react with an engineered cysteine in thechannel but it also can be cleaved off with a reducing agent. Thisallows the measurement of donor fluorescence (intracellularlylocated) in presence and absence of acceptor (in the extracel-lular regions of the channel) to compute the energy transferand the distance. These measurements were done in multiplesites of the S1–S2 loop, S3–S4 loop, and S4 segment underdepolarized and hyperpolarized conditions giving changes indistance that did not exceed 2 . In fact, some sites increasedwhile others decreased their distance to the intracellular donorupon depolarization, indicating that there is little translation ofthe S4 across the membrane.

The second method used the hydrophobic ion dipycrilamine(dpa) as an acceptor that distributes in the edges of the bilayeraccording to the membrane potential [52], [80]. The donor wasrhodamine attached to specific sites in the S4 segment. The ex-periment predicts a clear distinction for the outcome dependingwhether the S4 does or does not make a large translation acrossthe membrane. If the S4 segment undergoes a large translationacross the bilayer such that the donor crosses its midpoint, thena transient fluorescence decrease is expected because dipycril-amine and the fluorophore start and end in opposite sides of themembrane but there is a period where both donor and acceptorreside simultaneously in both sides of the membrane increasingtransfer and consequently decreasing donor fluorescence. Onthe other hand, if there is no crossing of the bilayer midline bythe donor, the fluorescence will increase if the donor is abovethe midline or decrease if it is below, but no transient should beobserved. The results of at least four sites are consistent with nocrossing of the midline strongly suggesting that the S4 does notmake a large translation upon depolarization [80].

Finally, one more experiment using LRET confirms the lackof large translation of the S4 [81]. In this case, the donor is Tb(in chelate form) attached in several sites of the S4 segment andthe S3–S4 linker while the acceptor is in a toxin that blocks thepore of Shaker from the extracellular side. Results show that thechanges in distance between the toxin and several tested sitesin the S3–S4 linker and S4 segment do not exceed 2 upondepolarization.

A recent molecular dynamic calculation of the voltagesensor of a K channel under the influence of an electric fieldalso supports a conformational change that involves minimumtranslation [82].

In summary, FRET experiments are inconsistent with a largetransmembrane displacement of the S4 segment in response to avoltage pulse. As many other experiments, presented above anddiscussed in recent papers and reviews [34], [40], [46], [83],[84], do not support the idea that the charged residues are in

the bilayer, the two main features of the paddle model becomeinconsistent with the available data.

VII. COUPLING OF THE SENSOR TO THE GATE

In contrast to a field-effect transistor where gating of thechannel is produced by a change in space charge, the gate involtage-gated channels seem to be a mechanical obstruction toflow [30]. The crystal structure of the bacterial channel KcSAreveals a closed state of the channel and Perozo et al. [85] foundthat when it opens the S6 makes a scissor-like action allowingions to go through. The crystal structure of MtsH, anotherprokaryot channel, shows an open pore where a glycine in theS6 segment is shown to break the S6 in two segments [86].This led MacKinnon to propose that the gate opens when theS6 segment is broken and the intracellular part is pulled apart[87]. In the crystal structure of the voltage-dependent prokaryotchannel, KvAP the pore is in the open conformation by a breakin the S6 segment in a glycine residue [32]. In the case of theeukaryotic Shaker K channel, there is a PVP sequence in theS6 segment that has been proposed to be the actual gate [88].In addition to the main gate formed by the bundle crossingof the S6 segments, there is now very good evidence that theselectivity filter can also stops conduction, thus introducinganother gate in series [89]. However, there is no evidence or aphysical mechanism to couple this filter gate to the movementof the sensor.

The question of how and what kind of physical movement ofthe sensor, mainly the S4 segment, couples the opening of thepore gate is far from resolved. Most of the proposals, includingthe paddle model, suggest that the change in position of the S4couples via the intracellular S4–S5 linker to change the positionof the S5 segment that in turn allows the opening of S6. In thetransporter model [42], [80] the change in tilt of the S4 segmentcarries the S5 segment away from S6, allowing the break at theglycine and thus opening the pore.

VIII. CONCLUSION

The main component of the sensor of voltage-gated channelsis the S4 segment with its basic residues moving in the field. Themovement of the sensor in response to changes in the electricfield produces the gating currents. The study of gating currents,single-channel currents, and macroscopic currents has gener-ated detailed kinetic models of channel operation. The sensorcouples to the gate possibly via the S4–S5 linker. The channelfully opens after all sensors have moved, and there is little coop-erativity in the early movements of the sensor in the K channelbut strong positive cooperativity in the Na channel. The resultsof a large variety of biophysical experiments have helped in de-lineating the conformational changes of the sensor. These datahas shown that the S4 segment does not undergo large move-ments. Rather, the charges are in water crevices, and they onlymove a small distance because the field is focused in a narrowregion within the protein core. In the absence of a crystal struc-ture representative of the channel in its native form, we believethat the data support the transporter model or some of the mostrecent versions of the helical screw model of the voltage sensor.

A detailed understanding of voltage gated channels meansthat we can represent the landscape of energy of the physical


states of the channel at atomic resolution together with the struc-tural changes evoked by the electric field. To achieve this goal, itwill not only require a static, high resolution 3-D structure, butalso a detailed description of the kinetics of the voltage-inducedconformational changes. The latter are expected to be obtainedwith spectroscopic and computational techniques. Fluorescenceand EPR spectroscopies have started to unravel some of the de-tails of conformational changes during gating but the study ofdetailed kinetics is still developing. For example, in the sameway that single-channel current recordings were critical in un-derstanding the operation of the pore and the gate, single-mole-cule fluorescence is expected to show the local conformationalchanges during voltage sensor operation and channel gating.These techniques are advancing very rapidly, and some resultshave already been published as just the conformational change[90], [91] or even correlated to current recordings [92], [93].

ACKNOWLEDGMENT

The author would like to thank Drs. R. Blunck and B. Chandafor reading the manuscript and for their helpful and insightfulcomments.

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Francisco Bezanilla received the Ph.D. degree fromthe Catholic University, Santiago, Chile, in 1968.

He held postdoctoral positions at the NationalInstitutes of Health, Bethesda, MD, and at theUniversity of Rochester, Rochester, NY. He helda position as a professor in The University ofChile. He is currently the Hagiwara Professor ofNeuroscience in the Departments of Physiologyand Anesthesiology at the D. Geffen School ofMedicine, University of California, Los Angeles.His primary research is aimed at the understanding

of the structure–function relation of ion channels and voltage-dependentmembrane processes.

voltage-gated ion channels - electrophysiology and the molecular

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