Correspondence to: Richard W. Aldrich, Howard Hughes Medical Institute and Department of Molecular and Cellular Physiology, Beckman Center B-171, Stanford University School of Medicine, Stanford, CA 94305-5426., raldrich{at}leland.stanford.edu (E-mail), Fax: 650-725-4463; (fax)
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Abstract |
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The best-known Shaker allele of Drosophila with a novel gating phenotype, Sh5, differs from the wild-type potassium channel by a point mutation in the fifth membrane-spanning segment (S5) (Gautam, M., and M.A. Tanouye. 1990. Neuron. 5:6773; Lichtinghagen, R., M. Stocker, R. Wittka, G. Boheim, W. Stühmer, A. Ferrus, and O. Pongs. 1990. EMBO [Eur. Mol. Biol. Organ.] J. 9:43994407) and causes a decrease in the apparent voltage dependence of opening. A kinetic study of Sh5 revealed that changes in the deactivation rate could account for the altered gating behavior (Zagotta, W.N., and R.W. Aldrich. 1990. J. Neurosci. 10:17991810), but the presence of intact fast inactivation precluded observation of the closing kinetics and steady state activation. We studied the Sh5 mutation (F401I) in ShB channels in which fast N-type inactivation was removed, directly confirming this conclusion. Replacement of other phenylalanines in S5 did not result in substantial alterations in voltage-dependent gating. At position 401, valine and alanine substitutions, like F401I, produce currents with decreased apparent voltage dependence of the open probability and of the deactivation rates, as well as accelerated kinetics of opening and closing. A leucine residue is the exception among aliphatic mutants, with the F401L channels having a steep voltage dependence of opening and slow closing kinetics. The analysis of sigmoidal delay in channel opening, and of gating current kinetics, indicates that wild-type and F401L mutant channels possess a form of cooperativity in the gating mechanism that the F401A channels lack. The wild-type and F401L channels' entering the open state gives rise to slow decay of the OFF gating current. In F401A, rapid gating charge return persists after channels open, confirming that this mutation disrupts stabilization of the open state. We present a kinetic model that can account for these properties by postulating that the four subunits independently undergo two sequential voltage-sensitive transitions each, followed by a final concerted opening step. These channels differ primarily in the final concerted transition, which is biased in favor of the open state in F401L and the wild type, and in the opposite direction in F401A. These results are consistent with an activation scheme whereby bulky aromatic or aliphatic side chains at position 401 in S5 cooperatively stabilize the open state, possibly by interacting with residues in other helices.
Key Words: gating current, ion channel, site-directed mutagenesis, activation, cooperativity
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Introduction |
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Potassium channels exert a stabilizing influence over the membrane potential of excitable cells, shaping the patterns of their electrical activity and serving as targets for modulators and drugs. To perform this role, many potassium channels have evolved exquisite sensitivity to transmembrane voltage. The Shaker channel, a member of the family of voltage-gated (Kv)1 potassium channels (
Voltage-dependent gating refers to the conformational transitions that the channel protein can undergo in which intrinsic charged or dipolar groups (gating charges) move in response to changes in the membrane voltage. Structurally, voltage-gated potassium channels exist as tetramers of like alpha subunits (
We have used a previously studied mutant Shaker allele as a starting point to help understand the role of the fifth membrane-spanning segment (S5) in activation gating and as a potential interacting partner for S4. Sh5 is the best known mutant Shaker allele that affects voltage-dependent gating in Drosophila. It differs from the wild-type sequence by a phenylalanine-to-isoleucine substitution located in the S5 transmembrane segment: F401I (
Using the background of an NH2 terminustruncated version of the wild-type Shaker channel free of fast N-type inactivation (
Several gating mechanisms for potassium channels that incorporate independent and cooperative steps in the activation process have been proposed (
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Materials and Methods |
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Terminology
All mutant channel constructs were made in the ShB6-46 background (
Site-directed Mutagenesis and Oocyte Expression
All conducting versions of constructs containing point substitutions in the S5 region were generated by synthetic oligonucleotide-directed cassette mutagenesis using the polymerase chain reaction. To record gating charge movement, a high-expression vector containing the W434F mutation (
Electrophysiology
Patch-clamp recordings from oocytes were carried out using the Axopatch 200A amplifier (Axon Instruments) with borosilicate glass pipettes (initial tip resistances between 0.4 and 2 M). Macroscopic ionic currents recorded in the inside-out and outside-out excised configurations (
To improve the signal-to-noise ratio for gating current experiments, we used a high-performance cut-open oocyte clamp (CA-1; Dagan Inc.) () glass microelectrodes were filled with 3 M KCl. Online series-resistance compensation was used. Linear leak and capacitative currents were subtracted using a P/-5 to P/-8 protocol from a holding voltage of -120 mV. Resulting traces were periodically compared with those obtained with a P/4 subtraction protocol from the holding voltage of +50 mV, and no consistent differences were noted. Records were low-pass filtered at 510 kHz.
A holding voltage of -100 mV was used except as noted in the text. All experiments were carried out at 20.0 ± 0.2°C, unless otherwise indicated, using a feedback temperature controller device.
Solutions
For patch-clamp recordings, we used chloride-containing solutions. The external solution contained (mM): 140 NaCl, 5 MgCl2, 2 KCl, 10 HEPES (NaOH), pH 7.1. The internal solution contained (mM): 140 KCl, 2 MgCl2, 11 EGTA, 1 CaCl2, 10 HEPES (N-methylglucamine), pH 7.2. To reduce the slowly activating native oocyte chloride conductances when using the cut-open clamp, we perfused nominally chloride-free solutions containing (top and guard chambers, mM): 110 NaOH, 2 KOH, 2 Mg(OH) 2, 5 HEPES (MES), pH 7.1; (bottom chamber, mM): 110 KOH, 2 Mg(OH) 2, 1 Ca(OH) 2, 10 EGTA, 5 HEPES (MES), pH 7.1.
Off-Line Analysis
Linear components of leak and capacitative currents were digitally subtracted. Macroscopic ionic and gating current records were analyzed further using Igor Pro (WaveMetrics) and custom-written software. Comparisons of the relative open probability versus voltage relationship among the wt and mutant channels were based on the isochronal (between 0.5 and 1 ms post-pulse) amplitude of their tail currents after variable test pulses because this approach does not rely on assumptions about the linearity of the open-channel i(V) or the reversal potential. This type of measurement is termed a steady state voltage dependence of the open probability [G(V)] relation in this paper. We fit the G(V) data with Boltzmann functions raised to the fourth power (see
where z is the apparent gating valence per channel subunit, V1/2 is the apparent mid-point of the voltage-dependent transition in each subunit, and R, T, and F have their usual thermodynamic meanings. The time course of current activation was fit with the exponential
beginning with the time of the half-maximal current amplitude. As a measure of delay in current turn-on, tdelay (the time-axis intercept of the fitted exponential function) was found to be widely variable between patches for the same channel species. Therefore, the independently measured time-to-half-maximum was used as an alternative indicator of the activation delay. Decaying exponential fits to the kinetics of tail currents were obtained using the equation
Fits to experimental data and model simulations were performed using a Levenberg-Marquardt nonlinear least squares optimization algorithm.
Model simulations were done using BigChannel software, courtesy of Toshinori Hoshi and Dorothy Perkins (Howard Hughes Medical Institute, Stanford University, Stanford, CA). In brief, simulated macroscopic ionic and gating currents were calculated numerically using an Euler integration method, digitally filtered to match the corner frequency of an eight-pole Bessel filter used in obtaining the corresponding data, and subsequently analyzed in the manner identical to experimental traces. Model parameters were allowed to vary slightly in fitting individual families of records. The goodness of fit was ultimately assessed by eye. Model parameters used in each simulation are given in the figure legends.
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Results |
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The Sh5 Replica Mutation Alters Activation Gating of Shaker
A point mutation converting the first phenylalanine of the fifth transmembrane segment to isoleucine served as a replica of the Sh5 mutation (Figure 1 C). Families from patches expressing either wild-type or mutant F401I currents activate over a similar range of voltages and deactivate completely at the relatively depolarized tail potential of -65 mV (Figure 1 A). Compared with the wt G(V) curve, F401I activation has a noticeably shallower voltage dependence (Figure 1 B). The fourth power Boltzmann fits to the G(V) curves yield values of z of approximately four elementary charges (e0) per wild-type subunit, which is similar to the estimated total charge displacement per channel of 12.514 e0, obtained from direct gating current measurements (
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If we consider a voltage-sensitive transition with associated charge displacement z in terms of transition-state theory, the voltage dependence of the forward and backward rates is determined by the charge movement before and after the transition state, respectively, and need not be equal. We asked if the diminished voltage dependence of the F401I mutant is associated primarily with forward or reverse transitions. A method to assess the forward rates in relative isolation from the backward transitions is illustrated in Figure 2. The currents from wt and F401I channels activate with a sigmoidal delay, reflecting a multistep opening process. As the test potential is stepped to more positive values, channel opening kinetics accelerate for both the wt and F401I families. With sufficiently depolarizing voltage steps (i.e., more positive than -10 mV where the probability of opening for both channels nears saturation), the reverse rates can be considered negligible and the kinetics of activation are almost entirely determined by the forward rates. In this voltage range, the time course of activation has a complex multiexponential behavior but, for a class of models commonly used to describe Shaker gating, the slowest exponential component has a time constant that is the inverse of the slowest forward rate (
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Whereas the voltage dependence of the forward rates and, therefore, the amount of charge movement before the transition state, appears unaffected by the F401I mutation, the voltage dependence of the closing (deactivation) transitions, reflecting the charge movement "after" the transition state, is very sensitive to this change. The kinetics of deactivation were studied from currents recorded during channel closing (tail currents) at hyperpolarized potentials (negative to -60 mV) after maximally activating prepulses (Figure 3A and Figure B). Deactivation follows a nearly single-exponential time course in both channels, with the time constants from the fits displayed in Figure 3 C against tail voltage. wt tail currents are not simply slower compared with F401I; the difference is greatest at -60 mV, but diminishes at very negative voltages and largely disappears below -160 mV (Figure 3 C, inset). Kinetics of the tail currents in the wt are steeply potential dependent, with the apparent charge associated with the backward transitions, zr , of 1.2 e0, consistent with a previous report (
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Comparison of S5 Phenylalanine Substitutions
F401 is one of five phenylalanines in the Shaker S5 sequence (at positions 401, 402, 404, 410, and 416; see Figure 1 C). To investigate whether other amino acid substitutions in S5 have similar effects on activation gating, we conducted alanine mutagenesis of the four phenylalanines downstream (towards the carboxyl terminus) of F401 as well as other S5 hydrophobic residues (leucines at positions 396, 398, 399, 403, and 409, and serines at positions 411 and 412), noting that it was an alanine substitution at F401 that resulted in the greatest effects (see below). Only F404A, F416A, L403A, S411A, and S412A gave rise to reliable ionic current expression. The results are shown in Figure 4. The mutants' steady state activation voltage dependence shows few differences from the wt other than a small 110-mV depolarizing shift in most of the G(V) curves. The apparent valence of activation was not altered in any of the mutants. For L403A, these findings confirm earlier observations on channels with intact inactivation (
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Because the F404 residue is the least well conserved of S5 phenylalanines among the family of potassium channels, with alanine occurring at the equivalent site in, for example, Kv2.1, fShal and fShab, we were not surprised that the F404A substitution did not significantly alter activation or deactivation kinetics. In contrast, the position equivalent to F416 in Shaker channels only contains aromatic amino acids among voltage-gated potassium channels. We noted small but consistent differences between the F416A mutant and the wild type. F416A currents have a more pronounced sigmoid delay in activation and more rapid deactivation kinetics. In summary, neither of the two downstream S5 phenylalanine-to-alanine mutations that produced functional channel expression, and none of the leucine and serine substitutions, influenced voltage-dependent gating to the degree evident for the F401 mutations.
Correlating Effects of F401 Mutations with Side Chain Properties
Because of the striking effects of the F401I substitution, we substituted other amino acids for the phenylalanine at 401 to investigate the role of side chain structure on gating. We introduced individually three progressively smaller aliphatic amino acids leucine, valine, and alanine at that site. Figure 5 A shows representative current families from these channels on different time scales to bring out the distinctive kinetic features of each channel type. In Figure 5 B, the range of change induced by these mutations in the steady state voltage dependence of the relative open probability is shown. For comparison, previously described fits of a fourth power of the Boltzmann function to the wt and F401I data are also included. The F401V G(V) relationship is shallower than that of the wt, and similar in slope (zapp = 2.5) to the F401I mutant. However, the V1/2 in F401V is positively shifted by ~5 mV compared with F401I. Steady state activation of the F401A mutant is the shallowest (zapp < 0.5 e0); in fact, the G(V) relationship fails to reach saturation at voltages in excess of +150 mV in five patches, and is therefore displayed on a dimensionless y axis. Unexpectedly, introduction at position 401 of a leucine, an amino acid chemically most similar to the isoleucine, carried nearly opposite consequences compared with the Sh5 replica mutation F401I. The F401L mutant has a G(V) relation as steep as that of the wt (zapp = 4.25) but with the midpoint of the activating transition shifted negatively (V1/2 = -69.7 mV), the only mutant in this study to do so.
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A look at the activation time course on the expanded time scale in Figure 6 A underscores that all channels bearing aliphatic substitutions for phenylalanine at position 401 activate more rapidly than the wt for a given voltage. Whereas F401L channels are least different from the wt, 401 isoleucine and valine channels are similar to each other and have faster kinetics than leucine channels; alanine channels are the fastest by far over all voltages. Quantitatively, Figure 2 B and 6 B show that the voltage dependence of activation time constants measured late in the activation process is similarly weak no matter which of the five residues is at position 401, with the apparent valence associated with the forward transitions, zf, ranging from 0.32 to 0.42 e0. The absolute values of the time constants, , are comparable except for F401A, in which they are significantly diminished. Regardless of whether F401 mutations diminish steady state voltage dependence of the currents, the voltage dependence of the forward rate, zf, remains in the wt range.
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We expected that, as in the case of Sh5 (F401I), the other aliphatic substitutions would preferentially perturb deactivating transitions. In Figure 7 A, time constants from fits to tail current relaxations are plotted for the leucine, valine, and alanine mutations. For comparison, fits to the voltage dependence of the deactivation time constant, , from wt and F401I are also included. The tail time constants of F401L currents are slower than those of the wt but have similarly steep voltage dependence. Deactivation kinetics of F401V (zr = 0.74 e0) are nearly the same as those of F401I, and F401A deactivation appears to be nearly voltage independent to the best of our ability to analyze its very rapid kinetics. This finding provides a ready explanation for the very shallow G(V) of F401A. In wild-type Shaker, a greater proportion of the total gating charge movement occurs after the transition state (
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Because several F401 mutants accelerate deactivation of macroscopic ionic currents, we hypothesized the faster rates for leaving the open state by deactivation should decrease the mean time spent in the open state. F401I has a unitary conductance similar to the wt but briefer open times (mean 2 vs. 4 ms in the wt), consistent with its faster deactivation kinetics. Single F401A channels show extremely brief, incompletely resolved openings that are seen promptly at the start of the test pulse (data not shown). Bandwidth limitations of the recording equipment did not allow us to pursue quantitative analysis of these channels, but qualitatively their behavior supports the hypothesis that isoleucine and especially alanine mutants accelerate transitions from the open state that reverse the activation sequence.
Gating Charge Movement in the F401 Mutants
One possible explanation for the reduction in the apparent valence of channel opening in Sh5 and related F401 mutants is an alteration in the coupling among charge-moving transitions. This could take the form of a transition (or transitions) that the channel must undergo during opening that has a voltage midpoint shifted far in the positive direction relative to the wt, such as has been proposed for several S4 and S4S5 linker mutations (
Families of gating currents from the wf and the wfF401L and wfF401A mutants are shown in Figure 8. The gating currents are shown superimposed and staggered to facilitate comparison of the development of kinetic features with changes in voltage. The wf ON gating currents (IgON) have a rising phase, appear at negative voltages, and show a slow decaying component in the voltage range where channels open. This latter component accelerates with further depolarizations. The overall time course of the IgON decay becomes faster in the order wf, wfF401L, and wfF401A, consistent with the faster time course of ionic current activation observed in the corresponding conducting species, although the F401A gating currents are accelerated to a lesser extent than the corresponding ionic currents. A prominent rising phase and slow decay appear in the wf OFF currents (IgOFF) at the voltages where there is a slow phase of the IgON decay, consistent with published observations from the cut-open oocyte clamp. (
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Steady state Q(V) relationships for the three channels were computed from the integral of the IgOFF after a test pulse. The integral of the IgON, while not shown, agreed closely. Figure 9 A shows that the wf Q(V) curve has a characteristically shallow base and steeper upper portion (see also
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We can compare the kinetics of the ON gating currents by fitting their decay phase with an exponential time constant. IgON is not well described by a single exponential at all voltages but, above -20 mV, these fits are useful as a way of assessing the overall kinetics of forward transitions in the channel. When these time constants are plotted against voltage for the wf and the wfF401L and wfF401A mutants (Figure 9 B), the rates of forward transitions are the fastest for wfF401A, followed by wfF401L and the wf channel. This mirrors the relationship among the activation time constants of ionic currents from the corresponding conducting channels. The voltage dependence for the movement of the charge "before" the transition states (zf) is conserved for the three channels, ranging between 0.57 and 0.63 e0. These values are similar to those estimated from ionic current measurements (Figure 6) and place important constraints on kinetic modeling of the early steps in channel activation.
The changes in both the ionic and gating currents reveal alterations in the voltage dependence and magnitude of the reverse rates out of the open state with the F401 residue replacements. The time course and voltage dependence of the forward activation rates are much less affected. These results suggest that the F401 mutations alter the energetic stability of the open state relative to closed states. Alterations in gating by changes of noncharged residues in the S4 segment have been interpreted in terms of a change in the energetics of a final cooperative opening step (
Behavior of Ionic Currents Indicates Nonindependence of Subunits
Shaker ionic currents activate upon depolarization with a delay, giving rise to a sigmoidal time course. This sigmoidicity arises from the multi-step nature of the activation process. Voltage dependence in the sigmoidicity of ionic currents is a diagnostic feature of deviation from subunit independence in activation (
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We sought to confirm further that the mutations at F401 alter the deviation from independence seen in wt channels upon entering the open state. As originally described by
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Gating Charge Movement Reveals Stability of the Open Conformation
Kinetics of the return of gating charge (IgOFF) upon stepping from a positive to a negative voltage provide important information about the voltage-dependent reverse transitions (those leading away from the open state). In potassium channels, their dependence on the duration and amplitude of the preceding depolarization has been extensively studied (
Figure 12 A contrasts the effect of test-pulse duration at three voltage levels on IgOFF in wf, wfF401L, and wfF401A channels. With pulses to -50 mV, charge return upon repolarization to -100 mV remains rapid in wf and wfF401A for pulse durations between 1 and 57 ms (wf) and 41 ms (wfF401A). F401L channels are significantly open at this voltage, however, and the OFF gating currents in wfF401L accordingly display progressively diminished amplitude and a prolonged declining phase as pulse length exceeds ~3 ms. A pulse amplitude of -30 mV (Figure 12 A, middle left) marks a transition zone for the kinetics of the wf IgOFF. Pulses of a few milliseconds duration do not impede subsequent rapid charge return, those longer than ~10 ms give rise to OFF currents with complex time courses in which at least three kinetic components can be recognized, and those >25 ms produce a rising phase and exponential decay. wfF401L OFF gating currents with all but the shortest -30 mV pulses are notable for the greatly slowed charge return that is incomplete after up to 30 ms at -100 mV (Figure 12 A, middle center). The families of wf and wfF401L channels show progression of the same trends when the pulse amplitude is 0 mV. In fact, IgOFF becomes nearly "immobilized" in wfF401L, displaying protracted decay after pulses lasting longer than 34 ms.
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wfF401A gating currents are unlike those of the other two species. With pulses to 0 and +100 mV, the latter of which are sufficient to activate many F401A channels, the time course of the OFF currents is rapid and unaffected by pulse length (Figure 12 A, right). The small steady state outward current seen at +100 mV is an ionic current contaminant, likely of native Xenopus oocyte origin because its appearance at that voltage is variable among different cells and does not depend on the level of channel expression. Its tail current also accounts for a very small slow phase on the OFF gating currents after longer pulse durations. The results for the wf (left), wfF401L (center), and wfF401A (right) channels are summarized in Figure 12 B, which plots the time constants from exponential fits to the decaying phase of IgOFF as a function of the length of pulses at the different pulse voltages. The time course of the OFF gating currents has a complex waveform, and these single-exponential fits are not meant to imply that there is an underlying first-order kinetic process; rather, they provide a ready means to document a transition from a predominantly fast to a slow process. For wfF401A, they additionally illustrate that as the probability of channel opening is changing over a voltage range of 150 mV, the kinetics of charge return at -100 mV are barely altered.
Kinetic Mechanisms for Nonindependent Gating
Ionic and gating current results described in the preceding section imply that mutating phenylalanine to alanine at position 401 drastically diminishes the cooperative stabilization of the open state characteristic of the wild type, whereas the leucine mutation augments it. In the following, we investigate this hypothesis quantitatively, using kinetic principles and models previously developed for Shaker gating.
Several general features of Shaker gating have been established by diverse means in different laboratories (e.g.,
One relatively simple formalism that has been put forth to account for these features of gating is the scheme of by which the first closing transition rate is divided. Otherwise, the activation pathway in this model is an independent process involving four gating subunits, each undergoing two sequential charge-moving transitions. The ZHA model is shown in an abbreviated form in Figure 13 A, emphasizing the fourfold symmetry. We used the parameters of the original ZHA model (
, which was either 1, 9.4, or 50. These were chosen to give the best overall approximations of the F401A, wt, and F401L channels' behavior, respectively. For F401A, the cooperativity factor (
) was set to a value of 1, the equivalent of complete subunit independence. The model succeeds in correctly describing the order of relative steepness of the G(V) curves, of the deactivation kinetics, and of the duration dependence of IgOFF. However, the extremely shallow voltage dependence of F401A channel opening (Figure 5) could not be reproduced by the model, even with the introduction of a modest amount of negative cooperativity (
= 0.4; Figure 13 B).
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The ZHA model provided a convenient starting point for arriving at kinetic descriptions of the wt and mutant currents. Manipulating only the degree of cooperative slowing of the first closing transition with changes to the factor does surprisingly well in describing the basic properties of the F401 mutant channels. However, the model proves inadequate for the quantitative agreement with the macroscopic ionic and gating current data. Proper fits to the kinetics of channel opening, steady state G(V) relations, ON gating currents, and the steady state Q(V) curves all require manipulation of additional parameters of the ZHA model and in a number of cases were unattainable without altering the fourfold symmetric structure that had made it so conceptually attractive. Therefore, we broadened our consideration of candidate models to include ones where a separate concerted transition (or transitions) connects the four parallel and independent activation pathways (one per subunit) to the open state. Precedents for this mechanism can be found in earlier models for potassium channels (
A detailed kinetic model of this class has been proposed for Shaker and a mutant channel (V2) ( Open transition, compared with 9 for the ZHA model and 20 for the 3+2' model of
and
and the two reverse rates ß and
for each of the four subunits and of the forward rate
and the reverse rate
for the concerted transition. The rates are assumed to be instantaneous exponential functions of voltage, according to:
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The total charge displacement for each channel is constrained to be ~14 e0 (14.47 e0 for the wt, 14.37 e0 for F401A, and 14.07 e0 for F401L), in agreement with previously published measurements in wt (
Kinetic transitions that follow channel opening at depolarized potentials are to states that are not obligatorily traversed during the activation process. These have been characterized using single-channel recordings of wt Shaker ( Cf... transition to the same values in all three channel species rather than let them vary among the wt, F401L, and F401A.
Testing Model Predictions in wt and Mutant Shaker Channels
Figure 15 A shows the fits of the model shown in Figure 14 for the wf, wfF401L, and wfF401A channel's steady state charge vs. voltage curves. Equilibrium constants for the two charge-moving transitions in each subunit and for the concerted step were optimized to obtain the desired steepness and position along the voltage axis. The total charge displacement for a given transition is the sum of the charges that move before and after the transition state or, equivalently, that are associated with the forward and backward rates of that transition. The three channels differ the most in their equilibria for the concerted opening transition. The zero-voltage equilibrium constants for this step are 55, 125, and 0.5 for the wf, wfF401L, and wfF401A, respectively. The marked decrease in wfF401A provides part of the explanation for the shallowness of the slope of its Q(V) curve, even though this transition carries only ~1/16 of the total charge displacement in the channel. To describe accurately the relatively shallow lower portion of each of the curves, it is necessary to make the charge displacement associated with the second of the two sequential subunit transitions (z) greater than that of the first (z
ß) (
is 2.02.1 e0. The first transition carries the charge of 1.35 e0. Models for wfF401L and wfF401A channels make the zero-voltage equilibrium constant for the first transition,
approximately twice as great, and that for the second transition,
nearly three times as great as those of the wf model in order to account for the more negative voltage range over which the initial gating charge movement occurs in the mutants.
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In Figure 15 B, model fits are shown superimposed on families of gating currents. The fits are a good description of the time course of IgON obtained at depolarized voltages and of the IgOFF. However, the models predict too rapid a rise and decay of the ON gating currents in the activating voltage range (approximately -80 to -40 mV for all three channels). The data suggest the presence of a rising phase in the IgON records even at these negative voltages. We were able to qualitatively improve on these fits by introducing a three-step per subunit activation sequence after 0 and
0. The wf time constant from such fits is ~2.5 ms (n = 11), which is over two times greater than the time constants for wfF401L and wfF401A. This provides the rationale for assigning the values of
0 = 560 s-1 and
0 = 1,340 s-1 for wt, or about one half of the corresponding zero-voltage rates for the two mutants. As described earlier, the kinetics of IgOFF are very sensitive to the amplitude of the voltage step in the wf and especially wfF401L, but not in wfF401A. The model is able, by the large differences in the first closing rate
, to account for the time course of the OFF gating current in the three families.
Inspection of the time course of the gating charge return as a function of pulse duration (Figure 12) reveals important differences among the three channel species. Model fits to these data, shown in Figure 16, indicate that our simulations are adequate to describe the time course of IgOFF for a variety of pulse durations at -50 and 0 mV. In particular, the observed slow decay of OFF gating currents observed in wfF401L at -50 mV at longer pulse durations as the consequence of significant open probability of the channels at that voltage is observed in simulated traces. The complex waveform of wf IgOFF after 0-mV pulses lasting ~10 ms deviates somewhat from the predictions of our model and would probably be better fit with the introduction of additional steps in the activation pathway (see above). The model predicts that the gating charge return will be rapid and independent of pulse duration in wfF401A, which is clearly a feature of our data.
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The predictions of the models for the macroscopic ionic currents are shown in Figure 17. Representative families of activating currents recorded from patches containing wt, F401L, and F401A channels are qualitatively comparable to model simulations at matching voltages in terms of the overall sigmoidal character and the voltage range over which activation kinetics are most noticeably changing. A consistent finding for all three channels is that the model traces appear to have a slower overall time course than the patch data. This discrepancy is quantified in Figure 17 B, which displays model predictions for the time constant of activation derived from fitting the late phase of current time course (
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In evaluating model predictions for the steady state open probability vs. voltage, we took notice of the observed differences in the voltage positions of G(V) curves obtained using wt cell-attached and excised patches. The former tended to be shifted positively by ~6 mV (data not shown). When displaying the G(V) curves for wt, F401L, and F401A with their respective models in Figure 18, we similarly offset the simulated curves by between -6 and -7 mV to compare them with the experimental results. With this correction, both the steepness and the midpoint of the voltage dependence for wt and F401L channels were well described by the model. As earlier, the shallowness of the G(V) relation in the F401A mutant precludes us from observing saturation of the open probability within the attainable voltage range. Therefore we cannot meaningfully normalize G(V) data from different patches for direct comparison. Instead, G(V) relations from a representative F401A patch obtained by two means [isochronal tail G(V) and pulse G(V)] are shown in Figure 18. Model traces for F401A were analyzed in the analogous manner, and the resulting G(V) curves are shown scaled to match F401A curves at +100 mV after a -6-mV shift along the x axis. The model, which postulates that the extremely shallow G(V) curve results from a destabilization of the open state by greatly speeding the initial closing transition, is qualitatively supported by these data.
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The proposed alterations in a backward transition leading away from the open state are expected to affect the time course of macroscopic ionic tail currents. Figure 19 (top) shows, for the wt and the two F401 mutants, the decay in the relative open probability as a function of time when voltage is stepped from a depolarized value of +50 mV to hyperpolarized potentials. All current traces are normalized to match their initial amplitudes. In Figure 19 (bottom), these data are re-plotted on a logarithmic time axis. These two transformations allow a closer examination of the kinetics of deactivation at the more hyperpolarized voltages at which tail currents are very small and rapid. Additionally, a logarithmic time scale would bring out the convergence of the open probability traces to an asymptotic value at very low voltages if a single closing transition were to become rate limiting. We used the models for the wt and F401L in which initial closing rates were modified somewhat to reflect the slower deactivation kinetics in excised patches. With 0 set to 50 s-1 for the wt and 29 s-1 for F401L, the open probability decay is well described by the model predictions for both channels over the range -60 to -160 mV (-180 mV for F401L). The valence of 0.6 e0 assigned to this rate is the minimum required to produce the necessary spacing of the traces at voltages below -120 mV (Figure 19, bottom). Less charge associated with the first closing step predicts a rate-limiting step that is not evident in the data. The analysis of F401A deactivation (Figure 19, right) is complicated by the extreme rapidity of its tail currents over a wide voltage range. Fitting exponential functions to their time course yields time constants in the range of 100500 µs, which are difficult to resolve well when the current amplitudes are small. Additionally, there is a prominent component of OFF gating current (note the lowermost trace in the panel which was taken at -90 mV) that is slower than the ionic tail in this mutant and significantly distorts its kinetics. Simulated F401A traces have a multi-exponential decay, with the very rapid component near the limit of resolution in our recordings (and perhaps more rapid than the excised patch data), and a slower component that at -30 and -50 mV cannot be reliably distinguished from the steady state component seen in the experimental data. Because of the nonionic components of the current decay (which the model does not take into account), more detailed comparison of the model simulations to the tail currents in F401A at hyperpolarized voltages was not undertaken.
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Sigmoidal activation kinetics are a cardinal feature of Shaker channel gating and, as shown in Figure 10, they remain present in the F401 mutants. The amount of sigmoidicity, as defined earlier, and the way it varies with pulse potential is a sensitive means to assess the presence of a slow first reverse transition from the open state (
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Discussion |
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In this paper, we have confirmed, with direct evidence obtained from channels without N-type inactivation, the hypothesis put forth by
We used F401, an important residue with known effects but a poorly understood mechanistic role in the activation process, as a molecular handle toward further elucidating the role of the S5 segment in gating. Progressively smaller aliphatic substitutions at this site produced more profound effects, with the important exception of leucine, as though the steric bulk of this residue is an important determinant of channel gating. A study of chimeras between Kv2.1 and Kv3.1 channels demonstrates that exchanging NH2 terminus ends of S5 transfers differences in both deactivation of ionic currents and the OFF gating currents ( carbon, as is the case for leucine, their more proximal branch point limits the flexibility of the main chain backbone (
The reduction in the apparent effective activating valence of the F401 mutations can be considered within the framework of several hypotheses: reduction in actual gating charge content, displacement of the voltage range over which some of the major charge-moving transitions take place, and altered coupling between a minor charge-moving transition intimately associated with channel opening and the rest of the activation pathway. Measurements of only steady state ionic currents are usually inadequate for distinguishing among these possibilities. Measurement of the limiting slope of activation (
With the aid of gating current recording, there are now two approaches available to count gating charges for testing the first hypothesis: one relies on ionic current fluctuation analysis (
An intriguing hypothesis for the role of F401 in wt gating is that the wt phenylalanine side chain is engaged in a cation-aromatic interaction with a basic amino acid in S4, reducing the energy penalty for having an unshielded gating charge buried in the membrane bilayer. While a few acidic residues in the S2 and S3 transmembrane segments appear to interact with some of the carboxy-terminus S4 basic amino acids, as indicated by second-site rescue mutations (
In this light, the very fast gating behavior of the F401A single channel is similar to the published description of the charge-conserving S4 mutant R377K (-electron ringguanidinium interaction. This mechanism of stabilization also provides an explanation for the behavior of the lysine mutant at 377 (
To explore this possibility further, we made a single point mutation, substituting a negatively charged glutamic acid at position 401 in the hope of observing effects of a salt bridge formation between it and R377. Also, we made a double mutant F401AR377Q with the expectation that the small side chain bulk of alanine at position 401 might rescue the expression of R377Q currents by permitting the latter residue greater steric freedom. Neither construct gave rise to functional channels as detected by the ability to measure ionic currents. We cannot exclude the possibility that channel protein was made but did not undergo correct trafficking or cell membrane insertion. Therefore, the possibility of an S4 arginineS5 aromatic interaction in gating remains an open question.
Upon initial inspection of the wt, F401A, and F401L ionic currents, their dissimilarities are quite striking. However, we observed considerable similarity among the channels in the movement of the gating charge among closed states at the low voltages where few channels open. Therefore, we sought to compare the properties of these channels that would be expected to change as the result of channel opening. Recent work on potassium channel gating mechanisms (
Our model for the activation of Shaker and the two F401 mutations succeeds in describing a common gating mechanism. The differences in the models for the three channels are few and are limited, primarily, to the concerted opening transition. We can compare the effects of the mutations on the stability of the open conformation of the channel by calculating the free energy difference (G) between the last closed state Cn and the open state from the rates of the final opening and first closing transitions in our model:
The value of G for the wt at 0 mV is -2.3 kcal/mol, which is increased to -2.8 kcal/mol by the F401L mutation. Correspondingly, both these channels are found overwhelmingly in the open state at 0 mV. The open state is, in contrast, less stable than the last closed state (Cn) by +0.4 kcal/mol in the F401A mutant. Therefore, the overall change in free energy difference (
G) between these conformations resulting from the F401A mutation is 2.7 kcal/mol. However, since the F401A mutation is present in all four subunits of the channel protein involved in the concerted transition, considerations of symmetry lead us to conclude that four weaker interactions, each contributing ~0.7 kcal/mol to the open channel stability, are disrupted in the mutant channel. It is intriguing to consider the energetic cost of the possible lost parallel stacking interactions between an aromatic ring and an S4 arginine side chain (
The free energy difference between a channel in the open and next-to-open state could be greater than expected from independence because of a favorable binding interaction between a channel and permeant (or blocking) ions (
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Footnotes |
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Portions of this work have been previously published in abstract form (Kanevsky, M., and R.W. Aldrich. 1994. Biophys. J. 66:A283; Kanevsky, M., and R.W. Aldrich. 1995. Biophys. J. 68:A136).
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Acknowledgements |
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We thank L. Toro for the kind gift of Shaker W434F high-expression vector, and T. Hoshi and D. Perkins for developing and perfecting modeling software. We acknowledge the help of C.A. Warren, J. Haab, and W.N. Zagotta in making several of the mutants. C.J. Smith-Maxwell and E.M. Ogielska made helpful comments on the manuscript, and Gargi Talukder helped with the figures.
M. Kanevsky was supported by Medical Scientist Training Program grant GM07365 from the National Institute of General Medical Sciences. R.W. Aldrich is an investigator with the Howard Hughes Medical Institute. This study was supported by the National Institutes of Health grant NS23294.
Submitted: December 24, 1998; Revised: June 14, 1999; Accepted: June 15, 1999.
1used in this paper: G(V), steady state voltage dependence of the open probability; Kv, voltage gated; Q(V), charge displacement versus voltage; S4, fourth transmembrane segment; S5, fifth membrane-spanning segment; wt, wild type
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