Mutations throughout the S6 region of the hKv1.5 channel alter the stability of the activation gate

Thomas C. Rich1,*, Sarita W. Yeola2,*, Michael M. Tamkun3, and Dirk J. Snyders2

Departments of 1 Biomedical Engineering, 2 Medicine and Pharmacology, and 3 Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee 37232


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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First published September 21, 2001; 10.1152/ ajpcell.00232.2001.---The S6 segment of voltage-gated K+ channels is thought to contribute to the gate that opens the central permeation pathway. Here we present evidence that mutations throughout the cytoplasmic end of S6 strongly influence hKv1.5 channel gating characteristics. Modification of hKv1.5 at positions T505, V512, and S515 resulted in large negative shifts in the voltage dependence of activation, whereas modifications at position Y519 resulted in negative (Y519N) and positive (Y519F) shifts. When adjusted for the altered voltage sensitivity, activation kinetics of mutated channels were similar to those of the wild-type (WT) channel; however, deactivation kinetics of mutations T505I, T505V, V512A, and V512M [time constant (tau ) = 35, 250, 170, and 420 ms, respectively] were still slower than WT (tau  = 8.3 ms). In addition, deactivation of WT channels was highly temperature sensitive. However, deactivation of T505I and V512A channels was largely temperature insensitive. Together, these data suggest that mutations in S6 decouple activation from deactivation by altering the open-state stability and that residues on both sides of the highly conserved Pro-X-Pro sequence influence the movement of S6 during channel gating.

potassium channels; structure-function analysis; temperature dependence of channel gating


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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MUCH RECENT WORK has focused on understanding the mechanisms that allow voltage-gated ion channels to sense changes in membrane potential. The fourth transmembrane segment (S4) is considered to be the voltage sensor of voltage-gated ion channels. Mutations that neutralize charged residues in this region reduce the voltage sensitivity (9, 17, 19). However, more conservative mutations that do not alter the number of charges in S4 also alter the voltage dependence of activation (17, 23, 25, 26). Recent studies have provided additional evidence for voltage-dependent conformational changes by monitoring the relative movements of S4 during the gating process (3, 7). The S2 region also has been implicated as part of the voltage sensor (14, 24).

Although these studies have revealed important mechanisms involved in the voltage sensitivity of K+ channels, much less information is known about how movement of S4 triggers the opening and closing of the permeation pathway. Early studies by Armstrong (1, 2) showed that tetraethylammonium blocked the K+ channels of squid axons when the channels were in an activated state. He proposed that conformational changes in the protein structure made the tetraethylammonium binding site accessible when the channel was activated and that the activation gate was located in the inner mouth of the pore. At the molecular level, the S6 region has been identified as a potential component of the inner mouth of the pore and the binding site for quaternary ammonium compounds (4, 13). Liu et al. (11) introduced cysteine residues at sites in the S6 region of Shaker to examine the accessibility of these residues to the methanethiosulfonate reagents. They found that several residues within the S6 region were accessible only when the channel was in an activated state, suggesting that the movement of an intracellular gate regulates access to the pore. Consistent with this result, modification of a cysteine residue in the S6 region of the Kv2.1 channel altered open-state stability and ion permeation (12).

The understanding of channel permeation and gating has been greatly advanced by the crystal structure of the bacterial K+-selective channel KcsA (6). In this channel the second transmembrane regions (equivalent to S6 in hKv1.5) of the four subunits intersect to form a teepee-like structure with the tip near the cytoplasmic end. A diaphragm-like movement of this region increases the diameter of the permeation pathway of the KcsA channel (18). Li-Smerin et al. (10) used tryptophan scanning to study the interactions of the S5-pore helix-S6 region of the Shaker channel. They found that when mutations that alter channel gating (found throughout these regions) are mapped onto the KcsA channel structure, they tend to cluster near the interface between pore domain subunits. Although the KcsA channel can be used as a model for the equivalent S5-S6 regions of voltage-gated K+ channels, there is evidence that, unlike KcsA, there is a sharp bend toward the cytoplasmic side of the S6 segment of the Shaker channel (5). Such a bend could be caused by the Pro-X-Pro sequence that is highly conserved among voltage-gated K+ channels and probably crucial for formation of functional homotetrameric channels. Indeed, subunits of the Kv1 through Kv4 subfamilies display this sequence and form functional homotetrameric channels, whereas those of the Kv5 through Kv9 subfamilies do not.

To further examine the role of S6 in channel gating, we made a series of mutations near the Pro-X-Pro sequence (P509-V510-P511) of the hKv1.5 voltage-gated K+ channel (Fig. 1). When adjusted for shifts in voltage-dependent gating, the activation kinetics of mutated channels were similar to those of the wild-type (WT) channel. Interestingly, even when adjusted for these shifts, the deactivation kinetics of T505I, T505V, V512A, and V512M channels were dramatically slower than those of WT, and those of Y519F were faster. We also tested for differences in the temperature dependence of activation and deactivation among WT channels and two of the slowly deactivating mutations, T505I and V512A. Activation of all three channels and deactivation of WT channels were highly sensitive to temperature. Surprisingly, deactivation of T505I and V512A was largely insensitive to temperature (20° <=  T <=  40°C). These data provide evidence that residues on both sides of the Pro-X-Pro sequence greatly affect the stability of the activation gate and that mutations to these residues can decouple channel activation from deactivation.


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Fig. 1.   Amino acid sequence of the S6 region of hKv1.5. A: alignment of the S6 region of several K+ channels. In the region of S6 examined, sequence variability occurs at T505, L508, V510, V512, S515, N518, and Y519. Except for V510 and N518, these residues can be aligned into an alpha -helical projection of the putative pore region (29). Asterisks indicate cysteine-substituted residues in Ref. 11 (homologous to hKv1.5 at positions I506, V510, P511, V512, and I513). B: two-dimensional schematic of hKv1.5 structure. The relevant amino acids in S6 are given as one-letter codes. Mutations that did not express functional channels were T505D, T505K, V512E, V512D, I513A, and F517V.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
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Cell preparation. The PCR-based method for site-directed mutagenesis in the S6 region and the mutations used to form stable cell lines expressing the mutated channels (29) and cell culture procedures (27) have been described elsewhere. Briefly, mouse Ltk- cells stably expressing WT or mutated hKv1.5 channels were cultured in DMEM supplemented with 10% horse serum and 0.25 mg/ml G418. The cultures were passed every 4-5 days by using a brief trypsin treatment. Before experimental use, cultures were incubated with 2 µM dexamethasone for 12-24 h to induce efficient channel expression. The cells were removed from the dish with a rubber policeman, stored at room temperature, and used within 12 h.

Electrical recording. Recordings were obtained using an Axopatch-200A patch-clamp amplifier (Axon Instruments, Foster City, CA) and the whole cell patch-clamp technique. Pipettes were pulled from starbore borosilicate glass (Radnoti, Monrovia, CA) and heat polished. Bath temperature (11° <=  T <=  45°C) was maintained within ±0.5°C of the set point by using a Peltier device. To ensure adequate voltage control in the whole cell configuration, pipette resistance was limited to 3 MOmega and averaged 2.4 ± 0.1 MOmega (n = 120). Voltage offsets were zeroed with the pipette in the bath solution. No additional corrections were done for the small liquid junction potential difference, which is estimated to be <5 mV with the solutions used (15). Pipettes were then lowered onto the cells, and gigaohm seals were formed by applying light suction (9.9 ± 0.7 GOmega ). After whole cell configuration was achieved, capacitive transients were elicited by applying 20-mV steps from the holding potential (-80 mV) and recorded at 40 kHz (filtered at 10 kHz) for calculation of access resistance and input impedance. Capacitance and series resistance compensation were then optimized to achieve 80% compensation. We calculated the residual resistance and excluded those experiments in which the voltage error due to series resistance exceeded 5 mV. Current records were sampled at 2-10 times the anti-alias filter setting, stored on a personal computer, and archived on an optical disk. Software for acquisition that allowed custom protocols (both stimulation and sampling) not available in commercial packages was designed in house (20, 27).

Solutions. The intracellular pipette filling solution contained (in mM) 110 KCl, 10 HEPES, 5 K4-BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid), 5 K2ATP, and 1 MgCl2 and was adjusted to a pH of 7.2 using KOH. This gave a final K+ concentration of ~145 mM. The bath solution contained (in mM) 130 NaCl, 4 KCl, 1.8 CaCl2, 1 MgCl2, 10 HEPES, and 10 glucose and was adjusted to pH of 7.35 with NaOH. The pH of the solutions decreased ~0.1 units with a 10°C increase in temperature due to the temperature dependence of the buffer. Thus the pH of the solutions at high temperature remained within 0.2 pH units of the pH at room temperature. Such small changes in pH did not significantly affect gating at room temperature.

Analysis. Results were assessed by calculating time constants of activation and deactivation and then calculating current-voltage relationships. All software for the analysis presented was written in house using Visual Basic, FORTRAN, and MATLAB. The curve-fitting procedure to calculate time constants (and their relative amplitudes) used either a nonlinear least-squares (Gauss-Newton) algorithm or a simplex algorithm. The number of exponentials required and goodness of fit were evaluated by inspecting the residuals for nonrandom trends and comparing chi 2 values statistically. All gating models were optimized with software written in MATLAB by using the least-squares error criterion.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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To examine the role of the inner pore in channel gating, we scanned the putative pore lining residues of the hKv1.5 channel with a series of single amino acid substitutions to the cytoplasmic end of the S6 region (Fig. 1). Currents elicited from functional mutations at positions T505, L508, V512, S515, and Y519 (capitalized residues in Fig. 1) were examined and compared with those of WT. Although these mutations were in the putative pore region, they did not significantly alter the reversal potential; thus K+ selectivity was maintained (Table 1). Two mutations that introduce charged residues at positions T505 (T505D and T505K) and V512 (V512E and V512D) as well as the mutations I513A and F517V failed to yield functional channels.

                              
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Table 1.   Characteristics of currents obtained from WT and mutated channels

Effects of S6 mutations on deactivation. To examine the time course of deactivation, we monitored current decay at membrane potentials between -30 and -110 mV following 50-ms depolarizations to +50 mV. In most cases deactivation was best fit with two exponentials (Fig. 2). In general, the amplitude of the dominant time constant was >70% of the total amplitude (Table 1). The deactivation of WT channels proceeded in a voltage-dependent manner with a dominant time constant of 8.3 ms at -100 mV (27). The deactivation of the mutated channels also proceeded in a voltage-dependent manner (Fig. 3). The deactivation of some mutations was similar to that of WT; however, the deactivation of other mutations was dramatically altered (Fig. 3 and Table 1). At position T505, the deactivation of T505S was 2-fold faster than that of WT, whereas the deactivation of T505A was almost 2-fold slower. The deactivation of T505I and T505V were 4- and 30-fold slower than that of WT, respectively. At position L508, L508M deactivation kinetics were similar to those of WT. Examination of L508A was limited by the low level of expression; however, deactivation kinetics appeared faster than those of WT (data not shown). At position V512, the mutations V512A and V512M both displayed a marked slowing of deactivation (20- and 50-fold slower, respectively). At position S515, introduction of both a positive (S515K) or a negative charge (S515E) was tolerated at this point, but currents for S515K were too small for detailed analysis. The deactivation kinetics of S515E were slower than those of WT at -50 mV; however, at more hyperpolarized potentials, the deactivation kinetics were similar to those of WT. At position Y519, both Y519F and Y519N displayed slower deactivation kinetics than WT.


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Fig. 2.   Exponential fits to deactivating currents of T505I channels measured at -50 mV after a 50-ms depolarization to +50 mV. Top: residual errors for the monoexponential (1 exp) and biexponential (2 exp) fits: monoexponential, tau  = 315 ms; biexponential, tau 1 = 322 ms, tau 2 = 51 ms, Atau 1 (amplitude of tau 1 component) = 80%. Bottom: fits to deactivating current (I) in semilogarithmic format. The two components of a biexponential fit are shown; the slow component is superimposed on the original data, and the fast component is superimposed on data points calculated by subtracting the slow component. Systematic deviations were absent from the biexponential fits.



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Fig. 3.   Slowing of channel closing by mutations in S6. Top left panel shows currents of wild-type (WT) channels from the entire pulse protocol (inset). Remaining panels show expanded views of the deactivating currents of WT and mutant channels measured at potentials of -50, -70, -90, and -110 mV. The mutations T505I, T505V, V512A, and V512M caused substantial slowing of channel closing. Vertical scale bar represents 300 pA. Note the changes in time scale for the various mutations.

The hKv1.5 channel is a strong outward rectifier; therefore, to more readily observe deactivation at potentials below -60 mV, we examined deactivation of some mutated channels in 145 mM external K+ (Fig. 4). Under these conditions large inward tails were observed. Deactivation of WT and mutant channels at -110 mV could be largely described by single exponentials with the following time constants: WT, 5 ms; T505I, 83 ± 5 ms (n = 6); T505V, 770 ± 110 ms (n = 5); V512A, 360 ± 20 ms (n = 4); and V512M, 730 ± 30 ms (n = 8). A problem with the analysis of deactivation kinetics for mutations T505I, T505V, V512A, and V512M was that the extremely slow deactivation time constants were similar to the voltage-independent inactivation time constants (240 and 2,700 ms) previously described (20, 27). Thus it is likely that both deactivation and slow inactivation contributed to the time course of channel closure, and the deactivation rates may be slower than indicated by the time constants.


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Fig. 4.   Representative traces of T505I (A), T505V (B), V512A (C), and V512M (D) currents measured in high extracellular K+ ([K+]o = 145 mM). The currents were elicited by 50-ms depolarizations to +50 mV followed by hyperpolarizations to -110 mV. Dashed lines depict scaled WT currents. Under these conditions, the slowing of channel closing caused by these mutations is obvious even at strong hyperpolarizations.

Effects of S6 mutations on the midpoint of activation. Activation of WT channels displayed a sigmoidal voltage dependence with a midpoint of activation (Eh) of -14 mV in the presence of the Kvbeta subunit endogenously expressed in mouse Ltk- cells (27, 28). We observed that the voltage dependence of activation of several of the mutations was similar to that of WT, whereas the voltage dependence of other mutations was dramatically altered (Table 1). T505A and T505S caused small shifts in the voltage dependence of activation (<5 mV). T505I caused a slightly larger shift (-8.5 mV). However, T505V caused a shift of -36 mV in the voltage dependence of activation. L508M displayed a shift of +8.5 mV in the voltage dependence of activation. L508A also caused a positive shift, but the current amplitudes were too small for accurate assessment of this shift. Both V512A and V512M caused large negative shifts (greater than -27 mV) in the voltage dependence of activation. The shift of -12 mV in the voltage dependence of S515E activation was opposite to that expected from a local charge effect on the voltage sensor. It is unlikely that this residue is located at a position extracellular to the voltage sensor; it seems more likely that this residue is not in close proximity to voltage sensor (e.g., it may be pointed toward the permeation pathway). This shift was much smaller than that caused by hydrophobic substitutions at nearby residues. Interestingly, mutations at position Y519 caused opposite shifts in Eh: +12 and -13.5 mV for Y519F and Y519N, respectively.

The kinetics of channel activation were characterized by a dominant exponential component with a time constant of ~2 ms (+60 mV) for WT channels. Figure 5, A-C, shows the time constant of the dominant exponential of channel kinetics for both WT (solid line) and mutant (dotted lines with symbols) channels. For each of the mutations the time constants of activation decreased with increasing membrane potential (Em). When the membrane potential was adjusted for shifts in the midpoint of activation, Em - Eh (Fig. 5, D-F), it became apparent that, for each mutation, the activation time constants displayed a similar voltage dependence to WT. Furthermore, the changes in activation kinetics at strong depolarizations were strongly and linearly correlated with shifts in Eh (Fig. 6A). Although the mutations with the slowest deactivation kinetics displayed negative shifts in Eh, the substantial slowing was disproportional to the observed shift in Eh (Fig. 6B).


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Fig. 5.   Voltage dependence of dominant activation and deactivation time constants (tau ). Time constants at membrane potentials above the midpoint of activation (Eh; see Table 1) were obtained by exponential fits of the time course of the activating current at indicated potentials (4 <=  n <=  10). Time constants at membrane potentials below Eh were obtained from deactivating currents (see Fig. 4) at indicated potentials. Solid line represents WT kinetics. Curves in A (T505V, T505I, T505A, and T505S), B (V512M and V512A), and C (Y519N, S515E, L508M, and Y519F) show the activation and deactivation time constants of mutated channels vs. the actual membrane potential (Em). Curves in D-F (same mutations as in A-C) show the time constants vs. a shifted membrane potential [Eh of each mutation subtracted from Em (Em - Eh)]. It is apparent that the impaired closing of mutations T505I, T505V, V512A, and V512M was not due to a pure shift in the voltage dependence of activation but, rather, that the shift in the voltage dependence of activation of these mutants was in part due to the slowed deactivation kinetics.



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Fig. 6.   Relationship between dominant activation and deactivation time constants and voltage dependence of activation. A: dominant time constants of activation obtained from exponential fits to currents measured at depolarizations to +60 mV (tau +60). The activation time constants varied linearly with the midpoint of activation (slope = 0.033 ± 0.007, intercept = 2.8 ± 0.2, P = 0.0028). The strong linear trend indicates that changes in forward transitions along the activation pathway are not responsible for observed shifts in the voltage dependence of activation. Eh was estimated by fitting the Boltzmann equation to currents obtained at -30 mV (-60 mV for T505V, V512A, and V512M) following 250-ms depolarizing steps to potentials between -80 and +60 mV. B: dominant time constants of deactivation were estimated from exponential fits to currents obtained at -100 mV (tau -100) following 50-ms depolarizations to +50 mV (note the logarithmic scale of the vertical axis). Each point represents the mean ± SE (n >=  4) for both Eh and tau .

Effects of S6 mutations on the temperature dependence of activation and deactivation. To further examine the relationship between channel activation and deactivation, we measured the temperature dependence of WT, T505I, and V512A kinetics. We used the dominant time constants of channel activation and deactivation rather than trying to dissect entropic and enthalpic contributions of individual transitions because of the difficulty in separating the effects of temperature on individual transitions. At increased temperature we observed that WT channels activated at more negative potentials. The midpoint of activation shifted from -12.8 ± 0.9 mV at 22°C to -25.1 ± 2.3 mV at 32°C. The dominant activation time constant also was temperature dependent (at +50 mV: tau 22°C = 2.0 ± 0.1, tau 32°C = 0.52 ± 0.04 ms). At temperatures above 28°C and strong depolarizations (+50 mV). the time constant of activation was best fit with a single exponential. As with WT, both T505I and V512A activated at more negative potentials at elevated temperatures. For T505I, the midpoint of activation shifted from -21.5 ± 1.5 mV at 22°C to -36.5 ± 1.6 at 32°C. Similarly, for V512A, the midpoint of activation shifted from -40.4 ± 1.0 mV at 22°C to -56.0 ± 3.0 at 29°C. The dominant activation time constants of T505I and V512A were also temperature dependent (T505I at +50 mV: tau 22°C = 2.6 ± 0.3 ms, tau 32°C = 0.64 ± 0.10 ms; V512A at +50 mV: tau 22°C = 2.4 ± 0.2 ms, tau 32°C 0.98 ± 0.02 ms). The activation time constants of WT, T505I, and V512A decreased in a log-linear fashion as temperature increased (Fig. 7, A-C). Thus the overall temperature dependence of the activation kinetics of the T505I and V512A channels was similar to that of WT. Deactivation of WT channels also was temperature dependent (at -60 mV: tau 22°C = 21.7 ± 3.4 ms, tau 37°C = 6.0 ± 0.4 ms). As with the activation time constant, the WT deactivation time constant decreased in a log-linear fashion with increased temperature (Fig. 8A). However, unlike the WT deactivation, T505I deactivation displayed little or no temperature dependence between 22 and 37°C (Fig. 8B). Deactivation of V512A also appeared temperature independent between 22 and 30°C (at -110: tau 22°C = 140 ± 31 ms, tau 28°C = 143 ± 12 ms). Although a more complete assessment of the temperature dependence was precluded by the very negative threshold of activation at elevated temperatures (especially of V512A), these data demonstrate that T505I and V512A channels close slowly, in a largely temperature-insensitive manner.


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Fig. 7.   Temperature dependence of channel activation. A: temperature dependence of WT hKv1.5 activation time constants (tau act) fit to data measured at depolarizations to +50 mV. Line represents an exponential fit to data with a "temperature coefficient" of 4.3°C-1 (corresponding to a Q10 of 3.3 for the activation rate constant 1/tau ). The holding potential was -80 mV for 10 < T < 28°C, -90 mV for 28 < T < 37°C, and -100 mV for T > 37°C, where T is temperature. B: temperature dependence of T505I tau act fit to data measured at depolarizations to +50 mV. Line represents an exponential fit to data with a temperature coefficient of 4.8°C-1 (corresponding to a Q10 of 3.2). The holding potential was -80 mV for 10 < T < 28°C, -90 mV for 28 < T < 32°C, and -100 mV for T > 32°C. C: temperature dependence of V512A tau act fit to data measured at depolarizations to +50 mV. Line represents an exponential fit to data with a temperature coefficient of 6.6°C-1 (corresponding to a Q10 of 3.0). The holding potential was -90 mV for 10 < T < 28°C, -100 mV for 28 < T < 37°C, and -110 mV for T > 37°C.



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Fig. 8.   Temperature dependence of channel closing. A: temperature dependence of WT hKv1.5 time constants (tau deact) fit to the deactivation time course measured at -60 mV. Line represents an exponential fit to data with a temperature coefficient of 8.3°C-1 (corresponding to a Q10 of 2.7). B: temperature dependence of T505I tau deact fit to the time course of channel closing measured at -60 () and -110 mV (open circle ). Lines represent exponential fits to data (for comparative purposes only; the temperature range is far too small for accurate fits) with temperature coefficients of 39°C-1 and -300°C-1 (corresponding to respective Q10 values of 1.2 and 0.35) at -60 and -110 mV, respectively. Holding potentials were as described in Fig. 7 legend.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We have observed several interesting results: 1) modifications of residues in the S6 region of the hKv1.5 channel dramatically slow the time course of channel closing; 2) these modifications alter the voltage dependence of activation; 3) the effects were similar for mutations on both sides of the conserved Pro-X-Pro sequence; 4) when shifts in the voltage dependence of activation are accounted for, the mutations did not cause significant changes in the activation time-course but still caused drastic changes in the deactivation time course; and 5) although the temperature dependence of activation of both T505I and V512A was similar to that of WT, the primary time constant of channel closing of these two mutations displayed little or no temperature dependence. These results suggest that the mutations alter a transition (or transitions) late in the activation pathway that, depending on the mutation, either facilitates or hinders channel opening and closing. Furthermore, they indicate that T505I and V512A cause a selective "deactivation failure" in the face of a preserved (near WT) activation gating.

Mutations to the S6 region alter channel gating. Several of the point mutations in the S6 region of hKv1.5 caused shifts in the voltage dependence of activation and dramatically altered the deactivation time course. These effects were dependent on both the amino acid position and the specific amino acid that replaced the WT residue. For example, the conservative substitution T505S resulted in faster deactivation than WT. However, when T505 was replaced by the smaller alanine, deactivation was slightly slower than that of WT, indicating that 1) the slowing of deactivation due to mutations at T505 was not a result of steric hindrance, and 2) the hydroxyl side chains of threonine and serine facilitated channel closing. When T505 was replaced by either valine or isoleucine, deactivation was dramatically slowed. The effects of the mutations at T505 were similar to those reported at the equivalent position of other channels (16, 30). Similarly, deactivation of V512A and V512M channels was dramatically slowed. These changes were not linearly correlated to shifts in the voltage dependence of activation. Thus the results suggest that the slowing of deactivation was, at a minimum, due to a stabilization of the open state and not a pure shift in voltage-dependent gating. This deactivation failure demonstrates that channel closing can be largely uncoupled from channel opening by altering the molecular properties of S6.

The ability of mutations before and after the Pro-X-Pro sequence to uncouple activation from deactivation suggests that this section of S6 is an integral part of the activation gate. As such, these results agree with the previous work by Liu et al. (11) demonstrating functionally that S6 comprises part of the activation gate of Shaker channels. They showed that cysteine-substituted residues (homologous to hKv1.5 at positions I506, V510, P511, V512, and I513; indicated by asterisks in Fig. 1A) were accessible to bulky thiol-specific reagents when channels were in an open state. In another study, Li-Smerin et al. (10) used tryptophan-scanning mutagenesis to show that modification of residues in S6 alters the gating properties of Shaker. Interestingly, when residues that altered Shaker channel gating were mapped onto the KcsA structure, the locations of these residues tended to cluster near the interface between pore domain subunits.

It has been difficult to dissect the molecular nature of the gating interactions that lead to channel opening and closing. This is due, in part, to the lack of homology in the S6 region between voltage-gated K+ channels and the crystallized KcsA channel. Also, investigations of the molecular interactions with site-directed mutagenesis are for the most part limited to 20 different natural amino acid side chains. However, some attempts have been made to probe the interactions of individual residues within their molecular environment. Zhou et al. (30) showed that changes in the rate of deactivation due to mutations at T529 in rKv1.4 (T505 in hKv1.5) correlated with the amino acid side chain partition energies of soluble proteins. They speculated that this might indicate the residue is exposed to the hydrophobic protein interior when the channel is closed and that, upon channel opening, the residue enters the more hydrophilic channel lumen. Our results do not indicate such a clear-cut correlation between hydrophobic interactions and channel deactivation. For example, if hydrophobic interactions at T505 were the primary determinants of changes in deactivation rate, then, on the basis of the partition energies (8), deactivation of T505I would be expected to be slower than that of T505V. However, at -100 mV the deactivation time constant of T505I was 35 ms, whereas the deactivation time constant of T505V was 250 ms. Although hydrophobic interactions may contribute to the changes in deactivation observed when T505 is modified, other interactions such as local helix flexibility and packing modifications caused by point mutations also may contribute. No correlation between partition energy and deactivation rate existed for mutations at position V512. It also is unlikely that changes in the side chain volume were a determining factor. Neither shifts in midpoint of activation nor deactivation time constants correlated with changes in side chain volume for mutations at T505 or V512 (using side chain volumes from Ref. 31).

Kinetic modeling of changes in open-state stability. We have demonstrated that mutations to S6 dramatically alter channel activation and deactivation. Can these results be described quantitatively by altering the stability of the activation gate? Consider a two-state model, C left-right-arrow O. Stabilization of the open state would not only slow channel deactivation, it would also shift the voltage dependence of activation toward more negative potentials, consistent with the results described above. However, such a simple model cannot describe a majority of voltage-gated K+ channel kinetics.

To determine whether changes in the stability of the final closed-to-open transition step can account for the data, we used the following kinetic model (Eqs. 1 and 2)
C<SUB>1</SUB> <LIM><OP><ARROW>⇄</ARROW></OP><LL><IT>&bgr;</IT></LL><UL>3<IT>&agr;</IT></UL></LIM> C<SUB>2</SUB> <LIM><OP><ARROW>⇄</ARROW></OP><LL>2<IT>&bgr;</IT></LL><UL>2<IT>&agr;</IT></UL></LIM> C<SUB>3</SUB> <LIM><OP><ARROW>⇄</ARROW></OP><LL>3<IT>&bgr;</IT></LL><UL><IT>&agr;</IT></UL></LIM> C<SUB>4</SUB> <LIM><OP><ARROW>⇄</ARROW></OP><LL><IT>&dgr;</IT></LL><UL><IT>&ggr;</IT></UL></LIM> O (1)
where alpha , beta , gamma , and delta  are the voltage-dependent rate constants described by
&agr;=<FR><NU>kT</NU><DE>h</DE></FR> exp<FENCE><IT>W<SUB>&agr;</SUB>−</IT><FR><NU><IT>zXeV</IT></NU><DE><IT>kT</IT></DE></FR></FENCE>

&bgr;=<FR><NU>kT</NU><DE>h</DE></FR> exp<FENCE><IT>W<SUB>&bgr;</SUB>−</IT><FR><NU><IT>z</IT>(1<IT>−X</IT>)<IT>eV</IT></NU><DE><IT>kT</IT></DE></FR></FENCE> (2)

&ggr;=<FR><NU>kT</NU><DE>h</DE></FR> exp<FENCE><IT>W<SUB>&ggr;</SUB>−</IT><FR><NU><IT>zXeV</IT></NU><DE><IT>kT</IT></DE></FR></FENCE>

&dgr;=<FR><NU>kT</NU><DE>h</DE></FR> exp<FENCE><IT>W<SUB>&dgr;</SUB>−</IT><FR><NU><IT>z</IT>(1<IT>−X</IT>)<IT>eV</IT></NU><DE><IT>kT</IT></DE></FR></FENCE>
where W is the activation energy (in units of kT), z is the net charge moved during the transition, X is the fraction of the membrane field through which the charge moves, e is the elementary charge unit, V is the membrane potential, T is the absolute temperature, k is the Boltzmann constant, and h is the Planck constant. Model parameters used to describe WT kinetics are given in Table 2. This model describes a majority of hKv1.5 activation and deactivation kinetics (the voltage dependence of activation and deactivation, the dominant activation and deactivation time constants). However, it does not reproduce the second component of activation or the multiple components of inactivation previously described for hKv1.5 (20). These aspects of hKv1.5 gating were neglected to illustrate the general results of the mutations to the S6 region.

                              
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Table 2.   Parameters of model describing kinetics of WT channels

Figure 9A shows that the model describes the overall deactivation time course of WT channels. We first examined whether simply stabilizing the open state (increasing the magnitude of Wdelta ) could explain both the shift in the voltage dependence of activation and the slow deactivation time course of T505I and V512A. Both the shift in the voltage dependence of T505I activation and the time course of channel closing were well fit by modifying Wdelta (Table 3 and Fig. 9B). However, neither the shift in the voltage dependence of V512A activation nor the time course of channel closing could be fit by modifying Wdelta (Table 3).


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Fig. 9.   Model fits (solid line) to the time course of channel closing (dot plots). Currents were elicited by 50-ms depolarizations to +50 mV followed by 500- (WT) or 5,000-ms (T505I and V512A) steps to membrane potentials of -50, -70, -90, and -110 mV. A: model fits to WT hKv1.5 deactivation kinetics. The model describes both activation (model Eh = -14 mV) and deactivation of WT channels. Parameters for this model are given in Table 2. B: model fits with stabilized open position of the activation gate describe T505I activation (model Eh = -20 mV) and deactivation. This model does not describe either V512A activation (model Eh = -20 mV) or deactivation well (see text). C: model fits with stabilized open position, destabilized closed position of the activation gate describe V512A activation (model Eh = -40 mV) and deactivation. Although it is uncertain whether the closed position of T505I is destabilized, it seems likely that the closed position of V512A is destabilized. Parameters for this model are given in Table 3.


                              
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Table 3.   Parameters of models describing kinetics of T505I and V512A channels

We next examined whether stabilizing the open state as well as destabilizing the preceding closed state (decreasing the magnitude of Wgamma ) could describe the effects of V512A. In this case, the model fit the time course of channel closing and also fit the shifts in the voltage dependence of activation of V512A (Fig. 9C and Table 3). Thus the large negative shifts in the voltage dependence of activation of V512A (as well as that of V512M and T505V) may have been due to both the destabilization of a closed-state prior channel opening and stabilization of an open state, relative to WT.

Decoupling activation from deactivation. Thus far we have provided experimental evidence suggesting that mutations in the S6 region of hKv1.5 decouple activation from deactivation and have presented a kinetic model consistent with this data. To further isolate the gating transitions that were affected by these mutations, we examined the temperature dependence of WT, T505I, and V512A activation and deactivation. We found that temperature influenced both the voltage dependence of activation and the activation time course of WT, T505I, and V512A to a similar extent. The temperature dependence of channel closing was markedly different between WT and the T505I and V512A channels. WT deactivation was highly temperature dependent, whereas deactivation of T505I and V512A was largely temperature independent. In fact, at increased temperatures, changes in the rates of channel closing were, in part contaminated by slow inactivation. These results suggest that T505I and V512A did not significantly alter temperature-dependent components of activation, but, rather, they stabilized the open state such that any temperature dependence of deactivation was masked. Thus these results are consistent with the hypothesis that mutations in the S6 region alter transitions late in the activation pathway (e.g., C right-arrow O and O right-arrow C) and that these transitions occur after the temperature-dependent activation transitions. These results also are consistent with the kinetic model presented above. In this model, the closed-to-open transition(s) would have little or no temperature dependence, whereas the open-to-closed transition(s) would have substantial temperature dependence. In studies of the temperature dependence of Shaker K+ channel gating, Rodriguez and coworkers (21, 22) provided strong evidence for such a model. They also observed that a net decrease in enthalpy occurs when the channel enters the open state from the final closed state, indicating that in some sense the channel becomes more ordered. In light of the data presented here, it seems likely that residues in S6 are crucial in this rearrangement. Although the exact nature of the molecular interactions remains unclear, the results did not correlate with changes in side chain volume or hydrophobic interactions.

In conclusion, we have shown that 1) mutations in the S6 region of hKv1.5 led to a shift of the open-closed equilibrium as a function of voltage; 2) these mutations caused a marked slowing of the deactivation of the channel; and 3) two of these mutations, T505I and V512A, have little effect on the temperature dependence of activation but dramatically reduce the temperature dependence of deactivation. The effects of these mutations suggest that this region of the S6 segment comprises part of the activation gate and that mutations in this region alter the stability of both the open and closed conformations of the gate. The effects of these mutations can be described quantitatively as changes in the energy barrier between the open and closed states. Thus these data are consistent with a scheme in which changes in membrane potential cause movement of the voltage sensor that, in turn, triggers a rearrangement of the inner mouth of the pore, allowing ion permeation. Residues throughout S6 are involved in this process, and changes in these residues lead to changes in the open-closed equilibrium. Once these channels are activated, they are locked into an open state, effectively decoupling activation from deactivation.


    ACKNOWLEDGEMENTS

This work was supported by National Heart, Lung, and Blood Institute Grants HL-59689 and HL-49330 and by Flanders Interuniversity Institute for Biotechnology Grant VIB0055.


    FOOTNOTES

* T. C. Rich and S. W. Yeola contributed equally to this work.

Present addresses: T. C. Rich, Dept. of Physiology and Biophysics, University of Colorado Health Sciences Center, Denver, CO 80262; S. W. Yeola, Bristol Myers Squibb, Wallingford, CT 06492; and M. M. Tamkun, Dept. of Physiology, Colorado State University, Ft. Collins, CO 80523.

Address for reprint requests and other correspondence: D. J. Snyders, Laboratory for Molecular Biophysics, Physiology, and Pharmacology, Dept. of Biomedical Sciences, Univ. of Antwerp, Universiteitsplein 1-T4.21, B-2610 Antwerp, Belgium (E-mail: dirk.snyders{at}ua.ac.be).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 29 May 2001; accepted in final form 17 September 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Am J Physiol Cell Physiol 282(1):C161-C171
0363-6143/02 $5.00 Copyright © 2002 the American Physiological Society




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