Labro AJ , Lacroix JJ , Villalba-Galea CA , Snyders DJ , Bezanilla F .

GM030376 NIGMS NIH HHS , R01 GM030376 NIGMS NIH HHS , R37 GM030376 NIGMS NIH HHS

	Figure 1. Ionic and gating currents from Shaker-H4-IR; behavior of the VSD and channel gate. (A) Activating ionic currents (Iac) recorded from Shaker-H4-IR using the pulse protocol shown on top. (B) Gating current recordings after depletion of K+ and external TEA block. The holding potential was −130 mV, and oocytes were depolarized in 5-mV increments from −120 to 80 mV. Background leak and capacitive currents were subtracted with a −P/4 protocol using a −120-mV holding potential. The inset is a scaled-up view of the IgOFF currents highlighting the gradual slowing in IgOFF decay when prepulse depolarization voltages became stronger (red trace is IgOFF for a −50-mV pulse). (C) Voltage dependence of both activating and deactivating charge movement (QV curve) and BC gate opening (GV curve). The GV curve (circles) was obtained from the ionic tail currents during the repolarization step of pulse protocols shown in A. The voltage dependence of gating charge activation (QV curve displayed with open triangles; Qac) was obtained by integrating the repolarizing gating currents (IgOFF) of the activation protocol shown in B. The voltage dependence of gating charge deactivation was obtained by integrating the repolarizing gating currents of the deactivation protocol shown in D (QV curve displayed with closed triangles; Qdeac). Both GV and QV values were normalized, and the curves shown are for both GV and QV the average fit to a Boltzmann equation. (D) Superposition of scaled ionic (red traces) and gating currents (black traces) obtained from the same oocyte using the voltage protocol shown on top. Scale bars for gating and ionic currents are shown in black and red, respectively. (E, top) A scaled-up view of the overlapping deactivation ionic (Ideac) and gating (IgOFF) currents from D at −110-mV repolarizing voltage. (bottom) The respective Ideac and IgOFF currents were normalized and superimposed. On the left, the IgOFF is inverted to highlight the overlap of the fast component in Ideac with the rising phase observed in IgOFF. On the right, a scaled-up view of the slow component in Ideac that matches the IgOFF decay. (F) Voltage dependency of the time constants ± SEM of IgON decay (open triangles; n = 8), IgOFF decay (closed triangles; n = 7), the rising phase of IgOFF (red circles; n = 7), Iac (gray circles; n = 7), the fast component of Ideac (yellow inverted triangles; n = 7), the slow component of Ideac (green inverted triangles; n = 7), and the weighted Ideac kinetics (blue circles; n = 7). Note the superposition of the slow Ideac component with IgOFF decay and the fast Ideac component with the rising phase in IgOFF. Error bars represent SEM.
	Figure 2. Slowing in IgOFF and Ideac kinetics of Shaker channels with prolonged depolarization. (A) Deactivating IgOFF currents upon different depolarization times at 20 mV. To avoid nonspecific effects or depletion effects, the pulse protocols were ran in random order, and, in the case of the represented currents, the protocol with a 10-s prepulse (bottom traces) was recorded before the 100-ms trace (top traces). (bottom) A superposition of IgOFF at different voltages upon 100-ms (black) and 10-s depolarizations (red). Note that IgOFF slows down and the total charge becomes more spread out when depolarizations are prolonged. (B) Ionic current deactivation after 100 ms (top traces) and 5 s (bottom traces) at 20 mV. (bottom) A superposition of scaled currents at −80 and −50 mV upon 100-ms (black) and 5-s (red) depolarizations. (C) IgOFF was fitted with a double exponential function, and the weighted time constants ± SEM are represented (n = 6). Note the gradual increase of the time constants with prolonged depolarization times. (D) Development of the ionic deactivation Ideac weighted time constant as a function of depolarization time, displaying, like IgOFF, a gradual slowing with prolonged depolarizations. (E, top) The weighted IgOFF time constant at −50 mV plotted as a function of depolarization time clearly displaying two slowing processes. The fit with a double exponential function yielded a fast τf of 8.5 ± 1.0 ms and a slow τs of 1,100 ± 100 ms (n = 6). (bottom) The behavior of Ideac kinetics at −50 mV showing a similar biphasic slowing process with a τf of 4.8 ± 0.6 ms and a τs of 670 ± 90 ms (n = 5). Error bars represent SEM.
	Figure 3. Direct correlation between VSD and gate movement in the Shaker-M356C channel using site-directed fluorimetry. (A) Simultaneous recording of Ideac (black traces) and recovery of fluorescence quenching (ΔF, gray traces) from an oocyte expressing TMRM-labeled M356C Shaker channels using the indicated protocol and various prepulse durations at 20 mV. For clarity, both traces have been normalized. The vertical scale bar indicates both ionic current amplitude (in microamps) and ΔF/F (in percentage). (B) The kinetics of the fluorescence recovery signal obtained at −60 mV is shown as a function of the prepulse duration. Note that like the gating current recordings, there were two slowing process in the kinetics with prolonged membrane depolarizations. (C) Development of the weighted Ideac time constant at −60 mV as a function of depolarization time, displaying, like the simultaneously recorded ΔF signal, a biphasic slowing of the kinetics. Error bars represent SEM.
	Figure 4. C-type inactivation properties of Shaker and Kv1.2. (A) Ionic current recordings from oocytes expressing Shaker (black trace) or Kv1.2 channels (gray trace) evoked by depolarizing the membrane potential from a holding of −80 to 20 mV. Upon prolonged depolarization, both channels display C-type inactivation, which is characterized by a reduction in current amplitude. At the end of the pulse, Kv1.2 channels display only 25 ± 3% (n = 4) current reduction, whereas Shaker channels display 52 ± 2% current decrease (n = 4). (B) Time constants of the inactivation process in Shaker (black circles) and Kv1.2 channels (gray circles) obtained by fitting the reduction in current amplitude with a single exponential function. Note that in both channels, the kinetics are voltage independent. Error bars represent SEM.
	Figure 5. Ionic and gating currents from Kv1.2; behavior of the VSD and BC gates. (A) Activating ionic currents from WT Kv1.2, a member of the Shaker family that displays a more modest C-type inactivation process. (B) Gating current recordings from Kv1.2 after depletion of K+. Current recordings were obtained using the activation pulse protocol shown on top. The holding potential was −130 mV, and oocytes were depolarized in 5-mV increments from −130 to 70 mV (for clarity only, current traces are shown every 10-mV increment). Similar to Shaker, there was a gradual slowing in IgOFF decay when prepulse depolarization voltages became stronger (red trace is IgOFF after a −40-mV prepulse). (C) Superposition of scaled deactivation ionic (red traces) and gating currents (black traces) from Kv1.2 obtained from the same oocyte using the voltage protocol shown on top. The scales for gating and ionic currents are shown in black and red, respectively. (D) Voltage dependence of BC gate opening (GV curve) and charge movement (QV curves) using an activation (Qac) or deactivation (Qdeac) protocol. Both GV and QV values were normalized, and for both GV and QV curves, the average fit to a Boltzmann equation is shown. (E) Mean time constant ± SEM for IgON decay (open triangles), IgOFF decay (closed triangles), rising phase of IgOFF (red circles), Iac (gray circles), fast component of Ideac (yellow inverted triangles), slow component of Ideac (green inverted triangles), and the weighted Ideac kinetics (blue circles) obtained in a similar way as described for Shaker (Fig. 1), with an n of at least six independent oocytes analyzed. Error bars represent SEM.
	Figure 6. Slowing in IgOFF and Ideac kinetics of Kv1.2 with prolonged depolarization times. (A) Deactivating gating (IgOFF) currents upon 200-ms (top traces) and 5-s (bottom traces) prepulse depolarization times at 20 mV. Like the experiments on Shaker, the pulse protocols were ran in random order to avoid nonspecific effects on channel kinetics. (bottom) A superposition of IgOFF at −120 and −90 mV upon 200-ms (black) and 5-s (red) depolarizations, respectively. (B) Ionic current deactivation after 200 ms (top traces) and 1 s (bottom traces) at 20 mV. (bottom) A superposition of scaled ionic currents at −80 and −50 mV upon 200-ms (black) and 1-s (red) depolarizations. (C) IgOFF kinetics as a function of depolarization duration at 20 mV. Values were obtained by weighting the time constants from a double exponential fit to IgOFF decay at different voltages (n = 5). (D) Voltage dependence of BC gate closure (Ideac) as a function of depolarization duration at 20 mV. The weighted Ideac kinetics obtained from a double exponential fit ± SEM (n = 7) are plotted. (E, top) The weighted IgOFF kinetics at −60 mV as a function of prepulse depolarization time. Like Shaker, there were two noticeable slowing processes in IgOFF kinetics, a fast τf of 13.5 ± 1.8 ms and a slow τs component of 1,120 ± 210 ms (n = 5). (bottom) Plot of the development of the weighted Ideac kinetics as a function of prepulse depolarization time. Like IgOFF, there appeared two clear slowing processes, and fitting the relation with a double exponential function yielded a τf of 16.5 ± 6.1 ms and a τs of 1,250 ± 160 ms (n = 7). Error bars represent SEM.
	Figure 7. Slowing in charge return of Ci-VSP sensing current Is. (A, top) Current tracings showing Is of Ci-VSP using the indicated activation protocol. The IsOFF from a moderate 30-mV test pulse is shown as the red trace in the inset. Note the rapid slowing down in IsOFF that accompanies the most positive depolarizations. (bottom) Current tracings are a superposition of IsOFF at −50 mV upon 200-ms (black) and 1-s (red) depolarizations to 80 mV. Note the slowing in IsOFF decay when depolarizations get prolonged. (B) Kinetics of activating IsON (open triangles) obtained in WT Ci-VSP from different activation voltage pulses (similar to the protocol shown in A) and of deactivating IsOFF obtained in WT Ci-VSP using 80-mV prepulses of the indicated durations from 200 ms to 5 s (colored symbols) and pulsing to different deactivation voltages. (C) The weighted time constant of IsOFF decay measured at 10 mV is plotted as a function of the duration of the conditioning prepulse. The obtained curve was fitted to a single exponential function and yielded a time constant of τ = 530 ± 80 ms (n = 7). Error bars represent SEM.
	Figure 8. VSD relaxation in the isolated VSD of Ci-VSP. (A, left) Cartoon representation of the full-length WT Ci-VSP and the truncated mutant Δ239–576 that lacks the phosphatase domain (red) attached to the VSD (blue). (right) Sensing current traces (Is) of the truncation mutant Δ239–576 recorded using the indicated activation protocol. (B) QV curves for the mutant Δ239–576 (red) and WT Ci-VSP (black) obtained by integrating the activating Is from pulse protocols shown in A and Fig. 7 A, respectively. (C) A superposition of IsOFF current traces at −20 mV after 10-ms (black) and 3-s (red) depolarizations to 80 mV is displayed. Note the slowing in IsOFF decay and the reduction in the fast component amplitude of the red trace. The inset shows the normalized tracings to better highlight the slowing in IsOFF decay. (D) Kinetics of activating IsON (open triangles) of the mutant Δ239–576 obtained from different activation voltage pulses (similar to the protocol shown in A) and of deactivating IsOFF of the same mutant obtained using 80-mV prepulses of the indicated durations from 3 ms to 3 s (colored symbols) and pulsing to different deactivation voltages. (E) Weighted time constant of IsOFF decay measured at −20 mV as a function of the duration of the conditioning prepulse at 80 mV. The obtained curve was fitted to a single exponential function and yielded a time constant of τ = 76 ± 11 ms (n = 5). (F) For both the full-length Ci-VSP (left) and the truncation mutant Δ239–576 (right), the normalized amount of sensing charges moved during the conditioning prepulse to 80 mV (QON, left axis) was superimposed on the depolarization-induced slowing in the kinetics of IsOFF decay (τw IsOFF, right axis). Fitting the normalized charge displacement as a function of depolarization duration with a single exponential function yielded τQONWT = 61.7 ± 0.1 ms (n = 4) for full-length Ci-VSP and τQONΔ239–576 = 18.8 ± 0.1 ms (n = 5) for the truncation mutant. Error bars represent SEM.
	Figure 9. State model and cartoon of the activation path in Shaker and Kv1.2. (A) Proposed simplified state diagram for Shaker and Kv1.2 channels whereby the state occupancy for both VSD (blue) and BC (red) gates are represented in two separate schemes. The stabilization imposed by the BC gate is termed the open stabilized (O-stabilized) state and results in the activated stabilized state at the VSD level. The stabilization that originates from the VSD relaxation process results in the relaxed and open relaxed (O-relaxed) state of the VSD and BC gate, respectively. (B) Cartoon of the structural path followed by Shaker and Kv1.2 channels upon a membrane depolarization. For clarity, only one out of four subunits is illustrated, and the specific change from one state to the next is indicated by an arrow. During a depolarization, the VSD (mostly the S4 segment) moves from its resting position (at the extreme left) to its preactivated state and in a subsequent concerted step to its activated state, resulting in the opening of the BC gate (open). Because in Shaker and Kv1.2 the stabilization imposed by the BC gate (depicted by the interaction between the S4S5L and S6 gate region) develops first, the BC gate evolves from the open to the open stabilized (O-stabilized) conformation. When the depolarization is prolonged, the VSD relaxes (illustrated with a tilt of the S4), and the channel populates its final open stabilized relaxed (O-stabilized-relaxed) state.

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