Capes DL , Goldschen-Ohm MP , Arcisio-Miranda M , Bezanilla F , Chanda B .

GM084140-01 NIGMS NIH HHS , GM30376 NIGMS NIH HHS , R01 GM084140 NIGMS NIH HHS , T32-HL 07936 NHLBI NIH HHS , T32 HL007936 NHLBI NIH HHS , R37 GM030376 NIGMS NIH HHS , R01 GM030376 NIGMS NIH HHS

	Figure 1. Domain-specific charge neutralizations. (A) Sequence alignments of the S4 voltage-sensing segments from each domain in Nav1.4. Mutations are marked in bold. (B) A representative family of ionic current traces for wild-type and mutant channels in response to 30-ms depolarizing pulses from −110 to 65 mV (10-mV steps), preceded by a 20-ms prepulse to −120 mV from a holding potential of −80 mV. (C) Normalized peak G-V relationship for wild-type and mutant channels. Solid lines are single Boltzmann fits to the mean for each construct (wild type: V1/2 = −23.8 mV and z = 5.8 e−; DI-CN: V1/2 = −36.5 mV and z = 3.8 e−; DII-CN: V1/2 = −25.1 mV and z = 3.3 e−; DIII-CN: V1/2 = −26.4 mV and z = 3.9 e−; DIV-CN: V1/2 = −23.1 mV and z = 3.8 e−).
	Figure 2. DIV voltage-sensor movement is sufficient for fast inactivation. (A) A representative family of current traces in response to a 30-ms test pulse to −30 mV to assay the fraction of non–steady-state inactivated channels after a 100-ms conditioning prepulse between −170 and 50 mV. See Results for a detailed description of the voltage-pulse protocol. (B) Normalized steady-state inactivation versus conditioning prepulse voltage for wild-type and mutant channels. Solid lines are single Boltzmann fits to the mean for each construct (wild type: V1/2 = −62.3 mV and z = 4.7 e−; DI-CN: V1/2 = −69.2 mV and z = 4.2 e−; DII-CN: V1/2 = −65.8 mV and z = 3.7 e−; DIII-CN: V1/2 = −68.7 mV and z = 2.7 e−; DIV-CN: V1/2 = −137.3 mV and z = 1.1 e−). Note that the relation does not saturate above −200 mV for DIV-CN, so the observed left-shift in the steady-state inactivation versus voltage relation reflects the minimum shift conferred by this mutant.
	Figure 3. DIV voltage-sensor movement is rate limiting for development of fast inactivation. (A) Representative families of traces illustrating current responses to a 0-mV test pulse after development of inactivation during a variable duration conditioning pulse at −30 mV. See Results for a detailed description of the voltage-pulse protocol. (B) Summary of the time course of development of fast inactivation for conditioning voltages spanning −120 to 0 mV. (C and D) For each conditioning voltage, the time course of development of fast inactivation shown in B was fit to the bi-exponential rise {A1(1−e−(t−t0)/τ1)+A2(1−e−(t−t0)/τ2)}H(t−t0), where t is the duration of the conditioning pulse, Ai and τi are the amplitudes and time constants of the rise (i = 1,2), H is the unit step function, and t0 is the delay after onset of the conditioning pulse to initiation of development of fast inactivation. The voltage dependence of the delay and fast time constant for each construct is summarized in C and D, respectively (mean ± SEM).
	Figure 4. DIV voltage-sensor movement is rate limiting for recovery from fast inactivation. (A) Representative families of traces illustrating current responses to a −20-mV test pulse after recovery from inactivation during a variable duration conditioning pulse at −140 mV after initially inactivating the channels for 30 ms at −20 mV. See Results for a detailed description of the voltage-pulse protocol. (B) Summary of the time course of recovery from fast inactivation for conditioning voltages spanning −180 to −110 mV. (C and D) For each conditioning voltage, the time course of recovery from fast inactivation shown in B was fit to the bi-exponential rise {A1(1−e−(t−t0)/τ1)+A2(1−e−(t−t0)/τ2)}H(t−t0), where t is the duration of the conditioning pulse, Ai and τi are the amplitudes and time constants of the rise (i = 1,2), H is the unit step function, and t0 is the delay after onset of the conditioning pulse to initiation of recovery from fast inactivation. The voltage dependence of the delay and fast time constant for each construct is summarized in C and D, respectively (mean ± SEM).
	Figure 5. DIV-CN eliminates most of the gating charge movement associated with DIV. (A) Comparison of normalized ON gating currents for wild-type (black) and DIV-CN (red) channels in response to depolarizing voltage steps to −30, −10, and 0 mV after a 50-ms prepulse to −130 mV. Pore currents were blocked with CTX, which we have recently shown does not affect DIV gating charge movement, unlike tetrodotoxin (Capes et al., 2012). (B and C) ON gating currents were fit with a bi-exponential decay. The voltage dependence of the fast and slow time constants and their relative amplitudes for wild-type and DIV-CN channels is summarized in B and C, respectively (mean ± SEM; n ≥ 4).
	Figure 6. A sodium channel gating model for the distinct role of DIV in fast inactivation. (A) Kinetic model for sodium channel gating. The horizontal transitions from left to right reflect activation of the DI–III voltage sensors followed by pore opening, and the vertical transitions from bottom to top reflect activation of the DIV voltage sensor followed by occlusion of the pore by the fast inactivation motif. Rate constants and associated charges are listed in Table 1. The effects of the DIV-CN mutant were explained solely by reducing the charge and varying the rates associated with movement of the DIV voltage sensor only such that DIV was biased toward its activated conformation (red arrow; see Table 1 for parameter values). The effects of the DI/II/III-CN mutants were qualitatively explained by reducing the charge and varying the rates for only the middle set of transitions associated with movement of the DI–III voltage sensors (blue arrow; see Table 1 for parameter values). (B) Simulated current responses to families of depolarizing voltage steps as described for Fig. 1 B. (C) Simulated current responses to a steady-state inactivation protocol as described for Fig. 2 A. (D) Simulated peak conductance (closed circles) and steady-state inactivation (open circles) as a function of voltage for wild-type (black), DIV-CN (red), and DI/II/III-CN (blue) channels. (E and F) Simulated time courses for development (−60 to 0 mV) and recovery (−180 to −110 mV) from inactivation for wild-type (black) and DIV-CN (red) channels. Note that fraction inactivated/recovered was not computed as the direct probability of being in one of the model’s inactivated states but rather was obtained from simulated current responses as described in Figs. 3 B and 4 B. Simulations for DI/II/III-CN channels were similar to wild type.
	Figure 7. DIV voltage-sensor movement as a mechanism for fast inactivation from closed states. Representative current traces for wild-type and DIV-CN channels in response to a depolarization to −20 mV as shown in Fig. 1 B overlaid with their respective time courses for development of fast inactivation at −20 mV as shown in Fig. 3 B. Currents were scaled to the fraction of fast inactivated channels at the time of peak current. Development of fast inactivation precedes current rise for DIV-CN but not wild-type channels.

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