Gating transitions in the selectivity filter region of a sodium channel are coupled to the domain IV voltage sensor

Abstract

Voltage-dependent ion channels are crucial for generation and propagation of electrical activity in biological systems. The primary mechanism for voltage transduction in these proteins involves the movement of a voltage-sensing domain (D), which opens a gate located on the cytoplasmic side. A distinct conformational change in the selectivity filter near the extracellular side has been implicated in slow inactivation gating, which is important for spike frequency adaptation in neural circuits. However, it remains an open question whether gating transitions in the selectivity filter region are also actuated by voltage sensors. Here, we examine conformational coupling between each of the four voltage sensors and the outer pore of a eukaryotic voltage-dependent sodium channel. The voltage sensors of these sodium channels are not structurally symmetric and exhibit functional specialization. To track the conformational rearrangements of individual voltage-sensing domains, we recorded domain-specific gating pore currents. Our data show that, of the four voltage sensors, only the domain IV voltage sensor is coupled to the conformation of the selectivity filter region of the sodium channel. Trapping the outer pore in a particular conformation with a high-affinity toxin or disulphide crossbridge impedes the return of this voltage sensor to its resting conformation. Our findings directly establish that, in addition to the canonical electromechanical coupling between voltage sensor and inner pore gates of a sodium channel, gating transitions in the selectivity filter region are also coupled to the movement of a voltage sensor. Furthermore, our results also imply that the voltage sensor of domain IV is unique in this linkage and in the ability to initiate slow inactivation in sodium channels.

Fig. 1.

Functionality of the charge-neutralized sodium channel mutants. (A) The sequence of the altered S4 segments of all four mutants. The sites that were mutated to glutamines are bold. (B) Current–voltage relationships for each of the mutants with a representative family of traces. Note that some of these current recordings were obtained in different ionic conditions (for details see Methods). Each graph represents the mean ± SE of at least three independent experiments. Also note that the ionic currents at more depolarized potentials are, in some cases, contaminated by gating currents.

Fig. 2.

Effect of TTX on gating pore currents through the individual voltage sensors. (A) Schematic diagram depicting the conformational processes that produce voltage-dependent gating pore currents. (Left) Channel where the main pore is closed and the voltage sensors are in a resting conformation. In this condition, the mutant voltage sensor is in a permissive position for gating pore currents. Currents through the central pore are blocked by a pore toxin. (Right) Channel with the voltage sensors in an activated conformation and the remaining charges in the mutant voltage sensor move into a position that blocks the flux of gating pore current. A family of gating pore currents before (Left; filled square) and after (Right; unfilled triangle) addition of TTX from the DI-CN (B), DII-CN (C), DIII-CN (D), and DIV-CN (E) mutants (Upper). Normalized current–voltage plots of the same (Lower). The currents at each voltage were measured at the end of 20 ms, marked as a dashed line. Each plot represents the mean ± SE of at least three independent experiments. *P value <0.05, statistical significance.

Fig. 3.

Effect of μ-CTX on gating pore currents through the individual voltage sensors. A family of hyperpolarization activated currents before (Left; filled square) and after (Right; unfilled triangle) addition of μ-CTX from the DI-CN (A), DII-CN (B), DIII-CN (C), and DIV-CN (D) mutants (Upper). Normalized current–voltage plots of the same (Lower). Currents were recorded and normalized following the protocol described in Methods. Currents at each voltage were measured at the end of 20 ms, marked as a dashed line. Each plot represents the mean ± SE of at least three independent experiments. *P value <0.05, statistical significance.

Fig. 4.

Off-gating currents from the wild-type sodium channel in absence and presence of outer pore blockers. (A) Wild-type off-gating currents before (black trace) and after (red trace) TTX. (Inset) Off-gating current traces on an enlarged scale. (B) Wild-type off-gating currents before (black trace) and after (red trace) μ-CTX. (Inset) Off-gating current traces are shown enlarged. (C) Charge–voltage curves of the wild-type channel in the presence (filled triangle) and absence (unfilled triangle) of TTX. Each plot represents the mean ± SE of at least four independent experiments. See Methods for a description of the protocol. *P < 0.01, statistical significance. (D) Charge–voltage curves of the wild-type channel before (filled triangle) and after (unfilled triangle) block of sodium currents by μ-CTX, shown as in C.

Fig. 5.

Effect of disulphide cross-bridge formation in the outer pore of the sodium channel on gating pore currents through DI, DII, DIII, and DIV voltage sensors. Above each panel, a schematic diagram depicting gating pore currents through individual voltage sensors is shown. Upon depolarization, the gating pores in individual domains close, which allowed us to monitor the conformational movements of individual domains. In all cases, the pore is trapped in an inactivated conformation by disulphide cross-linking. (A) Normalized current–voltage plots of D400C/E755C DI-CN gating pore currents in H₂O₂ (black squares) and DTT (red squares) compared with the DI-CN control in H₂O₂ (blue triangles) and DTT (green triangles). (B) Normalized current–voltage plots of D400C/E755C DII-CN gating pore currents compared with those of DII-CN. Symbols are as in A. (C) Normalized current–voltage curves of D400C/E755C DIII-CN gating pore currents compared with the DIII-CN control. The symbols are the same as in A. (D) Normalized current–voltage curves of D400C/E755C DIV-CN gating pore currents compared with those of the DIV-CN control. Each plot represents the mean ± SE of at least three independent experiments. I_o refers to the H₂O₂ traces collected at −160 mV. Data were normalized to I_o for the controls and for the double cysteine mutants. *P value <0.03, statistical significance.