Molecular determinants of coupling between the domain III voltage sensor and pore of a sodium channel

Abstract

In a voltage-dependent sodium channel, the activation of voltage sensors upon depolarization leads to the opening of the pore gates. To elucidate the principles underlying this conformational coupling, we investigated a putative gating interface in domain III of the sodium channel using voltage-clamp fluorimetry and tryptophan-scanning mutagenesis. Most mutations have similar energetic effects on voltage-sensor activation and pore opening. However, several mutations stabilized the activated voltage sensor while concurrently destabilizing the open pore. When mapped onto a homology model of the sodium channel, most localized to hinge regions of the gating interface. Our analysis shows that these residues are involved in energetic coupling of the voltage sensor to the pore when both are in resting and when both are in activated conformations, supporting the notion that electromechanical coupling in a voltage-dependent ion channel involves the movement of rigid segments connected by elastic hinges.

Figure 1. Membrane topology of a sodium channel and a sequence comparison of the skeletal muscle sodium channel with the Shaker and Kv 1.2/2.1 chimeric potassium channel

(a) Domain III of the sodium channel is enlarged for clarity. The approximate location of the fluorescent probe at the L1115C position is marked by a red symbol. The mutated regions in the S4–S5 linker and N-terminus of the S5 are highlighted in yellow and those in the S6 segment are highlighted in green. (b) Sequence alignment of the individual sodium channel domains with the two potassium channels. Only the region from the start of the S4 to the end of S6 without the extracellular pore loops are shown. The S4, S5, and S6 transmembrane segments are highlighted in light blue. The mutated regions are marked as described in (a).

Figure 2. Voltage-dependent conductance and fluorescence responses in the mutant sodium channels

(a) Families of ionic current and fluorescence traces corresponding to the WT (L1115C) and four representative tryptophan mutants. Ionic currents were elicited by pulsing to various test potentials (−100 to 65 mV at 5mV intervals) for 20 ms following a brief (50 ms) prepulse to −120 mV. Fluorescence traces were recorded by pulsing to various test potentials (−170 to 50 mV at 20 mV intervals) for 20 ms following a 50 ms prepulse to −120 mV. Each of fluorescence trace was obtained by averaging 10 trials with a 1 s inter pulse interval. Fluorescence traces are shown at 40 mV interval for clarity. (b) Normalized G–V and F–V curves corresponding to the wild type (L1115C) and the tryptophan mutants shown in (a). The conductance was normalized to the peak conductance (G_max) whereas the fluorescence response was normalized to maximum steady state fluorescence (F) values for each oocyte. The data points represent the mean ± standard error (S.E.) of at least three independent measurements and the smooth curves represent the best fits of the averaged data to a single Boltzmann function. (c) The difference in the half-maximal responses (ΔV_1/2) between the WT and mutants. The ΔV_1/2 of both fluorescence (grey) and conductance (blue) for each of the mutants are shown. Error bars represent propagation of errors. Statistical analysis of ΔV_1/2 of the F–V relationships was performed with a one-way ANOVA with Dunnett’s post-tests, *p<0.05.

Figure 3. Superposition of the homology model of sodium channel domain III with the structure of the Kv1.2/2.1 chimera and mapping the prominent class I positions on the homology model

(a) A superposed view of sodium channel domain III and the crystal structure of the potassium channel chimera (Kv1.2/2.1) (PDB code: 2R9R). The sodium channel is shown in blue while the chimeric potassium channel is shown in red. (b) Class I mutants that stabilize the open pore are in magenta whereas those that destabilize the open conformation are highlighted in purple. The other mutated regions in the S4–S5 linker and S5 are in green while those in the S6 are in yellow. (c) Enlarged view of the lower part of S5 and the middle part of S6 near the gating hinge. (d) Enlarged view of the S4–S5 linker and the bottom part of S6.

Figure 4

Normalized G–V (blue) and F–V relationships (red) for prominent class II mutants. The data points represent the mean ± SE of at least three independent measurements and smooth curves represent the best fits of the averaged data to a single Boltzmann function. The fitted WT data is shown as dashed curves (a) Class II mutants that cause a right shift in the G–V and a left shift in the F–V curve relative to the WT. (b) Class II mutants that cause a left shift in the G–V and a right shift in the F–V curve relative to the WT.

Figure 5. Mapping the prominent class II mutants on to the structural model of sodium channel domain III

(a) The sites of class II mutations are highlighted in red. The voltage-sensing S4 segment is in cyan. The other mutated positions in the S4–S5 linker and S5 are shown in yellow whereas those in the S6 segment are shown in green. (b) Enlarged view of the lower part of S5 and the middle part of S6 near the gating hinge. (c) Enlarged view of the S4–S5 linker and the bottom part of the S6.

Figure 6. The G–V and F–V curves upon perturbation of putative interaction pairs and mapping the positions of an interaction pair on to the homology model of sodium channel domain III

(a) Normalized G–V (top panel) and F–V (bottom panel) curves for the WT (dashed line), F1298A (filled circle), F1298W mutant (open circle), and R1135W-F1298R double mutant (filled square). The data points represent the mean ± SE of at least three independent experiments, and the smooth curves represent the best fits of the averaged data to a Boltzmann function. (b) The difference in half-maximal responses (ΔV_1/2) between the WT relative to the mutants R1135W, K1296W, K1296A, F1298W and R1135W-F1298R. The ΔV_1/2 of both fluorescence (grey) and conductance (blue) for each of the mutants are shown. The error bars represent propagation of errors. (c) The side chains of residues R1135 and F1298 are shown on the homology model. Inset, the region around the tail end of S6 and the beginning of S4–S5 linker is shown enlarged.

Figure 7. Two general models describing the coupled activation process involving the voltage-sensor and pore of a voltage-dependent ion channel

(a) Schemes IA and IB represent the canonical models of cooperativity, where a direct interaction between the open pore and the activated voltage-sensor mediates coupling of the two structural domains. Scheme II represents alternate model of voltage-sensor-pore coupling, where the closed pore interacts with the resting voltage-sensor and the open pore interacts with the activated voltage-sensor. K₁ and K₂ are the intrinsic activation constants of the voltage-sensor and the pore and θ is the coupling parameter. The intrinsic activation constants can be expressed as: $K_1=K_1^{0}exp(z_{1}F\beta )$ and $K_2=K_2^{0}exp(z_{2}F\beta )$ where $K_2^0$ and $K_1^0$ represent the contribution of chemical interactions to the activation process of the voltage-sensor and the pore, respectively. (b) and (c) The probabilities of voltage-sensor activation and pore opening when the three thermodynamic parameters are modified in Scheme IA. The expressions for P_VA and P_PO from Scheme I (Eqs. 1 and 2) were fitted to the WT F–V and G–V curves respectively, to obtain the values for the different parameters: $K_1^0=10000.01$, z₁ = 3.494, $K_2^0=0.079$, z₂= 4.219, θ = 999.916. $K_1^0,K_2^0$ and θ were varied over 10 orders of magnitude and the response to these changes on the P_VA values at −50mV and P_PO values at −20mV were plotted in (b) and (c) respectively. (d) and (e) The probabilities of voltage-sensor activation and pore opening when the three thermodynamic parameters are modified in Scheme IB. The initial thermodynamic parameters $K_1^0=9999.84$, z₁= 0.936, $K_2^0=73.938$, z₂= 3.914, θ = 1003.561 were obtained by fitting the expressions for P_VA and P_PO in Scheme IB (Eqs. 3 and 4) to the WT F–V and G–V curves respectively. $K_1^0,K_2^0$ and θ were varied over 10 orders of magnitude and the response to these changes on the P_VA values at −50mV and P_PO values at −20mV were plotted in (d) and (e) respectively. (f) and (g) The probabilities of voltage-sensor activation and pore opening when the three thermodynamic parameters are modified in Scheme II. The expressions for P_VA and P_PO from Scheme II (Eqs. 3 and 4) were fitted to the WT F–V and G–V curves respectively, to obtain the values for the different parameters: $K_1^0=10000.01$, z₁= 0.949, $K_2^0=0.074$, z₂= 3.905, θ= 999.913. $K_1^0,K_2^0$ and θ were varied over 10 orders of magnitude and the response to these changes on the P_VA values at −50mV and P_PO values at −20mV were plotted in (d) and (e) respectively.