Gating of skeletal and cardiac muscle sodium channels in mammalian cells

Abstract

1. Sodium channel ionic current (INa) and gating current (Ig) were compared for rat skeletal (rSkM1) and human heart Na+ channels (hH1a) heterologously expressed in cultured mammalian cells at approximately 13 C before and after modification by site-3 toxins (Anthopleurin A and Anthopleurin B). 2. For hH1a Na+ channels there was a concordance between the half-points (V ) of the peak conductance-voltage (G-V) relationship and the gating charge-voltage (Q-V) relationship with no significant difference in half-points. In contrast, the half-point of the Q-V relationship for rSkM1 Na+ channels was shifted to more negative potentials compared with its G-V relationship with a significant difference in the half-points of -8 mV. 3. Site-3 toxins slowed the decay of INa in response to step depolarizations for both rSkM1 and hH1a Na+ channels. The half-point of the G-V relationship in rSkM1 Na+ channels was shifted by -8.0 mV while toxin modification of hH1a Na+ channels produced a smaller hyperpolarizing shift of the V by -3.7 mV. 4. Site-3 toxins reduced maximal gating charge (Qmax ) by 33% in rSkM1 and by 31% in hH1a, but produced only minor changes in the half-points and slope factors of their Q-V relationships. In contrast to measurements in control solutions, after modification by site-3 toxin the half-points of the G-V and the Q-V relationships for rSkM1 Na+ channels demonstrated a concordance similar to that for hH1a. 5. Qmax vs. Gmax for rSkM1 and hH1a Na+ channels exhibited linear relationships with almost identical slopes, as would be expected if the number of electronic charges (e-) per channel was comparable. 6. We conclude that the faster kinetics in rSkM1 channels compared with hH1a channels may arise from inherently faster rate transitions in skeletal muscle Na+ channels, and not from major differences in the voltage dependence of the channel transitions.

Figure 1. rSkM1 (•) and hH1a (○) I_Na expressed in fused cells under control conditions

A shows a family of I_Na responses to step depolarizations between -70 and +30 mV for rSkM1 Na⁺ channels (top) and hH1a Na⁺ channels (bottom). The I_Na responses were capacity corrected but not leak corrected, and digitally filtered at 5 kHz. (Cells T4.01 and P2.01.) B shows the time to peak I_Na (mean ±s.e.m.) for rSkM1 (14 cells) and hH1a (10 cells) isoforms. Values were significantly different at P < 0.05 except at test potentials of -65, -55 and -45 mV. C shows the mean peak I-V relationships for 6 fused cells transfected with rSkM1 and for 5 cells transfected with hH1a. The lines connect the mean values. D shows the peak G-V relationships for the same cells as in C with the values for each cell normalized to the G_max value of each cell. The lines represent the best fits to the grouped data with parameters in the table below to a transform of a Boltzmann distribution:(1)where I_Na is the peak current in the depolarizing step and V_t is the test potential. The parameters estimated by the fit were V_rev, the reversal potential; G_max, the maximum peak conductance; V_½, the half-point of the relationship; and s, the slope factor (mV). [Table: see text]

Figure 2. Time dependent shift of I_Na kinetics

Time dependent shift of the half-point (V_½) of the G-V relationship for 10 cells transfected with rSkM1 (A) and 9 cells transfected with hH1a (B). The line in each panel represents the mean of the individual linear fits to the V_½ of each cell. The mean slope for rSkM1 was -0.15 ± 0.07 mV min⁻¹ and for hH1a it was -0.13 ± 0.05 mV min⁻¹.

Figure 3. Family of Na⁺ channel gating currents and their integrals

Family of gating currents (top) and their integrals (bottom) from fused cells expressing rSkM1 Na⁺ channels (A) and hH1a Na⁺ channels (B). Step depolarizations ranged from -120 to +40 mV from a holding potential of -150 mV. Data are capacity and leak corrected, digitally filtered at 15 kHz, and with every sixth point plotted. (Cells T4.10 and Q7.03.) C shows the single exponential fits to I_g relaxations for rSkM1 (•) and hH1a (○) Na⁺ channels. The data are the means ±s.e.m. for 10 cells expressing rSkM1 and 6 cells expressing hH1a. Time values (τ) were significantly different at P < 0.05 when V_t≥ 0 mV.

Figure 4. Superimposed peak G-V and Q-V relationships in control conditions

Superimposed peak Q-V (•) and G-V (dotted lines, from Fig. 1D) relationships for rSkM1 Na⁺ channels (A) and for hH1a Na⁺ channels (B). The Q-V relationships are from the same cells as shown in the G-V relationships from Fig. 1D. The data plotted in each panel are the means ±s.e.m. and the charge was normalized to the Q_max of each cell. The continuous lines represent the means of the best fits to each cell by a Boltzmann distribution:(2)where fractional Q_max is the normalized charge during depolarizing step; V_t is the test potential; V_½ is the half-point of the relationship; and s is the slope factor (mV). The parameters from the best fits are: [Table: see text]

Figure 5. Effects of site-3 toxins on I_Na

Effects of site-3 toxins on a family of I_Na responses to step depolarizations between -70 and +30 mV for fused cells expressing rSkM1 (A) and hH1a Na⁺ channels (B). These are the same cells as shown in Fig. 1A. The data are shown capacity corrected but not leak corrected, and digitally filtered at 5 kHz. (Cells T4.01 and P2.01.)

Figure 6. Effect of site-3 toxins on peak I_Na voltage and G-V relationships

Effect of site-3 toxins on peak I_Na-V relationships (A and C) and peak G-V relationships (B and D) for 6 fused cells transfected with rSkM1 (A and B), and for 5 cells transfected with hH1a (C and D). All control (○) data are the mean values of each cell value measured before toxin modification and after wash of toxin except for one cell transfected with hH1a which had no wash. Data obtained after modification by site-3 toxins are shown (•). For B and D control data were normalized to G_max for each cell in control, and toxin data to G_max for each cell in toxin. The lines in A and C connect the data points. The lines in C and D represent the means of the best fits of each cell to eqn (1) with the parameters listed in Table 1.

Figure 7. Effect of site-3 toxins on Q-V relationships of transfected cells

Effect of site-3 toxins on Q-V relationships of 6 cells transfected with rSkM1 (A and C) and 5 cells transfected with hH1a (B and D). A and B show families of gating currents (top) and their integrals (bottom) after leak correction and digitally filtering at 15 kHz. Data plotted are means ±s.e.m. for cells in control (○), toxin (•) and after wash (□), except one cell from each isoform had no wash. Gating charge in toxin and wash were normalized to the Q_max determined for each cell in control solution. The continuous lines represent the best fits (see Table 2) to a Boltzmann distribution (eqn (2)).

Figure 8. Superimposed peak G-V and Q-V relationships for modified Na⁺ channels

Superimposed peak G-V (•) and Q-V (○) relationships for site-3 toxin modified rSkM1 Na⁺ channels (A) and for site-3 toxin modified hH1a Na⁺ channels (B). The results plotted in each panel are the means ±s.e.m. for 6 cells expressing rSkM1 Na⁺ channels and 5 cells expressing hH1a Na⁺ channels. Data were normalized either to G_max for G-V relationships or to Q_max for Q-V relationships. The lines represent the means of the best fits (seeTable 3) of each cell to a Boltzmann distribution (eqns (1) and (2)).

Figure 9. Relationship of Q_maxvs.G_max

Relationship of Q_maxvs.G_max for rSkM1 (•) and hH1a (○) under control conditions. Q_max and G_max were obtained from the best fit of Boltzmann distributions to Q-V and G-V relationships for 14 fused cells expressing rSkM1 Na⁺ channels, and 13 cells expressing hH1a Na⁺ channels. The continuous line is the best fit by least squares regression with an intercept set to zero for rSkM1 (slope = 5.3 pC nS⁻¹, r²= 0.99), and the dotted line is the least squares regression for hH1a (slope = 5.4 pC nS⁻¹, r²= 0.97).