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Sodium channel inactivation is altered by substitution of voltage sensor positive charges.
Kontis KJ
,
Goldin AL
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The role of the voltage sensor positive charges in fast and slow inactivation of the rat brain IIA sodium channel was investigated by mutating the second and fourth conserved positive charges in the S4 segments of all four homologous domains. Both charge-neutralizing mutations (by glutamine substitution) and charge-conserving mutations were constructed in a cDNA encoding the sodium channel alpha subunit. To determine if fast inactivation altered the effects of the mutations on slow inactivation, the mutations were also constructed in a channel that had fast inactivation removed by the incorporation of the IFMQ3 mutation in the III-IV linker (West, J.W., D.E. Patton, T. Scheuer, Y. Wang, A.L. Goldin, and W.A. Catterall. 1992. 89:10910- 10914). Most of the mutations shifted the vof fast inactivation in the negative direction, with the largest effects resulting from mutations in domains I and II. These shifts were in the opposite direction compared with those observed for activation. The effects of the mutations on slow inactivation depended on whether fast inactivation was intact or not. When fast inactivation was eliminated, most of the mutations resulted in positive shifts in the v of slow inactivation. The largest effects again resulted from mutations in domains I and II. When fast inactivation was intact, the mutations in domains II and III resulted in negative shifts in the v of slow inactivation. Neutralization of the fourth charge in domain I or II resulted in the appearance of a second component in the voltage dependence of slow inactivation that was only observable when fast inactivation was intact. These results suggest the S4 regions of all four domains of the sodium channel are involved in the voltage dependence of inactivation, but to varying extents. Fast inactivation is not strictly coupled to activation, but it derives some independent voltage sensitivity from the charges in the S4 domains. Finally, there is an interaction between the fast and slow inactivation processes.
Figure 2. Effects of S4 mutations on the voltage dependence of fast inactivation time constants. Time constants (Ïh) of fast inactivation were determined for all of the mutants as described in materials and methods. The time constants are plotted on a log scale versus voltage for the mutants in domains I (A), II (B), III (C), and IV (D). Data for the double mutants are shown in B and D. The data points represent the means of at least three determinations and the error bars show the standard deviations.
Figure 3. Voltage command protocol and representative current traces showing the voltage dependence of slow inactivation of the IFMQ3 channel. Xenopus oocytes were coinjected with in vitro transcribed RNA encoding the sodium channel α and β1 subunits. After 2 d incubation at 20°C, data were recorded using the two-electrode whole cell voltage clamp as described in materials and methods. Oocytes were held at â100 mV, and slow inactivation was induced by 60-s depolarizations from â90 to +5 mV in 5-mV steps. This was followed by a 20-ms hyperpolarization to â150 mV to allow recovery from fast inactivation (if any, since the channels all contain the IFMQ3 mutation) and a 100-ms test depolarization to â10 mV. (A) Representative traces of the command potential are shown for depolarizations to â90, â40, â35, and +5 mV. (B) The current recorded during the same series of depolarizations is shown. The largest inward current was obtained at a prepulse of â90 mV and essentially no current can be elicited after a +5-mV depolarization.
Figure 4. Effects of S4 mutations on steady state slow inactivation when fast inactivation is eliminated. Xenopus oocytes were injected with RNA encoding the IFMQ3 sodium channel or each of the S4 mutants in the IFMQ3 background, along with RNA encoding the β1 subunit. Currents were recorded as described in Fig. 3. The normalized peak current during the test pulse is plotted against the prepulse potential. Data are shown for the mutants in domains I (A), II (B), III (C), and IV (D). Data for the double mutants are shown in B and D. The data points represent the means of at least three determinations and the error bars show the standard deviations. The smooth lines are fits to a two-state Boltzmann function, as described in materials and methods. The parameters of the fits are included in Table II.
Figure 5. Effects of S4 mutations on steady state slow inactivation when fast inactivation is intact. Xenopus oocytes were injected with RNA encoding the wild-type sodium channel or each of the S4 mutants in the wild-type background, along with RNA encoding the β1 subunit. Currents were recorded as described in Fig. 3. The normalized peak current during the test pulse is plotted against the prepulse potential. Data are shown for the mutants in domains I (A), II (B), III (C), and IV (D). Data for the double mutants are shown in B and D. The data points represent the means of at least three determinations and the error bars show the standard deviations. The smooth lines are fits to a two-state Boltzmann function, as described in materials and methods. The parameters of the fits are included in Table III.
Figure 6. The 1K4Q mutation results in two components of slow inactivation when fast inactivation is intact. Steady-state slow inactivation of the wild-type sodium channel is compared with that of the 1K4Q and 1K4Q:2K4Q mutants. The smooth lines are fits to a single Boltzmann function for the wild-type channel, and the sum of two Boltzmann functions for the mutants. The parameters of the Boltzmann fits are shown in Table III.
Figure 7. The 1K4Q mutation affects one component of slow inactivation. The kinetics of slow inactivation for the 1K4Q mutant channel were compared with those of the wild-type and IFMQ3 channels. (A) Oocytes injected with RNA encoding either the IFMQ3 channel or the 1K4Q mutant channel in the IFMQ3 (noninactivating) background were examined by two-electrode voltage clamping. The oocytes were held at â100 mV and depolarized to 0 mV for 60 s (only the first 20 s of the records are shown). The kinetics of inactivation for both channels were best fit with two exponential time constants. For the IFMQ3 channel, Ï1 = 4.9 ± 0.9 s, A1 = 67 ± 9%, Ï2 = 1.0 ± 0.2 s, A2 = 33 ± 9% (n = 5). For the 1K4Q channel, Ï1 = 5.0 ± 0.5 s, A1 = 51 ± 8%, Ï2 = 0.43 ± 0.06 s, A2 = 49 ± 8% (n = 3). (B) Oocytes injected with RNA encoding either the wild-type channel or the 1K4Q mutant channel in the inactivating background were examined by two-electrode voltage clamping. The protocol was similar to that shown in Fig. 3 except that the conditioning pulse was to a constant value of 0 mV and the duration of that pulse varied from 1 to 30 s. The amplitude of the current during the test pulse (normalized to the current obtained without any conditioning pulse) is plotted versus the conditioning pulse duration. The kinetics were best fit with two exponential time constants. For the wild-type channel, Ï1 = 32.5 ± 15.3 s, A1 = 71 ± 12%, Ï2 = 3.6 ± 0.9 s, A2 = 36 ± 9%, C = â0.07 ± 0.19 (n = 5). For the 1K4Q channel, Ï1 = 14.2 ± 2.2 s, A1 = 62 ± 4%, Ï2 = 0.2 ± 0.1 s, A2 = 14 ± 7%, C = 0.27 ± 0.05 (n = 4). Symbols represent means and error bars indicate standard deviations.
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