Kontis KJ et al. (1997), Sodium channel activation gating is affected by...

XB-ART-15986

J Gen Physiol 1997 Oct 01;1104:391-401.

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Sodium channel activation gating is affected by substitutions of voltage sensor positive charges in all four domains.

Kontis KJ , Rounaghi A , Goldin AL .

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The role of the voltage sensor positive charges in the activation and deactivation gating 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 (by glutamine substitution) and -conserving mutations were constructed in a cDNA encoding the sodium channel alpha subunit 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.). A total of 16 single and 2 double mutants were constructed and analyzed with respect to voltage dependence and kinetics of activation and deactivation. The most significant effects were observed with substitutions of the fourth positive charge in each domain. Neutralization of the fourth positive charge in domain I or II produced the largest shifts in the voltage dependence of activation, both in the positive direction. This change was accompanied by positive shifts in the voltage dependence of activation and deactivation kinetics. Combining the two mutations resulted in an even larger positive shift in half-maximal activation and a significantly reduced gating valence, together with larger positive shifts in the voltage dependence of activation and deactivation kinetics. In contrast, neutralization of the fourth positive charge in domain III caused a negative shift in the voltage of half-maximal activation, while the charge-conserving mutation resulted in a positive shift. Neutralization of the fourth charge in domain IV did not shift the half-maximal voltage of activation, but the conservative substitution produced a positive shift. These data support the idea that both charge and structure are determinants of function in S4 voltage sensors. Overall, the data supports a working model in which all four S4 segments contribute to voltage-dependent activation of the sodium channel.

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	Figure 2. Representative current traces showing the activation and deactivation kinetics of the RBIIA channel containing the IFMQ3 mutation. Xenopus oocytes were co-injected with in vitro transcribed RNA encoding the sodium channel Î± and Î²1 subunits. After a 2-d incubation at 20Â°C, data were recorded using the cut-open oocyte voltage clamp method as described in materials and methods. (A) Current traces recorded during 57.5-ms depolarizations ranging from â50 to +50 mV. The IFMQ3 mutation removes fast inactivation, and all of the S4 mutations were made in this background. The tail currents occurring after repolarization are indicated by the vertical arrow in A and are shown in greater detail in B. (C) Instantaneous tail currents from the same oocyte were recorded after activation of the channels with a prepulse to +20 mV for 2 ms, followed by 75-ms steps to voltages ranging from â90 to 0 mV in 10-mV increments.
	Figure 3. Effects of S4 mutations on the voltage dependence of activation. Xenopus oocytes were injected with RNA encoding the IFMQ3 channel or each of the S4 mutants, along with RNA encoding the Î²1 subunit. Currents were recorded from a holding potential of â100 mV by depolarizations ranging from â90 to +55 mV, as described in materials and methods. The fraction of sodium channels activated at each potential was determined, and is plotted as a function of voltage. 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 I.
	Figure 4. Determination of the voltage dependence of activation time constants in the IFMQ3 sodium channel. Sodium currents were recorded from oocytes expressing IFMQ3, as described in Fig. 3. The rising phase of the inward sodium currents was fit with a single exponential, as described in materials and methods. Each data point represents a single determination and the smooth lines were generated by a least-squares fit to an equation (inset) describing the voltage-dependent variation of the activation time constant (Ïm). The parameters of the fits are shown in Table II.
	Figure 5. Effects of S4 mutations on the voltage dependence of activation time constants. Time constants of activation (Ïm) were determined for all of the mutants as described in Fig. 4. The values for potentials from â30 to +30 mV 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 parameters of the fits are shown in Table II.
	Figure 6. The voltage dependence of deactivation time constants can be fit by a single exponential. Sodium currents were recorded from oocytes expressing IFMQ3, as described in Fig. 3. Instantaneous tail currents were acquired by a depolarization to +20 mV for 1â3 ms, followed by a 33.7-ms depolarization ranging from â90 to â45 mV in 5-mV increments. The smooth lines represent the best fits to a voltage-dependent single exponential equation (inset). The parameters of the fits are shown in Table III.
	Figure 7. Effects of S4 mutations on the voltage dependence of deactivation time constants. Time constants of deactivation (Ïd) were determined for all of the mutants as described in Fig. 6. The values 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 parameters of the fits are shown in Table III.

References [+] :

Aggarwal, Contribution of the S4 segment to gating charge in the Shaker K+ channel. 1996, Pubmed, Xenbase