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

Kontis KJ, Rounaghi A, Goldin AL - J. Gen. Physiol. (1997)

Bottom Line: The most significant effects were observed with substitutions of the fourth positive charge in each domain.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, University of California, Irvine, California 92697-4025, USA.

ABSTRACT
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.

Show MeSH

Related in: MedlinePlus

Effects of S4 mutations on the voltage dependence  of activation time constants.  Time constants of activation (τm)  were determined for all of the  mutants as described in Fig. 4.  The values for potentials from  −30 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.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2229375&req=5

Figure 5: Effects of S4 mutations on the voltage dependence of activation time constants. Time constants of activation (τm) were determined for all of the mutants as described in Fig. 4. The values for potentials from −30 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.

Mentions: Data for all of the mutants were similarly analyzed, and the activation time constants are plotted on a log scale against step potential in Fig. 5, with the parameters of the fits shown in Table II. Differences in τ0 between each of the mutants and IFMQ3 can be expressed as a shift along the voltage axis, assuming that there is no difference in k between the mutant and IFMQ3 channels. Although this assumption is not completely correct for most of the mutants, the values for k are similar enough between the mutants and IFMQ3 so that the calculated shifts are generally valid. That is, the value of the shift changed by <1.7 mV depending on whether the mutant or IFMQ3 value for k was used for all of the mutants except 1K4Q:2K4Q. For that double mutant, the calculated voltage shift was 24.7 mV if the mutant value for k was used.


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

Kontis KJ, Rounaghi A, Goldin AL - J. Gen. Physiol. (1997)

Effects of S4 mutations on the voltage dependence  of activation time constants.  Time constants of activation (τm)  were determined for all of the  mutants as described in Fig. 4.  The values for potentials from  −30 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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2229375&req=5

Figure 5: Effects of S4 mutations on the voltage dependence of activation time constants. Time constants of activation (τm) were determined for all of the mutants as described in Fig. 4. The values for potentials from −30 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.
Mentions: Data for all of the mutants were similarly analyzed, and the activation time constants are plotted on a log scale against step potential in Fig. 5, with the parameters of the fits shown in Table II. Differences in τ0 between each of the mutants and IFMQ3 can be expressed as a shift along the voltage axis, assuming that there is no difference in k between the mutant and IFMQ3 channels. Although this assumption is not completely correct for most of the mutants, the values for k are similar enough between the mutants and IFMQ3 so that the calculated shifts are generally valid. That is, the value of the shift changed by <1.7 mV depending on whether the mutant or IFMQ3 value for k was used for all of the mutants except 1K4Q:2K4Q. For that double mutant, the calculated voltage shift was 24.7 mV if the mutant value for k was used.

Bottom Line: The most significant effects were observed with substitutions of the fourth positive charge in each domain.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, University of California, Irvine, California 92697-4025, USA.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus