Abstract
The S6 segment of domain IV (DIV-S6) of the voltage-gated Na channel is considered to be a key player in gating and local anesthetic drug block. Thus, some mutations in DIV-S6 substantially alter the channel's inactivation properties. In order to get a comprehensive picture of the kinetic role of DIV-S6 in fast inactivation we performed a cysteine scanning analysis of sites 1575-1591 in the DIV-S6 of the rNav1.4 channel. In addition, we produced the same cysteine replacements in the background of the mutation K1237E. K1237 is located in the P-loop of domain III and mutations at this site have dramatic effects both on permeation and gating properties. Hence, K1237E most likely causes a complex conformational change of the channel. We sought to explore whether K1237E changes the pattern of gating perturbations by the serial cysteine replacements in DIV-S6. The constructs were expressed in Xenopus laevis oocytes and studied by means of two electrode voltage-clamp. The half-point of availability following a 50 ms conditioning prepulse (V05) was -44 ± 1 mV and -51 ± 1 mV in wild-type and K1237E, respectively (P < 0.001) . Most serial amino acid replacements in DIV-S6 produced shifts in V05, both in wild-type and in K1237E background, ranging from +17 ± 1 mV to -9 ± 2 mV. A plot of the shifts in V05 by single DIV-S6 mutants relative to wild-type versus the shifts in V05 by double mutants relative to K1237E showed a significant positive correlation (R= 0.72, P=0.002). This indicates that the general pattern of gating perturbations in DIV-S6 is not affected by K1237E, suggesting a high conformational stability of the DIV-S6 segment during the fast inactivated state. Support: FWF P21006-B11
Highlights
The S6 segment of domain IV (DIV-S6) of voltage-gated Na+ channels is considered to be a key player in gating and local anesthetic drug block
For a comprehensive analysis of the kinetic role of DIV-S6 in fast inactivation we performed a cysteine scanning analysis of sites 1575-1591 in the DIV-S6 of the rNaV1.4 channel. These mutations were engineered into the wildtype channel and into rNaV1.4 carrying the mutation K1237E
We sought to explore whether K1237E changes the pattern of gating perturbations produced by the serial cysteine replacements in DIV-S6
Summary
The S6 segment of domain IV (DIV-S6) of voltage-gated Na+ channels is considered to be a key player in gating and local anesthetic drug block. Double mutant gating perturbation analysis predicts a high conformational stability of the domain IV S6 segment of the voltage-gated Na+ channel Address: Institute of Pharmacology, Center of Biomolecular Medicine and Pharmacology, Medical University of Vienna, 1090 Vienna, Austria
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