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Double mutant gating perturbation analysis predicts a high conformational stability of the domain IV S6 segment of the voltage-gated Na+ channel


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. Thus, mutations at several sites of DIV-S6 are known to substantially alter the channel's inactivation properties.


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 wild-type channel and into rNaV1.4 carrying 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 produced 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 by cysteines in DIV-S6 produced shifts in V05, both in the background of wild-type and in the background of K1237E, 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 vs. the shifts in V05 by double mutants relative to K1237E showed a significant positive correlation (r = 0.92, 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.


Funding support: Austrian Science Fund P210006-B11.

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Correspondence to Hannes Todt.

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Open Access This article is published under license to BioMed Central Ltd. This is an Open Access article is distributed under the terms of the Creative Commons Attribution License ( ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Cervenka, R., Zarrabi, T., Lukács, P. et al. Double mutant gating perturbation analysis predicts a high conformational stability of the domain IV S6 segment of the voltage-gated Na+ channel. BMC Pharmacol 9 (Suppl 2), A25 (2009).

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