Skip to main content

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

Background

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.

Methods

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.

Results

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).

Conclusion

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.

Acknowledgements

Funding support: Austrian Science Fund P210006-B11.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Hannes Todt.

Rights and permissions

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 ( https://creativecommons.org/licenses/by/2.0 ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Reprints and Permissions

About this article

Cite this article

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, A25 (2009). https://doi.org/10.1186/1471-2210-9-S2-A25

Download citation

Keywords

  • Xenopus Laevis
  • Drug Block
  • Anesthetic Drug
  • Amino Acid Replacement
  • Xenopus Laevis Oocyte