Time- and state-dependent effects of methanethiosulfonate ethylammonium (MTSEA) exposure differ between heart and skeletal muscle voltage-gated Na + channels

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Abstract

The substituted-cysteine scanning method (SCAM) is used to study conformational changes in proteins. Experiments using SCAM involve site-directed mutagenesis to replace native amino acids with cysteine and subsequent exposure to a methanethiosulfonate (MTS) reagent such as methanethiosulfonate ethylammonium (MTSEA). These reagents react with substituted-cysteines and can provide functional information about relative positions of amino acids within a protein. In the human heart voltage-gated Na + channel hNav1.5 there is a native cysteine at position C373 that reacts rapidly with MTS reagents resulting in a large reduction in whole-cell Na + current (I Na). Therefore, in order to use SCAM in studies in this isoform, this native cysteine is mutated to a non-reactive residue, e.g., tyrosine. This mutant, hNav1.5-C373Y, is resistant to the MTS-mediated decrease in I Na. Here we show that this resistance is time- and state-dependent. With relatively short exposure times to MTSEA (<4 min), there is little effect on I Na. However, with longer exposures (4-8 min), there is a large decrease in I Na, but this effect is only found when hNav1.5-C373Y is inactivated (fast or slow) - MTSEA has little effect in the closed state. Additionally, this long-term, state-dependent effect is not seen in human skeletal muscle Na + channel isoform hNav1.4, which has a native tyrosine at the homologous site C407. We conclude that differences in molecular determinants of inactivation between hNav1.4 and hNav1.5 underlie the difference in response to MTSEA exposure. © 2011 Elsevier B.V. All rights reserved.

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O&apos;Reilly, J. P., & Shockett, P. E. (2012). Time- and state-dependent effects of methanethiosulfonate ethylammonium (MTSEA) exposure differ between heart and skeletal muscle voltage-gated Na + channels. Biochimica et Biophysica Acta - Biomembranes, 1818(3), 443–447. https://doi.org/10.1016/j.bbamem.2011.11.031

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