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

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 (INa). 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 INa. 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 INa. However, with longer exposures (4–8 min), there is a large decrease in INa, 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.

Basal late sodium current is a significant contributor to the duration of action potential of guinea pig ventricular myocytes

“…In this study, the selective Na V 1.5 channel blocker MTSEA (Haufe et al. ; O'Reilly and Shockett ) decreased the amplitude of basal I NaL by 90 ± 5%, indicating that under the conditions of our experiments, the Na V 1.5 channel isoform is a major contributor to basal I NaL of guinea pig ventricular myocytes.…”

“…MTSEA is a selective blocker of Na V 1.5 channels (Haufe et al. ; O'Reilly and Shockett ). In this study, MTSEA (2 mmol/L) was added to the bath solution to determine whether the basal I NaL of myocytes was generated from the Na V 1.5 channels.…”

“…In addition, we examined whether the basal I NaL is generated from Na V 1.5 channels, and whether the basal I NaL is regulated by Ca 2+ /calmodulin‐dependent protein kinase II (CaMKII), respectively, by applying the selective Na V 1.5 channel blocker MTSEA (methanethiosulfonate ethylammonium) (Haufe et al. ; O'Reilly and Shockett ) and the CaMKII inhibitors KN‐93 and AIP (autocamtide‐2‐related inhibitory peptide).…”

MTSET modification of D4S6 cysteines stabilize the fast inactivated state of Nav1.5 sodium channels

“…In the absence of reagent, the peak currents of wild-type and Y1767C channels increased an average of 29.1 ± 3.6% ( n = 15) and 21.6 ± 2.6% ( n = 9), respectively. Including MTSET in the patch pipette resulted in increased wild-type currents similar to reagent-free controls ( Figure 1B ) indicating that in the absence of inserted D4S6 cysteines the Na v 1.5 channels are insensitive to internally applied MTSET ( O’Reilly and Shockett, 2012 ). This contrasted with the Y1767C mutant where internal MTSET produced a 42.1 ± 4.6% decrease in the Na + current amplitude ( Figure 1D ).…”

Enhanced basal late sodium current appears to underlie the age-related prolongation of action potential duration in guinea pig ventricular myocytes

“…In addition, we examined whether the increased basal I NaL of ventricular myocytes from old hearts is generated from Na V 1.5 channels and is regulated by CaMKII, using the selective Na V 1.5-channel blocker methanethiosulfonate ethylammonium (MTSEA) (12,28) and the specific CaMKII blockers KN-93 and autocamtide-2-related inhibitory peptide (AIP), respectively.…”