The long-term objective of this project is to acquire a fundamental understanding of the relationship between molecular structure and physiological function in voltage-gated Na+ channels (Navs). Specifically, this project will focus on the human cardiac Nav isoform hNav1.5. Understanding the relationship between normal structure and function in hNav1.5 will provide insight into the relationship between abnormal structure and function such as that found in cardiac diseases resulting from heritable mutations in Navs (channelopathies).
The specific aims of this project will focus on slow inactivation in hNav1.5, a kinetic process that is important in action potential firing properties and setting membrane excitability. Site-directed mutagenesis, electrophysiological recordings from wild-type and mutant hNav1.5 expressed in HEK cells, and the substituted-cysteine accessibility method (SCAM) will be used to determine the functional role of the inner pore region (of D1-S6 and D2-S6) in hNav1.5 slow inactivation. The following specific aims will be addressed:
Specific Aim 1 will determine the effect on slow inactivation in hNav1.5 of substituting cysteine (C) and glutamine (Q), which vary in size and hydrophobicity, for the native amino acids in the inner pore region of D1-S6 and D2-S6. The substitutions will span the region from N406 in D1-S6 and V930 in D2-S6 to the putative gating-hinges in S6 of the respective domains. Electrophysiological recordings of whole-cell Na+ current will be used to determine the functional effect of the substitutions. The hypothesis is that this region of the inner pore is critical in slow inactivation gating and therefore, that mutagenesis in this region will disrupt normal slow inactivation.
Specific Aim 2 will determine if there is molecular rearrangement in the inner pore regions in D1-S6 and/or in D2-S6 during slow inactivation in hNav1.5. The hypothesis is that conformational changes in this region are an important molecular mechanism of slow inactivation and that this molecular rearrangement alters the relative positions of critical amino acids in these regions. This hypothesis will be tested with the substituted-cysteine accessibility method (SCAM) using the cysteine-substituted mutant channels from Specific Aim 1. The cysteine-substituted mutants will be exposed to methanethiosulfonate (MTS) reagents at rest, during fast inactivation, and while in the slow-inactivated state. MTS-accessibility will be used as an indicator of relative positional changes and movement in D1-S6 and D2-S6 during slow inactivation. This proposal will provide novel information on the molecular mechanism of Nav slow inactivation, which will enhance our understanding of human heart diseases such as long QT and Brugada Syndrome that are characterized by disruption of Nav kinetic processes such as slow inactivation. PUBLIC HEALTH RELEVENCE: More than 80 mutations in human heart sodium channels have been identified in patients with conditions such as long QT and Brugada syndrome. These mutations can produce changes in the normal electrophysiological function of the heart, which can lead to sudden cardiac death in adults and infants (i.e., sudden infant death syndrome, SIDS). Understanding the relationship between molecular structure and physiological function in cardiac sodium channels will provide us with an understanding of abnormal function (pathophysiology) of the human heart, which may provide useful information for diagnosis, therapeutic intervention, and/or prevention of sudden cardiac death. ? ? ?
| O'Reilly, John P; Shockett, Penny E (2012) Time- and state-dependent effects of methanethiosulfonate ethylammonium (MTSEA) exposure differ between heart and skeletal muscle voltage-gated Na(+) channels. Biochim Biophys Acta 1818:443-7 |
| Chancey, Jessica Hotard; Shockett, Penny E; O'Reilly, John P (2007) Relative resistance to slow inactivation of human cardiac Na+ channel hNav1.5 is reversed by lysine or glutamine substitution at V930 in D2-S6. Am J Physiol Cell Physiol 293:C1895-905 |
| O'Reilly, John P; Shockett, Penny E (2006) Slow-inactivation induced conformational change in domain 2-segment 6 of cardiac Na+ channel. Biochem Biophys Res Commun 345:59-66 |
