The long term objective of this laboratory is to understand the mechanisms of antiarrhythmic drug modification of the cardiac sodium channel. The goals of this proposal continue to pursue that objective by using an experimental approach that combines the measurements of Na channel gating currents, that are a direct measure of the movement of the channel's voltage sensors reflecting its molecular conformational states, with modification of Na channels by local anesthetic drugs and specific channel toxins to investigate antiarrhythmic drug interaction with the cardiac Na channel. We have applied this approach during the previous grant period, and have made important advances in the understanding of Na channel inactivation and its role in the mechanism of action by local anesthetic drugs. For the next grant period we expand upon our previous studies of cardiac Na channels with new studies on the Na channel's putative voltage-sensors associated with channel activation and inactivation and their modification by antiarrhythmic drugs. The experiments will use both wild-type and mutated cardiac Na channels heterologously expressed in tissue-cultured mammalian cells. Wild-type and mutant cardiac Na channel function will be probed by a combination of antiarrhythmic drugs, site-3 peptide toxins and MTS reagents. 1. Determine if modification of the Na channel gating charge-voltage (Q-V) relationship by lidocaine and its quarternary amine analogs results principally from inhibition of movement of the S4 of Domain III. 2. Demonstrate that the intracellular linker between domains III & IV (i.e. the inactivation lid) is not required for local anesthetic drug modification of the putative voltage sensors in cardiac Na channels. 3. Demonstrate that lidocaine and local anesthetic drugs do not cause gating charge to become """"""""immobilized"""""""" similar to that found for fast inactivated wild-type hHla channels. 4. Determine if the S4 in domain III contributes to charge immobilization (i.e. the slow return of gating charge during repolarization) only after binding of an intact inactivation lid in Na channels. 5. Determine if the voltage sensor formed by the S4 segment in domains III contributes approximately 22 percent to the total Qmax of the cardiac Na channel. The results of the studies in this grant proposal should further our understanding of the molecular mechanisms of cardiac Na channel function, and help determine which components of the channel's voltage sensors are modified by lidocaine.
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