Ca2+ entry though TTX-sensitive Na+ channels was discovered by the PI in heart cells [154] and called """"""""slip-mode conductance"""""""". Activated by protein kinase A (PKA), Ca2+ permeability relative to Na+ (P/Ca/P/Na) increased from near zero to approximately 1.0. While slip-mode conductance was confirmed in HEK293 cells expressing cardiac alpha subunits, beta subunits had to be co-expressed [38] and alpha subunits from neither brain nor skeletal muscle could replace cardiac (see Preliminary Results). This proposal will examine slip-mode conductance of the cardiac Na+ channel, quantify its physiological and determine its molecular basis. The planned experiments will test the hypothesis that slip-mode conductance provides significant Ca2+ influx under physiological conditions. Using confocal Ca2+ imaging and patch clamp methods, the PI will test the hypothesis in cardiac myocytes and in HEK293 cells expressing Na+ channels by addressing two experimental questions. (1). What fraction of the [Ca2+]i transient in heart that is due to slip mode conductance? Slip-mode conductance will be examined quantitatively to determine Ca2+ influx and establish how it is affected by physiologic modulators of Ca2+ signaling in heart (e.g. pH, the amount of Ca2+ in the SR protein kinase C). Cardiac myocytes from rat, mouse and human hearts will be compared. Heart cells from transgenic and knockout mice will enable the investigation of A-kinase anchoring proteins (AKAPs) and the beta subunits in slip-mode conductance. (2). Why is the cardiac Na+ channel uniquely abuse to activate slip-mode conductance? Cardiac-skeletal muscle chimeras of the alpha subunit will be used to determine what part(s) of the cardiac alpha subunit is(are) necessary for slip-mode conductance. Mutations that affect channel kinetics (e.g. fast inactivation) will be used to examine the effects of channel gating on slip-mode conductance. The proposed experiments should broaden our understanding of Ca2+ signaling in heart. We should identify the molecular basis of Ca2+ permeation of cardiac Na+ channels and characterize its physiological importance. The planned work thus supports the PI's long-term plan to broaden our understanding of heart function.
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