Ion channels are critical regulators of excitability in neurons and muscle. The cardiac sodium channel, Nav1.5, is responsible for the initial upstroke of the action potential in ventricular myocytes. Post-translational modifications, such as phosphorylation, modulate Nav1.5. During physiological events, constitutive activation of one or more enzymes results in the integration of signal transduction pathways, thereby altering channel modulation. Specifically, previous studies implicate the integration of PKC and Fyn kinase signal transduction pathways. I studied the effects of dual kinase modulation in Nav1.5 by using Fyn kinase (Fyn) and a partially-selective PKC inhibitor, Bisindolylmaleimide-1 (BIM1). Whole-cell voltage clamp experiments were performed using HEK293 cells co-expressing Nav1.5 and either FynCA (constitutively active) or FynKD (kinase dead, which exerts a dominant-negative effect on tyrosine phosphorylation). Cells co-expressing Nav1.5 + FynCA (without BIM1) showed (i) a significant left shift in the mid-point of steady-state fast inactivation, (ii) accelerated rate of fast inactivation, and (iii) increased non-inactivating sodium current, all of which were not seen in Nav1.5 + FynKD (without BIM1), control or Nav1.5 + FynCA + BIM1 experiments. These results indicate that constitutive activation of Fyn (i) confers dual kinase modulation of Nav1.5 and (ii) leads to the hypoexcitability of cells, which may be pro-arrthythmogenic.
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Thesis advisor: Ruben, Peter C.
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