L-type Ca2+ current (ICa) is a critical source of inward current for triggering oscillations in cell membrane potential, called early afterdepolarizations (EADs) that lead to life-threatening arrhythmias, including Torsade de Pointes (TdP). Conventional ion channel antagonist drugs are ineffective for treating TdP, so improved therapies are needed. ICa is an inward current that appears essential for many EADs, but ICa also provides intracellular Ca2+ ([Ca2+]i) for activating signaling molecules linked to EAD initiation. During the initial period of this award, it has become clear that intracellular Ca2+ ([Ca2+]i) bound to the Ca2+-sensing protein, calmodulin (CAM), and the Ca2+/CaM-activated protein kinase II (CaMKII) are proarrhythmic signals for EADs and TdP. The proarrhythmic mechanism for Ca2+/CaM and CaMKII in EADs is thought to be increases in ICa, termed facilitation. At the same time, our laboratory found that CaM binding domains on the L-type Ca2+ channel (LTCC) also cause facilitation (a process we call auto-facilitation). We have developed exciting new evidence that these CaM binding domains are ICa auto-facilitation ligands, which require coexpression of an auxilliary beta subunit. Our preliminary studies show that CaMKII-mediated ICa facilitation also requires this same beta subunit, suggesting the novel hypothesis that CaMKII and CaM binding domains are signals that converge upon a shared LTCC facilitation apparatus. The proposed experiments are designed to test the hypothesis that CaMKII, and LTCC CaM-binding domains are beta subunit-dependent signaling elements for ICa facilitation and EAD initiation. This hypothesis will be tested using the following Specific Aims. ? ? 1. Determine the mechanim of CaMKII-dependent facilitation and EAD initiation ? 2. Determine the mechanism of IQ and CB domain-dependent auto-facilitation and EAD initiation ? 3. Determine the relationship of CaM binding domain auto-facilitation and CaMKII-mediated facilitation. ? ? Understanding these molecular mechanisms for Ca2+/CaM-dependent ICa facilitation will likely be necessary to develop improved therapies for TdP. ? ?
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