The work outlined in this application stems from our recent discovery that dihydropyridine-sensitive, voltage- gated CaV1.2 and CaV1.3 channels form clusters that undergo dynamic allosteric interactions, which allow cooperative gating of these channels in cardiac myocytes. The significance of these findings is underscored by our demonstration that coupled activation of these channels modulates pace-making activity in sinoatrial node (SAN) cells (CaV1.2 and CaV1.3) and contraction in ventricular myocytes (CaV1.2) under physiological and pathological conditions. The experiments proposed in this application test a novel model for the regulation of CaV1.2 and CaV1.3 channel activity in SAN and ventricular myocytes. In this model, CaV1.2 and CaV1.3 channels undergo reciprocal physical and functional interactions that are initiated by increases in intracellular Ca2+ concentration ([Ca2+]i). During the action potential, channel-to-channel coupling is initiated when membrane depolarization opens CaV1.2 and CaV1.3 channels, allowing a small amount of Ca2+ to enter the cell. The incoming Ca2+ binds to calmodulin (CaM), thereby promoting physical coupling of adjacent channels via the pre-IQ domains located in the C-tails of the channels. Physical contact increases the activity of adjoined channels. As individual channels within a cluster inactivate and close, [Ca2+]i decreases and coupling fades, but persists longer than the current that evoked it, serving as a type of `molecular memory'. A new concept in our model is that the overall activity of CaV1.2 and CaV1.3 channels within a cluster depends on the number of channels that couple and the duration of these interactions. The project will test the physiological and pathological implications of this model in three specific aims.
Specific aim 1 tests the hypothesis that coupling between CaV1.2 and CaV1.3 channels in SAN cells regulates pace-making activity.
Specific aim 2 tests the hypothesis that persistent CaV1.2 channel coupling in ventricular myocytes induces long-term potentiation of Ca2+ currents and increases contractility.
Specific aim 3 tests the hypothesis that long-QT syndrome CaM mutants increase the probability of arrhythmogenesis by altering functional coupling between CaV1.2 channels. Diverse, state-of-the-art methods, including patch-clamp electrophysiology, optical clamping, optogenetics and confocal, TIRF, and super-resolution microscopy, will be used to achieve these aims.
This project investigates the mechanisms by which small clusters of voltage-gated, dihydropyridine-sensitive CaV1.2 and CaV1.3 channels gate coordinately during the cardiac cycle, thus regulating heart rate and contractility. Furthermore, we will investigate how specific mutations in the Ca2+-binding protein calmodulin that are known to cause long-QT syndrome alter this process and thereby tune cardiac excitability.
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|Ghosh, Debapriya; Nieves-Cintrón, Madeline; Tajada, Sendoa et al. (2018) Dynamic L-type CaV1.2 channel trafficking facilitates CaV1.2 clustering and cooperative gating. Biochim Biophys Acta Mol Cell Res 1865:1341-1355|
|Gentil, Benoit J; O'Ferrall, Erin; Chalk, Colin et al. (2017) A New Mutation in FIG4 Causes a Severe Form of CMT4J Involving TRPV4 in the Pathogenic Cascade. J Neuropathol Exp Neurol 76:789-799|
|Vivas, Oscar; Moreno, Claudia M; Santana, Luis F et al. (2017) Proximal clustering between BK and CaV1.3 channels promotes functional coupling and BK channel activation at low voltage. Elife 6:|
|Li, Lei; Li, Jing; Drum, Benjamin M et al. (2017) Loss of AKAP150 promotes pathological remodelling and heart failure propensity by disrupting calcium cycling and contractile reserve. Cardiovasc Res 113:147-159|
|Nieves-Cintrón, Madeline; Hirenallur-Shanthappa, Dinesh; Nygren, Patrick J et al. (2016) AKAP150 participates in calcineurin/NFAT activation during the down-regulation of voltage-gated K(+) currents in ventricular myocytes following myocardial infarction. Cell Signal 28:733-40|
|Dickson, Eamonn J; Jensen, Jill B; Vivas, Oscar et al. (2016) Dynamic formation of ER-PM junctions presents a lipid phosphatase to regulate phosphoinositides. J Cell Biol 213:33-48|
|Moreno, Claudia M; Dixon, Rose E; Tajada, Sendoa et al. (2016) Ca(2+) entry into neurons is facilitated by cooperative gating of clustered CaV1.3 channels. Elife 5:|
|Drum, Benjamin M L; Yuan, Can; Li, Lei et al. (2016) Oxidative stress decreases microtubule growth and stability in ventricular myocytes. J Mol Cell Cardiol 93:32-43|
|Dixon, Rose E; Moreno, Claudia M; Yuan, Can et al. (2015) Graded Ca²?/calmodulin-dependent coupling of voltage-gated CaV1.2 channels. Elife 4:|
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