Project Summary/Abstract Atrial fibrillation (AF) is the most common atrial arrhythmia affecting veteran population, and is associated with a significant risk of embolism and stroke. The problem is further exacerbated by the fact that treatment strategies have proven largely inadequate. During the last funding period, we uncovered surprising, yet insightful findings that have broad therapeutic ramifications. In contrast to previous reports that suggested that Cav1.3 (a1D) L-type Ca2+ channel (LTCC) is expressed mainly in neurons and neuroendocrine cells, we demonstrated significant expression of Cav1.3 Ca2+ channel in atrial myocytes. Indeed, Cav1.3 Ca2+ channel is preferentially expressed in atrial compared to ventricular myocytes. Additionally, the importance of Cav1.3 Ca2+ channels in atria is underpinned by the revelation that null deletion of the channel results in significant alteration in atrial excitability, atrial arrhythmias as well as profound sinoatrial (SA) and atrioventricular (AV) nodes dysfunction. We further demonstrated that Cav1.2 and Cav1.3 channels form multimeric protein complexes with small conductance Ca2+-activated K+ channels (SK channels) in the heart, which were first uncovered recently in our laboratory. Of major intriguing and functional importance are the findings that SK channels are also preferentially expressed in atrial myocytes as well as pacemaking tissues of the heart. We further demonstrated that SK2 channels associate with Cav1.3 and Cav1.2 through a physical bridge, a- actinin2 in cardiac myocytes. In addition, we have obtained new preliminary data, which demonstrate that cytoskeletal proteins are critical in the proper membrane localization of SK2 channel. Given these relevant data, we will directly examine the molecular determinants of SK channel trafficking. Moreover, the functional roles of the newly described SK channels in pacemaking tissues will be directly delineated. Our findings represent the beginning of a unified molecular and cellular mechanism that demonstrates a functional spatiotemporal cross talk between Cav channels and Ca2+-activated K+ channels (KCa) in atrial cells. Embedded in these findings are relevant paradigm shifts that may be exploited in developing atrial-specific drugs for the treatment of atrial arrhythmia. Hence, the overall thrust of the proposal is to deploy new molecular and functional strategies, many inspired from channel mechanistic studies, for the discovery of fundamental and newly accessible arenas of Ca2+ and K+ channels in the heart. We will directly test the hypothesis using a combination of in vitro interaction assay, confocal microscopic imaging, electron microscopic analyses, gene silencing, biochemical studies and functional analyses. Finally, we hypothesize that all 3 isoforms of SK channels are expressed in SA and AV node cells and contribute critically to the firing and action potential durations of nodal cells. We propose to directly test the hypothesis using null mutant models of SK1, SK2 and SK3 channels. Our proposed studies will substantially expand our understanding of the specific functions of individual Ca2+ and SK channels. Indeed, novel insights into the atrial-specific and pacemaking tissue-specific ion channels may provide new means to target these channels without interfering with the excitability of ventricular tissues.