Dysfunction of the Na current (INa) flowing through the ? subunit of the cardiac Na channel encoded by SCN5A participates in pathogenic mechanisms for arrhythmia, heart failure, and ischemia. Over the past 9 years we have been an active participant and contributor as the field progressed from discovery of arrhythmia mutations in ? subunits of ion channels to discovery of mutations in other subunits and associated proteins that form ion channel macromolecular complexes (MMCs). The MMC for INa now has at least 21 associated components that we will call MMCCs. These include subunits, ChIPs, scaffolding, and adapter and regulatory proteins. In the past 4-year period we identified ten new clinical syndromes involving arrhythmia genes encoding INa- MMCCs: CAV3 in LQT9 and SIDS, SCN4B in LQT10 and SIDS, SNTA1 in LQT12 and SIDS, GPD1L in SIDS, and SCNB3 in IVF and SIDS; all of these we showed to cause dysfunction of INa. We also made progress on mechanism showing that GPD1L mutations decrease INa through a mechanism of direct SCN5A phosphorylation and that SNTA1 mutations increase late INa through a mechanism of direct nitrosylation of SCN5A. In the next project period we propose to more thoroughly investigate mechanisms of CAV3 and SNTA1 action on INa involving nitrosylation of SCN5A and the MMCC Nedd4.2L. In Aim 1 we will elucidate mechanisms for INa dysfunction (gain of function or late INa) involving direct nitrosylation of SCN5A. In Aim 2 we will elucidate mechanisms for INa dysfunction (loss of function or INa) involving nitrosylation of Nedd4.2L. In Aim 3 we will determine compositions and locations of different types of INa-MMCs in mouse and human heart with a focus on CAV3-MMCCs. And we will continue the gene discovery efforts under Aim 4 where we will characterize INa from novel mutations in INa-MMCCs identified in screens of arrhythmia patients who are not linked to known genotypes and determine the mechanism of action that affect INa. We already have two novel candidate genes with two mutations in SAP97 and three mutations in Nedd4.2L from patients with Brugada syndrome. The progress on this project on arrhythmia gene discovery will have impact on increasing the diagnostic yield for patients with inherited arrhythmias. The elucidation of mechanisms by which these gene products affect INa will produce insights into both physiological regulation of INa, and pathophysiological causes of INa dysfunction for both inherited arrhythmia and commonly acquired cardiac diseases such as heart failure and ischemia.