Abstract 1512: Heteromeric Assembly of HCN4 and C-Terminal Truncated HCN2 Subunits in the Generation of Cardiac Pacemaker (If) Channels

Abstract

In sino-atrial and atrio-ventricular nodal cells, hyperpolarization- activated cyclic nucleotide-gated (HCN) cationic channels are expressed that carry inward currents, I f , contributing importantly to the diastolic depolarization critical for cardiac pacemaker functioning. Although previous studies have demonstrated myocardial expression of HCN2 and HCN4 subunits, the roles of these subunits in the generation of mature, functional I f channels remains unclear. In preliminary experiments, completed on transiently transfected HEK293 cells expressing wild type and dominant negative mutant HCN2 and HCN4 subunits, we find that HCN2 and HCN4 preferentially co-assemble to form heteromeric channels. In further studies focused on defining the molecular compositions of functional cardiac I f channels, we developed and validated C-and N-terminal HCN2 and HCN4 subunit specific antibodies and exploited these to identify the HCN subunits expressed in adult (mouse) heart and to immunoprecipitate cardiac HCN-encoded I f channel complexes. These experiments revealed that the HCN2 and HCN4 proteins co-immunoprecipitate from heart, In addition, Western blot experiments revealed that, although the full length HCN2 (105Da) and HCN4 (160Da) proteins are expressed in transiently transfected HEK293 cells and in adult (mouse) brain, the molecular weight of the HCN2 protein in the heart is reduced to about 60 kDa. In addition, the mature, 60 kDa HCN2 protein lacks the C-terminal cAMP binding domain. These observations likely explain the results of previous studies demonstrating that the targeted deletion of HCN2 does not affect the cAMP sensitivity of I f . In addition, taken together, these results demonstrate that functional myocardial I f channels reflect the heteromeric assembly of HCN2 and HCN4 subunits and further that the HCN4 subunit regulates cAMP-mediated regulation of I f channels. This research has received full or partial funding support from the American Heart Association, AHA National Center.