Calmodulin and Fibroblast Growth Factor Homologous Factor Co-Regulation of the NaV1.5 Channel in LQT3 Mutations

Abstract

Long QT syndrome type 3 (LQT3) results from mutations in the cardiac sodium channel NaV1.5 that increase the “late” Na+ current. Previous studies implicated the NaV1.5 auxiliary subunit apocalmodulin (apoCaM), a Ca2+ binding protein that interacts with the channel's cytosolic C-terminal domain (CTD), in the regulation of late Na+ current since a mutant channel unable to bind apoCaM displayed increased late current. Recent structural studies defined the precise apoCaM interaction sites on the CTD, which also binds a fibroblast growth factor homologous factor (FHF). How CaM influences the late Na+ current and whether the nearby FHF also regulates late current is unknown. Here, we queried the roles of both channel auxiliary subunits with biochemical and functional approaches by examining LQT3 mutations within the CTD.We found that: 1) FHF and CaM interaction with the NaV1.5 CTD is cooperative; 2) The LQT3 mutations E1901Q, Q1909R, and R1913H lower the affinity of the CTD for CaM; and 3) When expressed in HEK293 cells, all three mutant NaV1.5 channels had increased late Na+ current. We hypothesized that increased late current results from decreased affinity for CaM and/or the FHF, which we tested by re-measuring late current after overexpressing either CaM or FGF13, an FHF expressed in heart. Our rationale was that CaM overexpression would, by mass action, restore CaM binding to the mutant or that FHF expression, via its cooperative influence on CaM binding, would increase the mutant channel's affinity for CaM. Indeed, for all three mutants over-expression of an FHF or CaM decreased late current. Thus, we propose that CaM and an FHF co-operate to regulate NaV1.5 late Na+ current and that LQT3 mutations perturbing interaction of CaM or the FHF will enhance pathologic late Na+ current.