Abstract 17319: Distinct Effects of Human Calmodulinopathy on the Function of Small Conductance Ca 2+ -Activated K + (SK) Channels

Abstract

Background: Calmodulin (CaM) plays a critical role in intracellular signaling and regulation of Ca 2+ -dependent ion channels. Mutations in CALM1, CALM2, and CALM3 have recently been linked to cardiac arrhythmias, such as Long QT Syndrome (LQTS), catecholaminergic polymorphic ventricular tachycardia (CPVT), and familial idiopathic ventricular fibrillation (IVF). Small-conductance Ca 2+ - activated K + channels (SK) are voltage-independent channels that are regulated solely from beat-to-beat changes in intracellular calcium. CaM regulates the function of multiple ion channels, including SK channels, although the effect of CaM mutations on these channels is not yet understood. We hypothesize that human CaM mutations linked to sudden cardiac death disrupt SK channel function by distinct mechanisms. Methods and Results: We tested the effects of LQTS (CaM D96V , CaM D130G ), CPVT (CaM N54I , CaM N98S ), and IVF (CaM F90L ) CaM mutants compared to CaM WT on SK channel function. Using whole-cell voltage-clamp recordings, we found that CaM D96V and CaM D130G mutants significantly inhibited apamin-sensitive currents. Similarly, action potential studies in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) also revealed significant knockdown of apamin-sensitive currents. Immunofluorescent confocal microscopy confirmed that this effect was not due to changes in SK channel trafficking. Rather, co-immunoprecipitation studies showed a significant decrease in the association of these CaM mutants with the SK channel. Rosetta molecular modeling was used to identify a conformational change in CaM F90L structure compared to that of CaM WT . Conclusions: We found that CaM D96V and CaM D130G mutants significantly reduced apamin-sensitive currents, through a dominant negative effect on SK channel function. Consistent with our hypothesis, CaM F90L resulted in the least inhibitory effects. The data suggests that specific mutations with phenylalanine to leucine (CaM F90L ) may disrupt the interaction between apo-CaM with CaMBD on the SK2 channel.