ERG1a K+Channel Increases Intracellular Calcium Concentration through Modulation of Calsequestrin 1 in C2C12Myotubes

Abstract

ABSTRACTThe ERG1A K+channel modulates the protein degradation that contributes to skeletal muscle atrophy by increasing intracellular calcium concentration ([Ca2+]i) and enhancing calpain activity, but the mechanism by which the channel regulates the [Ca2+]iis not known. Here, we have investigated the effect of human ERG1A (HERG) on [Ca2+]iin C2C12myotubes, using fura-2 calcium assays, immunoblot, RT-qPCR, and electrophysiology. We hypothesized that HERG would modulate L-type calcium channel activity, specifically the Cav1.1 channel known to carry signal from the sarcoplasmic membrane of skeletal muscle to the sarcomeres of the myofibrils. However, we find that HERG has no effect on the amplitude of L-type channel current nor does it affect the mRNA levels nor protein abundance of the Cav1.1 channel. Instead we find that, although the rise in [Ca2+]i(induced by depolarization) is greater in myotubes over-expressing HERG relative to controls, it is not sensitive to the L-type channel blocker nifedipine, which suggests that HERG modulates excitation coupled calcium entry (ECCE). Indeed, the HERG-enhanced increase in [Ca2+]iinduced by depolarization is blocked by 2-APB, an inhibitor of ECCE. Further we discovered that HERG also modulates store operated calcium entry and the activity of ryanodine receptors. Therefore, we decided to investigate the effect of HERG on calsequestrin 1, a calcium buffering/binding protein known to modulate ryanodine receptor 1 and store operated Ca2+entry activities. Indeed, we find that calsequestrin 1 mRNA levels are decreased by 17% (p<0.05) and the total protein abundance is lowered 77% (p<0.05) in myotubes over-expressing HERG relative to controls. In summary, the data show that ERG1A overexpression modulates [Ca2+]iin skeletal muscle cells by lowering the abundance of the calcium buffering/binding protein calsequestrin 1.