ERG1A K+Channel Increases Intracellular Calcium Concentration through Modulation of Calsequestrin1 in C2C12Myotubes

ABSTRACT The ERG1A K + channel modulates the protein degradation that contributes to skeletal muscle atrophy by increasing intracellular calcium concentration ([Ca 2+ ]i) and enhancing calpain activity, but the mechanism by which the channel regulates the [Ca 2+ ]i is not known. Here, we have investigated the effect of human ERG1A (HERG) on [Ca 2+ ]i in C 2 C 12 myotubes, 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 [Ca 2+ ]i (induced by depolarization) is greater in myotubes over-expressing HERG relative to controls, the difference between the KCl-stimulated Ca 2+ increase in control and HERG over-expressing cells cannot be accounted for by L-type channel mediated Ca 2+ influx, which suggests that HERG could modulate excitation coupled calcium entry (ECCE). Indeed, the HERG-enhanced increase in [Ca 2+ ]i induced by depolarization is blocked by 2-APB, an inhibitor of ECCE (and SOCE). Further, we show data suggesting that HERG also modulates the activity of ryanodine receptors, a component of ECCE, as well as store operated calcium entry (SOCE). Therefore, we investigated the effect of HERG on calsequestrin1, a calcium buffering/binding protein known to modulate ryanodine receptor 1 and store operated Ca 2+ entry activities. Indeed, we find that calsequestrin1 mRNA levels are decreased 0.83-fold (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 [Ca 2+ ]i in skeletal muscle cells by lowering the abundance of the calcium buffering/binding protein calsequestrin1.