ERG1A K⁺ channel increases intracellular calcium concentration through modulation of calsequestrin1 in C₂C₁₂ myotubes

The 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⁺]i is not known. Here, we have investigated the effect of human ERG1A (HERG) on [Ca²⁺]i in C₂C₁₂ myotubes, using Fura-2 calcium assays, immunoblot, RT-qPCR, and electrophysiology. The data show that the rise in [Ca²⁺]_i induced by KCl-stimulated depolarization is of greater amplitude in C₂C₁₂ myotubes over-expressing HERG relative to controls, but this difference does not result from an increase in L-type channel (Ca_v1.1) Ca²⁺ influx because there is no statistical difference in the nifedipine-sensitive response upon depolarization between the expression groups. Indeed, HERG overexpression in C₂C₁₂ myotubes 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. This finding suggests that HERG modulates excitation coupled calcium entry (ECCE). Indeed, the HERG-enhanced increase in [Ca²⁺]i induced by depolarization is blocked by 2-aminoethoxydiphenyl borate, an inhibitor of ECCE. Further, HERG also modulates the activity of ryanodine receptors (RYR1, 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 RYR1 and SOCE 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 conclusion, the data show that ERG1A overexpression modulates [Ca²⁺]i in skeletal muscle cells by lowering the abundance of the calcium buffering/binding protein calsequestrin1 which interacts with RyR1 and SOCE pathways. Indeed, we report that overexpression of HERG in myotubes increases [Ca²⁺]_i by modulation of RyR1 as well as ECCE and SOCE activities. It is likely that HERG enhancement of RyR1 activity, through decreased Casq1 abundance, is increasing [Ca²⁺]i. This study provides a potential mechanism to explain how upregulation of ERG1A contributes to increased [Ca²⁺]i and, thus, atrophy in skeletal muscle.