Persistent Na influx drives L-type channel resting Ca entry in rat melanotrophs.

Rat melanotrophs express several types of voltage-gated and ligand-gated calcium channels, although mechanisms involved in the maintenance of the resting intracellular Ca concentration ([Ca]) remain unknown. We analyzed mechanisms regulating resting [Ca] in dissociated rat melanotrophs by Ca-imaging and patch-clamp techniques. Treatment with antagonists of L-type, but not N- or P/Q-type voltage-gated Ca channels (VGCCs) as well as removal of extracellular Ca resulted in a rapid and reversible decrease in [Ca], indicating constitutive Ca influx through L-type VGCCs. Reduction of extracellular Na concentration (replacement with NMDG) similarly decreased resting [Ca]. When cells were champed at -80 mV, decrease in the extracellular Na resulted in a positive shift of the holding current. In cell-attached voltage-clamp and whole-cell current-clamp configurations, the reduction of extracellular Na caused hyperpolarisation. The holding current shifted in negative direction when extracellular K concentration was increased from 5 mM to 50 mM in the presence of K channel blockers, Ba and TEA, indicating cation nature of persistent conductance. RT-PCR analyses of pars intermedia tissues detected mRNAs of TRPV1, TRPV4, TRPC6, and TRPM3-5. The TRPV channel blocker, ruthenium red, shifted the holding current in positive direction, and significantly decreased the resting [Ca]. These results indicate operation of a constitutive cation conductance sensitive to ruthenium red, which regulates resting membrane potential and [Ca] in rat melanotrophs.