Persistent Na ⁺ influx drives L-type channel resting Ca ²⁺ 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 ²⁺ concentration ([Ca ²⁺ ] _i ) remain unknown. We analyzed mechanisms regulating resting [Ca ²⁺ ] _i in dissociated rat melanotrophs by Ca ²⁺ -imaging and patch-clamp techniques. Treatment with antagonists of L-type, but not N- or P/Q-type voltage-gated Ca ²⁺ channels (VGCCs) as well as removal of extracellular Ca ²⁺ resulted in a rapid and reversible decrease in [Ca ²⁺ ] _i , indicating constitutive Ca ²⁺ influx through L-type VGCCs. Reduction of extracellular Na ⁺ concentration (replacement with NMDG ⁺ ) similarly decreased resting [Ca ²⁺ ] _i . 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 ²⁺ 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 ²⁺ ] _i . These results indicate operation of a constitutive cation conductance sensitive to ruthenium red, which regulates resting membrane potential and [Ca ²⁺ ] _i in rat melanotrophs.