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Persistent Na+ influx drives L-type channel resting Ca2+ entry in rat melanotrophs

Publication at Faculty of Medicine in Pilsen |
2019

Abstract

Rat melanotrophs express several types of voltage-gated and ligand-gated calcium channels, although mechanisms involved in the maintenance of the resting intracellular Ca2+ concentration ([Ca2+](i)) remain unknown. We analyzed mechanisms regulating resting [Ca2+](i) in dissociated rat melanotrophs by Ca2+-imaging and patch-clamp techniques.

Treatment with antagonists of L-type, but not N- or P/Q-type voltage-gated Ca2+ channels (VGCCs) as well as removal of extracellular Ca2+ resulted in a rapid and reversible decrease in [Ca2+](i), indicating constitutive Ca2+ influx through L-type VGCCs. Reduction of extracellular Na+ concentration (replacement with NMDG(+)) similarly decreased resting [Ca2+](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, Ba2+ 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 [Ca2+](i). These results indicate operation of a constitutive cation conductance sensitive to ruthenium red, which regulates resting membrane potential and [Ca2+](i) in rat melanotrophs.