Journal
CELL CALCIUM
Volume 66, Issue -, Pages 1-9Publisher
ELSEVIER SCI LTD
DOI: 10.1016/j.ceca.2017.05.008
Keywords
L-type channels; Cav channels; PKG; cGMP; Insulin; Rin-m5F cells
Categories
Funding
- Conacyt [221660, R00MH099405]
- Conacyt
- Dr. John P. and Therese Mulcahy Endowed Professorship in Ophthalmology
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cGMP is a second messenger widely used in the nervous system and other tissues. One of the major effectors for cGMP is the serine/threonine protein kinase, cGMP-dependent protein kinase (PKG), which catalyzes the phosphorylation of a variety of proteins including ion channels. Previously, it has been shown that the cGMP-PKG signaling pathway inhibits Ca2+ currents in rat vestibular hair cells and chromaffin cells. This current allegedly flow through voltage-gated Ca(v)1.3L-type Ca2+ channels, and is important for controlling vestibular hair cell sensory function and catecholamine secretion, respectively. Here, we show that native L-type channels in the insulin-secreting RIN-m5F cell line, and recombinant Ca(v)1.3 channels heterologously expressed in HEK-293 cells, are regulatory targets of the cGMP-PKG signaling cascade. Our results indicate that the Cavai ion-conducting subunit of the Ca(v)1.3 channels is highly expressed in RIN-m5F cells and that the application of 8-Br-cGMP, a membrane-permeable analogue of cGMP, significantly inhibits Ca2+ macroscopic currents and impair insulin release stimulated with high K+. In addition, KT-5823, a specific inhibitor of PKG, prevents the current inhibition generated by 8-Br-cGMP in the heterologous expression system. Interestingly, mutating the putative phosphorylation sites to residues resistant to phosphorylation showed that the relevant PKG sites for Ca(v)1.3 L-type channel regulation centers on two amino acid residues, Ser793 and Ser860, located in the intracellular loop connecting the II and III repeats of the Ca-v alpha(1) pore-forming subunit of the channel. These findings unveil a novel mechanism for how the cGMP-PKG signaling pathway may regulate Ca(v)1.3 channels and contribute to regulate insulin secretion. (C) 2017 Elsevier Ltd. All rights reserved.
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