Regulation of L-type CaV1.3 channel activity and insulin secretion by the cGMP-PKG signaling pathway.

Academic Article

Abstract

  • 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 CaV1.3L-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 CaV1.3 channels heterologously expressed in HEK-293 cells, are regulatory targets of the cGMP-PKG signaling cascade. Our results indicate that the CaVα1 ion-conducting subunit of the CaV1.3 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 CaV1.3 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 CaVα1 pore-forming subunit of the channel. These findings unveil a novel mechanism for how the cGMP-PKG signaling pathway may regulate CaV1.3 channels and contribute to regulate insulin secretion.
  • Authors

  • Sandoval, Alejandro
  • Duran, Paz
  • Gandini, María A
  • Andrade, Arturo
  • Almanza, Angélica
  • Kaja, Simon
  • Felix, Ricardo
  • Status

    Publication Date

  • September 2017
  • Published In

  • Cell Calcium  Journal
  • Keywords

  • Animals
  • Calcium Channels, L-Type
  • Carbazoles
  • Cav channels
  • Cell Line
  • Cyclic GMP
  • Cyclic GMP-Dependent Protein Kinases
  • HEK293 Cells
  • Humans
  • Insulin
  • L-type channels
  • Membrane Potentials
  • Mutagenesis, Site-Directed
  • Nitric Oxide
  • PKG
  • Patch-Clamp Techniques
  • Phosphorylation
  • Protein Subunits
  • Rats
  • Rin-m5F cells
  • Signal Transduction
  • cGMP
  • Digital Object Identifier (doi)

    Start Page

  • 1
  • End Page

  • 9
  • Volume

  • 66
  • Issue

  • Cold Spring Harb. Protoc. 2014 2014