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

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 Ca_V1.3L-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_V1.3 channels heterologously expressed in HEK-293 cells, are regulatory targets of the cGMP-PKG signaling cascade. Our results indicate that the Ca_Vα₁ ion-conducting subunit of the Ca_V1.3 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_V1.3 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_Vα₁ pore-forming subunit of the channel. These findings unveil a novel mechanism for how the cGMP-PKG signaling pathway may regulate Ca_V1.3 channels and contribute to regulate insulin secretion.

Keywords: Cav channels; Insulin; L-type channels; PKG; Rin-m5F cells; cGMP.

Fig. 1

Expression of PKG and Ca_V channel subunits in RIN-m5F cells. A) RT-PCR analysis of PKG cDNA expression in RIN-m5F cells. Total RNA from mouse whole brain was used as a positive control. Actin was used as an internal control. Primer sequences are indicated in Supplemental Table S1. B) Levels of different Ca_V channel subunit mRNAs expressed in RIN-m5F cells estimated from gene quantitative RT-PCR data and expressed as a number of copies of β-actin mRNA expression.

Fig. 2

PKG inhibit L-type Ca_V channels in RIN-m5F cells and contributes to determine insulin secretion. A) Representative traces of Ba2+ currents (I_Ba) through Ca2+ channels in RIN-m5F cells in the control condition and after 8-Br-cGMP (1 mM) application. B) Average current density-voltage relationships for I_Ba recorded from RIN-m5F cells in the absence and presence of 8-Br-cGMP (1 mM). C) High K⁺-induced insulin secretion from RIN-m5F cells in the absence and the presence of 8-Br-cGMP as indicated. RIN-m5F cells were incubated with KRB buffer containing 40 mm KCl. Insulin content in the supernatants was measured by ELISA. The mean ± S.E. of four independent experiments is shown.

Fig. 3

The NO-cGMP-PKG pathway inhibited recombinant L-type Ca_V 1.3 channel activity. A) RT-PCR analysis of PKG cDNA expression in the HEK-293 cell line. Actin was used as an internal control. Primer sequences are indicated in Supplemental Table S1. B) Bar chart of the percentage inhibition ± S.E. of I_Ba measured with test pulses to −30 mV at different concentrations of SNP alone, or after 500 μM CPTIO application. The number of recorded cells is indicated in parenthesis. C) Comparison of the of the percentage inhibition ± S.E. of I_Ba measured with test pulses to −30 mV at different concentrations of 8-Br-cGMP alone or in combination of KT-5823, as indicated.

Fig. 4

PKG activation modifies current density through recombinant L-type Ca_V 1.3 channels. A) Representative superimposed current traces recorded in HEK-293 cells expressing Ca_V 1.3/Ca_V α₂ δ-1/Ca_V β₃ channels in the absence and presence of 8-Br-cGMP (1 mM). Currents were evoked by 140-ms depolarizing pulses from a V_h of −80 to −30 mV. B) Average current density-voltage relationships for I_Ba recorded from HEK-293 cells expressing Ca_V 1.3/Ca_V α₂ δ-1/Ca_V β₃ channels in the absence and presence of 8-Br-cGMP (1 mM), Calyculin (10 nM) and KT-5823 (10 μM), as indicated.

Fig. 5

Mutation of two serine residues (S763 and S860) to delete PKG-mediated phosphorylation sites in the Ca_V 1.3α₁ subunit alters channel functional expression. A) Average current density-voltage relationships for I_Ba recorded from HEK-293 cells expressing the wild-type Ca_V 1.3 channel and its mutant variant (Ser793Ala) in the control condition and after incubation with 1 mM 8- Br-cGMP plus 10 nM Calyculin A (B). I_Ba density was calculated at a series of test pulses applied from a V_h of −80 mV in 5 mV steps between −70 and 30 mV. C) Average current density-voltage relationships for I_Ba recorded from HEK-293 cells expressing the wild-type Ca_V 1.3 channel and its mutant variant (Ser860Ala) in the control condition and after incubation with 1 mM 8- Br-cGMP plus 10 nM Calyculin A (D).

Fig. 6

Phosphorylation at sites S763 and S860 of Ca_V 1.3α₁ subunit influences channel functional expression. A) Representative traces of Ba2+ currents recorded from HEK-293 cells expressing DPM mutant channels in absence and presence of 8-Br-cGMP (1 mM) plus 10 nM Calyculin A. B) Average current density-voltage relationships for I_Ba from HEK-293 cells expressing the wild-type Ca_V 1.3 channel and its double phosphorylation mutant (DPM) variant in the control condition and after incubation with 1 mM 8-Br-cGMP plus 10 nM Calyculin A (C). I_Ba density was calculated at a series of test pulses applied from a V_h of −80 mV in 5 mV steps between −70 and 30 mV.

Fig. 7

Schematic overview of the mechanisms involved in PKG-induced regulation of L-type Ca_V channel activity and insulin secretion. A) The L-type channel complex is composed of the pore-forming Ca_V α₁ and auxiliary subunits (Ca_V α₂ δ, Ca_V β, and Ca_V γ). The pore-forming Ca_V α₁ subunits consist of four transmembrane domains (I–IV) and the linker joining II and III encompasses the putative PKG phosphorylation sites (S793 and S860). B) NO is synthesized by the enzyme NO synthase (NOS) through the oxidation of L-arginine to NO and L-citrulline, with the assistance of cofactors. NO endogenously produced by NOS or released from exogenously applied NO donors (sodium nitroprusside, SNP) activates NO-sensitive guanylate cyclase (GC) leading to increased synthesis of cGMP which activates PKG. The protein kinase catalyzes the phosphorylation of the L-type Ca_V channels which cause a decrease in Ca2+ influx through the cell membrane resulting in reduced release of insulin in RIN-m5F cells. The inhibitory action of PKG is blocked by the selective inhibitor KT-5823 and terminated by protein phosphatases (PPh). Calyculin A, is a serine/threonine phosphatase inhibitor.