Gαi2- and Gαi3-Specific Regulation of Voltage-Dependent L-Type Calcium Channels in Cardiomyocytes

Abstract

BackgroundTwo pertussis toxin sensitive Gi proteins, Gi2 and Gi3, are expressed in cardiomyocytes and upregulated in heart failure. It has been proposed that the highly homologous Gi isoforms are functionally distinct. To test for isoform-specific functions of Gi proteins, we examined their role in the regulation of cardiac L-type voltage-dependent calcium channels (L-VDCC).MethodsVentricular tissues and isolated myocytes were obtained from mice with targeted deletion of either Gαi2 (Gαi2 −/−) or Gαi3 (Gαi3 −/−). mRNA levels of Gαi/o isoforms and L-VDCC subunits were quantified by real-time PCR. Gαi and Cavα1 protein levels as well as protein kinase B/Akt and extracellular signal-regulated kinases 1/2 (ERK1/2) phosphorylation levels were assessed by immunoblot analysis. L-VDCC function was assessed by whole-cell and single-channel current recordings.ResultsIn cardiac tissue from Gαi2 −/− mice, Gαi3 mRNA and protein expression was upregulated to 187±21% and 567±59%, respectively. In Gαi3 −/− mouse hearts, Gαi2 mRNA (127±5%) and protein (131±10%) levels were slightly enhanced. Interestingly, L-VDCC current density in cardiomyocytes from Gαi2 −/− mice was lowered (−7.9±0.6 pA/pF, n = 11, p<0.05) compared to wild-type cells (−10.7±0.5 pA/pF, n = 22), whereas it was increased in myocytes from Gαi3 −/− mice (−14.3±0.8 pA/pF, n = 14, p<0.05). Steady-state inactivation was shifted to negative potentials, and recovery kinetics slowed in the absence of Gαi2 (but not of Gαi3) and following treatment with pertussis toxin in Gαi3 −/−. The pore forming Cavα1 protein level was unchanged in all mouse models analyzed, similar to mRNA levels of Cavα1 and Cavβ2 subunits. Interestingly, at the cellular signalling level, phosphorylation assays revealed abolished carbachol-triggered activation of ERK1/2 in mice lacking Gαi2.ConclusionOur data provide novel evidence for an isoform-specific modulation of L-VDCC by Gαi proteins. In particular, loss of Gαi2 is reflected by alterations in channel kinetics and likely involves an impairment of the ERK1/2 signalling pathway.

Knockdown of Inhibitory Guanine Nucleotide Binding Protein Giα‐2 by Antisense Oligodeoxynucleotides Attenuates the Development of Hypertension and Tachycardia in Spontaneously Hypertensive Rats

“…Several studies have shown that knocking out one isoform of Giα protein upregulates the expression of other isoforms of Giα protein in knockout mice . In the current study, however, the effect of antisense was very specific because Giα‐2 antisense attenuated the expression of the Giα‐2 protein without affecting the expression of Giα‐3 protein, and Giα‐3 antisense attenuated the expression of Giα‐3 and not Giα‐2 protein.…”

Insulin secretion stimulated by l-arginine and its metabolite l-ornithine depends on Gαi2

“…Our detailed analysis of G protein levels in the G␣ i2 genetargeted mouse lines corroborates significant upregulation of G␣ i3 (about 45%) in the global G␣ i2 -deficient mice, which has also been described for other tissues and organs (6,17,39,40). In contrast to the global G␣ i2 -deficient islets, the G␣ i3 protein levels were unchanged in G␣ i2 ␤cko islets.…”

Gαi2 Is the Essential Gαi Protein in Immune Complex–Induced Lung Disease

“…2), and this may also account for some of the enhanced responses observed in Ga i3 deficiency. Further studies will be needed to test the possibility of an antagonistic interplay between Ga i2 and Ga i3 in the transendothelial migration of neutrophils, as has been suggested previously for CXCR3-mediated signaling in T cells (41) and for the G i -specific regulation of calcium channels in cardiomyocytes (42,43).…”