G&agr;i2- and G&agr;i3-Specific Regulation of Voltage-Dependent L-Type Calcium Channels in Cardiomyocytes.

Background: Two pertussis toxin sensitive G_i proteins, G_i2 and G_i3, are expressed in cardiomyocytes and upregulated in heart failure. It has been proposed that the highly homologous G_i isoforms are functionally distinct. To test for isoformspecific functions of G_i proteins, we examined their role in the regulation of cardiac L-type voltage-dependent calcium channels (L-VDCC). Methods: Ventricular tissues and isolated myocytes were obtained from mice with targeted deletion of either G&agr;_i2 (Ga&agr;_i2^-/-) or G&agr;_i3 (Ga&agr;_i3^-/-)). mRNA levels of Gai/o isoforms and L-VDCC subunits were quantified by real-time PCR. Gai and Cava1 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. Results: In cardiac tissue from G&agr;_i2^-/- mice, G&agr;_i3 mRNA and protein expression was upregulated to 187±21% and 567±59%, respectively. In G&agr;_i3^-/- mouse hearts, G&agr;_i2 mRNA (127±5%) and protein (131±10%) levels were slightly enhanced. Interestingly, L-VDCC current density in cardiomyocytes from G&agr;_i2^-/- 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 G&agr;_i2^-/- 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 G&agr;_i2 (but not of G&agr;_i3) and following treatment with pertussis toxin in G&agr;_i3^-/-. The pore forming Ca_v&agr;₁ protein level was unchanged in all mouse models analyzed, similar to mRNA levels of Ca_v&agr;₁ and Ca_v&bgr;₂ subunits.Interestingly, at the cellular signalling level, phosphorylation assays revealed abolished carbachol-triggered activation of ERK1/2 in mice lacking G&agr;_i2. Conclusion: Our data provide novel evidence for an isoform-specific modulation of L-VDCC by Gai proteins. In particular, loss of G&agr;_i2 is reflected by alterations in channel kinetics and likely involves an impairment of the ERK1/2 signalling pathway. [ABSTRACT FROM AUTHOR]