Gαi2- and Gαi3-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 isoform-specific 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 alpha(i2) (G alpha(-/-)(i2)) or G alpha(i3) (G alpha(-/-)(i3)). mRNA levels of G alpha(i/o) isoforms and L-VDCC subunits were quantified by real-time PCR. G alpha(i) and Ca-v alpha(1) 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 alpha(-/-)(i2) mice, G alpha(i3) mRNA and protein expression was upregulated to 187 +/- 21% and 567 +/- 59%, respectively. In G alpha(-/-)(i3) mouse hearts, G alpha(i2) mRNA (127 +/- 5%) and protein (131 +/- 10%) levels were slightly enhanced. Interestingly, L-VDCC current density in cardiomyocytes from G alpha(-/-)(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 alpha(-/-)(i3) 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 alpha(i2) (but not of G alpha(i3)) and following treatment with pertussis toxin in G alpha(-/-)(i3). The pore forming Ca-v alpha(1) protein level was unchanged in all mouse models analyzed, similar to mRNA levels of Ca-v alpha(1) and Ca-v beta(2) subunits. Interestingly, at the cellular signalling level, phosphorylation assays revealed abolished carbachol-triggered activation of ERK1/2 in mice lacking G alpha(i2). Conclusion: Our data provide novel evidence for an isoform-specific modulation of L-VDCC by G alpha(i) proteins. In particular, loss of G alpha(i2) is reflected by alterations in channel kinetics and likely involves an impairment of the ERK1/2 signalling pathway.