Modulation of rat cardiac sodium channel by the stimulatory G protein α subunit

1. Modulation of cardiac sodium currents (I-Na) by the G protein stimulatory alpha subunit (Gs alpha) was studied using patch-clamp techniques on freshly dissociated rat ventricular myocytes. 2. Whole-cell recordings showed that stimulation of beta-adrenergic receptors with 10 mu M isoprenaline (isoproterenol, ISO) enhanced I-Na by 68.4 +/- 9.6% (mean +/- S.E.M.; n = 7, P<0.05 vs. baseline). With the addition of 22 mu g ml(-1) protein kinase A inhibitor (PKI) to the pipette solution, 10 mu M ISO enhanced I-Na by 30.5 +/- 7.0% (n = 7, P<0.05 vs. baseline). With the pipette solution containing both PKI and 20 mu g ml(-1) anti-as alpha IgG or 20 mu g ml(-1) anti-Gs alpha IgG alone, 10 mu M ISO produced no change in I-Na. 3. The effect of Gs alpha on I-Na was not due to changes ire the steady-state activation or inactivation curves, the time course of current decay, the development of inactivation, or the recovery from inactivation. 4. Whole-cell I-Na was increased by 45.2 +/- + 5.3% (n = 13, P < 0.05 vs. control) with pipette solution containing 1 mu M Gs alpha 27-42 peptide (amino acids 27-42 of rat brain Gs alpha) without altering the properties of Na+ channel kinetics. Furthermore, application of 1 nM Gs alpha 27-42 to Na+ channels in inside-out macropatches increased the ensemble-averaged I-Na by 32.5 +/- 6.8% (n=8, P<0.05 vs. baseline). The increase in I-Na was reversible upon Gs alpha 27-42 peptide washout. Single channel experiments showed that the Gs alpha 27-42 peptide did not alter the Na+ single channel current amplitude, the mean open time or the mean closed time, but increased the number of functional channels (N) in the patch. 5. Application of selected short amino acid segments (Gs alpha 27-36, Gs alpha 33-42 and Gs alpha 30-39) of the 16 amino acid Gs alpha peptide (Gs alpha 27-42 peptide) showed that only the C-terminal segment of this peptide (Gs alpha 33-42) significantly increased I-Na in a dose-dependent fashion. These results show that cardiac I-Na is regulated by Gs alpha via a mechanism independent of PKA that results in an increase in the number of functional Na+ channels. In addition, a 10 residue domain (amino acids 33-42) near the N-terminus of Gs alpha is important in modulating cardiac Na+ channels.