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

Abstract

1. Modulation of cardiac sodium currents (INa) by the G protein stimulatory alpha subunit (Gsalpha) 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 microM isoprenaline (isoproterenol, ISO) enhanced INa by 68.4 +/- 9.6 % (mean +/- s.e.m.; n = 7, P < 0.05 vs. baseline). With the addition of 22 microgram ml-1 protein kinase A inhibitor (PKI) to the pipette solution, 10 microM ISO enhanced INa by 30.5 +/- 7.0 % (n = 7, P < 0.05 vs. baseline). With the pipette solution containing both PKI and 20 microgram ml-1 anti-Gsalpha IgG or 20 microgram ml-1 anti-Gsalpha IgG alone, 10 microM ISO produced no change in INa. 3. The effect of Gsalpha on INa was not due to changes in 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 INa was increased by 45.2 +/- 5.3% (n = 13, P < 0.05 vs. control) with pipette solution containing 1 microM Gsalpha27-42 peptide (amino acids 27-42 of rat brain Gsalpha) without altering the properties of Na+ channel kinetics. Furthermore, application of 1 nM Gsalpha27-42 to Na+ channels in inside-out macropatches increased the ensemble-averaged INa by 32.5 +/- 6.8 % (n = 8, P < 0.05 vs. baseline). The increase in INa was reversible upon Gsalpha27-42 peptide washout. Single channel experiments showed that the Gsalpha27-42 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 (Gsalpha27-36, Gsalpha33-42 and Gsalpha30-39) of the 16 amino acid Gsalpha peptide (Gsalpha27-42 peptide) showed that only the C-terminal segment of this peptide (Gsalpha33-42) significantly increased INa in a dose-dependent fashion. These results show that cardiac INa is regulated by Gsalpha 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 Gsalpha is important in modulating cardiac Na+ channels.

Amyloid Precursor Protein Enhances Nav1.6 Sodium Channel Cell Surface Expression

“…The G o protein binding domain in the intracellular domain of APP is known to selectively activate the heterotrimeric G protein G o (8 -10). Although G protein modulation of Nav1.6 has not been reported previously, our findings are consistent with G proteinmediated modulation of various other sodium channels (37)(38)(39). The increase in cell surface expression was dependent upon phosphorylation of APP695 at Thr-668.…”

“…APP-mediated Increases in Nav1.6 Cell Surface Expression Are G Protein-dependent-Various sodium channels are modulated by G proteins (37)(38)(39). To investigate whether a G o protein-linked mechanism could contribute to the increase in Nav1.6 surface expression on overexpression of APP, we conducted experiments in HEK293 Nav1.6 cells, which have strong endogenous expression of APP.…”

Regulation of caveolar cardiac sodium current by a single Gsα histidine residue

“…Under these conditions, G s ␣ increases Na v 1.5 current amplitude through a parallel PKA-independent pathway without altering voltage dependence or kinetics of the sodium current (I Na ). Overall, these studies suggest that the increase in current amplitude is mediated by activating additional channels from a cellular reservoir of Na v 1.5 channels (15). Furthermore, the addition of a short NH 2 -terminal G s ␣-derived peptide (residues 28 -42) to the pipette solution used in whole cell patch-clamp recordings can mimic the effect of ␤-AR stimulation and is prevented by the addition of anti-G s ␣ antibodies (15,33).…”

“…PKA-mediated phosphorylation of voltage-gated Na ϩ channel subunit 1.5 (Na v 1.5) channels typically results in increases in both current amplitude and the rate of current decay (10,15,17,21,34). In addition to this classic paradigm, another pathway has been identified through studies in which pipette dialysis was used to apply the PKA inhibitor (PKI) peptide to adult ventricular myocytes (15). Under these conditions, G s ␣ increases Na v 1.5 current amplitude through a parallel PKA-independent pathway without altering voltage dependence or kinetics of the sodium current (I Na ).…”

Genesis of the monophasic action potential: role of interstitial resistance and boundary gradients

“…In all simulations, the Pandit et al (27) model was used to describe the transmembrane ionic flux (I ion) with a single modification, in which gNa was increased from 1.064 to 1.5 mS/cm 2 to more closely approximate the upstroke rate of rise observed for the mouse action potential (1,23). The modification enabled a better comparison with experimental data because the time course of the Pandit et al model (developed for the rat) resembles that of a mouse cardiac action potential.…”