Hot Topics: Cardiac Ca2+ Channel Regulation in the Fight-or-Flight Response: the monomeric small G-protein as another piece in the puzzle

Thirty eight years after the discovery that injection of the catalytic subunits of cyclic AMP-dependent protein kinase A (PKA) into isolated pig ventricular cardiac myocytes (1) increases the amplitude of L-type Ca²⁺ current (today known to be formed by Ca_v1.2 channels, (2)), we are now getting close to understand the responsible molecular mechanism. Initial research that has focused on direct regulatory mechanisms, such as the activation by the α-subunit of the stimulatory heterotrimeric G-protein Gs (3), and direct phosphorylation of the pore-forming α1- or accessory β2-subunit of the Ca_v1.2 channel has not provided a conclusive answer. β-adrenergic modulation was not (α1 Ser-1928, (4)) or only partially (α1 Ser-1700/Thr-1704, (5)) prevented in mutant mice lacking predicted phosphorylation sites in α1 or β2 subunits (6). This was further supported by an elegant transgenic approach showing that expression of channel complexes with all PKA consensus sites mutated to alanine in the Ca_v1.2 α1 and the β2 subunits (7) has no effects on β-adrenergic modulation.

Assuming that other proteins may be involved in this regulatory pathway, Liu et al. (7) used a biotinylation-based proteomic proximity assay to identify proteins in the Ca_v1.2 neighborhood regulated by isoproterenol treatment in cardiomyocytes. This led to the discovery of the small Ras-like G protein Rad as a potential candidate. It belongs to the family of the Rad, Rem, Rem2 and Gem/Kir (RGK) Ras-like GTP-binding proteins, which were shown earlier to inhibit high-voltage-activated Ca²⁺ channels by binding to their β-subunits. Indeed, Rad appears to be the long-sought missing piece in the puzzle: in HEK293T Rad-mediated inhibition of cardiac Ca_v1.2 channels was relieved by PKA phosphorylation. Modulation was prevented either by the simultaneous mutation of the known cardiac PKA phosphorylation sites of Rad to alanine or by inhibiting the interaction of Rad with the β-subunit.

This mechanism may be universal and may also explain the PKA modulation of other types of β-subunit associated high-voltage activated Ca²⁺ channels in Rad expressing cells. It also provides evidence that the mechanism of PKA modulation of Ca_v1.2 Ca²⁺ channels appears to differ in different cell types. Clearly, direct phosphorylation of Ser-1928 of the α1-subunit is not required for β-adrenergic regulation of Ca_v1.2 in the heart (4,7). However, this residue is required for β-adrenergic stimulation of Ca²⁺ channels in hippocampal neurons and for hyperglycaemia-induced stimulation of Ca²⁺ currents in arterial smooth muscle cells (8,9).

Despite compelling evidence for this novel mechanism, several questions remain: First, what is the role of Rad-dependent modulation of the cardiac L-type channel in intact cardiomyocytes, for example in cardiomyocytes expressing Rad with mutated PKA phosphorylation sites. Second, what is the relative importance of Rad versus modulation by direct phosphorylation of Ser-1700 (and Thr-1704), which has also been found to confer some of the PKA activation of L-type current in cardiomyocytes of Ser1700Ala mutant mice (5).

Despite these open questions, we are now getting close to understand the molecular mechanisms underlying the fight-or-flight response.

Comments by Jörg Striessnig, University of Innsbruck, Chair for NC-IUPHAR Subcommitee for Voltage-gated calcium channels, Liaison for NC-IUPHAR subcommittees on Voltage-gated ion channels

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7. Liu G, Papa A, Katchman AN, Zakharov SI, Roybal D, Hennessey JA, et al. (2020) Mechanism of adrenergic CaV1.2 stimulation revealed by proximity proteomics. Nature 577, 695–700 [PMID: 31969708].
8. Qian H, Patriarchi T, Price JL, Matt L, Lee B, Nieves-Cintrón M, et al. (2017) Phosphorylation of Ser1928 mediates the enhanced activity of the L-type Ca²⁺ channel Cav1.2 by the β2-adrenergic receptor in neurons. Sci Signal. 10 (463), pii: eaaf9659
9. Nystoriak MA, Nieves-Cintrón M, Patriarchi T, Buonarati OR, Prada MP, Morotti S, et al. (2017) Ser1928 phosphorylation by PKA stimulates the L-type Ca2+ channel Cav1.2 and vasoconstriction during acute hyperglycemia and diabetes. Sci Signal. 10(463), pii: eaaf9647