S-Nitrosylation-Mediated Reduction of Ca V 1.2 Surface Expression and Open Probability Underlies Attenuated Vasoconstriction Induced by Nitric Oxide

Abstract

Background: L-type Ca V 1.2 calcium channel, the primary gateway for Ca 2+ influx in smooth muscles, is widely regulated by multiple posttranslational modifications, such as protein kinase-mediated phosphorylation and nitric oxide-induced S-nitrosylation. However, the effect of S-nitrosylation on Ca V 1.2 channel function and its role in arterial contractility are not well understood. Methods: Electrophysiological recordings, Ca 2+ and confocal imaging, and biochemical assays were used to functionally characterize S-nitrosylated Ca V 1.2 channels in vitro, while pressure myography and tail-cuff blood pressure measurement were conducted to evaluate the physiological effects of Ca V 1.2 S-nitrosylation ex vivo and in vivo. Results: S-nitrosylation significantly reduced the Ca V 1.2 current density by promoting lysosomal degradation that leads to decreased levels of total and surface Ca V 1.2 channel proteins in a Ca V β-independent manner and reducing the open probability of Ca V 1.2 channel. Mechanistically, the Cys1180 and Cys1280 residues within Ca V 1.2 channel have been determined as the molecular targets for S-nitrosylation as substitution of either Cys1180 or Cys1280 for serine resulted in substantial reduction of S-nitrosylation levels. Of note, Ca V 1.2 S-nitrosylation levels were significantly reduced in arteries isolated from both spontaneously hypertensive rats and patients with pulmonary hypertension. Moreover, mouse resistance arteries incubated with S-nitrosocysteine displayed much lower contractility and spontaneously hypertensive rats injected with S-nitrosocysteine also showed significantly reduced blood pressure, suggesting that reduced S-nitrosylation contributes to the upregulation of Ca V 1.2 channel activity in hypertensive arteries. Conclusions: This study provides strong evidence that S-nitrosylation-mediated downregulation of Ca V 1.2 channels is via 2 distinctive mechanisms and the findings offer potential pathways for therapeutic inventions in hypertension.