Rationale: The cardiac inwardly rectifying K⁺ current (I_K1) plays a critical role in modulating excitability by setting the resting membrane potential and shaping phase 3 of the cardiac action potential.

Objective: This study aims to analyze the effects of nitric oxide (NO) on human atrial I_K1 and on Kir2.1 channels, the major isoform of inwardly rectifying channels present in the human heart.

Methods and Results: Currents were recorded in enzymatically isolated myocytes and in transiently transfected CHO cells, respectively. NO at myocardial physiological concentrations (25 to 500 nmol/L) increased inward and outward I_K1 and I_Kir2.1. These effects were accompanied by hyperpolarization of the resting membrane potential and a shortening of the duration of phase 3 of the human atrial action potential. The I_Kir2.1 increase was attributable to an increase in the open probability of the channel. Site-directed mutagenesis analysis demonstrated that NO effects were mediated by the selective S-nitrosylation of Kir2.1 Cys76 residue. Single ion monitoring experiments performed by liquid chromatography/tandem mass spectrometry suggested that the primary sequence that surrounds Cys76 determines its selective S-nitrosylation. Chronic atrial fibrillation, which produces a decrease in NO bioavailability, decreased the S-nitrosylation of Kir2.1 channels in human atrial samples as demonstrated by a biotin-switch assay, followed by Western blot.

Conclusions: The results demonstrated that, under physiological conditions, NO regulates human cardiac I_K1 through a redox-related process.