Effects of Nitric Oxide on Voltage-Gated K⁺ Currents in Human Cardiac Fibroblasts through the Protein Kinase G and Protein Kinase A Pathways but Not through S-Nitrosylation

Abstract

This study investigated the expression of voltage-gated K⁺ (K_V) channels in human cardiac fibroblasts (HCFs), and the effect of nitric oxide (NO) on the K_V currents, and the underlying phosphorylation mechanisms. In reverse transcription polymerase chain reaction, two types of K_V channels were detected in HCFs: delayed rectifier K⁺ channel and transient outward K⁺ channel. In whole-cell patch-clamp technique, delayed rectifier K⁺ current (I_K) exhibited fast activation and slow inactivation, while transient outward K⁺ current (I_to) showed fast activation and inactivation kinetics. Both currents were blocked by 4-aminopyridine. An NO donor, S-nitroso-N-acetylpenicillamine (SNAP), increased the amplitude of I_K in a concentration-dependent manner with an EC₅₀ value of 26.4 µM, but did not affect I_to. The stimulating effect of SNAP on I_K was blocked by pretreatment with 1H-(1,2,4)oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) or by KT5823. 8-bromo-cyclic GMP stimulated the I_K. The stimulating effect of SNAP on I_K was also blocked by pretreatment with KT5720 or by SQ22536. Forskolin and 8-bromo-cyclic AMP each stimulated I_K. On the other hand, the stimulating effect of SNAP on I_K was not blocked by pretreatment of N-ethylmaleimide or by DL-dithiothreitol. Our data suggest that NO enhances I_K, but not I_to, among K_V currents of HCFs, and the stimulating effect of NO on I_K is through the PKG and PKA pathways, not through S-nitrosylation.

Keywords: S-nitrosylation; delayed rectifier K+ channel; human cardiac fibroblasts; nitric oxide; protein kinase A; protein kinase G; transient outward K+ channel; voltage-gated K+ channels.