Nitric Oxide Stimulates a Large-Conductance Ca²⁺-Activated K⁺ Channel in Human Skin Fibroblasts through Protein Kinase G Pathway

Abstract

In order to investigate the large-conductance Ca²⁺-activated K⁺ (BK_Ca) channel and determine the effects of nitric oxide (NO) on the channel in human skin fibroblasts, we performed electrophysiological patch clamp recordings on 5th-passage cells of human genital skin cultures. The whole-cell outward K⁺ current was increased with depolarization, and proved to be sensitive to NS1619 (a selective BK_Ca channel activator) and iberiotoxin (a specific BK_Cachannel inhibitor). The single-channel currents showed 226 pS of mean conductance in symmetrical K⁺. Sodium nitroprusside (SNP; an NO donor) significantly increased the K⁺ current amplitude in the whole-cell mode, and open probability of the channel (NPo) in the cell-attached mode, but not in the inside-out mode. S-nitroso-N-acetylpenicillamine (an NO donor) and 8-Br-cGMP (a membrane-permeant cGMP analogue) also increased the BK_Cachannel activity. The stimulatory effect of SNP on BK_Ca channels was inhibited by pretreatment with 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxalin-1-one (a soluble guanylyl cyclase inhibitor), or KT5823 [a specific protein kinase G (PKG) inhibitor]. Cytoplasmic PKG also increased the channel activity in inside-out patches. In conclusion, the present data indicate that BK_Ca channels constitute a significant fraction of K⁺ current in human skin fibroblasts, and that NO increases NPo of BK_Ca channels, which are mediated via the cGMP/PKG pathway, without direct effects on the channel.

Effects of nitric oxide on large‐conductance Ca2+‐activated K+ currents in human cardiac fibroblasts through PKA and PKG‐related pathways

“…For example, when sGC becomes activated, and sGC catalyses the production of cGMP, which in turn activates PKG. NO activates BKCa channels through a cGMP‐dependent mechanism in rabbit colonic smooth muscle cells and human dermal fibroblasts . However, the signal transduction mechanisms underlying the effects of NO on BKCa channels are poorly understood, and NO can directly activate BKCa channels in cell‐free membrane patches in the absence of cGMP in rat vascular smooth muscle cells conflicts with previous results .…”

“…NO activates BKCa channels through a cGMP-dependent mechanism in rabbit colonic smooth muscle cells 18 and human dermal fibroblasts. 33 However, the signal transduction mechanisms underlying the effects of NO on BKCa channels are poorly understood, and NO can directly activate BKCa channels in cell-free membrane patches in the absence of cGMP in rat vascular smooth muscle cells conflicts with previous results. 34 Additionally, cross-activation of the PKA pathway, which stimulates currents through cGMP, has been detected in human dermal fibroblasts.…”

The Stimulating Effects of Nitric Oxide on Intermediate Conductance Ca^(2+)-Activated K^+ Channels in Human Dermal Fibroblasts through PKG Pathways but not the PKA Pathways

“…In smooth muscle cells (19) and in rat aortic endothelium (27), NO modulates the BK channels through PKG activation via the cGMP pathway. In a previous study, we also reported that NO stimulates the BK channels in human dermal fibroblasts through the cGMP/PKG pathway (18) and also through partial cross-activation of the cGMP/ PKA pathway (28).…”

Dependence of the Excitability of Pituitary Cells on Cyclic Nucleotides

“…In numerous cell types, it is well established that cGMP activates the BK channels (107–111). The α subunit of BK channels is directly phosphorylated by PKG, causing the activation of these channels (74).…”