Expression of Ca ²⁺-Activated K ⁺ Channels in Human Dermal Fibroblasts and Their Roles in Apoptosis

Abstract

Backgrounds/Aims: Cell proliferation and apoptosis are responsible for maintaining normal tissue homeostasis, and K⁺ currents play important roles in regulating the physiological balance between them. This function of Ca²⁺-activated K⁺ (K_Ca) channels has been demonstrated in many types of tissues, but not in dermal fibroblasts. We investigated the expression of K_Ca channels and their effects on proliferation and apoptosis in human dermal fibroblasts. Methods: We used discoidin domain receptor 2 immunostaining to identify human dermal fibroblasts, and reverse transcription polymerase chain reaction, Western blot analysis and electrophysiological patch clamp recordings to evaluate the expression and characteristics of 3 members of the K_Ca channel family, large-conductance K_Ca (BK), intermediate-conductance K_Ca (IK) and small-conductance K_Ca channels. We also used the 3-(4,5-dimethyl-2-yl)-2,5-diphenyltetrazolium bromide assay, flow cytometry, Hoechst 33258 staining and Depsipher staining to investigate the effects of K_Ca channels on cell proliferation and the mechanisms involved. Results and Conclusions: All 3 members of the K_Ca channel family were found in fibroblasts. 1,3-Dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one (NS1619, a BK channel activator) or 1-ethyl-2-benzimidazolinone (EBIO, an IK channel activator) decreased the proliferation of fibroblasts and induced apoptotic changes by mitochondrial membrane potential disruption. However, a pan-caspase inhibitor (Z-VAD-fmk) failed to prevent the apoptotic changes. Our findings indicate that 3 types of functional K_Ca channels are expressed in human dermal fibroblasts and are involved in apoptosis of the cells through the mitochondrial apoptotic pathway, but seemingly in a caspase-independent manner.