Apical SK potassium channels and Ca²⁺-dependent anion secretion in endometrial epithelial cells

Apical uridine triphosphate (UTP) stimulation was shown to increase short circuit current (I_sc) in immortalized porcine endometrial gland epithelial monolayers. Pretreatment with the bee venom toxin apamin enhanced this response. Voltage-clamp experiments using amphotericin B-permeablized monolayers revealed that the apamin-sensitive current increased immediately after UTP stimulation and was K⁺ dependent. The current-voltage relationship was slightly inwardly rectifying with a reversal potential of -52 ± 2 mV, and the P_K/_PNa ratio was 14, indicating high selectivity for K⁺. Concentration-response relationships for apamin and dequalinium had IC₅₀ values of 0.5 nM and 1.8 μM, respectively, consistent with data previously reported for SK3 channels in excitable cells and hepatocytes. Treatment of monolayers with 50 μM BAPTA-AM completely blocked the effects of UTP on K⁺ channel activation, indicating that the apamin-sensitive current was also Ca²⁺ dependent. Moreover, channel activation was blocked by calmidazolium (IC₅₀ = 5 μM), suggesting a role for calmodulin in Ca²⁺-dependent regulation of channel activity. RT-PCR experiments demonstrated expression of mRNA for the SK1 and SK3 channels, but not SK2 channels. Treatment of monolayers with 20 nM oestradiol-17β produced a 2-fold increase in SK3 mRNA, a 2-fold decrease in SK1 mRNA, but no change in GAPDH mRNA expression. This result correlated with a 2.5-fold increase in apamin-sensitive K⁺ channel activity in the apical membrane. We speculate that SK channels provide a mechanism for rapidly sensing changes in intracellular Ca²⁺ near the apical membrane, evoking immediate hyperpolarization necessary for increasing the driving force for anion efflux following P2Y receptor activation.