P2Y 2 receptor stimulation increases K secretion by activating K Ca 3.1 potassium channels in human mammary epithelial cells

Abstract

Human mammary epithelial (HME) cells express several P2Y receptor subtypes located in both apical and basolateral membranes. Apical UTP (10 μM) or ATP-γ-S (10 μM) stimulation of monolayers mounted in Ussing chambers evoked a rapid, but transient decrease in short circuit current (Isc), consistent with activation of an apical K conductance. In contrast, basolateral stimulation with UTP (10 μM) produced a longer duration increase in Isc that was blocked by 5 μM benzamil. Inhibition of Ca2+ mobilization using BAPTA-AM abolished these changes in Isc. Moreover, apical pretreatment with charybdotoxin (200 nM), blocked the effect on Isc by >80% and a similar degree of inhibition was observed with clotrimazole (10 μM) and TRAM-34 (0.5 μM). Silencing expression of KCa3.1 by transfection of HME cells with an shRNA containing lentivirus construct targeting the channel produced ~80% inhibition of mRNA expression and 71% inhibition of the apical Isc response. In addition, silencing the P2Y2 receptor by expression of an shRNA containing lentivirus construct reduced the level of P2Y2 mRNA by 75% and inhibited the apical effects of 10 μM ATP-γ-S by 65%. These results suggest that P2Y2 receptors mediate the effects of purinoceptor agonists on K secretion by regulating the activity of KCa3.1 channels expressed in the apical membrane of HME cells.