Inhibitory Role of PIP2 on Calcium-Activated Chloride Channel Activity

Abstract

Ca2+-activated Cl- channels (CaCCs) in vascular and many non-vascular smooth muscle cells are thought to be encoded by the gene TMEM16A. Our group recently showed that the amplitude and pharmacology of CaCCs currents (IClca) are influenced by cholesterol depletion (Sones et al. Cardiovasc Res 2010;87:476-484). The aim of this study was to investigate the mechanism by which native CaCCs are regulated by cholesterol depletion and the possible role of phosphatidylinositol 4, 5-bisphosphate (PIP2). Freshly isolated pulmonary artery myocytes were obtained from male Wistar rats (200-225g), sacrificed in accordance with Schedule 1 of the United Kingdom Animals Act (1986). In whole cell patch clamp recording mode, macroscopic IClca were elicited by dialysing the cells with a pipette solution set to 500nM ‘free’ [Ca2+]. Single channel inside-out recordings were also induced by 500nM [Ca2+] and displayed similar characteristics to earlier characterisation. These currents displayed the distinct voltage- and time-dependent properties described in previous studies. Cholesterol depletion from the cell membrane through the application of 3mg.ml−1 methyl-β-cyclodextrin (mβcd) augmented IClca, which was prevented by application of the TMEM16A specific inhibitor T16Ainh-A01. Augmentation by mβcd was also sensitive to the phospholipase C inhibitor U73312, but not the inactive analogue U73343. Inhibiting PIP2 synthesis by incubation with 20µM wortmannin, a PI-4 kinase inhibitor augmented IClca and prevented the stimulatory effects of mβcd. Enrichment of the pipette solution with 1μM diC-8 PIP2 attenuated IClcaand suppressed the effect of mβcd. Wortmannin and PIP2 effects on IClca were replicated in single channel inside-out recordings. Furthermore, the application of the poly-cation Poly-L-lysine (50µg/ml), which scavenges PIP2, augmented IClca. This study suggests that cholesterol depletion of the cell membrane increases TMEM16A/CaCC activity, partly through the removal of the inhibitory effect of PIP2.