Inhibitory role of phosphatidylinositol 4,5-bisphosphate on TMEM16A-encoded calcium-activated chloride channels in rat pulmonary artery.
OBJECTIVE
Calcium-activated chloride channels (CaCCs) are key depolarizing mechanisms that have an important role in vascular smooth muscle contraction. Here, we investigated whether these channels are regulated by phosphatidylinositol (4,5) bisphosphate [P(4,5)P2 ], a known regulator of various ion channels.


METHODS
Calcium-activated Cl(-) currents (IClCa ) were recorded by patch clamp electrophysiology of rat isolated pulmonary artery smooth muscle cells. TMEM16A protein-phosphoinositide interaction was studied by co-immunoprecipitation and phosphoinositide binding arrays on protein lysates from whole pulmonary arteries and HEK293 cells overexpressing TMEM16A, the molecular correlate.


RESULTS
PI(4,5)P2 and other phospholipids were shown to bind directly to TMEM16A isolated from whole pulmonary artery (PA) and TMEM16A-eGFP expressed in HEK293 cells. Agents that reduced PI(4,5)P2 levels through different routes [PLC activation, PI4K inhibition, PI(4,5)P2 scavenging and absorption] all increased IClCa evoked by solutions containing clamped-free [Ca(2+) ], whereas enrichment of activating solutions with PI(4,5)P2 inhibited IClca in PA smooth muscle cells with approximately 50% reduction at 1 μM.


CONCLUSIONS
These data are the first to show a negative regulation of TMEM16A-encoded CaCCs by PI(4,5)P2 and propose that control of PI(4,5)P2 levels is a key determinant of arterial physiology.
Vascular Biology Research Centre, Institute of Cardiovascular and Cell Sciences, St George's, University of London, London, UK.
H A T
HA
Pritchard
N
N
Leblanc
A P
AP
Albert
I A
IA
Greenwood
D000818:Animals; D000075369:Anoctamin-1; D018118:Chloride Channels / Q000502:physiology*; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D018345:Mice, Knockout; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000502:physiology*; D011651:Pulmonary Artery / Q000166:cytology / Q000502:physiology*; D017208:Rats, Wistar
C552942:ANO1 protein, rat; D000075369:Anoctamin-1; D018118:Chloride Channels; D019269:Phosphatidylinositol 4,5-Diphosphate