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Inhibitory role of phosphatidylinositol 4,5‐bisphosphate on TMEM16A ‐encoded calcium‐activated chloride channels in rat pulmonary artery
Author(s) -
Pritchard H A T,
Leblanc N,
Albert A P,
Greenwood I A
Publication year - 2014
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1111/bph.12778
Subject(s) - chloride channel , phosphatidylinositol , hek 293 cells , patch clamp , microbiology and biotechnology , pi , chemistry , depolarization , phosphatidylinositol 4,5 bisphosphate , gq alpha subunit , calcium , biophysics , biochemistry , biology , signal transduction , g protein , receptor , organic chemistry
Background and Purpose 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) P 2 ], a known regulator of various ion channels. Experimental Approach Calcium‐activated Cl − currents ( I ClCa ) 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 HEK 293 cells overexpressing TMEM16A , the molecular correlate. Key Results PI (4,5) P 2 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) P 2 levels through different routes [PL C activation, PI4K inhibition, PI (4,5) P 2 scavenging and absorption] all increased I ClCa evoked by solutions containing clamped‐free [ Ca 2+ ], whereas enrichment of activating solutions with PI (4,5) P 2 inhibited I Clca in PA smooth muscle cells with approximately 50% reduction at 1 μM. Conclusions and Implications These data are the first to show a negative regulation of TMEM16A ‐encoded CaCCs by PI (4,5) P 2 and propose that control of PI (4,5) P 2 levels is a key determinant of arterial physiology.