title: Ca2+ release from the sarcoplasmic reticulum is required for sustained TRPM4 activity in cerebral artery smooth muscle cells.
abstract: The melastatin transient receptor potential (TRP) channel TRPM4 is a critical regulator of vascular smooth muscle cell membrane potential and contractility. Activation of the channel is Ca(2+)-dependent, but prolonged exposure to high (>1 microM) levels of intracellular Ca(2+) causes rapid (within approximately 2 min) desensitization of TRPM4 currents under conventional whole cell and inside-out patch-clamp conditions. The goal of the present study was to establish a novel method to record sustained TRPM4 currents in smooth muscle cells under near-physiological conditions. Using the amphotericin B-perforated patch-clamp technique, we recorded and characterized sustained (up to 30 min) transient inward cation currents (TICCs) in freshly isolated cerebral artery myocytes. In symmetrical cation solutions, TICCs reversed at 0 mV and had an apparent unitary conductance of 25 pS. Replacement of extracellular Na(+) with the nonpermeable cation N-methyl-d-glucamine abolished the current. TICC activity was attenuated by the TRPM4 blockers fluflenamic acid and 9-phenanthrol. Selective silencing of TRPM4 expression using small interfering RNA diminished TICC activity, suggesting that the molecular identity of the responsible ion channel is TRPM4. We used the perforated patch-clamp method to test the hypothesis that TRPM4 is activated by intracellular Ca(2+) signaling events. We found that TICC activity is independent of Ca(2+) influx and ryanodine receptor activity but is attenuated by sarco(endo)plasmic reticulum Ca(2+)-ATPase inhibition and blockade of inositol 1,4,5-trisphosphate receptor-mediated Ca(2+) release from the sarcoplasmic reticulum. Our findings suggest that TRPM4 channels in cerebral artery myocytes are regulated by Ca(2+) release from inositol 1,4,5-trisphosphate receptor on the sarcoplasmic reticulum.
authors.affiliation: Vascular Physiology Research Group, Department of Biomedical Sciences, Colorado State University, Fort Collins, Colorado 80523-1617, USA.
authors.forename: Albert L
authors.initials: AL
authors.lastname: Gonzales
authors.forename: Gregory C
authors.initials: GC
authors.lastname: Amberg
authors.forename: Scott
authors.initials: S
authors.lastname: Earley
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology; D002536:Cerebral Arteries / Q000378:metabolism*; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000378:metabolism; D032389:Myocytes, Smooth Muscle / Q000378:metabolism*; D051381:Rats; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D050053:TRPM Cation Channels / Q000378:metabolism*; D013997:Time Factors
chemical_list: D050053:TRPM Cation Channels; C500239:TRPM4 protein, rat; D002118:Calcium