Direct effect of Ca2+-calmodulin on cGMP-activated Ca2+-dependent Cl-channels in rat mesenteric artery myocytes.
Recently a novel cGMP-activated Ca2+-dependent Cl- channel has been described in rat mesenteric artery smooth muscle cells. In the present work we have investigated the actions of calmodulin (CaM) on single channel cGMP-activated Ca2+-dependent Cl- current (ICl(cGMP,Ca) in inside-out patches. When 1 microm CaM was applied to the intracellular surface of inside-out patches bathed with 10 microm cGMP and 100 nm [Ca2+]i there was approximately a 10-fold increase in channel open probability (NPo). This effect of CaM was not observed with lower [Ca2+]i and 100 nm [Ca2+]i with 1 microm CaM did not activate Cl- channels in the absence of cGMP. The unitary conductance, reversal potential and mean open time of the single-channel currents were similar in the absence or presence of CaM. With 10 microm cGMP and 100 nm [Ca2+]i the relationship between NPo and CaM concentration was well fitted by the Hill equation yielding an equilibrium constant for CaM of about 1.9 nm and a Hill coefficient of 1.7. With 1 microm CaM (+10 microm cGMP) the relationship between [Ca2+]i and NPo was also fitted by the Hill equation which yielded an apparent equilibrium constant of 74 nm [Ca2+]i and a Hill coefficient of 4.8. When [Ca2+]i was increased from 300 nm to 1 microm there was a decrease in NPo. The potentiating effect of CaM was markedly reduced by the selective CaM binding peptide Trp (5 nm) but not by the Ca2+/CaM-dependent protein kinase II (CaMKII) inhibitor autocamtide II related inhibitory peptide (AIP). It is concluded that CaM potentiates the activity of single channel ICl(cGMP,Ca) by increasing the probability of channel opening via a CaMKII-independent mechanism.
Piper
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Department of Basic Medical Sciences, Pharmacology and Clinical Pharmacology, Cardiovascular Research Group, St George's Hospital Medical School, Cranmer Terrace, London SW17 0RE, UK. a.piper@sghms.ac.uk
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D000200:Action Potentials / Q000187:drug effects / Q000502:physiology; D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002147:Calmodulin / Q000494:pharmacology*; D065101:Chloride Channel Agonists; D018118:Chloride Channels / Q000502:physiology*; D006152:Cyclic GMP / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D005260:Female; D008297:Male; D008638:Mesenteric Arteries / Q000187:drug effects / Q000502:physiology; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000502:physiology; D032389:Myocytes, Smooth Muscle / Q000187:drug effects* / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley
D002147:Calmodulin; D065101:Chloride Channel Agonists; D018118:Chloride Channels; D006152:Cyclic GMP; D002118:Calcium