Calmodulin reverses rundown of L-type Ca(2+) channels in guinea pig ventricular myocytes |
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Authors: | Xu Jian-Jun Hao Li-Ying Kameyama Asako Kameyama Masaki |
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Institution: | Department of Physiology, Graduate School of Medical and Dental Sciences, Kagoshima University, Sakuragaoka, Kagoshima 890-8544, Japan. |
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Abstract: | Calmodulin (CaM) is implicated in regulation of Ca2+ channels as a Ca2+ sensor. The effect of CaM on rundown of L-type Ca2+ channels in inside-out patch form was investigated in guinea pig ventricular myocytes. Ca2+ channel activity disappeared within 13 min and did not reappear when the patch was excised and exposed to an artificial intracellular solution. However, application of CaM (0.03, 0.3, 3 µM) + 3 mM ATP to the intracellular solution within 1 min after patch excision resulted in dose-dependent activation of channel activity. Channel activity averaged 11.2%, 94.7%, and 292.9%, respectively, of that in cell-attached mode. Channel activity in inside-out patch mode was induced by CaM + ATP at nanomolar Ca2+ concentrations (Ca2+]); however, increase to micromolar Ca2+] rapidly inactivated the channel activity induced, revealing that the effect of CaM on the channel was Ca2+ dependent. At the 2nd, 4th, 6th, 8th, and 10th minutes after patch excision, CaM (0.75 µM) + ATP induced Ca2+ channel activity to 150%, 100%, 96.9%, 29.3%, and 16.6%, respectively, revealing a time-dependent action of CaM on the channel. CaM added with adenosine 5'-( , -imido)triphosphate (AMP-PNP) also induced channel activity, although with much lower potency and shorter duration. Protein kinase inhibitors KN-62, CaM-dependent protein kinase (CaMK)II 281-309, autocamtide-related CaMKII inhibitor peptide, and K252a (each 110 µM) did not block the effect of CaM, indicating that the effect of CaM on the Ca2+ channel was phosphorylation independent. Neither CaM nor ATP alone induced Ca2+ channel activity, showing a cooperative effect of CaM and ATP on the Ca2+ channel. These results suggest that CaM is a crucial regulatory factor of Ca2+ channel basal activity. cardiac myocyte; calcium channel; patch clamp |
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