Calmodulin reverses rundown of L-type Ca(2+) channels in guinea pig ventricular myocytes.
Calmodulin (CaM) is implicated in regulation of Ca(2+) channels as a Ca(2+) sensor. The effect of CaM on rundown of L-type Ca(2+) channels in inside-out patch form was investigated in guinea pig ventricular myocytes. Ca(2+) channel activity disappeared within 1-3 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 microM) + 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 Ca(2+) concentrations ([Ca(2+)]); however, increase to micromolar [Ca(2+)] rapidly inactivated the channel activity induced, revealing that the effect of CaM on the channel was Ca(2+) dependent. At the 2nd, 4th, 6th, 8th, and 10th minutes after patch excision, CaM (0.75 microM) + ATP induced Ca(2+) 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'-(beta,gamma-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 1-10 microM) did not block the effect of CaM, indicating that the effect of CaM on the Ca(2+) channel was phosphorylation independent. Neither CaM nor ATP alone induced Ca(2+) channel activity, showing a cooperative effect of CaM and ATP on the Ca(2+) channel. These results suggest that CaM is a crucial regulatory factor of Ca(2+) channel basal activity.
Xu
Jian-Jun
JJ
Department of Physiology, Graduate School of Medical and Dental Sciences, Kagoshima University, Sakuragaoka, Kagoshima 890-8544, Japan.
Hao
Li-Ying
LY
Kameyama
Asako
A
Kameyama
Masaki
M
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism; D020746:Calcium Channels, L-Type / Q000378:metabolism*; D002147:Calmodulin / Q000494:pharmacology*; D004357:Drug Synergism; D005260:Female; D006168:Guinea Pigs; D006352:Heart Ventricles / Q000166:cytology; D008564:Membrane Potentials / Q000187:drug effects; D032383:Myocytes, Cardiac / Q000187:drug effects* / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology
D020746:Calcium Channels, L-Type; D002147:Calmodulin; D047428:Protein Kinase Inhibitors; D000255:Adenosine Triphosphate; D002118:Calcium