The distinct roles of calmodulin and calmodulin kinase II in the reversal of run-down of L-type Ca(2+) channels in guinea-pig ventricular myocytes.
In this study, we investigated the roles of calmodulin kinase II (CaMKII) and calmodulin (CaM) in the reversal of run-down of L-type Ca(2+) channels. Single Ca(2+)-channel activities in guinea-pig ventricular myocytes were recorded using the patch-clamp technique, and run-down of the channel activities was induced by inside-out patch formation in the basic internal solution. At 1 min after patch excision, 1 - 30 muM CaMKII mutant T286D (CaMKIIT286D), a constitutively active type of CaMKII, induced the Ca(2+)-channel activities to only 2% - 10% of that recorded in the cell-attached mode. However, in the presence of CaMKIIT286D, the time-dependent attenuation of CaM's effects in the reversal of run-down was abolished. A GST-fusion protein containing amino acids 1509 - 1789 of the C-terminal region of guinea-pig Cav1.2 (CT1) was prepared. In pull-down assays, CT1 treated with CaMKIIT286D showed a higher affinity for CaM compared with CT1 treated with phosphatase. We propose a model in which CaMKII-mediated phosphorylation of the channels regulates the binding of CaM to the channels in the reversal of run-down of L-type Ca(2+) channels.
Hao
Li-Ying
LY
Department of Pharmaceutical Toxicology, School of Pharmacological Sciences, China Medical University, Shenyang 110001, China. lyhao@mail.cmu.edu.cn
Wang
Wu-Yang
WY
Minobe
Etsuko
E
Han
Dong-Yun
DY
Xu
Jian-Jun
JJ
Kameyama
Asako
A
Kameyama
Masaki
M
D000818:Animals; D020746:Calcium Channels, L-Type / Q000502:physiology*; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000235:genetics / Q000502:physiology*; D002147:Calmodulin / Q000378:metabolism*; D002460:Cell Line; D006168:Guinea Pigs; D015640:Ion Channel Gating / Q000502:physiology; D032383:Myocytes, Cardiac / Q000502:physiology*; D018408:Patch-Clamp Techniques; D010766:Phosphorylation; D051381:Rats; D013997:Time Factors; D016276:Ventricular Function / Q000502:physiology*
D020746:Calcium Channels, L-Type; D002147:Calmodulin; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2