title: PKA and phosphatases attached to the Ca(V)1.2 channel regulate channel activity in cell-free patches.
abstract: Calmodulin (CaM) + ATP can reprime voltage-gated L-type Ca(2+) channels (Ca(V)1.2) in inside-out patches for activation, but this effect decreases time dependently. This suggests that the Ca(V)1.2 channel activity is regulated by additional cytoplasmic factors. To test this hypothesis, we examined the role of cAMP-dependent protein kinase A (PKA) and protein phosphatases in the regulation of Ca(V)1.2 channel activity in the inside-out mode in guinea pig ventricular myocytes. Ca(V)1.2 channel activity quickly disappeared after the patch was excised from the cell and recovered to only 9% of that in the cell-attached mode on application of CaM + ATP at 10 min after the inside out. However, immediate exposure of the excised patch to the catalytic subunit of PKA + ATP or the nonspecific phosphatase inhibitor okadaic acid significantly increased the Ca(V)1.2 channel activity recovery by CaM + ATP (114 and 96%, respectively) at 10 min. Interestingly, incubation of the excised patches with cAMP + ATP also increased CaM/ATP-induced Ca(V)1.2 channel activity recovery (108%), and this effect was blocked by the nonspecific protein kinase inhibitor K252a. The channel activity in the inside-out mode was not maintained by either catalytic subunit of PKA or cAMP + ATP in the absence of CaM, but was stably maintained in the presence of CaM for more than 40 min. These results suggest that PKA and phosphatase(s) attached on or near the Ca(V)1.2 channel regulate the basal channel activity, presumably through modulation of the dynamic CaM interaction with the channel.
authors.affiliation: Department of Physiology, Graduate School of Medical & Dental Sciences, Kagoshima University, Kagoshima, Japan;
authors.forename: Jianjun
authors.initials: J
authors.lastname: Xu
authors.affiliation: Department of Physiology, Graduate School of Medical & Dental Sciences, Kagoshima University, Kagoshima, Japan; Department of Pharmaceutical Toxicology, School of Pharmacy, China Medical University, Shenyang, China; Department of Ethnopharmacology, School of Pharmacy, China Medical University, Shenyang, China; and.
authors.forename: Lifeng
authors.initials: L
authors.lastname: Yu
authors.affiliation: Department of Physiology, Graduate School of Medical & Dental Sciences, Kagoshima University, Kagoshima, Japan;
authors.forename: Etsuko
authors.initials: E
authors.lastname: Minobe
authors.affiliation: Department of Physiology, Graduate School of Medical & Dental Sciences, Kagoshima University, Kagoshima, Japan; Laboratory of Environmental Biology, Northeastern University, Shenyang, China.
authors.forename: Liting
authors.initials: L
authors.lastname: Lu
authors.affiliation: Department of Physiology, Graduate School of Medical & Dental Sciences, Kagoshima University, Kagoshima, Japan; Department of Pharmaceutical Toxicology, School of Pharmacy, China Medical University, Shenyang, China;
authors.forename: Ming
authors.initials: M
authors.lastname: Lei
authors.affiliation: Department of Physiology, Graduate School of Medical & Dental Sciences, Kagoshima University, Kagoshima, Japan; kame@m.kufm.kagoshima-u.ac.jp.
authors.forename: Masaki
authors.initials: M
authors.lastname: Kameyama
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D020746:Calcium Channels, L-Type / Q000502:physiology*; D002147:Calmodulin / Q000378:metabolism*; D002474:Cell-Free System; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D006168:Guinea Pigs; D015640:Ion Channel Gating / Q000502:physiology; D032383:Myocytes, Cardiac / Q000502:physiology*; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*
chemical_list: D020746:Calcium Channels, L-Type; D002147:Calmodulin; C419385:L-type calcium channel alpha(1C); D000255:Adenosine Triphosphate; D017868:Cyclic AMP-Dependent Protein Kinases; D010744:Phosphoric Monoester Hydrolases; D002118:Calcium
keywords: calcium channel; cardiac myocytes; patch clamp