Role of alpha1 2.3 subunit I-II linker sites in the enhancement of Ca(v) 2.3 current by phorbol 12-myristate 13-acetate and acetyl-beta-methylcholine.
Potentiation of Ca(v) 2.3 currents by phorbol 12-myristate 13-acetate (PMA) or acetyl-beta-methylcholine (MCh) may be due to protein kinase C (PKC)-mediated phosphorylation of the alpha1 2.3 subunit. Mutational analysis of potential PKC sites unique to the alpha1 2.3 subunit revealed several sites in the II-III linker that are specific to MCh (Kamatchi, G., Franke, R., Lynch, C., III, and Sando, J. (2004) J. Biol. Chem. 279, 4102-4109). To identify sites responsive to PMA, Ser/Thr --> Ala mutations were made in potential PKC sites homologous to the alpha1 2.3 and 2.2 subunits, both of which respond to PMA. Wild type alpha1 2.3 or mutants were expressed in Xenopus oocytes in combination with beta1b and alpha2/delta subunits and muscarinic M1 receptors. Inward current (I(Ba)) was recorded using Ba2+ as the charge carrier. Thr-365 of the I-II linker was identified as the primary site of PMA action, and this site also was required, along with the previously identified MCh-selective sites, for the MCh response. Ser-369 and Ser-1995 contributed to current enhancement only if Thr-365 also was available. Mutation of the essential sites to Asp increased the basal I(Ba) and caused a corresponding decrease in the PMA or MCh responses, consistent with possible regulation of these sites by phosphorylation. These results suggest that PMA and MCh both activate a pathway that can regulate the common PMA-sensitive sites in the I-II linker but that MCh also activates an additional pathway required for regulation of the MCh-unique sites, especially in the II-III linker.
Fang
Hongyu
H
Department of Anesthesiology, University of Virginia Health Sciences Systems, Charlottesville, Virginia 22908, USA.
Franke
Ruthie
R
Patanavanich
Saharat
S
Lalvani
Amrita
A
Powell
Natalie K
NK
Sando
Julianne J
JJ
Kamatchi
Ganesan L
GL
D019943:Amino Acid Substitution; D000818:Animals; D015220:Calcium Channels / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D027682:Cation Transport Proteins / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D005260:Female; D016210:Methacholine Chloride / Q000494:pharmacology*; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014982:Xenopus laevis
D015220:Calcium Channels; D027682:Cation Transport Proteins; D016210:Methacholine Chloride; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate