Identification of sites responsible for potentiation of type 2.3 calcium currents by acetyl-beta-methylcholine.
To address mechanisms for the differential sensitivity of voltage-gated Ca2+ channels (Cav) to agonists, channel activity was compared in Xenopus oocytes coexpressing muscarinic M(1) receptors and different Cav alpha1 subunits, all with beta1B,alpha2/delta subunits. Acetyl-beta-methylcholine (MCh) decreased Cav 1.2c currents, did not affect 2.1 or 2.2 currents, but potentiated Cav 2.3 currents. Phorbol 12-myristate 13-acetate (PMA) did not affect Cav 1.2c or 2.1 currents but potentiated 2.2 and 2.3 currents. Comparison of the amino acid sequences of the alpha1 subunits revealed a set of potential protein kinase C phosphorylation sites in common between the 2.2 and 2.3 channels that respond to PMA and a set of potential sites unique to the alpha1 2.3 subunits that respond to MCh. Quadruple Ser --> Ala mutation of the predicted MCh sites in the alpha1 2.3 subunit (Ser-888, Ser-892, and Ser-894 in the II-III linker and Ser-1987 in the C terminus) caused loss of the MCh response but not the PMA response. Triple Ser --> Ala mutation of just the II-III linker sites gave similar results. Ser-888 or Ser-892 was sufficient for the MCh responsiveness, whereas Ser-894 required the presence of Ser-1987. Ser --> Asp substitution of Ser-888, Ser-892, Ser-1987, and Ser-892/Ser-1987 increased the basal current and decreased the MCh response but did not alter the PMA response. These results reveal that sites unique to the II-III linker of alpha1 2.3 subunits mediate the responsiveness of Cav 2.3 channels to MCh. Because Cav 2.3 channels contribute to action potential-induced Ca2+ influx, these sites may account for M1 receptor-mediated regulation of neurotransmission at some synapses.
Kamatchi
Ganesan L
GL
Department of Anesthesiology, University of Virginia Health Sciences Systems, Charlottesville, Virginia 22908-0710, USA. gk3p@virginia.edu
Franke
Ruthie
R
Lynch
Carl
C
Sando
Julianne J
JJ
D000818:Animals; D001665:Binding Sites; D020908:Calcium Channels, R-Type / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D005260:Female; D066298:In Vitro Techniques; D016210:Methacholine Chloride / Q000494:pharmacology*; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D021122:Protein Subunits; D051381:Rats; D011994:Recombinant Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014982:Xenopus laevis
D020908:Calcium Channels, R-Type; D021122:Protein Subunits; D011994:Recombinant Proteins; D016210:Methacholine Chloride; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate