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 Ca super(2+) channels (Ca sub(v)) to agonists, channel activity was compared in Xenopus oocytes coexpressing muscarinic M sub(1) receptors and different Ca sub(v) [alpha]1 subunits, all with [beta]1B,[alpha]2/[delta] subunits. Acetyl-[beta]-methylcholine (MCh) decreased Ca sub(v) 1.2c currents, did not affect 2.1 or 2.2 currents, but potentiated Ca sub(v) 2.3 currents. Phorbol 12-myristate 13-acetate (PMA) did not affect Ca sub(v) 1.2c or 2.1 currents but potentiated 2.2 and 2.3 currents. Comparison of the amino acid sequences of the [alpha] sub(1) 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 [alpha] sub(1) 2.3 subunits that respond to MCh. Quadruple Ser [->] Ala mutation of the predicted MCh sites in the [alpha] sub(1) 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 [alpha] sub(1) 2.3 subunits mediate the responsiveness of Ca sub(v) 2.3 channels to MCh. Because Ca sub(v) 2.3 channels contribute to action potential-induced Ca super(2+) influx, these sites may account for M sub(1) receptor-mediated regulation of neurotransmission at some synapses.