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J. Biol. Chem., Vol. 280, Issue 25, 23559-23565, June 24, 2005

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Role of ₁ 2.3 Subunit I-II Linker Sites in the Enhancement of Ca_v 2.3 Current by Phorbol 12-Myristate 13-Acetate and Acetyl--methylcholine^*

Hongyu Fang, Ruthie Franke, Saharat Patanavanich, Amrita Lalvani, Natalie K. Powell, Julianne J. Sando, and Ganesan L. Kamatchi

From the Department of Anesthesiology, University of Virginia Health Sciences Systems, Charlottesville, Virginia 22908

Potentiation of Ca_v 2.3 currents by phorbol 12-myristate 13-acetate (PMA) or acetyl--methylcholine (MCh) may be due to protein kinase C (PKC)-mediated phosphorylation of the ₁ 2.3 subunit. Mutational analysis of potential PKC sites unique to the ₁ 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 ₁ 2.3 and 2.2 subunits, both of which respond to PMA. Wild type ₁ 2.3 or mutants were expressed in Xenopus oocytes in combination with 1b and 2/ 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.

Received for publication, February 9, 2005 , and in revised form, April 14, 2005.

^* This work was supported by National Institutes of Health Grant GM65214 (to G. L. K.) and GM31184 (to J. J. S.) and by the Department of Anesthesiology at the University of Virginia. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Anesthesiology, University of Virginia Health Sciences System, P. O. Box 800710, Charlottesville, VA 22908-0710. Tel.: 434-924-2924; Fax: 434-982-0019; E-mail: gk3p{at}virginia.edu.

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