HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) All Versions of this Article: 293/6/C1983    most recent 00308.2007v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kang, M. Articles by Akbarali, H. I. Search for Related Content PubMed PubMed Citation Articles by Kang, M. Articles by Akbarali, H. I.

MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

COOH-terminal association of human smooth muscle calcium channel Ca_v1.2b with Src kinase protein binding domains: effect of nitrotyrosylation

Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, Virginia

Submitted 19 July 2007 ; accepted in final form 13 October 2007

The carboxyl terminus of the calcium channel plays an important role in the regulation of calcium entry, signal transduction, and gene expression. Potential protein-protein interaction sites within the COOH terminus of the L-type calcium channel include those for the SH3 and SH2 binding domains of c-Src kinase that regulates calcium currents in smooth muscle. In this study, we examined the binding sites involved in Src kinase-mediated phosphorylation of the human voltage-gated calcium channel (Ca_v) 1.2b (hCav1.2b) and the effect of nitrotyrosylation. Cotransfection of human embryonic kidney (HEK)-293 cells with hCa_v1.2b and c-Src resulted in tyrosine phosphorylation of the calcium channel, which was prevented by nitration of tyrosine residues by peroxynitrite. Whole cell calcium currents were reduced by 58 + 5% by the Src kinase inhibitor PP2 and 64 + 6% by peroxynitrite. Nitrotyrosylation prevented Src-mediated regulation of the currents. Glutathione S-transferase fusion protein of the distal COOH terminus of hCa_v1.2b (1809-2138) bound to SH2 domain of Src following tyrosine phosphorylation, while binding to SH3 required the presence of the proline-rich motif. Site-directed mutation of Y²¹³⁴ prevented SH2 binding and resulted in reduced phosphorylation of hCa_v1.2b. Within the distal COOH terminus, single, double, or triple mutations of Y¹⁸³⁷, Y¹⁸⁶¹, and Y²¹³⁴ were constructed and expressed in HEK-293 cells. The inhibitory effects of PP2 and peroxynitrite on calcium currents were significantly reduced in the double mutant Y^1837-2134F. These data demonstrate that the COOH terminus of hCa_v1.2b contains sites for the SH2 and SH3 binding of Src kinase. Nitrotyrosylation of these sites prevents Src kinase regulation and may be importantly involved in calcium influx regulation during inflammation.

Src kinase; SH2; SH3; peroxynitrite; tyrosine; calcium channel

This article has been cited by other articles:

				M. S. Reifenberger, K. L. Arnett, C. Gatto, and M. A. Milanick The reactive nitrogen species peroxynitrite is a potent inhibitor of renal Na-K-ATPase activity Am J Physiol Renal Physiol, October 1, 2008; 295(4): F1191 - F1198. [Abstract] [Full Text] [PDF]