Colonic inflammation alters Src kinase-dependent gating properties of single Ca2+ channels via tyrosine nitration.
Nitration of L-type calcium channels during colonic inflammation impairs phosphorylation by the tyrosine kinase, Src kinase. This results in decreased calcium currents. The purpose of this study was to determine the mechanism of the downregulation of Ca2+ currents in colonic inflammation. In whole cell voltage clamp of mouse single smooth muscle cells, long-duration depolarization produced noninactivating calcium currents that were significantly reduced by the Src kinase inhibitor, protein phosphatase 2 (PP2). Unitary Ba2+ currents were recorded upon repolarization from positive potentials in cell-attached patches of smooth muscle and hCa(v)1.2b-transfected cells to assess the properties of the single channels attributed to the noninactivating open state. Repolarization to -40 mV from 0 mV resulted in single-channel events with conductance of approximately 23 pS. The ensemble average of the tail currents from 1,000 sweeps was 337 +/- 27 fA in control and 218 +/- 49 fA (P < 0.05) in inflamed cells. Neither open-probability nor open-time constants were significantly different between control and inflamed cells. However, the transition to the open state measured as channel availability was significantly reduced from 19 +/- 3% to 6.4 +/- 1%. Similarly, peak ensemble average current and channel availability were significantly reduced by PP2 and treatment with peroxynitrite in control cells. Mutation of COOH-terminal tyrosine residues in hCa(v)1.2b Chinese hamster ovarian cells also decreased peak ensemble average tail currents and availability. The present findings suggest that the transition of Ca2+ channels to the noninactivating open state is Src kinase dependent. Tyrosine nitration prevents Src-mediated transitions, leading to decreased calcium currents.
Ross
Gracious R
GR
Dept. of Pharmacology and Toxicology, Virginia Commonwealth Univ., 1112 E. Clay St., McGuire Hall 317, Richmond, VA 23298, USA.
Kang
Minho
M
Akbarali
Hamid I
HI
D000818:Animals; D016466:CHO Cells; D015220:Calcium Channels / Q000378:metabolism*; D003092:Colitis / Q000139:chemically induced* / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D015640:Ion Channel Gating / Q000502:physiology*; D008297:Male; D051379:Mice; D014443:Tyrosine / Q000378:metabolism*; D019061:src-Family Kinases / Q000235:genetics / Q000378:metabolism*
D015220:Calcium Channels; D014443:Tyrosine; D019061:src-Family Kinases