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 down-regulation of Ca2+ currents in colonic inflammation. In whole cell voltage clamp of mouse single smooth muscle cells, long duration depolarization produced non-inactivating calcium currents that were significantly reduced by the src kinase inhibitor, PP2. Unitary Ba2+ currents were recorded upon repolarization from positive potentials in cell-attached patches of smooth muscle and hCav1.2b transfected cells to assess the properties of the single channels attributed to the non-inactivating open state. Repolarization to -40 mV from 0 mV resulted in single channel events with conductance of ~23pS. The ensemble average of the tail currents from 1000 sweeps was 337 ± 27fA in control and 218 ± 49fA (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 currents and channel availability were significantly reduced by PP2 and treatment with peroxynitrite in control cells. Mutation of c-terminus tyrosine residues in hCav1.2b CHO cells decreased peak ensemble average tail currents and availability. The present findings suggest that the transition of Ca2+ channels to the non-inactivating open state is src-kinase dependent. Tyrosine nitration prevents src-mediated transitions leading to decreased calcium currents.
- calcium channel
- tyrosine kinase
- smooth muscle
- Copyright © 2010, American Journal of Physiology- Gastrointestinal and Liver Physiology