Src kinase‐dependent gating properties of single Ca2+ channels are altered by tyrosine nitration in colitis

Abstract

Reduced L‐type Ca2+ ‐channel (LTCC) function contributes to colonic dysmotility during inflammation. We have previously shown that nitration of tyrosine residues within the carboxy terminus of LTCC (Y1837 and Y2134) impairs c‐Src kinase regulation during colitis. We examined the gating properties of single LTCC, using cell‐attached patch clamp mode, in mouse colon smooth muscle cells (control vs colitis‐TNBS) and CHO cells transfected with hCaV1.2b (wild type) or mutated hCaV1.2b (Y1837F:Y2134F). Inflammation significantly reduced LTCC ensemble average current (−0.33 vs −0.22 pA, P=0.05), without affecting the single‐channel properties: mean amplitude (−2.4pA), slope conductance (23 vs 27pS) or the open‐time constants (τ1: 1.6 vs 1.2; τ2: 6.1 vs 3.9 ms). However, the availability (slow gating) of LTCC was significantly reduced by inflammation (0.19 vs 0.06; P=0.02) without affecting the open probability of modal gating. Similar effects were seen with inhibition of c‐Src kinase by PP2 or by tyrosine nitration induced by SIN‐1+ONOO− combination in smooth muscle cells. Mutation of tyrosine residues (Y1837/2134F) in hCaV1.2b, which prevents c‐Src kinase regulation, resulted in diminished channel availability. This study demonstrates that src kinase‐ phosphorylation enhances the availability of LTCC, that is impaired by tyrosine nitration during colonic inflammation. Supported by NIH DK046367.