Modulation of Voltage-dependent Ca2+Channels in Rabbit Colonic Smooth Muscle Cells by c-Src and Focal Adhesion Kinase

Xiang-Qun Hu,Namita Singh,Debabrata Mukhopadhyay,Hamid I Akbarali

doi:10.1074/jbc.273.9.5337

Abstract

There is emerging evidence indicating that smooth muscle contraction and Ca2+ influx through voltage-dependent L-type Ca2+ channels are regulated by tyrosine kinases; however, the specific kinases involved are largely unknown. In rabbit colonic muscularis mucosae cells, tyrosine-phosphorylated proteins of approximately 60 and 125 kDa were observed in immunoblots using an anti-phosphotyrosine antibody and were identified as c-Src and focal adhesion kinase (FAK) by immunoblotting with specific antibodies. FAK co-immunoprecipitated with c-Src, and the phosphorylation of the c-Src.FAK complex was markedly enhanced by platelet-derived growth factor (PDGF) BB. The presence of activated c-Src in unstimulated cells was identified in cell lysates by immunoblotting with an antibody recognizing the autophosphorylated site (P416Y). In whole-cell patch-clamp studies, intracellular dialysis of a Src substrate peptide and anti-c-Src and anti-FAK antibodies suppressed Ca2+ currents by 60, 62, and 43%, respectively. In contrast, intracellular dialysis of an anti-mouse IgG or anti-Kv1.5 antibody did not inhibit Ca2+ currents. Co-dialysis of anti-c-Src and anti-FAK antibodies inhibited Ca2+ currents (63%) equivalent to dialysis with the anti-c-Src antibody alone. PDGF-BB enhanced Ca2+ currents by 43%, which was abolished by the anti-c-Src and anti-FAK antibodies. Neither the MEK inhibitor PD 098059 nor an anti-Ras antibody inhibited basal Ca2+ currents or PDGF-stimulated Ca2+ currents. The alpha1C subunit of the L-type Ca2+ channel co-immunoprecipitated with anti-c-Src and anti-phosphotyrosine antibodies, indicating direct association of c-Src kinase with the Ca2+ channel. These data suggest that c-Src and FAK, but not the Ras/mitogen-activated protein kinase cascade, modulate basal Ca2+ channel activity and mediate the PDGF-induced enhancement of L-type Ca2+ currents in differentiated smooth muscle cells.

Highlights

The influx of extracellular Ca2ϩ is a prerequisite for many cellular functions including cell proliferation and motility
We have evaluated the roles of c-Src and Focal adhesion kinase (FAK) as well as their downstream components Ras and mitogen-activated protein (MAP) kinase in the regulation of basal Ca2ϩ channel activity and their involvement in platelet-derived growth factor (PDGF)-induced enhancement of Ca2ϩ currents
Inhibition of Ca2ϩ Currents by Src Substrate Peptide and Anti-c-Src and Anti-FAK Antibodies—To demonstrate whether c-Src and FAK may be involved in the regulation of Ca2ϩ currents, we examined the effects of intracellular dialysis of a c-Src substrate peptide and anti-c-Src and anti-FAK antibodies on Ca2ϩ currents in single smooth muscle cells using the patchclamp technique

Summary

Introduction

The influx of extracellular Ca2ϩ is a prerequisite for many cellular functions including cell proliferation and motility. Recent studies in vascular smooth muscle cells indicate that c-Src may be involved in the regulation of Ca2ϩ channels based on the finding that intracellular dialysis of c-Src enhances Ca2ϩ currents [17].

Results

Conclusion