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Modulation of voltage-dependent Ca2+ channels in rabbit colonic smooth muscle cells by c-Src and focal adhesion
X Q Hu1, N Singh, D Mukhopadhyay
1Division of Gastroenterology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215, USA.
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.
Insights
Tyrosine kinases c-Src and focal adhesion kinase (FAK) regulate calcium channel activity in smooth muscle. These kinases mediate platelet-derived growth factor (PDGF)-induced calcium influx, independent of the Ras/MAPK pathway.
Area of Science:
- Cellular biology
- Molecular physiology
- Smooth muscle research
Background:
- Emerging evidence suggests tyrosine kinases regulate smooth muscle contraction and calcium (Ca2+) influx via L-type Ca2+ channels.
- The specific tyrosine kinases involved in this process remain largely unidentified.
Purpose of the Study:
- To identify the specific tyrosine kinases regulating L-type Ca2+ channels in smooth muscle cells.
- To elucidate the role of identified kinases in basal and growth factor-stimulated Ca2+ currents.
Main Methods:
- Immunoblotting and co-immunoprecipitation to identify tyrosine-phosphorylated proteins.
- Whole-cell patch-clamp electrophysiology to measure Ca2+ currents.
- Inhibition studies using specific antibodies and peptides.
Main Results:
- c-Src and focal adhesion kinase (FAK) were identified as key tyrosine-phosphorylated proteins.
- Intracellular dialysis with anti-c-Src and anti-FAK antibodies significantly suppressed basal Ca2+ currents.
- Platelet-derived growth factor (PDGF)-BB enhanced Ca2+ currents, an effect abolished by anti-c-Src and anti-FAK antibodies.
- The alpha1C subunit of the L-type Ca2+ channel directly associated with c-Src.
Conclusions:
- c-Src and FAK modulate basal L-type Ca2+ channel activity in smooth muscle cells.
- These kinases mediate PDGF-induced enhancement of Ca2+ currents.
- The Ras/mitogen-activated protein kinase cascade is not involved in these processes.