pp60^src increases L-type Ca²⁺ channel current through upregulation of β₃ subunit in cultured rat vascular smooth muscle cells

Abstract

We investigated whether pp60^src increases L-type Ca²⁺ channel current (I_Ca(L)) in cultured rat VSM cells (A7r5). A7r5 cells were transfected with a plasmid of wild type mouse src cDNA/pUSE expression vector using lipofectamine. Negative control cells were prepared by transfection with an empty vector, (-)/pUSE. After transfection, the cells were treated with 1 mg/ml of G418 to select the positive cells stably expressing src cDNA or (-)/pUSE. I_Ca(L) was recorded by whole-cell voltage clamp using a patch-clamp technique. 5 mM BaCl₂ was used as a charge carrier for recording of I_Ca(L). Western blot demonstrated that expression of pp60^src was increased by 34-fold and that β₃ subunit of L-type Ca²⁺ channel current was significantly increased in src cDNA/pUSE expressing cells as compared with (-)/pUSE expressing cells. Current/voltage relationships demonstrated that although voltages for peak amplitude of I_Ca(L) were 20 mV for both src cDNA and (-)/pUSE expressing cells, peak amplitude of I_Ca(L) in src cDNA/pUSE expressing cells was increased by 2-fold (src, 6.1 ± 0.96; (-), 3.1 ± 0.38 pA/pF). From steady-state inactivation and activation analyses of I_Ca(L), voltages for half-activation and -inactivation and slope factors did not differ between src cDNA and (-)/pUSE expressing cells. Thus, overexpression of pp60^src increases I_Ca(L) through upregulation of β₃ subunit of L-type Ca²⁺ channel without affecting voltage sensors of L-type Ca²⁺ channels. [J Physiol Sci. 2008;58 Suppl:S176]