FIGURE 3.

Potentiation of mSlo Ca²⁺ sensitivity by overexpression of active c-src but not inactive c-src. A, Western blots comparing relative levels of c-src in nontransfected cells and after transient transfection with WT c-src or catalytically inactive c-src in addition to mSlo. Immunoreactive bands at ∼60 kDa (2 s exposure) are prominent in lane 3 (co-expression of mSlo and WT c-src) and lane 4 (co-expression of mSlo and inactive c-src) but not in lane 1 (co-transfection of mSlo with empty vector), lane 2 (transfection of mSlo alone), and lane 5 (nontransfected cells). α-Tubulin mouse monoclonal IgM (∼53 kDa) was used as a loading control. With longer time exposure (15 s), a faint band consistent with endogenous expression of c-src was observed in nontransfected cells (lane 6). B, summary of G-V relations from patches expressing mSlo with or without co-expression of WT c-src. G-V curves were left-shifted after co-expression of WT c-src at all Ca²⁺ levels except 0 Ca²⁺. The largest shifts occurred at 1 and 5 μm cytosolic free Ca²⁺. C, calculated V_0.5 values from B are plotted as a function of free Ca²⁺ and show that a significant enhancement in Ca²⁺ sensitivity occurs in patches excised from cells co-expressing WT c-src but not inactive c-src, compared with mSlo alone. Co-expression of inactive c-src blocked the G-V curve shifts induced by α5β1 integrin activation (compare curve with mSlo + α5β1 Ab in Fig. 2B).