Fig. 4.

Effect of PIP₂ depletion with carbachol on NCX activities of WT and mutant cells at 140 mM Na⁺. Cells expressing WT (A), K229Q (B), or F223E (C) exchanger were transiently transfected to express the human M₁ receptor and assayed after 72 h. Cells were incubated for 8 min in Na-PSS containing 1 μg/ml gramicidin and then treated with 100 μM carbachol or 100 μM ATP; NCX activity was initiated 90 s later by application of 4–5 ml of 0.1 mM CaCl₂ in Na-PSS. D: difference in V_max values between ATP-treated WT and K229Q (K) cells was not significant (P ∼ 0.09); corresponding difference between F223E (F) and WT cells was highly significant (P ∼ 0.003). Values are means, with SE bars for every other point; n = 5 coverslips each for carbachol and ATP in A, n = 7 for carbachol and 4 for ATP in B, and n = 9 for carbachol and 5 for ATP in C. Experiments were carried out at 37°C.