FIGURE 5.

Bafilomycin-sensitive, V-ATPase-dependent extracellular acidification is modulated by Ser-175 A subunit mutants and PKA activators in HEK-293T cells. Cells were transfected with vector alone, WT, S175A, or S715D mutant FLAG-tagged A subunit for 28–31 h, and the rate of extracellular acidification in each set of transfected cells was measured in a low buffering capacity solution before and after the addition of bafilomycin A1, a specific V-ATPase inhibitor (see “Experimental Procedures”). The mean (±S.E.) rate of extracellular acidification (−[final buffer pH − initial buffer pH]/Δt) was obtained in the absence or presence of bafilomycin, for cells incubated either with (A) or without (B) PKA activators (n = 6 for each transfection condition; n = 6 for each treatment condition) (*, p < 0.005 relative to vector alone; #, p < 0.02 relative to WT).