Swelling-induced activation of the outwardly rectifying
anion current, ICl, swell, is modulated by intracellular
ATP. The mechanisms by which
ATP controls channel activation, however, are unknown. Whole cell patch clamp was employed to begin addressing this issue. Endogenous
ATP production was inhibited by dialyzing N1E115
neuroblastoma cells for 4-5 min with solutions containing (microM): 40
oligomycin, 5 iodoacetate, and 20
rotenone. The effect of
ATP on current activation was observed in the absence of intracellular Mg2+, in cells exposed to extracellular metabolic inhibitors for 25-35 min followed by intracellular dialysis with
oligomycin, iodoacetate, and
rotenone, after substitution of
ATP with the nonhydrolyzable analogue
AMP-PNP, and in the presence of
AMP-PNP and
alkaline phosphatase to dephosphorylate intracellular
proteins. These results demonstrate that the
ATP dependence of the channel requires
ATP binding rather than hydrolysis and/or phosphorylation reactions. When cells were swollen at 15-55%/min in the absence of intracellular
ATP, current activation was slow (0.3-0.8 pA/pF per min).
ATP concentration increased the rate of current activation up to maximal values of 4-6 pA/pF per min, but had no effect on the sensitivity of the channel to cell swelling. Rate of current activation was a saturable, hyperbolic function of
ATP concentration. The EC50 for
ATP varied inversely with the rate of cell swelling. Activation of current was rapid (4-6 pA/pF per min) in the absence of
ATP when cells were swollen at rates >/=65%/min. Intracellular
ATP concentration had no effect on current activation induced by high rates of swelling. Current activation was transient when endogenous
ATP was dialyzed out of the cytoplasm of cells swollen at 15%/min. Rundown of the current was reversed by increasing the rate of swelling to 65%/min. These results indicate that the channel and/or associated regulatory
proteins are capable of sensing the rate of cell volume increase. We suggest that channel activation occurs via
ATP-dependent and -independent mechanisms. Increasing the rate of cell swelling appears to increase the proportion of channels activating via the
ATP-independent pathway. These findings have important physiological implications for understanding ICl, swell regulation, the mechanisms by which cells sense volume changes, and volume homeostasis under conditions where cell metabolism is compromised.