Abstract
Squid giant axons recover from acid loads by activating a Na+-driven Cl–HCO3 exchanger. We internally dialyzed axons to an intracellular pH (pH i ) of 6.7, halted dialysis and monitored the pH i recovery (increase) in the presence of ATP or other nucleotides, using cyanide to block oxidative phosphorylation. We computed the equivalent acid-extrusion rate (J H) from the rate of pH i increase and intracellular buffering power. In experimental series 1, we used dialysis to vary [ATP] i , finding that Michaelis-Menten kinetics describes J H vs. [ATP] i , with an apparent V max of 15.6 pmole cm−2 s−1 and K m of 124 μM. In series 2, we examined ATPγS, AMP-PNP, AMP-PCP, AMP-CPP, GMP-PNP, ADP, ADPβS and GDPβS to determine if any, by themselves, could support transport. Only ATPγS (8 mM) supported acid extrusion; ATPγS also supported the HCO −3 -dependent 36Cl efflux expected of a Na+-driven Cl–HCO3 exchanger. Finally, in series 3, we asked whether any nucleotide could alter J H in the presence of a background [ATP] i of ∼230 μM (control J H = 11.7 pmol cm−2 s−1). We found J H was decreased modestly by 8 mM AMP-PNP (J H = 8.0 pmol cm−2 s−1) but increased modestly by 1 mM ADPβS (J H = 16.0 pmol cm−2 s−1). We suggest that ATPγS leads to stable phosphorylation of the transporter or an essential activator.
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Acknowledgments
This work was supported by National Institutes of Health grants NS18400 and NS11946. We thank Mr. Mike Hernandez for assistance in performing the ATP assays and Mr. Duncan Wong for technical assistance.
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Davis, B.A., Hogan, E.M., Russell, J.M. et al. ATP Dependence of Na+-Driven Cl–HCO3 Exchange in Squid Axons. J Membrane Biol 222, 107–113 (2008). https://doi.org/10.1007/s00232-008-9100-1
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DOI: https://doi.org/10.1007/s00232-008-9100-1