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.
The Journal of Membrane Biology – Springer Journals
Published: May 14, 2008
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