pH-sensitive activation of the intracellular-pH regulation system in squid axons by ATP-γ-S

Abstract

The regulation of intracellular pH (pH_i) is essential for normal cell function¹, and controlled changes in pH_i may play a central role in cell activation². Sodium-dependent Cl–HCO₃ exchange is the dominant mechanism of pH_i regulation in the invertebrate cells examined^3–6, and also occurs in mammalian cells^7,8. The transporter extrudes acid from the cell by exchanging extracellular Na⁺ and HCO₃⁻ (ref. 9) (or a related species) for intracellular Cl⁻ (refs 3, 4). It is blocked by the stilbene derivatives DIDS (4,4′-diisothiocyano-stilbene-2,2′-disulphonate, ref. 10) and SITS (4-acetamido-4′-isothiocyano-stilbene-2,2′-disulphonate, ref. 3), and has a stoichiometry of two intracellular H⁺ neutralized for each Na⁺ taken up and each Cl^– extruded by the axon¹¹. Because the inwardly-directed Na⁺ concentration gradient is sufficiently large to energize both the HCO₃⁻ influx and Cl⁻ efflux, this electroneutral exchanger could be a classic secondary active transporter, thermodynamically independent of ATP hydrolysis. However, at least in the squid axon, the exchanger has an absolute requirement for ATP (ref. 3). Thus, a major unresolved issue is whether this Na-dependent Cl–HCO₃ exchanger stoichiometri-cally hydrolyses ATP (the pump hypothesis), or whether ATP activates the transporter by a mechanism such as phosphorylation or simple binding (the activation hypothesis). We have now explored the role of ATP in pH_i regulation by dialysing axons with the ATP analogue ATP-γ-S. In many systems, ATP–γ–S is an acceptable substrate for protein kinases^12,13, whereas the resulting thiophosphorylated proteins are not as readily hydrolysed by phosphatases as are phosphorylated proteins^14,15. Our results rule out the pump hypothesis, and show that the basis of the axon's ATP requirement is the pH-dependent activation (by, for instance, phosphorylation or ATP binding) of the exchanger itself, or of an essential activator.