Effects of vanadate on MgATP stimulation of Na-Ca exchange support kinase-phosphatase modulation in squid axons

Abstract

We have proposed that in squid axons MgATP stimulation of Na-Ca exchange involves a phosphorylation-dephosphorylation process catalyzed by a kinase-phosphatase system. In the present work, we used vanadate as a tool to gather further evidence about the mechanism of metabolic control of the Na-Ca exchanger in internally dialyzed and voltage-clamped squid axons. Vanadate, at concentrations up to 100 microM, stimulated extracellular Na (Nao)-dependent Ca efflux only in the presence of MgATP but failed to do so when the axons were dialyzed with the nonhydrolyzable ATP analogue beta, gamma-methyleneadenosine 5'-triphosphate or with CrATP, a MgATP analogue that completely abolishes MgATP stimulation of the Na-Ca exchange. In axons fully activated by Mg-adenosine 5'-O-(3-thiotriphosphate), vanadate had no effect on Na-Ca exchange. The dose-response curve for vanadate stimulation followed Michaelian kinetics with a Km of 5.6 +/- 0.4 microM and a maximum velocity of 216 +/- 10 fmol.cm-2.s-1 (intracellular Ca concentration = 0.8 microM). This coincides with the high affinity of vanadate in inhibiting the in vitro phosphatase activity of an alkaline phosphatase extracted from rat liver. In addition, vanadate increased fivefold the apparent affinity for MgATP (Km from 220 +/- 14 to 40 +/- 4 microM). Concentrations of vanadate in the millimolar range inhibited the MgATP-stimulated Na-Ca exchange (apparent Ki of 5.7 +/- 0.3 mM) and the in vitro phosphorylation by the catalytic subunit of a adenosine 3',5'-cyclic monophosphate protein kinase (apparent Ki 2.64 +/- 0.04 mM). We conclude that MgATP stimulation of Na-Ca exchange is proportional to the levels of phosphorylation that result from the balance of the activity of a kinase and a phosphatase activity.