Micromolar free calcium exposes ouabain-binding sites in digitonin-permeabilized Xenopus laevis oocytes

Abstract

As demonstrated previously, digitonin-permeabilized Xenopus oocytes have a large internal pool of sodium pumps which are inaccessible to cytosolic ouabain [Schmalzing, Kröner & Passow (1989) Biochem. J. 260, 395-399]. Access to internal ouabain-binding sites required permeabilization of inner membranes with SDS. In the present study, micromolar free Ca2+ was found to stimulate ouabain binding in the digitonin-permeabilized cells (K0.5 0.5 microM-Ca2+, h 1.9, average of seven experiments) without disrupting intracellular membranes. Sustained incubation at 9 microM-Ca2+ was as effective as SDS in inducing access to the ouabain-binding sites of the internal sodium pumps. Omission of either Mg2+ or ATP completely abolished the Ca2+ effect. Half-maximal stimulation by Ca2+ required approx. 0.4 mM-MgATP. Of a variety of nucleotides tested, none was as effective as ATP (rank order ATP greater than ADP greater than ATP[S] (adenosine 5'-[gamma-thio]triphosphate) greater than CTP greater than UTP greater than ITP = XTP greater than GTP). Pi, AMP, cyclic AMP, cyclic GMP, GTP[S] (guanosine 5'-[gamma-thio]triphosphate) and a stable ATP analogue p[NH]ppA (adenosine 5'-[beta gamma-imido]triphosphate), were ineffective. The metalloendoproteinase inhibitor carbobenzoxy-Gly-Phe-amide reduced the Ca2+ effect by some 50%. Inhibitors of chymotrypsin and the Ca2+ proteinase calpain had no effect. Ca2+ ionophores (A23187 and ionomycin) and the polycations neomycin and polymixin B blocked the Ca2+ response entirely. Neomycin also abolished a Ca2(+)-independent stimulation of ouabain binding by the wasp venom mastoparan. The requirements for increasing the accessibility of ouabain-binding sites are remarkably similar to those for exocytosis in secretory cells, suggesting that oocytes and eggs possess a Ca2(+)-regulated pathway for the plasma membrane insertion of sodium pumps.

Up-regulation of sodium pump activity in Xenopus laevis oocytes by expression of heterologous β1 subunits of the sodium pump

“…Recent evidence suggests that the Na+ pump of Xenopus oocytes is regulated by endocytosis [39] and exocytosis [40]. In principle, up-regulation of Na+ pump activity after microinjection of cRNAs encoding /31 subunits (Figs.…”

“…[3H]Ouabain (1.1, 0.62 and 0.67 TBq/mmol), 86RbCl and ['4C]sucrose (0.37 GBq/mmol) were purchased from Amersham-Buchler; L-[35S]methionine (approx. 40 TBq/mmol) was from New England Nuclear. Restriction enzymes were from Biolabs, except for NheI (Boehringer Mannheim) and SmaI (Stratagene).…”