The cardiac glycoside, ouabain, inhibits alkali-cation transport in HeLa cells. It binds to 0.75 X 106 sites per cell, and the half-time for its dissociation is 16 hr. After partial blockade by ouabain, the cell generates new ouabain-binding sites, with total restoration of transport in 10% of a cell cycle (-3 hr). This recovery requires protein snythesis and appears to be a response to altered cell-electrolyte content, since growth of cells in media with low K + concentration enhances the titer of the transport enzyme in a fashion similar to the effect of ouabain. Totally blocked cells do not recover.The interaction of any exogenous chemical with a cell begins at the cell surface; in fact many biologically active chemicals penetrate no further into the cell. Since some of these agents have deleterious effects on the cell, cell survival may depend on the mitigation of these surface effects, either by their reversal or repair. Treatment of cultured cells with hydrolytic enzymes results in loss of antigens and of viral or other receptors on the cell surface that, upon subsequent incubation of the cells, return to their initial titer within a few hours (1-4). In such studies, it is not clear whether the reappearance of the surface properties represents repair above and beyond normal membrane turnover. The rate of bulk turnover of surface proteins, carbohydrates, and lipids does not seem to be stimulated after treatment with hydrolytic enzymes (4, 5).We investigated membrane repair more specifically by use of alkali-cation transport (Na+-K+-Mg2+-ATPase) in HeLa cells. Our rationale for this selection was as follows: (i) the proper functioning of the cation-transport system, in association with maintenance of normal cell electrolyte composition, is recognized as a principal factor in the regulation of cell volume (6, 7). Also, K+ is a cofactor for several intracellular enzymes, and for their activity intracellular K+ must be maintained at appropriate concentrations (see, e.g., refs. 8-10). Thus, the integrity of the alkali-cation transport system is of known and demonstrable importance to cell viability. (ii) Alkali-cation transport can be specifically blocked by cardiac glycosides (11-13). In HeLa cells, ouabain, in low concentrations, binds tightly to the cells and is an effective inhibitor. (iii) By the use of [3H Jouabain it is possible to monitor the extent of binding of the inhibitor, while at the same time the activity of alkali-cation transport can be assayed by measurement of transport of labeled cations. In sum, these characteristics enable us to make specific measurements of an essential enzymatic function localized at the cell surface and subject to specific inhibition.In this paper, we show that subtotal blockade of transport
