Passive membrane permeability to small molecules and ions in transformed mammalian cells: Probable role of surface phosphorylation

Abstract

Addition of ATP to medium surrounding intact, transformed 3T3 cells causes the formation of aqueous channels in the plasma membrane. This effect of extracellular ATP is sharply dependent on the pH and temperature of the incubation medium, and is inhibited by low levels of La3+ or ruthenium red; inhibition is also obtained with concentrations of Mg2+ ions that exceed a ratio of Mg/ATP of one. The effect of ATP on membrane channel formation is unaffected by chelators of metal ions or by prior modification of the cell surface with various surface-active enzymes or sulfhydryl reagents. Under conditions which favor aqueous channel formation, incubation of intact 3T6 cells with ATP (gamma-32P) leads to phosphorylation of two membrane components with apparent molecular weight of 40,000 (40K) and 110,000 (110K) daltons; the 110K component which is unaffected by trypsin under normal conditions is rendered trypsin-sensitive by the phosphorylation reaction, probably as a result of a conformational change. Conditions which inhibit aqueous channel formation also inhibit phosphorylation of the 110K protein and decrease the labeling of the 40K component. These results indicate the probable role of cell surface phosphorylation, involving one or both of these components, in the formation of aqueous channels in transformed 3T3 cells. Aqueous channel formation by extracellular ATP is not associated with gross unfolding of the cell surface as revealed by lactoperoxidase-catalyzed iodination of the 3T6 cell surface.

Permeabilization of transformed cells in culture by external ATP

“…Several studies yielded data consistent with this idea [28,59]. In a later investigation the phosphorylation of certain proteins by exogenous ATP was confirmed, but it was established that this was unrelated to the permeabilization process [93].…”

“…The divalent cation, Ca '-+, appears to have a specific inhibitory effect which is not shared by Mg 2+. First of atl, Ca 2+, but not Mg 2+ is able to seal permeabilized cells at an alkaline pH, although both cations function at a neutral pH [59]. Furthermore, it was found that La 3~-and Tb 3 § inhibit permeabilization when present at one-fifth the concentration of external ATP [26,59].…”

“…First of atl, Ca 2+, but not Mg 2+ is able to seal permeabilized cells at an alkaline pH, although both cations function at a neutral pH [59]. Furthermore, it was found that La 3~-and Tb 3 § inhibit permeabilization when present at one-fifth the concentration of external ATP [26,59]. These trivalent cations have been reported to replace Ca 2+ in modulating many enzymatic reactions [32,40,79,91].…”

Exogenous ATP and other nucleoside phosphates modulate epidermal growth factor receptors of A-431 epidermoid carcinoma cells.

“…Receptor from TPA-treated cells was phosphorylated largely on serine, and receptor from ATP-treated cells was phosphorylated on both serine and threonine (Fig. 5) Nucleosides and nucleoside phosphates, particularly ATP, have many effects on cells (3,5), changing inositol phosphate levels (5) and intracellular Ca2+ concentrations (7,26) and inducing phosphorylation of surface membrane proteins (27,28). Most of these effects appear to be moderated through nucleoside phosphate-specific receptors.…”

Ca2+ homeostasis and cytotoxicity in isolated hepatocytes: Studies with extracellular adenosine 5′‐triphosphate

“…In addition, the similarity between the molecular radius (110 kDa) of this intermediate to that of the plasma membrane Ca2+-ATPase (40,41) supports the hypothesis that the Ca2+-ATPase may represent one of the specific targets of the extracellular ATP action. Other investigators have already reported the phosphorylation of other membrane proteins induced by extracellular ATP, but these occurred under different conditions of incubation (alkaline pH, medium lacking Ca2+ and Mg2+) and with less detailed characterization of the phosphorylated intermediate (42,43). The inhibition of active Ca2 + extrusion would result in the observed Ca2+ accumulation, although the precise mechanism by which enzyme phosphorylation prevents active Ca2 + transport is presently unknown.…”