ATP-induced pore formation in the plasma membrane of rat peritoneal mast cells

Abstract

We have investigated the ATP-induced permeabilization of rat peritoneal mast cells using three different techniques: (a) by measuring uptake of fluorescent membrane and DNA marker dyes, (b) by voltage-clamp measurements using the patch-clamp technique, and (c) by measurements of exocytosis in response to entry of Ca2+ and GTPγS into permeabilized cells. In the absence of divalent cations cells become highly permeable at ATP concentrations as low as 3 μM. In normal saline containing 1 mM MgCl2 and 2 mM CaCl2, dye uptake and electric conductance are detectable at 100 μM ATP corresponding to 4 μM ATP4-. The permeabilization is half-maximal at an ATP4- concentration of 5-20 μM with a Hill coefficient near 2. The ATP-induced whole-cell conductance at saturating ATP concentrations was 35-70 nS, exhibiting only weak cation selectivity. The activation is very fast with a time constant ≤65 ms. Pores which are large enough to allow for permeation of substances of 300-900 D are expected to have a unit conductance of ∼200-400 pS. However, in whole cells as well as outside-out patches, discrete openings and closings of channels could not be observed at a resolution of ∼40 pS and the single-channel conductance obtained from noise analysis is ∼2-10 pS. Entry of Ca2+ into cells permeabilized with ATP stimulates exocytosis at low but not at high ATP concentrations indicating loss of an essential intracellular component or components at a high degree of permeabilization. This inactivation is removed when GTPγS is provided in the medium and this leads to enhanced exocytosis. The enhancement only occurs at high ATP concentrations. These results strongly suggest that the ATP-induced pores are of variable size and can increase or decrease by very small units.