ATP-induced P2X₇-associated uptake of large molecules involves distinct mechanisms for cations and anions in macrophages

Macrophages express the P2X₇ receptor and other nucleotide (P2) receptors, and display the phenomenon of extracellular ATP (ATP_e)-induced P2X₇-dependent membrane permeabilization, which occurs through a poorly understood mechanism. We used patch-clamp recordings, cytoplasmic Ca²⁺ measurements and fluorescent dye uptake assays to compare P2X₇-associated transport phenomena of macrophages and HEK-293 cells transfected with P2X₇ receptors (HEK-P2X₇ cells). Both cell types showed inward currents, increase of free cytoplasmic Ca²⁺ concentration and the uptake of cationic dyes upon exposure to ATP_e, as previously described. However, in contrast to the macrophages, HEK-P2X₇ cells did not take up anionic dyes and did not display the 440 pS channels (Z pores) under cell-attached patch-clamping conditions. In addition, the transport mechanism of anionic dyes displayed by macrophages was also able to support dye efflux and, once activated at 37°C, it remained active at 4°C, whereas uptake of cationic dyes was temperature-dependent and unidirectional. Our results indicate that the mechanism of ATP_e-induced dye uptake, usually called a 'permeabilization phenomenon' and associated with a 'permeabilization pore' can be ascribed to at least two distinct mechanisms in macrophages: a diffusional pathway, possibly associated with the 440 pS Z pores, and a cation uptake mechanism that is not diffusional and should be ascribed to an, as yet, unidentified transport mechanism.