Leukocyte P(2) purinergic receptors: Expression and characterization in Xenopus oocytes

Extracellular ATP activates two distinct P{dollar}{bsol}sb2{dollar}-purinergic receptor-mediated signaling pathways in leukocytes: (1) the formation of non-selective pores and/or channels in the plasma membrane and (2) a G protein-dependent stimulation of phospholipase C and increase in cytosolic calcium. 3{dollar}{bsol}sp{bsol}prime{dollar}-O-(4-benzoyl)benzoyl ATP (BzATP) and UTP are selective agonists for the P{dollar}{bsol}sb{lcub}{bsol}rm 2z{rcub}{dollar} and P{dollar}{bsol}sb{lcub}{bsol}rm 2u{rcub}{dollar} purinergic receptors, respectively, in BAC1.2F5 macrophages. We demonstrate that BzATP and ATP activate electrophysiological responses in BAC1.2F5 poly(A){dollar}{bsol}sp{lcub}+{rcub}{dollar}RNA-injected Xenopus oocytes, consistent with expression of the P{dollar}{bsol}sb{lcub}{bsol}rm 2z{rcub}{dollar} purinergic receptor. BzATP and ATP induce a multiphasic current in voltage-clamped oocytes which is characterized by a rapid phase, followed by a delayed, steady increase in conductance. This response is potentiated in the absence of extracellular divalent cations, consistent with the specificity of the P{dollar}{bsol}sb{lcub}{bsol}rm 2z{rcub}{dollar} purinergic receptor for tetrabasic nucleotides. The BzATP-induced current is distinct from the transient increase in Ca{dollar}{bsol}sp{lcub}2+{rcub}{dollar}-dependent Cl{dollar}{bsol}sp{lcub}-{rcub}{dollar} current stimulated by P{dollar}{bsol}sb{lcub}{bsol}rm 2u{rcub}{dollar} purinergic receptor-dependent mobilization of intracellular Ca{dollar}{bsol}sp{lcub}2+{rcub}{dollar} stores in poly(A){dollar}{bsol}sp{lcub}+{rcub}{dollar}RNA-injected oocytes. N,N-hexamethylene amiloride (HMA) selectively and reversibly i nhibits the delayed component of the BzATP-induced inward current. This HMA-sensitive current is carried by large organic cations, such as N-methylglucamine (NMG{dollar}{bsol}sp{lcub}+{rcub}{dollar}) and Tris{dollar}{bsol}sp{lcub}+{rcub}{dollar}, in addition to small inorganic cations. In contrast, the HMA-insensitive current is readily carried by Na{dollar}{bsol}sp{lcub}+{rcub}{dollar}, Li{dollar}{bsol}sp{lcub}+{rcub}{dollar} and K{dollar}{bsol}sp{lcub}+{rcub}{dollar}, but not by NMG{dollar}{bsol}sp{lcub}+{rcub}{dollar} or Tris{dollar}{bsol}sp{lcub}+{rcub}.{dollar} Reduced temperature discriminates two BzATP-activated permeability pathways in the native BAC1.2F5 macrophages. At 20{dollar}{bsol}sp{bsol}circ{lcub}{bsol}rm C{rcub},{dollar} BzATP does not significantly increase membrane permeability to NMG{dollar}{bsol}sp{lcub}+{rcub}{dollar} (Mr 195 Da), ethidium{dollar}{bsol}sp{lcub}+{rcub}{dollar} (Mr 314 Da), or fura-2 (Mr 831 Da), but does stimulate an ion conducting pathway competitively permeated by Na{dollar}{bsol}sp{lcub}+{rcub}{dollar} or Ca{dollar}{bsol}sp{lcub}2+{rcub}{dollar}. At 37{dollar}{bsol}sp{bsol}circ{lcub}{bsol}rm C{rcub},{dollar} BzATP increases membrane permeability also to NMG{dollar}{bsol}sp{lcub}+{rcub}{dollar}, ethidium{dollar}{bsol}sp{lcub}+{rcub}{dollar}, and fura-2. These data indicate that macrophage P{dollar}{bsol}sb{lcub}{bsol}rm 2z{rcub}{dollar} purinergic receptors can differentially activate: (1) a rapidly gated cation channel, and (2) a time- and temperature-dependent formation of non-selective pores. These events can be distinguished by their rates of activation, ion-selectivities, sensitivities to HMA, and temperature-dependence.