Functional evidence of distinct ATP activation sites at the human P2X7 receptor

摘要: The effect of the agonist ATP on whole cell currents Xenopus oocytes expressing either wild-type human P2X7 receptor (hP2X7), an N-terminally hexahistidyl-tagged hP2X7 (His-hP2X7), or a truncated His-hP2X7 (His-hP2X7ΔC) lacking C-terminal 156 amino acids was investigated using two-microelectrode voltage clamp technique. The activation time course can be described as sum exponentially growing and additional almost linearly activating current component. The amplitude component displayed biphasic dependence concentration, which could best approximated by model two equal high-sensitivity low-sensitivity non-cooperative sites with apparent dissociation constants about 4 200 μm free ATP4-, respectively. The monophasically dependent concentration constant μm. The contribution to kinetics reduced abolished in His-hP2X7ΔC. Our data indicate that possesses at least types sites, differ ATP4- sensitivity factor 50. degree occupation these influences both deactivation kinetics. Both N- domains appear important determinants elicited low sensitivity, but not for mediated highly ATP-sensitive sites. Many effects extracellular cells immune system have been attributed presence so-called P2Z receptor. Recent work has shown one members P2X family ATP-gated receptors (Buell et al. 1996; Soto 1997; Ralevic & Burnstock, 1998; MacKenzie 1999), designated P2X7, shares many phenotypical properties upon heterologous expression, suggesting are identical. is therefore also referred P2Z/P2X7 (Di Virgilio, 1995; Di Virgilio 1998). A peculiarity subunit its very long intracellular tail, 196 132 longer than P2X1 P2X2 isoform, respectively. Several studies attempting characterise recombinant provided information complex function, yet fully understood. During short applications lasting few seconds, behaves like typical member, exhibiting permeability small cations only. However, prolonged repeated ATP, large non-selective pores formed plasma membrane some (Surprenant Rassendren Virginio itself. On other hand, it suggested represent distinct entities, become activated subsequent stimulation (Coutinho-Silva Persechini, Schilling 1999). In native cells, elicits different over wide range. For instance, increases T lymphocytes, does cause lysis concentrations below 100 (El-Moatassim 1989), whereas millimolar range induces cytolysis death lymphocytes 1989; Filippini 1990; Zanovello 1990). Furthermore, immunomodulatory effects, such inhibition natural killer reactivity (Schmidt 1984) macrophage-mediated cytotoxicity (Cameron, 1984), observed occur < 500 necessary ATP-induced killing mycobacteria macrophages (Lammas 1997). In addition induction cytolytic system, receptor-dependent phospholipase D (Dubyak El-Moatassim, 1993), NFkB (Ferrari 1997) interleukin-1β-converting enzyme 1998) described, loss l-selectin from (Jamieson 1996). The molecular basis behaviour broad still unclear may simply reflect modification internal milieu concentration-dependent permeabilising activation. various cellular responses thus triggered quantitatively changes Ca2+, Na+ K+ due stimulation. Thus breakdown ectoATPases, amount expression divalent reducing ligand, 1998), taken into account. possible involvement considered. Here we provide electrophysiological evidence their 50 influence differently permeation characteristics existence provides explanation variable pattern signalling hP2X7-expressing ATP.