Virginio C, Church D, North R A, Surprenant A
Geneva Biomedical Research Institute, Glaxo Wellcome, Switzerland.
Neuropharmacology. 1997 Sep;36(9):1285-94. doi: 10.1016/s0028-3908(97)00141-x.
The P2X7 receptor is a uniquely bifunctional molecule through which ATP can open a small cationic channel typical of ionotropic receptors and also induce a large pore permeable to high molecular weight molecules (> 600 Da). Activation of this large pore can lead to cell lysis within 1-2 min. We asked whether pharmacological differences existed between the cationic channel and the cell permeabilizing pore by measuring whole-cell currents and uptake of a propidium dye (YO-PRO; Mw 629) in HEK293 cells stably expressing the rat P2X7 receptor, and comparing the actions of divalent cations and protons in these two assays. Currents in response to 2'-3'-(O)-(4-benzoyl benzoyl) ATP (BzATP, 30 microM) were inhibited by extracellular calcium, magnesium, zinc, copper and protons with half-maximal inhibitory concentrations (IC50) of 2.9 mM, 0.5 mM, 11 microM, 0.5 microM and 0.4 microM, respectively. The inhibition was voltage independent in each case. YO-PRO uptake induced by BzATP was also inhibited with similar IC50 values. The rank order of potency of a range of divalents was Cu2+ > Cd2+ = Zn2+ > Ni2+ >> Mg2+ = Co2+ > Mn2+ > Ca2+ = Ba2+ >> Sr2+. These results suggest that these divalent cations and protons all act primarily as allosteric modulators to alter the affinity of ATP binding to the P2X7 receptor. In contrast, extracellular (but not intracellular) calmidazolium inhibited the BzATP-evoked current by up to 90% (IC50 = 15 nM) but had no effect on YO-PRO uptake. Thus, calmidazolium can block activation of the ionic channel but this does not prevent the formation of the large permeabilizing pore.
P2X7受体是一种独特的双功能分子，通过它ATP可以打开离子型受体典型的小阳离子通道，还能诱导形成对高分子量分子（>600 Da）通透的大孔。这种大孔的激活可在1 - 2分钟内导致细胞裂解。我们通过测量稳定表达大鼠P2X7受体的HEK293细胞中的全细胞电流以及一种碘化丙啶染料（YO-PRO；分子量629）的摄取情况，并比较这两种检测中不同二价阳离子和质子的作用，来探究阳离子通道与使细胞通透的孔之间是否存在药理学差异。对2'-3'-(O)-(4-苯甲酰苯甲酰)ATP（BzATP，30 μM）产生的电流受到细胞外钙、镁、锌、铜和质子的抑制，其半数最大抑制浓度（IC50）分别为2.9 mM、0.5 mM、11 μM、0.5 μM和0.4 μM。每种情况下这种抑制均与电压无关。BzATP诱导的YO-PRO摄取也以相似的IC50值受到抑制。一系列二价阳离子的效力排序为：Cu2+ > Cd2+ = Zn2+ > Ni2+ >> Mg2+ = Co2+ > Mn2+ > Ca2+ = Ba2+ >> Sr2+。这些结果表明，这些二价阳离子和质子主要作为变构调节剂来改变ATP与P2X7受体结合的亲和力。相比之下，细胞外（而非细胞内）的钙调蛋白抑制剂可将BzATP诱发的电流抑制高达90%（IC50 = 15 nM），但对YO-PRO摄取没有影响。因此，钙调蛋白抑制剂可阻断离子通道的激活，但这并不妨碍大的通透孔的形成。
