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

Abstract

Many effects of extracellular ATP on cells of the immune system have been attributed to the presence of a so-called P2Z receptor. Recent work has shown that one of the members of the P2X family of ATP-gated receptors Soto et al. 1997;Ralevic & Burnstock, 1998;MacKenzie et al. 1999), designated P2X 7 , shares many phenotypical properties with the P2Z receptor upon heterologous expression, suggesting that the two are identical. The P2X 7 receptor is therefore also referred to as P2Z/P2X 7 receptor (Di Virgilio, 1995;Di Virgilio et al. 1998). A peculiarity of the P2X 7 subunit is its very long intracellular C-terminal tail, which is 196 or 132 amino acids longer than that of the P2X 1 or P2X 2 subunit isoform, respectively.Several studies attempting to characterise the recombinant P2X 7 receptor have provided information about its complex function, which is not yet fully understood. During short applications of ATP lasting a few seconds, the P2X 7 receptor behaves like a typical P2X family member, exhibiting permeability to small cations only. However, upon prolonged or repeated applications of ATP, large non-selective pores are formed in the plasma membrane of some cells expressing P2X 7 (Surprenant et al. 1996;Rassendren et al. 1997;Virginio et al. 1999), which have been attributed to the receptor itself. On the other hand, it has also been suggested that the pores represent distinct entities, which become activated subsequent to the stimulation of P2X 7 receptors (Coutinho-Silva & Persechini, 1997;Schilling et al. 1999).In native cells, ATP elicits different effects over a wide concentration range. For instance, ATP increases the cell 1. The effect of the agonist ATP on whole cell currents of Xenopus oocytes expressing either the wild-type human P2X 7 receptor (hP2X 7 ), an N-terminally hexahistidyl-tagged hP2X 7 receptor (His-hP2X 7 ), or a truncated His-hP2X 7 receptor (His-hP2X 7 ∆C) lacking the C-terminal 156 amino acids was investigated using the two-microelectrode voltage clamp technique.2. The activation time course of the wild-type hP2X 7 receptor can be described as the sum of an exponentially growing and an additional almost linearly activating current component.3. The amplitude of the exponentially activating current component of the wild-type hP2X 7 receptor displayed a biphasic dependence on the agonist concentration, which could be best approximated by a model of two equal high-sensitivity and two equal low-sensitivity noncooperative activation sites with apparent dissociation constants of about 4 and 200 µM free ATP 4_ , respectively.4. The linearly activating current was monophasically dependent on the agonist concentration with an apparent dissociation constant of about 200 µM.5. The contribution of the low-sensitivity sites to current kinetics was reduced or almost abolished in oocytes expressing His-hP2X 7 or His-hP2X 7 ∆C.6. Our data indicate that the hP2X 7 receptor possesses at least two types of activation sites, which differ in ATP 4_ sensitivity by a factor of 50. The degree of occupatio...

Rat submandibular glands secrete nanovesicles with NTPDase and 5′-nucleotidase activities

“…Purinergic receptor agonist-stimulated salivation was suppressed by more than 70 % in submandibular glands from P2X7 null mice [10]. At least one EC 50 is >100 μM ATP for this receptor [8,41], which is consistent with the high ATP Km we measured for microsomal NTPDases.…”

The P2X7 receptor from Xenopus laevis: formation of a large pore in Xenopus oocytes

“…3A, lower right). Apart from the slowly increasing conductance following washout of the agonist, this current behavior matches rather closely the behavior of human P2X 7 receptor expressed in oocytes [14,18]. In contrast, the rat subtype, when expressed in oocytes, undergoes a strong current run-down following activation [13].…”

“…3A, upper right). Following washout of ATP, the inward current deactivated with a fast and a slowly deactivating component as it has recently been described for the human receptor [18]. But usually, after washout of ATP, a slowly increasing conductance appeared (Fig.…”

P2X7 Receptor Cell Surface Expression and Cytolytic Pore Formation Are Regulated by a Distal C-terminal Region

“…This long C-terminal domain (ϳ240 amino acids) appears to modulate the function of the P2X7R, because the removal of this region tempers the receptor's response to ATP. In particular, the removal of the last 177 amino acids (to yield P2X7-(1-418)) abolishes pore formation without affecting channel properties (1,14). Investigation of a 1-439 truncated human P2X7 receptor in Xenopus oocytes revealed an altered kinetic response to ATP applications, thought to be related to a difference in the agonist binding site of the truncated receptor compared with the full-length P2X7R (14).…”

“…In particular, the removal of the last 177 amino acids (to yield P2X7-(1-418)) abolishes pore formation without affecting channel properties (1,14). Investigation of a 1-439 truncated human P2X7 receptor in Xenopus oocytes revealed an altered kinetic response to ATP applications, thought to be related to a difference in the agonist binding site of the truncated receptor compared with the full-length P2X7R (14). Furthermore, when the human P2X7R is truncated at residue 415, it does not exhibit cell surface expression (15).…”