Contribution of Individual Subunits to the Multimeric P2X₂ Receptor: Estimates based on Methanethiosulfonate Block at T336C

Abstract

P2X receptors are membrane proteins that incorporate a cation-selective ion channel that can be opened by the binding of extracellular ATP. They associate as hetero- and homo-multimers of currently unknown stoichiometry. In this study, we have used Xenopus laevis oocytes to express rat P2X(2) receptor subunits, which carry a cysteine mutation at position 336. ATP-induced currents at this mutant receptor subunit were blocked by more than 90% when exposed to [2-(trimethylammonium) ethyl] methanethiosulfonate (MTSET), whereas currents from wild-type subunits were not affected. To compare mutant and wild-type channel expression, we introduced an epitope in their extracellular domains and found for both channels a similar linear relationship between antibody binding and currents induced by ATP. To study the contribution of the individual subunits to the block by MTSET, we coinjected different mixtures of wild-type and mutant-encoding mRNAs. We found that the inhibition by MTSET depended linearly on the proportion of mutant subunits, which was clearly contrary to the hypothesis that a single mutant subunit could act in a dominant fashion. Subsequent concatenation of wild-type and mutant-encoding cDNAs resulted in an inhibition by MTSET that also depended linearly on the number of mutant subunits and was independent of the position of the mutant subunit, as long as only two or three P2X(2) subunits were joined. With four or six subunits joined, however, the inhibition by MTSET became strongly position-dependent. The present results show that a "per-subunit" channel block causes the blocking effects of MTSET and they suggest that not four but maximally three subunits actively participate in the channel formation.

Identification of a Trafficking Motif Involved in the Stabilization and Polarization of P2X Receptors

“…Receptors-Tags were introduced by overlapping PCR as described previously (8) in the extracellular loop of P2X subunits, all at similar positions; FLAG tags were inserted in P2X 2 and P2X 6 between Asp 78 -Lys 79 and Glu 83 -Lys 84 , respectively; P2X 3 has a Myc tag inserted between residues Asn 104 and Arg 105 ; P2X 4 has an HA tag between Gln 78 and Leu 79 ; and P2X 5 has either a Myc or an HA tag inserted between Thr 78 and Thr 79 . In all cases tag insertions did not alter the function of the different P2X subunits measured electrophysiologically.…”

“…P2X subunits have a membrane topology with two transmembrane domains linked by a large extracellular loop; N and C termini are localized intracellularly (5,6). To form a channel, P2X subunits are thought to associate as homo-or heterooligomers, composed of three subunits (7,8) organized in a head-to-tail orientation around a central pore (9). This model is consistent with studies that show that the permeation pathway is associated with transmembrane regions (10 -12), and with the fact that the gating of the channel is in part due to movements of the subunits relative to the each other (12).…”

Purinergic receptors in the splanchnic circulation

“…Often native cell types express multiple P2X subunit mRNA transcripts and multiple P2X subunits. It is not clear how many subunits are required in each heteromer, which ones dominate, or if a greater number of heteromers can be formed [78,121,163]. P2X 6 subunits form only heteromeric channels and P2X 7 subunits only homomeric channels [173].…”

“…P2X receptors form as multimeric assemblies of channel subunits. The exact stoichiometry of the receptor remains unclear although studies have suggested that the channel may be comprised of at least three subunits [163].…”

P2X receptors

“…Several lines of evidence indicated that the functional protein was a trimer: indeed, three ATP-binding sites had been suggested by Bean [14] on the basis of his ATP dose-response curves from bullfrog sensory neurons. This evidence included biochemical approaches using blue native polyacrylamide gel electrophoresis [30] and functional approaches with co-expression and concatenation of subunits carrying reporter mutations [31][32][33][34][35]. The demonstration that P2X receptors were trimers set them in clear distinction from the tetrameric glutamate-gated ion channels and the pentameric nicotinic superfamily (which also includes channels gated by glycine, g-aminobutryic acid and 5-hydroxytryptamine; [22]).…”