title: Contribution of individual subunits to the multimeric P2X(2) 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.
authors.affiliation: Geneva Biomedical Research Institute, Glaxo Wellcome Research and Development, Geneva, Switzerland. ron.stoop@ibcm.unil.ch
authors.forename: R
authors.initials: R
authors.lastname: Stoop
authors.forename: S
authors.initials: S
authors.lastname: Thomas
authors.forename: F
authors.initials: F
authors.lastname: Rassendren
authors.forename: E
authors.initials: E
authors.lastname: Kawashima
authors.forename: G
authors.initials: G
authors.lastname: Buell
authors.forename: A
authors.initials: A
authors.lastname: Surprenant
authors.forename: R A
authors.initials: RA
authors.lastname: North
mesh_terms: D000818:Animals; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D008698:Mesylates / Q000494:pharmacology*; D009154:Mutation; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism; D051381:Rats; D018048:Receptors, Purinergic P2 / Q000096:biosynthesis / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D058476:Receptors, Purinergic P2X2; D014982:Xenopus laevis
chemical_list: D018076:DNA, Complementary; D008698:Mesylates; C546403:P2rx2 protein, rat; D018048:Receptors, Purinergic P2; D058476:Receptors, Purinergic P2X2; C074502:methanethiosulfonate