title: Polar residues in the second transmembrane domain of the rat P2X2 receptor that affect spontaneous gating, unitary conductance, and rectification.
abstract: Membrane ion channels activated by extracellular ATP (P2X receptors) are widely distributed in the nervous system. Their molecular architecture is fundamentally distinct from that of the nicotinic or glutamate receptor families. We have measured single-channel currents, spontaneous gating, and rectification of rat P2X2 receptor in which polar and charged residues of the second transmembrane domain (TM2) were systematically probed by mutagenesis. The results suggest that Asn(333) and Asp(349) lie respectively in external and internal vestibules. Substitutions at Asn(333), Thr(336), and Ser(340) were particularly likely to cause spontaneously active channels. At Thr(336), Thr(339), and Ser(340), the introduction of positive charge (Arg, Lys, or His, or Cys followed by treatment with 2-aminoethyl methanethiosulphonate) greatly enhanced outward currents, suggesting that side-chains of these three residues are exposed in the permeation pathway of the open channel. These functional findings are interpreted in the context of the recently reported 3.1 A crystal structure of the zebrafish P2X4.1 receptor in the closed state. They imply that the gate is formed by residues Asn(333) to Thr(339) and that channel opening involves a counter-clockwise rotation and separation of the TM2 helices.
authors.affiliation: Faculty of Medical and Human Sciences, University of Manchester, Manchester M13 9PL, UK.
authors.forename: Lishuang
authors.initials: L
authors.lastname: Cao
authors.forename: Helen E
authors.initials: HE
authors.lastname: Broomhead
authors.forename: Mark T
authors.initials: MT
authors.lastname: Young
authors.forename: R Alan
authors.initials: RA
authors.lastname: North
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D006801:Humans; D008564:Membrane Potentials / Q000502:physiology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D018408:Patch-Clamp Techniques; D011487:Protein Conformation; D051381:Rats; D018048:Receptors, Purinergic P2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D058476:Receptors, Purinergic P2X2; D017386:Sequence Homology, Amino Acid; D013997:Time Factors
chemical_list: C546401:P2RX2 protein, human; C546403:P2rx2 protein, rat; D018048:Receptors, Purinergic P2; D058476:Receptors, Purinergic P2X2; D000255:Adenosine Triphosphate