title: The second transmembrane domain of P2X7 contributes to dilated pore formation.
abstract: Activation of the purinergic receptor P2X7 leads to the cellular permeability of low molecular weight cations. To determine which domains of P2X7 are necessary for this permeability, we exchanged either the C-terminus or portions of the second transmembrane domain (TM2) with those in P2X1 or P2X4. Replacement of the C-terminus of P2X7 with either P2X1 or P2X4 prevented surface expression of the chimeric receptor. Similarly, chimeric P2X7 containing TM2 from P2X1 or P2X4 had reduced surface expression and no permeability to cationic dyes. Exchanging the N-terminal 10 residues or C-terminal 14 residues of the P2X7 TM2 with the corresponding region of P2X1 TM2 partially restored surface expression and limited pore permeability. To further probe TM2 structure, we replaced single residues in P2X7 TM2 with those in P2X1 or P2X4. We identified multiple substitutions that drastically changed pore permeability without altering surface expression. Three substitutions (Q332P, Y336T, and Y343L) individually reduced pore formation as indicated by decreased dye uptake and also reduced membrane blebbing in response to ATP exposure. Three others substitutions, V335T, S342G, and S342A each enhanced dye uptake, membrane blebbing and cell death. Our results demonstrate a critical role for the TM2 domain of P2X7 in receptor function, and provide a structural basis for differences between purinergic receptors.
authors.affiliation: Department of Immunology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States of America.
authors.forename: Chengqun
authors.initials: C
authors.lastname: Sun
authors.forename: Michelle E
authors.initials: ME
authors.lastname: Heid
authors.forename: Peter A
authors.initials: PA
authors.lastname: Keyel
authors.forename: Russell D
authors.initials: RD
authors.lastname: Salter
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D016923:Cell Death / Q000187:drug effects; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002463:Cell Membrane Permeability / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D057809:HEK293 Cells; D006801:Humans; D007211:Indoles / Q000378:metabolism; D015640:Ion Channel Gating / Q000187:drug effects; D051379:Mice; D008810:Mice, Inbred C57BL; D017354:Point Mutation / Q000235:genetics; D017434:Protein Structure, Tertiary; D051381:Rats; D058474:Receptors, Purinergic P2X1 / Q000737:chemistry / Q000378:metabolism; D058848:Receptors, Purinergic P2X4 / Q000737:chemistry / Q000378:metabolism; D058486:Receptors, Purinergic P2X7 / Q000737:chemistry* / Q000378:metabolism*; D013329:Structure-Activity Relationship
chemical_list: D007211:Indoles; D058474:Receptors, Purinergic P2X1; D058848:Receptors, Purinergic P2X4; D058486:Receptors, Purinergic P2X7; C007293:DAPI; D000255:Adenosine Triphosphate