Dataset Information

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.

SUBMITTER: Sun C

PROVIDER: S-EPMC3629090 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

The second transmembrane domain of P2X7 contributes to dilated pore formation.

Sun Chengqun C Heid Michelle E ME Keyel Peter A PA Salter Russell D RD

PloS one 20130417 4

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 expre ...[more]

PMID: 23613968

Similar Datasets

Project description:We propose mechanisms by which the transmembrane domain of vesicular stomatitis virus (VSV-TMD) promotes both initiation of fusion and formation of a fusion pore. Time courses of polyethyleneglycol (PEG)-mediated fusion of 25 nm small unilamellar vesicles composed of dioleoylphosphatidylcholine, dioleoylphosphatidylethanolamine (DOPE), bovine brain sphingomyelin, and cholesterol (35:30:15:20 molar ratio) were recorded at pH 7.4 at five different temperatures (from 17°C to 37°C) and compared with time courses obtained with the same vesicles containing the fusion-active TMD of the G protein of VSV. Multiple time courses were fitted globally to a one-intermediate ensemble kinetic model to estimate the rate constants for conversion of the aggregated state to an intermediate hemifused state (k1, stalk, or I1) that rapidly transits to an unstable intermediate (I2 state) that converts to a final fusion pore state with a combined rate k3. The probabilities of lipid mixing, contents mixing, and contents leakage in the three states were also obtained from this analysis. The activation thermodynamics for each step were consistent with previously published models of lipid rearrangements during intermediate and pore formation. The influences of VSV-TMD, hexadecane, and VSV-TMD + hexadecane on the kinetics, activation thermodynamics, and membrane structure support the hypothesis that these two agents do not catalyze fusion by a common mechanism, except possibly at the lowest temperatures examined. VSV-TMD primarily catalyzed initial intermediate formation, although it substantially increased the probability of contents mixing in the intermediate state. Our results support the hypothesis that the catalytic influence of VSV-TMD on the initial-intermediate- and pore-forming steps of PEG-mediated fusion derives from its ability to impose a positive intrinsic curvature and thereby stress small unilamellar vesicle outer leaflets as well as the periphery of intermediate microstructures.

Dataset Information

The second transmembrane domain of P2X7 contributes to dilated pore formation.

Publications

The second transmembrane domain of P2X7 contributes to dilated pore formation.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets