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Lipid raft association and cholesterol sensitivity of P2X1-4 receptors for ATP: chimeras and point mutants identify intracellular amino-terminal residues involved in lipid regulation of P2X1 receptors.
J Biol Chem 2010; 285(43):32770-7JB

Abstract

Cholesterol-rich lipid rafts act as signaling microdomains and can regulate receptor function. We have shown in HEK293 cells recombinant P2X1-4 receptors (ATP-gated ion channels) are expressed in lipid rafts. Localization to flotillin-rich lipid rafts was reduced by the detergent Triton X-100. This sensitivity to Triton X-100 was concentration- and subunit-dependent, demonstrating differential association of P2X1-4 receptors with lipid rafts. The importance of raft association to ATP-evoked P2X receptor responses was determined in patch clamp studies. The cholesterol-depleting agents methyl-β-cyclodextrin or filipin disrupt lipid rafts and reduced P2X1 receptor currents by >90%. In contrast, ATP-evoked P2X2-4 receptor currents were unaffected by lipid raft disruption. To determine the molecular basis of cholesterol sensitivity, we generated chimeric receptors replacing portions of the cholesterol-sensitive P2X1 receptor with the corresponding region from the insensitive P2X2 receptor. These chimeras identified the importance of the intracellular amino-terminal region between the conserved protein kinase C site and the first transmembrane segment for the sensitivity to cholesterol depletion. Mutation of any of the variant residues between P2X1 and P2X2 receptors in this region in the P2X1 receptor (residues 20-23 and 27-29) to cysteine removed cholesterol sensitivity. Cholesterol depletion did not change the ATP sensitivity or cell surface expression of P2X1 receptors. This suggests that cholesterol is normally needed to facilitate the opening/gating of ATP-bound P2X1 receptor channels, and mutations in the pre-first transmembrane segment region remove this requirement.

Authors+Show Affiliations

Department of Cell Physiology and Pharmacology, University of Leicester, Leicester LE1 9HN, United Kingdom.No affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

20699225

Citation

Allsopp, Rebecca C., et al. "Lipid Raft Association and Cholesterol Sensitivity of P2X1-4 Receptors for ATP: Chimeras and Point Mutants Identify Intracellular Amino-terminal Residues Involved in Lipid Regulation of P2X1 Receptors." The Journal of Biological Chemistry, vol. 285, no. 43, 2010, pp. 32770-7.
Allsopp RC, Lalo U, Evans RJ. Lipid raft association and cholesterol sensitivity of P2X1-4 receptors for ATP: chimeras and point mutants identify intracellular amino-terminal residues involved in lipid regulation of P2X1 receptors. J Biol Chem. 2010;285(43):32770-7.
Allsopp, R. C., Lalo, U., & Evans, R. J. (2010). Lipid raft association and cholesterol sensitivity of P2X1-4 receptors for ATP: chimeras and point mutants identify intracellular amino-terminal residues involved in lipid regulation of P2X1 receptors. The Journal of Biological Chemistry, 285(43), pp. 32770-7. doi:10.1074/jbc.M110.148940.
Allsopp RC, Lalo U, Evans RJ. Lipid Raft Association and Cholesterol Sensitivity of P2X1-4 Receptors for ATP: Chimeras and Point Mutants Identify Intracellular Amino-terminal Residues Involved in Lipid Regulation of P2X1 Receptors. J Biol Chem. 2010 Oct 22;285(43):32770-7. PubMed PMID: 20699225.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Lipid raft association and cholesterol sensitivity of P2X1-4 receptors for ATP: chimeras and point mutants identify intracellular amino-terminal residues involved in lipid regulation of P2X1 receptors. AU - Allsopp,Rebecca C, AU - Lalo,Ulyana, AU - Evans,Richard J, Y1 - 2010/08/10/ PY - 2010/8/12/entrez PY - 2010/8/12/pubmed PY - 2010/12/14/medline SP - 32770 EP - 7 JF - The Journal of biological chemistry JO - J. Biol. Chem. VL - 285 IS - 43 N2 - Cholesterol-rich lipid rafts act as signaling microdomains and can regulate receptor function. We have shown in HEK293 cells recombinant P2X1-4 receptors (ATP-gated ion channels) are expressed in lipid rafts. Localization to flotillin-rich lipid rafts was reduced by the detergent Triton X-100. This sensitivity to Triton X-100 was concentration- and subunit-dependent, demonstrating differential association of P2X1-4 receptors with lipid rafts. The importance of raft association to ATP-evoked P2X receptor responses was determined in patch clamp studies. The cholesterol-depleting agents methyl-β-cyclodextrin or filipin disrupt lipid rafts and reduced P2X1 receptor currents by >90%. In contrast, ATP-evoked P2X2-4 receptor currents were unaffected by lipid raft disruption. To determine the molecular basis of cholesterol sensitivity, we generated chimeric receptors replacing portions of the cholesterol-sensitive P2X1 receptor with the corresponding region from the insensitive P2X2 receptor. These chimeras identified the importance of the intracellular amino-terminal region between the conserved protein kinase C site and the first transmembrane segment for the sensitivity to cholesterol depletion. Mutation of any of the variant residues between P2X1 and P2X2 receptors in this region in the P2X1 receptor (residues 20-23 and 27-29) to cysteine removed cholesterol sensitivity. Cholesterol depletion did not change the ATP sensitivity or cell surface expression of P2X1 receptors. This suggests that cholesterol is normally needed to facilitate the opening/gating of ATP-bound P2X1 receptor channels, and mutations in the pre-first transmembrane segment region remove this requirement. SN - 1083-351X UR - https://www.unboundmedicine.com/medline/citation/20699225/Lipid_raft_association_and_cholesterol_sensitivity_of_P2X1_4_receptors_for_ATP:_chimeras_and_point_mutants_identify_intracellular_amino_terminal_residues_involved_in_lipid_regulation_of_P2X1_receptors_ L2 - http://www.jbc.org/cgi/pmidlookup?view=long&pmid=20699225 DB - PRIME DP - Unbound Medicine ER -