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.
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.
Allsopp
Rebecca C
RC
From the Department of Cell Physiology and Pharmacology, University of Leicester, Leicester LE1 9HN, United Kingdom.
Lalo
Ulyana
U
From the Department of Cell Physiology and Pharmacology, University of Leicester, Leicester LE1 9HN, United Kingdom.
Evans
Richard J
RJ
From the Department of Cell Physiology and Pharmacology, University of Leicester, Leicester LE1 9HN, United Kingdom. Electronic address: rje6@le.ac.uk.
D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D002460:Cell Line; D002784:Cholesterol / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology; D021962:Membrane Microdomains / Q000235:genetics / Q000378:metabolism*; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D009154:Mutation; D058911:Purinergic P2X Receptor Agonists; D058474:Receptors, Purinergic P2X1 / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D058476:Receptors, Purinergic P2X2 / Q000235:genetics / Q000378:metabolism*; D058477:Receptors, Purinergic P2X3 / Q000235:genetics / Q000378:metabolism*; D058848:Receptors, Purinergic P2X4 / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000819:agonists / Q000235:genetics / Q000378:metabolism
D058911:Purinergic P2X Receptor Agonists; D058474:Receptors, Purinergic P2X1; D058476:Receptors, Purinergic P2X2; D058477:Receptors, Purinergic P2X3; D058848:Receptors, Purinergic P2X4; D011993:Recombinant Fusion Proteins; D000255:Adenosine Triphosphate; D002784:Cholesterol