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
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
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DOI：
10.1074/jbc.m110.148940
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发表时间：
2010-10-22
影响因子：
4.8
通讯作者：
Evans, Richard J.
中科院分区：
文献类型：
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作者：
Allsopp, Rebecca C.;Lalo, Ulyana;Evans, Richard J.
富含胆固醇的脂筏作为信号微域，可以调节受体功能。我们已经证明在HEK293细胞中重组P2X1-4受体（atp门控离子通道）在脂筏中表达。洗涤剂Triton X-100减少了对富浮蛋白脂筏的定位。这种对Triton X-100的敏感性是浓度和亚基依赖性的，表明P2X1-4受体与脂筏的差异关联。在膜片钳研究中确定了筏关联对atp诱发的P2X受体反应的重要性。降低胆固醇的药物甲基- β -环糊精或filipin破坏脂质筏，使P2X1受体电流减少bbb90 %。相反，atp诱导的P2X2-4受体电流不受脂筏破坏的影响。为了确定胆固醇敏感性的分子基础，我们生成了嵌合受体，用不敏感的P2X2受体的相应区域取代胆固醇敏感的P2X1受体的部分。这些嵌合体确定了保守的蛋白激酶C位点和第一跨膜段之间的细胞内氨基末端区域对胆固醇消耗的敏感性的重要性。P2X1受体区域P2X1和P2X2受体之间的任何变异残基（残基20-23和27-29）突变为半胱氨酸可消除胆固醇敏感性。胆固醇消耗不改变ATP敏感性和P2X1受体的细胞表面表达。这表明通常需要胆固醇来促进atp结合的P2X1受体通道的打开/门化，而第一跨膜段区域的突变消除了这一要求。
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-beta-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.