Blanton M P, McCardy E A, Huggins A, Parikh D
Department of Pharmacology, Texas Tech University Health Sciences Center, Lubbock 79430, USA.
Biochemistry. 1998 Oct 13;37(41):14545-55. doi: 10.1021/bi981435q.
The hydrophobic photoreactive compound 3-trifluoromethyl-3-(m-[125I]iodophenyl) diazirine ([125I]TID) has revealed important structural information about the pore of the ion channel and lipid-protein interface of the nicotinic acetylcholine receptor (AChR). To further characterize the structure of the AChR, we have mapped the sites of photoincorporation of a benzoic acid ester analogue of TID ([125I]TID-BE) and a phospholipid analogue ([125I]TIDPC/16). For each photoreactive probe, labeled sites were identified by amino-terminal sequencing of purified tryptic fragments of individual receptor subunits. [125I]TID-BE reacted with alphaCys-412, alphaMet-415, and alphaCys-418 in the M4 segment of the alpha-subunit and gammaCys-451 and gammaSer-460 in gammaM4. In the M1 segment of the alpha- and beta-subunits, [125I]TID-BE labeled alphaPhe-227, alphaLeu-228, and betaLeu-234, betaAla-235, respectively. The labeling pattern in the M1 and M4 segments indicate that TID and TID-BE interact with the AChR lipid-protein interface in a similar fashion, revealing the same lipid-exposed face of each transmembrane segment. In contrast to TID, there was, however, no detectable incorporation of [125I]TID-BE into the channel lining betaM2 segment when the AChR was labeled in the resting state conformation. In the presence of agonist (desensitized state), [125I]TID-BE reacted with betaLeu-257, betaVal-261, and beta-Leu-264 in betaM2; a labeling pattern which indicates that, in comparison to TID, the binding loci for TID-BE is located closer to the extracellular end of the channel. For [125I]TIDPC/16, receptor labeling was insensitive to the presence of agonist and the sites of incorporation mapped to the confines of the transmembrane segments alphaM4, alphaM1, and gammaM4, validating previous results found with small lipophilic probes.
疏水性光反应性化合物3 - 三氟甲基 - 3 -（间 - [¹²⁵I]碘苯基）重氮丙啶（[¹²⁵I]TID）已揭示了有关离子通道孔以及烟碱型乙酰胆碱受体（AChR）脂质 - 蛋白质界面的重要结构信息。为了进一步表征AChR的结构，我们绘制了TID的苯甲酸酯类似物（[¹²⁵I]TID - BE）和磷脂类似物（[¹²⁵I]TIDPC/16）的光掺入位点。对于每个光反应性探针，通过对各个受体亚基纯化的胰蛋白酶片段进行氨基末端测序来鉴定标记位点。[¹²⁵I]TID - BE与α亚基M4段中的αCys - 412、αMet - 415和αCys - 418以及γM4中的γCys - 451和γSer - 460发生反应。在α和β亚基的M1段中，[¹²⁵I]TID - BE分别标记αPhe - 227、αLeu - 228以及βLeu - 234、βAla - 235。M1和M4段中的标记模式表明，TID和TID - BE以相似的方式与AChR脂质 - 蛋白质界面相互作用，揭示了每个跨膜段相同的脂质暴露面。然而，与TID不同的是，当AChR在静息状态构象下被标记时，未检测到[¹²⁵I]TID - BE掺入通道内衬βM2段。在激动剂存在下（脱敏状态），[¹²⁵I]TID - BE与βM2中的βLeu - 257、βVal - 261和β - Leu - 264发生反应；这种标记模式表明，与TID相比，TID - BE的结合位点更靠近通道的细胞外端。对于[¹²⁵I]TIDPC/16，受体标记对激动剂的存在不敏感，掺入位点映射到跨膜段αM4、αM1和γM4的范围内，证实了先前用小亲脂性探针获得的结果。
