Gadbut A P, Riccardi D, Wu L, Hebert S C, Galper J B
Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts 02115, USA.
J Biol Chem. 1996 Mar 15;271(11):6398-402. doi: 10.1074/jbc.271.11.6398.
The G-protein-gated inward-rectifying K+ channel GIRK1 has been demonstrated in heart and brain. These tissues also both express the M2, M3, and M4, muscarinic acetylcholine receptors (mAChR) (Gadbut, A.P., and Galper, J.B. (1994),J. Biol. Chem. 269,25823-25829). Only the M2 mAChR has been demonstrated to couple to GIRK1 (Kubo, Y., Reuveny, E., Slesinger, P. A., Jan, Y. N., and Jan, L. Y. (1993) Nature 264, 802-806). In this study we determined the specificity of coupling of the M3 and M4 mAChR to a new GIRK1 cloned from a chick brain cDNA library. This clone codes for a 492-amino acid protein that is 93% identical to rat GIRK1 and is expressed in brain, atrium, and ventricle, but not skeletal muscle. In Xenopus laetis oocytes co-expression of GIRK1 with either the chick M2 or M4 mAChR gave carbamylcholine (10 microm)-stimulated K+ currents of 308 +/-26 nA and 298 +/-29 nA, respectively, which were both Ba2+- and pertussis toxin-sensitive. Activation of the M3 receptor produced 2382 +/-478 nA of current which was insensitive to Ba2+ and pertussis toxin, but was 85% inhabitable by the Cl channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (10-20 microm) consistent with coupling to an endogenous Ca2+-activated Cl- channel via a phosphatidylinositol-dependent mechanism. Co-expression of the cardiac inward rectifier CIR with chick M2 or M4 mAChR and GIRK1 increased currents more than 10-fold, but had no effect on specificity of coupling. These data demonstrate a new function for the M4 mAChR and a high degree of specificity for coupling of each receptor subtype to GIRK1.
G蛋白门控内向整流钾通道GIRK1已在心脏和大脑中得到证实。这些组织也都表达M2、M3和M4毒蕈碱型乙酰胆碱受体（mAChR）（加德布特，A.P.，和加尔珀，J.B.（1994年），《生物化学杂志》269，25823 - 25829）。仅M2 mAChR已被证明可与GIRK1偶联（久保，Y.，鲁韦尼，E.，斯莱辛格，P.A.，扬，Y.N.，和扬，L.Y.（1993年）《自然》264，802 - 806）。在本研究中，我们确定了M3和M4 mAChR与从鸡脑cDNA文库克隆的一种新的GIRK1的偶联特异性。该克隆编码一种492个氨基酸的蛋白质，与大鼠GIRK1有93%的同一性，在脑、心房和心室中表达，但在骨骼肌中不表达。在非洲爪蟾卵母细胞中，GIRK1与鸡M2或M4 mAChR共表达分别产生了氨甲酰胆碱（10微摩尔）刺激的钾电流，分别为308±26纳安和298±29纳安，这两种电流对钡离子和百日咳毒素均敏感。M3受体的激活产生了2382±478纳安的电流，该电流对钡离子和百日咳毒素不敏感，但85%可被氯离子通道阻滞剂5 - 硝基 - 2 -（3 - 苯基丙基氨基）苯甲酸（10 - 20微摩尔）抑制，这与通过磷脂酰肌醇依赖性机制与内源性钙激活氯离子通道偶联一致。心脏内向整流器CIR与鸡M2或M4 mAChR和GIRK1共表达使电流增加了10倍以上，但对偶联特异性没有影响。这些数据证明了M4 mAChR的一种新功能以及每种受体亚型与GIRK1偶联的高度特异性。
