Burgmer U, Schulz U, Tränkle C, Mohr K
Pharmacology and Toxicology, Institute of Pharmacy, University of Bonn, Germany.
Naunyn Schmiedebergs Arch Pharmacol. 1998 Apr;357(4):363-70. doi: 10.1007/pl00005180.
Mg2+-ions have been suspected to attenuate the inhibitory effect of allosteric modulators on the dissociation of orthosteric ligands from muscarinic M2 receptors. It was aimed to gain more insight into the molecular events underlying the effect of Mg2+. The interaction of Mg2+ with the allosteric model compounds W84 (hexane-1,6-bis [dimethyl-3'-phthalimidopropylammonium bromide]) and Chin3/6 (hexane-1 ,6-bis[dimethyl-3'-¿4-oxo-2-phenyl-3,4-dihydro-2H-quinazolin-1-yl propylammonium bromide]) was studied in porcine heart muscarinic receptors, the primary binding site of which was occupied by the ligand [3H]N-methylscopolamine ([3H]NMS). The incubation buffer was composed of 4 mM Na2HPO4 and 1 mM KH2PO4 (pH 7.4, 23 degrees C). The retardation of [3H]NMS dissociation (control t1/2=5.6 min) induced by the allosteric test compounds was diminished by 3 mM Mg2+ to a greater extent than to be expected with regard to its contribution to the ionic strength of the buffer solution. Concentration-effect curves for the allosteric retardation of [3H]NMS dissociation by W84 (half maximal effective concentration EC0.5=24 nM in the absence of Mg2+) and by Chin3/6 (EC0.5=28 nM) were shifted by Mg2+ to the right in a parallel fashion. The curve-shift was compatible with a competitive interplay between Mg2+ and the modulators. The pKb-values as a measure of the antagonistic potency of Mg2+, however, differed depending on the modulator, i.e. pKb=3.4 with W84 and pKb=2.8 with Chin3/6. Mg2+ itself was capable of slowing the dissociation of [3H]NMS; the maximal retardation of [3H]NMS dissociation was about 3 fold, the concentration-effect relationship was compatible with a two-site model using the above-mentioned pKb-values as affinity constants. Since the equilibrium-binding of [3H]NMS remained unchanged up to a Mg2+-concentration of 3 mM, the cation appears to inhibit the association and dissociation of [3H]NMS to the same extent in this concentration range. Taken together, the findings indicate that Mg2+ may bind to the allosteric region of muscarinic M2 receptors and that more than one site is involved in this interaction. The sites of action may represent divalent cation binding sites.
镁离子一直被怀疑会减弱变构调节剂对正构配体从毒蕈碱M2受体解离的抑制作用。目的是更深入地了解镁离子作用背后的分子事件。研究了镁离子与变构模型化合物W84（己烷-1,6-双[二甲基-3'-邻苯二甲酰亚胺基丙基溴化铵]）和Chin3/6（己烷-1,6-双[二甲基-3'-（4-氧代-2-苯基-3,4-二氢-2H-喹唑啉-1-基丙基溴化铵]）在猪心脏毒蕈碱受体中的相互作用，其主要结合位点被配体[3H]N-甲基东莨菪碱（[3H]NMS）占据。孵育缓冲液由4 mM磷酸氢二钠和1 mM磷酸二氢钾组成（pH 7.4，23℃）。变构测试化合物诱导的[3H]NMS解离延迟（对照t1/2 = 5.6分钟）被3 mM镁离子减弱的程度大于根据其对缓冲溶液离子强度的贡献所预期的程度。W84（在无镁离子时半数最大有效浓度EC0.5 = 24 nM）和Chin3/6（EC0.5 = 28 nM）对[3H]NMS解离的变构延迟的浓度-效应曲线被镁离子以平行方式向右移动。曲线移动与镁离子和调节剂之间的竞争性相互作用一致。然而，作为镁离子拮抗效力衡量指标的pKb值因调节剂而异，即与W84时pKb = 3.4，与Chin3/6时pKb = 2.8。镁离子本身能够减缓[3H]NMS的解离；[3H]NMS解离的最大延迟约为3倍，浓度-效应关系与使用上述pKb值作为亲和常数的双位点模型一致。由于在镁离子浓度高达3 mM时[3H]NMS的平衡结合保持不变，在该浓度范围内阳离子似乎对[3H]NMS的结合和解离具有相同程度的抑制作用。综上所述，这些发现表明镁离子可能与毒蕈碱M2受体的变构区域结合，并且这种相互作用涉及多个位点。作用位点可能代表二价阳离子结合位点。
