Rosati A M, Traversa U
Basic Research and Integrative Neuroscience Centre-Department of Biomedical Sciences, University of Trieste, Italy.
Biochem Pharmacol. 1999 Aug 15;58(4):623-32. doi: 10.1016/s0006-2952(99)00135-5.
The dose-dependent inhibition of zinc and cadmium ions of agonist binding to A1 adenosine receptors in rat brain is prevented by histidine and cysteine, respectively. In the present study, the possible different mechanisms of Zn2+ and Cd2+ inhibitions were examined. The effects of Zn2+ and Cd2+ on equilibrium binding parameters of the agonists N6-cyclohexyl-[2,8-3H]-adenosine ([3H]CHA) or chloro-N6-cyclopentyl-adenosine ([3H]CCPA) and the antagonist cyclopentyl-1,3-dipropylxanthine ([3H]DPCPX) were compared with those effects of reagents or binding conditions which altered histidyl or cysteinyl residues of the A1 receptor. Zn2+ pretreatment did not change A1 agonist or antagonist affinity, but did reduce the Bmax. The inhibitory effects of Zn2+ pretreatments were also maintained after several membrane washings. Diethylpyrocarbonate, a histidine-specific alkylating reagent, behaved like zinc ions: pretreatment with A1 agonist protected the histidyl residues of the [3H]CHA binding site against modification by Zn2+, while the modification of the protonation state of the nitrogen of the imidazole group of histidines by changing pH indicated that the interactions of Zn2+ with the histidyl residues were feasible with their unprotonated form. These findings suggest the formation of coordination bonds between Zn2+ and histidines critical for [3H]CHA or [3H]DPCPX binding, which may prevent the ligand interaction with the specific sites without modifying the binding kinetics of radioligand to the non-chelated recognition sites. Cd2+ pretreatment reduced the [3H]CCPA affinity, but did not modify the affinity of the antagonist [3H]DPCPX, the Bmax remaining unaffected. As with cadmium effects, the oxidation of the thiol group of cysteine by dithionitrobenzoic acid (DTNB) reduced [3H]CCPA affinity without changing the number of binding sites. The reducing reagent dithiothreitol, which alone was unable to modify [3H]CCPA binding, overcame the inhibiting effects of both Cd2+ and DTNB. These findings suggest that cadmium ions may oxidize SH groups of cysteines localized on the A1 receptor molecule or a cysteine localized in the region of G(i)alpha subunit involved in the coupling with receptors. This mechanism can justify potential conformational modifications of the receptor molecule producing the decrease in affinity.
组氨酸和半胱氨酸分别可防止锌离子和镉离子对大鼠脑内激动剂与A1腺苷受体结合的剂量依赖性抑制。在本研究中，我们检测了Zn2+和Cd2+抑制作用可能存在的不同机制。比较了Zn2+和Cd2+对激动剂N6-环己基-[2,8-3H]-腺苷（[3H]CHA）或氯-N6-环戊基腺苷（[3H]CCPA）以及拮抗剂环戊基-1,3-二丙基黄嘌呤（[3H]DPCPX）平衡结合参数的影响，以及改变A1受体组氨酸或半胱氨酸残基的试剂或结合条件的影响。Zn2+预处理未改变A1激动剂或拮抗剂的亲和力，但降低了Bmax。多次洗涤膜后，Zn2+预处理的抑制作用仍得以维持。焦碳酸二乙酯是一种组氨酸特异性烷基化试剂，其作用类似于锌离子：用A1激动剂预处理可保护[3H]CHA结合位点的组氨酸残基不被Zn2+修饰，而通过改变pH值改变组氨酸咪唑基团氮的质子化状态表明，Zn2+与组氨酸残基的相互作用在其未质子化形式下是可行的。这些发现表明Zn2+与组氨酸之间形成了对[3H]CHA或[3H]DPCPX结合至关重要的配位键，这可能会阻止配体与特定位点的相互作用，而不会改变放射性配体与未螯合识别位点的结合动力学。Cd2+预处理降低了[3H]CCPA的亲和力，但未改变拮抗剂[3H]DPCPX的亲和力，Bmax不受影响。与镉的作用一样，二硫代硝基苯甲酸（DTNB）氧化半胱氨酸的巯基会降低[3H]CCPA的亲和力，而不改变结合位点的数量。单独使用时无法改变[3H]CCPA结合的还原剂二硫苏糖醇克服了Cd2+和DTNB的抑制作用。这些发现表明，镉离子可能会氧化位于A1受体分子上的半胱氨酸的SH基团，或位于与受体偶联的G(i)α亚基区域内的半胱氨酸。这种机制可以解释受体分子潜在的构象修饰导致亲和力降低的原因。
