Thompson Andrew J, Lummis Sarah C R
Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW, UK.
Neuropharmacology. 2009 Jan;56(1):285-91. doi: 10.1016/j.neuropharm.2008.07.009. Epub 2008 Jul 16.
Calcium modulates the 5-HT3 receptor response by reducing peak current amplitude and increasing rates of activation, deactivation and desensitisation, but the binding site(s) and mechanism(s) of this modulation are unknown. Here we study residues that may be involved in calcium binding in two partially overlapping regions of the extracellular domain (E213-E215-E218 and D204-E218-V219). The modulatory effects of calcium were assessed by radioligand binding and whole-cell patch-clamp. Comparisons of [3H]granisetron binding showed an increase in Kd in 10mM calcium that was abolished by the substitutions E213Q, E215Q, D204N and V219L. E218Q mutant receptors displayed no specific binding or function, and immunofluorescence showed that they did not reach the cell surface. E213Q increased inherent rates of desensitisation, but the relative effects of calcium on these rates, and on the reduction in current amplitude, were similar to wild type receptors. Current responses and calcium-mediated effects at E215Q mutant receptors were indistinguishable from wild type. D204N and V219L mutants were non-functional. A calcium impermeable mutant (E277A/S297R) revealed no changes in peak amplitude or kinetics with increased calcium. Our results are consistent with residues D204, E218 and V219 participating in receptor assembly, structure and/or trafficking to the plasma membrane, and we speculate that this might rely upon the stabilising effect of bound calcium. E213, E215, D204 and V219 may contribute to a calcium binding site that is responsible for the calcium-mediated effects on ligand binding. However, the major site for calcium-dependent modulation of the 5-HT3 current is located within the ion channel or cell interior.
钙通过降低峰值电流幅度、提高激活、失活和脱敏速率来调节5-羟色胺3（5-HT3）受体反应，但这种调节的结合位点和机制尚不清楚。在此，我们研究了细胞外结构域两个部分重叠区域（E213-E215-E218和D204-E218-V219）中可能参与钙结合的残基。通过放射性配体结合和全细胞膜片钳评估钙的调节作用。[3H]格拉司琼结合的比较显示，在10mM钙存在下解离常数（Kd）增加，而E213Q、E215Q、D204N和V219L替代可消除这种增加。E218Q突变体受体未显示特异性结合或功能，免疫荧光显示它们未到达细胞表面。E213Q增加了内在脱敏速率，但钙对这些速率以及对电流幅度降低的相对作用与野生型受体相似。E215Q突变体受体的电流反应和钙介导的效应与野生型无差异。D204N和V219L突变体无功能。钙不可渗透突变体（E277A/S297R）显示，随着钙增加，峰值幅度或动力学无变化。我们的结果表明，残基D204、E218和V219参与受体组装、结构和/或向质膜的转运，我们推测这可能依赖于结合钙的稳定作用。E213、E215、D204和V219可能有助于形成一个钙结合位点，该位点负责钙介导的对配体结合的效应。然而，5-HT3电流钙依赖性调节的主要位点位于离子通道或细胞内部。
