Rings of Charge within the Extracellular Vestibule Influence Ion Permeation of the 5-HT3A Receptor
Rings of Charge within the Extracellular Vestibule Influence Ion Permeation of the 5-HT3A Receptor
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DOI：
10.1074/jbc.m111.219618
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发表时间：
2011-05-06
影响因子：
4.8
通讯作者：
Peters, John A.
中科院分区：
文献类型：
--
作者：
Livesey, Matthew R.;Cooper, Michelle A.;Peters, John A.
真核五聚体配体门控离子通道内的单通道电导（γ）和离子选择性的决定因素传统上归因于第二跨膜结构域内的氨基酸残基及其组成亚基的侧翼序列。然而，最近的证据表明，γ是另外控制的胞内和胞外结构域内的残基。我们研究了高电导的人突变型5-羟色胺3A（5-HT（3）A）受体（5-HT（3）A（QDA））细胞外前庭内两个阴离子残基（Asp（113）和Asp（127））对γ的影响、细胞外Ca ~（2+）对后者的调节以及Ca ~（2+）相对于Cs ~+的渗透性（P-Ca/P-Cs）。突变中和（Asp -> Asn），或逆转（Asp -> Lys），在113位点的电荷减少内向γ分别为46%和58%，但外向电流不受影响。D127 N突变减少了82%的内向γ，也抑制了外向电流，而D127 K突变导致可观察到的单通道电流的损失。上述突变，除了D127 K，这是无法评估，改善抑制内向单通道电流的细胞外Ca 2+。先前报道的5-HT（3）A（QDA）构建体的P-Ca/P-Cs为3.8，通过D127 N和D127 K突变分别降低至0.13和0.06，D113 N（1.04）和D113 K（0.60）取代引起的影响较小，但明显显著。一价阳离子和阴离子（P-Na/P-Cl）之间的电荷选择性不受任何突变的影响。这些数据确定了5-HT（3）A受体细胞外前庭中的两个关键残基，它们显著影响γ，P-Ca/P-Cs，以及Ca 2+对γ的抑制。
The determinants of single channel conductance (gamma) and ion selectivity within eukaryotic pentameric ligand-gated ion channels have traditionally been ascribed to amino acid residues within the second transmembrane domain and flanking sequences of their component subunits. However, recent evidence suggests that gamma is additionally controlled by residues within the intracellular and extracellular domains. We examined the influence of two anionic residues (Asp(113) and Asp(127)) within the extracellular vestibule of a high conductance human mutant 5-hydroxytryptamine type-3A (5-HT(3)A) receptor (5-HT(3)A(QDA)) upon gamma, modulation of the latter by extracellular Ca2+, and the permeability of Ca2+ with respect to Cs+ (P-Ca/P-Cs). Mutations neutralizing (Asp -> Asn), or reversing (Asp -> Lys), charge at the 113 locus decreased inward gamma by 46 and 58%, respectively, but outward currents were unaffected. The D127N mutation decreased inward gamma by 82% and also suppressed outward currents, whereas the D127K mutation caused loss of observable single channel currents. The forgoing mutations, except for D127K, which could not be evaluated, ameliorated suppression of inwardly directed single channel currents by extracellular Ca2+. The P-Ca/P-Cs of 3.8 previously reported for the 5-HT(3)A(QDA) construct was reduced to 0.13 and 0.06 by the D127N and D127K mutations, respectively, with lesser, but clearly significant, effects caused by the D113N (1.04) and D113K (0.60) substitutions. Charge selectivity between monovalent cations and anions (P-Na/P-Cl) was unaffected by any of the mutations examined. The data identify two key residues in the extracellular vestibule of the 5-HT(3)A receptor that markedly influence gamma, P-Ca/P-Cs, and additionally the suppression of gamma by Ca2+.