Haghighi A P, Cooper E
Department of Physiology, McGill University, Montréal, Québec, Canada H3G 1Y6.
J Neurosci. 2000 Jan 15;20(2):529-41. doi: 10.1523/JNEUROSCI.20-02-00529.2000.
Many nicotinic acetylcholine receptors (nAChRs) expressed by central neurons are located at presynaptic nerve terminals. These receptors have high calcium permeability and exhibit strong inward rectification, two important physiological features that enable them to facilitate transmitter release. Previously, we showed that intracellular polyamines act as gating molecules to block neuronal nAChRs in a voltage-dependent manner, leading to inward rectification. Our goal is to identify the structural determinants that underlie the block by intracellular polyamines and govern calcium permeability of neuronal nAChRs. We hypothesize that two ring-like collections of negatively charged amino acids (cytoplasmic and intermediate rings) near the intracellular mouth of the pore mediate the interaction with intracellular polyamines and also influence calcium permeability. Using site-directed mutagenesis and electrophysiology on alpha(4)beta(2) and alpha(3)beta(4) receptors expressed in Xenopus oocytes, we observed that removing the five negative charges of the cytoplasmic ring had little effect on either inward rectification or calcium permeability. However, partial removal of negative charges of the intermediate ring diminished the high-affinity, voltage-dependent interaction between intracellular polyamines and the receptor, abolishing inward rectification. In addition, these nonrectifying mutant receptors showed a drastic reduction in calcium permeability. Our results indicate that the negatively charged glutamic acid residues at the intermediate ring form both a high-affinity binding site for intracellular polyamines and a selectivity filter for inflowing calcium ions; that is, a common site links inward rectification and calcium permeability of neuronal nAChRs. Physiologically, this molecular mechanism provides insight into how presynaptic nAChRs act to influence transmitter release.
许多由中枢神经元表达的烟碱型乙酰胆碱受体（nAChRs）位于突触前神经末梢。这些受体具有高钙通透性并表现出强烈的内向整流特性，这两个重要的生理特征使它们能够促进神经递质释放。此前，我们发现细胞内多胺作为门控分子以电压依赖性方式阻断神经元nAChRs，导致内向整流。我们的目标是确定细胞内多胺阻断作用的结构决定因素，并调控神经元nAChRs的钙通透性。我们假设，在孔道细胞内口附近的两个带负电荷氨基酸的环状集合（胞质环和中间环）介导与细胞内多胺的相互作用，并影响钙通透性。通过对非洲爪蟾卵母细胞中表达的α(4)β(2)和α(3)β(4)受体进行定点诱变和电生理学研究，我们观察到去除胞质环的五个负电荷对内向整流或钙通透性几乎没有影响。然而，部分去除中间环的负电荷会减弱细胞内多胺与受体之间的高亲和力、电压依赖性相互作用，消除内向整流。此外，这些非整流突变受体的钙通透性大幅降低。我们的结果表明，中间环带负电荷的谷氨酸残基既形成了细胞内多胺的高亲和力结合位点，又形成了流入钙离子的选择性过滤器；也就是说，一个共同位点将神经元nAChRs的内向整流和钙通透性联系起来。在生理上，这种分子机制为突触前nAChRs如何影响神经递质释放提供了深入了解。
