Chicoine Linda M, Suppiramaniam Vishnu, Vaithianathan Thirumalini, Gianutsos Gerald, Bahr Ben A
Department of Pharmaceutical Sciences and the Neurosciences Program, University of Connecticut, Storrs, USA.
J Neurosci Res. 2004 Feb 1;75(3):408-16. doi: 10.1002/jnr.10871.
Previous work found evidence that alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptors interact with and are functionally regulated by the glycosaminoglycan heparin. The present study tested whether dextran species affect ligand binding, channel kinetics, and calcium permeability of AMPA receptors. Dextran sulfate of 500 kDa markedly reduced high affinity [3H]AMPA binding in solubilized hippocampal membranes. In isolated receptors reconstituted in a lipid bilayer, the same dextran sulfate prolonged the lifetime of open states exhibited by AMPA-induced channel fluctuations. The large polysaccharide further changed the single channel kinetics by increasing the open channel probability five- to sixfold. Such modulation of channel activity corresponded with enhanced levels of calcium influx as shown in hippocampal neurons loaded with Fluo3AM dye. With an exposure time of <1 min, AMPA produced a dose-dependent increase in intracellular calcium that was blocked by 6-cyano-7-nitroquinoxaline-2,3-dione disodium (CNQX). Dextran sulfate, at the same concentration range that modified ligand binding (EC50 of 5-10 nM), enhanced the AMPA-induced calcium influx by as much as 60%. The enhanced influx was blocked by CNQX, although unchanged by the N-methyl-D-aspartate (NMDA) receptor antagonist AP5. Confocal microscopy showed that the increase in calcium occurred in neuronal cell bodies and their processes. Interestingly, smaller 5-8-kDa dextran sulfate and a non-sulfated dextran of 500 kDa had little or no effect on the binding, channel, and calcium permeability properties. Together, these findings suggest that synaptic polysaccharide species modulate hippocampal AMPA receptors in a sulfate- and size-dependent manner.
先前的研究发现，α-氨基-3-羟基-5-甲基异恶唑-4-丙酸（AMPA）型谷氨酸受体与糖胺聚糖肝素相互作用并受到其功能调节。本研究测试了不同种类的葡聚糖是否会影响AMPA受体的配体结合、通道动力学和钙通透性。500 kDa的硫酸葡聚糖显著降低了溶解的海马体膜中高亲和力的[3H]AMPA结合。在脂质双分子层中重组的分离受体中，相同的硫酸葡聚糖延长了AMPA诱导的通道波动所表现出的开放状态的持续时间。这种大的多糖通过将开放通道概率提高五到六倍，进一步改变了单通道动力学。通道活性的这种调节与钙内流水平的提高相对应，如在加载了Fluo3AM染料的海马神经元中所示。暴露时间<1分钟时，AMPA使细胞内钙呈剂量依赖性增加，这被6-氰基-7-硝基喹喔啉-2,3-二酮二钠（CNQX）阻断。在改变配体结合的相同浓度范围内（EC50为5-10 nM），硫酸葡聚糖使AMPA诱导的钙内流增加了多达60%。增强的内流被CNQX阻断，尽管N-甲基-D-天冬氨酸（NMDA）受体拮抗剂AP5对其没有影响。共聚焦显微镜显示，钙的增加发生在神经元细胞体及其突起中。有趣的是，较小的5-8 kDa硫酸葡聚糖和500 kDa的非硫酸化葡聚糖对结合、通道和钙通透性特性几乎没有影响。总之，这些发现表明，突触多糖种类以硫酸化和大小依赖的方式调节海马AMPA受体。
