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Comput Struct Biotechnol J
2025 Mar 18;27:1148-1157. doi: 10.1016/j.csbj.2025.03.028.
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Molecular investigation of ergot alkaloid ergotamine's modulatory effects on glycine receptors expressed in Xenopus oocytes.
Eom S
,
Pyeon M
,
Moon M
,
Yun J
,
Yang J
,
Yun J
,
Yeom HD
,
Lee MH
,
Lee G
,
Lee JH
.
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The relationship between oxidative stress and glycine receptors is complex, involving multiple mechanisms through which reactive oxygen species can modify glycine receptor function. Understanding these interactions is essential for developing therapeutic strategies to mitigate the effects of oxidative stress on inhibitory neurotransmission in various neurological disorders. Inhibitory glycine receptors play a critical role in regulating the final grand postsynaptic potential by attenuating excitatory postsynaptic potentials through inhibitory postsynaptic potentials in postsynaptic neurons. This is particularly important in rapid signal transmission systems, where it determines whether the grand postsynaptic potential exceeds the activation threshold. Glycine receptors are known to be expressed not only in the spinal cord and brainstem but also in the hippocampus, as evidenced by studies conducted over the past decade. Interestingly, these regions share a common cellular architecture, predominantly composed of pyramidal neurons. In hippocampal pyramidal neurons, glycine receptors contribute to the regulation of synapse formation and plasticity, and they are crucial in motor neuron control within the pyramidal tract. However, there is limited research on glycine receptor antagonism, which is necessary to fully understand their biological functions in these regions. We conducted a comprehensive molecular-level analysis of the pharmacological properties of glycine receptors, examined their interaction mechanisms through electrophysiological studies, and identified binding sites using structural modeling and site-directed mutagenesis. Our findings suggest that ergotamine may serve as a promising antioxidant candidate to address issues associated with excessive or prolonged inhibitory postsynaptic potentials, offering a potential new therapeutic pathway.
Fig. 1. Schematic Overview of Ergotamine Regulation through Glycine Receptors in Situations of Prolonged or Excessive IPSP. The left-central nervous system (CNS) diagram illustrates the expression sites of glycine receptors in the hippocampus's pyramidal neurons and pyramidal tract. While glycine receptors are conventionally known to be expressed in the spinal cord and brainstem, this image highlights their expression in the hippocampus and the pyramidal tract (encompassing the corticospinal tract and corticobulbar tract). These regions share a commonality in the presence of pyramidal neurons, and the diagram on the right elucidates the mechanism of glycine receptors expressed in pyramidal neurons, modulating signal transmission. Ultimately, the failure to form action potentials may lead to functional impairment. In situations characterized by persistent or excessive Inhibitory Postsynaptic Potentials (IPSP), ergotamine demonstrates the potential to improve functional deficits.
Fig. 2. Chemical structure and inhibitory effects of ergotamine on glycine receptor α1 in Xenopus oocytes. (A) This is the secondary structure of ergotamine. (B) Running two-electrode voltage-clamp recording in oocytes expressing the glycine receptor α1, glycine (100 µM) evoked a large inward current (IGly) at a holding potential of −80 mV for the indicated times. Ergotamine (100 µM) were supplemented into recording solution for 1 min (bar in figure). The IGly current treated by co-application of ergotamine (EG) and glycine (Gly) was inhibited. In the presence of ergotamine (10 µM and 30 µM) with the consistence concentration of glycine (100 µM), the inhibition effect was showed reversibly. Tracings are representative of six to eight separate oocytes from four different frogs. (C) Current–voltage relationships of IGly on ergotamine-stimulated inhibition in oocytes. The representative current–voltage relationship was obtained using 1 s-duration voltage ramps from −100 to + 60 mV at a holding potential of −80 mV. Voltage ramps were applied before and after the application of 100 µM glycine in the presence or absence of ergotamine (10 and 100 µM). Each point represents the mean ± S.E.M. (n = 6–8 oocytes from four different frogs). (D) Competition test between glycine and ergotamine for binding with glycine receptors α1. Exposing with the absence (■ Con) or presence of ergotamine (● 10 µM and ▲100 µM) for 1 min and then evoked with glycine from 0.3 to 100 µM, glycine-induced inward currents were voltage clamped at a holding potential of −80 mV for indicated times. Each point represents the mean ± S.E.M. (n = 6–8/group). The additional Imax, IC50, and Hill coefficient values are summarized in Table 1.
Fig. 3. Computational molecular modeling of ergotamine docked to glycine α1 channel. A and C Side views of the docked ergotamine in complex with glycine α1 channel. B and D Top views of docking model. The PDB 3JAE model, which is a glycine-bound state, was used and it was found that ergotamine binds to the upper (N-terminal) of the ECD.
Fig. 4. Virtual docking of ergotamine to wild-type glycine α1 receptor and mutations. (A and B) Binding pocket and docking results of ergotamine and glycine receptor α1 channel. (A) Ergotamine located in binding pocket in extracellular area. (B) 2D schematic presentation of the predicted binding mode of ergotamine in the ligand binding pocket. Ligands and important residues are shown. (C and D) Pose view analysis and binding interface of ergotamine of wild-type (C) and mutant channels (D), whose mutations can disturb the interaction with ergotamine at varying degrees. The dotted lines show the distance between atoms as Å unit.
Fig. 5. Effects of ergotamine on IGly in wild-type and various mutant glycine receptor α1 channels. (A-C) Representative traces on IGly stimulated by glycine 100 µM with and without ergotamine (30 and 100 μM) in α1 F124A (A), α1 D130A (B) and α1 F124A and D130A (C). Traces are representative of six to eight separate oocytes from four different frogs. (D) Concentration-dependent curves of ergotamine on glycine–evoked inward current from oocytes expressing wild-type and various mutant glycine receptor α1 channels. Ergotamine reduced IGly in a concentration-dependent manner in wild-type receptors. The effect of ergotamine on IGly was presented following the rank order of potency: α1 wild >> α1 F124A > α1 D130A > α1 F124A and D130A.
