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A triad of residues is functionally transferrable between 5-HT3 serotonin receptors and nicotinic acetylcholine receptors.

ABSTRACT: Cys-loop receptors are pentameric ligand-gated ion channels that facilitate communication within the nervous system. Upon neurotransmitter binding, these receptors undergo an allosteric activation mechanism connecting the binding event to the membrane-spanning channel pore, which expands to conduct ions. Some of the earliest steps in this activation mechanism are carried out by residues proximal to the binding site, the relative positioning of which may reflect functional differences among members of the Cys-loop family of receptors. Herein, we investigated key side-chain interactions near the binding site via mutagenesis and two-electrode voltage-clamp electrophysiology in serotonin-gated 5-HT3A receptors (5-HT3ARs) and nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus laevis oocytes. We found that a triad of residues aligning to Thr-152, Glu-209, and Lys-211 in the 5-HT3AR can be exchanged between the homomeric 5-HT3AR and the muscle-type nAChR ?-subunit with small functional consequences. Via triple mutant cycle analysis, we demonstrated that this triad forms an interdependent network in the muscle-type nAChR. Furthermore, nAChR-type mutations of the 5-HT3AR affect the affinity of nicotine, a competitive antagonist of 5-HT3ARs, in a cooperative manner. Using mutant cycle analyses between the 5-HT3A triad, loop A residues Asn-101 and Glu-102, ?9 residue Lys-197, and the channel gate at Thr-257, we observed that residues in this region are energetically linked to the channel gate and are particularly sensitive to mutations that introduce a net positive charge. This study expands our understanding of the differences and similarities in the activation mechanisms of Cys-loop receptors.

SUBMITTER: Mosesso R

PROVIDER: S-EPMC5827437 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature

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A triad of residues is functionally transferrable between 5-HT<sub>3</sub> serotonin receptors and nicotinic acetylcholine receptors.

Mosesso Richard R Dougherty Dennis A DA

The Journal of biological chemistry 20180103 8

Cys-loop receptors are pentameric ligand-gated ion channels that facilitate communication within the nervous system. Upon neurotransmitter binding, these receptors undergo an allosteric activation mechanism connecting the binding event to the membrane-spanning channel pore, which expands to conduct ions. Some of the earliest steps in this activation mechanism are carried out by residues proximal to the binding site, the relative positioning of which may reflect functional differences among membe ...[more]

PMID: 29298898

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Project description:Substantial colocalization of functionally independent alpha4 nicotinic acetylcholine receptors and 5-HT(3) serotonin receptors on presynaptic terminals has been observed in brain. The present study was aimed at addressing whether nicotinic acetylcholine receptors and 5-HT(3) serotonin receptors interact on the same presynaptic terminal, suggesting a convergence of cholinergic and serotonergic regulation.Ca(2+) responses in individual, isolated nerve endings purified from rat striatum were measured using confocal imaging.Application of 500 nmol/L nicotine following sustained stimulation with the highly selective 5-HT(3) receptor agonist m-chlorophenylbiguanide at 100 nmol/L resulted in markedly reduced Ca(2+) responses (28% of control) in only those striatal nerve endings that originally responded to m-chlorophenylbiguanide. The cross-regulation developed over several minutes. Presynaptic nerve endings that had not responded to m-chlorophenylbiguanide, indicating that 5-HT(3) receptors were not present, displayed typical responses to nicotine. Application of m-chlorophenylbiguanide following sustained stimulation with nicotine resulted in partially attenuated Ca(2+) responses (49% of control). Application of m-chlorophenylbiguanide following sustained stimulation with m-chlorophenylbiguanide also resulted in a strong attenuation of Ca(2+) responses (12% of control), whereas nicotine-induced Ca(2+) responses following sustained stimulation with nicotine were not significantly different from control.These results indicate that the presynaptic Ca(2+) increases evoked by either 5-HT(3) receptor or nicotinic acetylcholine receptor activation regulate subsequent responses to 5-HT(3) receptor activation, but that only 5-HT(3) receptors cross-regulate subsequent nicotinic acetylcholine receptor-mediated responses. The findings suggest a specific interaction between the two receptor systems in the same striatal nerve terminal, likely involving Ca(2+)-dependent intracellular pathways that regulate these signaling systems at one or more levels.

Project description:Several novel bisbenzylisoquinoline alkaloids (BBIQAs) have recently been isolated from a Matis tribe arrow poison and shown by two-electrode voltage-clamp to inhibit mouse muscle nicotinic acetylcholine receptors (nAChR). Here, using radioligand assay with Aplysia californica AChBP and radioiodinated ?-bungarotoxin ([125I]-?Bgt), we show that BBIQA1, BBIQA2, and d-tubocurarine (d-TC) have similar affinities to nAChR orthosteric site. However, a competition with [125I]-?Bgt for binding to the Torpedo californica muscle-type nAChR revealed that BBIQAs1, 2, and 3 are less potent (IC50s = 26.3, 8.75, and 17.0 ?M) than d-TC (IC50 = 0.39 ?M), while with ?7 nAChR in GH4C1 cells, BBIQA1 was less potent that d-TC (IC50s = 162 ?M and 7.77 ?M, respectively), but BBIQA2 was similar (IC50 = 5.52 ?M). In inhibiting the Ca2+ responses induced by acetylcholine in Neuro2a cells expressing the mouse adult ?1?1?? nAChR or human ?7 nAChR, BBIQAs1 and 2 had similar potencies to d-TC (IC50s in the range 0.75-3.08 ?M). Our data suggest that BBIQA1 and BBIQA2 can inhibit adult muscle ?1?1?? nAChR by both competitive and noncompetitive mechanisms. Further experiments on neuronal ?3?2, ?4?2, and ?9?10 nAChRs, expressed in Xenopus laevis oocytes, showed that similar potencies for BBIQAs1, 2, and d-TC. With ?3?2?2 GABAAR currents were almost completely inhibited by d-TC at a high (100 ?M) concentration, but BBIQAs1 and 2 were less potent (only 40-50% inhibition), whereas in competition with Alexa Fluor 546-?-cobratoxin for binding to ?1?3?2 GABAAR in Neuro2a cells, d-TC and these analogs had comparable affinities. Especially interesting effects of BBIQAs1 and 2 in comparison with d-TC were observed for 5-HT3AR: BBIQA1 and BBIQA2 were 5- and 87-fold less potent than d-TC (IC50 = 22.63 nM). Thus, our results reveal that these BBIQAs differ from d-TC in their potencies towards certain Cys-loop receptors, and we suggest that understanding the reasons behind this might be useful for future drug design.

Dataset Information

A triad of residues is functionally transferrable between 5-HT3 serotonin receptors and nicotinic acetylcholine receptors.

Publications

A triad of residues is functionally transferrable between 5-HT<sub>3</sub> serotonin receptors and nicotinic acetylcholine receptors.

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