Identification of Novel Functionally Important Aromatic Residue Interactions in the Extracellular Domain of the Glycine Receptor

Tang, Bijun; Devenish, Steven O.; Lummis, Sarah C. R.

doi:10.1021/acs.biochem.8b00425

Cited by 5 publications

(7 citation statements)

References 26 publications

(39 reference statements)

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“…Studies have indicated several possible routes by which M4 residues could affect channel function, and 2 of these have been more extensively investigated. The first is through direct interaction with the M1/M3 helices, a route which is supported by a range of studies demonstrating, in particular, the importance of aromatic interactions of M4 with M1/M3. ,,− In addition, specific links for some residues have been identified, as in the case of the C418W mutation in the Torpedo nAChR, which couples energetically with residues in M1 to stabilize the open state, decreasing EC 50 . The second route involves the interaction of the tip of M4 with the Cys-loop.…”

Section: Discussionmentioning

confidence: 94%

M4, the Outermost Helix, is Extensively Involved in Opening of the α4β2 nACh Receptor

Mesoy

Lummis

2020

ACS Chem. Neurosci.

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Nicotinic acetylcholine receptors (nAChR) are the archetypal members of the pentameric ligand-gated ion channel (pLGIC) family, an important class of cell signaling proteins. In all members of this family, each of the five subunits has four transmembrane α-helices (M1–M4), with M2 lining the pore, then M1 and M3, and with M4 outermost and adjacent to the membrane lipids. Despite its remote location, M4 contributes both to receptor assembly and gating in pLGICs where it has been examined. This study probes the role of M4 residues in the α4β2 nAChR using site-directed mutagenesis to individually mutate each residue to alanine, followed by expression in HEK293 cells and then characterization using membrane potential sensitive dye and radioligand binding. Two of the resulting mutant receptors showed altered EC50s, while 13 were nonfunctional, although coexpression with the chaperones RIC3 and nAChO resulted in 4 of these responding to agonist. Of the remaining 9, radioligand binding with epibatidine showed that 8 were expressed, suggesting these residues may play a role in channel opening. These data differ from similar studies in other pLGIC, where few or no Ala mutants in M4 ablate function, and they suggest that the α4β2 nAChR M4 may play a more significant role than in related receptors.

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Section: Discussionmentioning

confidence: 94%

M4, the Outermost Helix, is Extensively Involved in Opening of the α4β2 nACh Receptor

Mesoy

Lummis

2020

ACS Chem. Neurosci.

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“…In the context of LGICs, this means that mutations in the ECD, including those at or near the subunit interface, tend to result in increased EC 50 values for ligands binding at the orthosteric binding site or more generally disrupt channel function. This has been observed for a variety of LGICs, such as GlyR α1, nAChR α7 and iGluRs (Braun et al, 2016; Iacobucci et al, 2021; Tang et al, 2018; Tang & Lummis, 2018; Weston et al, 2006). In fact, even mutational scans in the ECD of a close cousin of the P2X2R, the P2X1R, have established that the vast majority of mutations result in increased EC 50 values (Ennion et al, 2000; Roberts & Evans, 2004, 2006).…”

Section: Discussionmentioning

confidence: 59%

P2X2 receptor subunit interfaces are missense variant hotspots where mutations tend to increase apparent ATP affinity

Gasparri

Bielickaite

Poulsen

et al. 2021

Preprint

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P2X receptors (P2XRs) are trimeric ligand-gated ion channels that open a cation-selective pore in response to ATP binding to their large extracellular domain (ECD). The seven known P2XR subtypes typically assemble as homo- or heterotrimeric complexes and they contribute to numerous physiological functions, including nociception, inflammation and hearing. Both the overall structure of P2XRs and the details of how ATP is coordinated at the subunit interface are well established. By contrast, little is known about how inter-subunit interactions in the ECD contribute to channel function. Here we investigate both single and double mutants at the subunit interface of rP2X2Rs using electrophysiological and biochemical approaches. Our data demonstrate that the vast majority of mutations that disrupt putative inter-subunit interactions result in channels with higher apparent ATP affinity and that double mutants at the subunit interface show significant energetic coupling, especially if the mutations are located in close proximity. Overall, we show that inter-subunit interactions, as well as possibly interactions in other parts of the receptor, stabilize WT rP2X2Rs in the closed state. This suggests that, unlike other ligand-gated ion channels, P2X2 receptors have not evolved for an intrinsically low threshold for activation, possibly to allow for additional modulation or as a cellular protection mechanism against overstimulation.

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“…In the context of ligand‐gated ion channels, this means that mutations in the extracellular domain, including those at or near the subunit interface, tend to result in increased EC 50 values for ligands binding at the orthosteric binding site or more generally disrupt channel function. This has been observed for a variety of ligand‐gated ion channels, such as glycine receptor α1 subunit, nicotinic acetylcholine receptor α7 subunit and ionotropic glutamate receptors (Braun et al, 2016; Iacobucci et al, 2021; Tang et al, 2018; Tang & Lummis, 2018; Weston et al, 2006). In fact, even mutational scans in the extracellular domain of a close cousin of the P2X2 receptor, the P2X1 receptor, have established that the vast majority of mutations result in increased EC 50 values (Ennion et al, 2000; Roberts & Evans, 2004, 2006).…”

Section: Discussionmentioning

confidence: 79%

P2X2 receptor subunit interfaces are missense variant hotspots, where mutations tend to increase apparent ATP affinity

Gasparri

Sarkar

Bielickaite

et al. 2022

British J Pharmacology

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Background and Purpose P2X receptors are trimeric ligand‐gated ion channels that open a cation‐selective pore in response to ATP binding to their large extracellular domain. The seven known P2X subtypes can assemble as homotrimeric or heterotrimeric complexes and contribute to numerous physiological functions, including nociception, inflammation and hearing. The overall structure of P2X receptors is well established, but little is known about the range and prevalence of human genetic variations and the functional implications of specific domains. Experimental Approach Here, we examine the impact of P2X2 receptor inter‐subunit interface missense variants identified in the human population or by structural predictions. We test both single and double mutants through electrophysiological and biochemical approaches. Key Results We demonstrate that predicted extracellular domain inter‐subunit interfaces display a higher‐than‐expected density of missense variations and that the majority of mutations that disrupt putative inter‐subunit interactions result in channels with higher apparent ATP affinity. Lastly, we show that double mutants at the subunit interface show significant energetic coupling, especially if located in close proximity. Conclusion and Implications We provide the first structural mapping of the mutational distribution across the human population in a ligand‐gated ion channel and show that the density of missense mutations is constrained between protein domains, indicating evolutionary selection at the domain level. Our data may indicate that, unlike other ligand‐gated ion channels, P2X2 receptors have evolved an intrinsically high threshold for activation, possibly to allow for additional modulation or as a cellular protection mechanism against overstimulation.

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Identification of Novel Functionally Important Aromatic Residue Interactions in the Extracellular Domain of the Glycine Receptor

Cited by 5 publications

References 26 publications

M4, the Outermost Helix, is Extensively Involved in Opening of the α4β2 nACh Receptor

M4, the Outermost Helix, is Extensively Involved in Opening of the α4β2 nACh Receptor

P2X2 receptor subunit interfaces are missense variant hotspots where mutations tend to increase apparent ATP affinity

P2X2 receptor subunit interfaces are missense variant hotspots, where mutations tend to increase apparent ATP affinity

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