Thermal Unfolding of a Mammalian Pentameric Ligand-gated Ion Channel Proceeds at Consecutive, Distinct Steps*

Tol, Menno B.; Deluz, Cédric; Hassaı̈ne, Ghérici; Graff, Alexandra; Stahlberg, Henning; Vogel, Horst

doi:10.1074/jbc.m112.422287

Cited by 22 publications

(15 citation statements)

References 72 publications

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“…Moreover, they highlight the heightened structural sensitivity of ELIC to its surrounding environment compared with GLIC, consistent with the hypothesis that intramembrane aromatic interactions enhance TMD stability. Previous studies have shown that TMD stability impacts the overall stability of a pLGIC (38).…”

Section: Resultsmentioning

confidence: 99%

Intramembrane Aromatic Interactions Influence the Lipid Sensitivities of Pentameric Ligand-gated Ion Channels

Carswell

Sun

Baenziger

2015

Journal of Biological Chemistry

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Background: Channel gating is uncoupled from agonist binding when nicotinic receptors lack activating lipids. Results: Gating is also uncoupled with ELIC, but engineered aromatic interactions at the M4-M1/M3 interface restore coupling. Conclusion:The strength of M4-M1/M3 interactions impacts both coupling and lipid sensitivity. Significance: Variable levels of intramembrane aromatics in eukaryotic homologs likely lead to variable lipid sensitivities.

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Section: Resultsmentioning

confidence: 99%

Intramembrane Aromatic Interactions Influence the Lipid Sensitivities of Pentameric Ligand-gated Ion Channels

Carswell

Sun

Baenziger

2015

Journal of Biological Chemistry

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“…6d, inset) showed that after thermal unfolding, hisPS2 formed SDS-resistant aggregates that did not enter the separation gel. This kind of thermal unfolding-induced aggregation has also been reported for other membrane proteins [67,68].…”

Section: Thermal Stability Of the Secondary And Tertiary Structure Ofmentioning

confidence: 86%

Expression, purification, and preliminary characterization of human presenilin-2

Yang

Kubíček³

et al. 2018

Process Biochemistry

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A B S T R A C TPresenilins (PS1 and PS2) exhibit similar γ-secretase-dependent and −independent functions with subtle variations. In this study, we established a cost-effective process to overexpress and purify full-length human PS2 in sufficient quantities and quality for structural studies. Upon optimization, milligram quantities of homogeneous trimeric hisPS2 were purified, which enabled the preliminary characterization of human hisPS2 zymogen. Far-UV and near-UV CD as well as fluorescence spectroscopy revealed that purified hisPS2 contained the expected secondary structure and was folded into a defined tertiary structure. Thermal stability analysis revealed a T m value of ∼55°C for secondary structure while cholesterol significantly increased the stability. The low melting temperature of ∼34°C for the tertiary structure was able to explain the purity and aggregation problems observed during purification. Additionally, the occurrence of calcium ions induced structural changes to different extents for PS2WT and PS2-D263A/D366A was observed, which is consistent with previous studies.

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“…Remarkable high melting temperatures could be observed, all of them were in the range of 60 to 80°C. The melting temperature of purified Alpo6-eGFP for example is 77 ± 1°C, not only exceeding the melting temperatures of eukaryotic members of the CLR family [27,35,53,57], but also clearly higher than the melting temperature of GLIC, a prokaryotic homologue with a T m of 52°C [58]. However, we have to consider the presence of eGFP in the intracellular loop.…”

Section: Discussionmentioning

confidence: 96%

Functional and Biochemical Characterization of Alvinella Pompejana Cys-Loop Receptor Homologues

Wijckmans

Nys

Debaveye

et al. 2016

Biophysical Journal

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Cys-loop receptors are membrane spanning ligand-gated ion channels involved in fast excitatory and inhibitory neurotransmission. Three-dimensional structures of these ion channels, determined by X-ray crystallography or electron microscopy, have revealed valuable information regarding the molecular mechanisms underlying ligand recognition, channel gating and ion conductance. To extend and validate the current insights, we here present promising candidates for further structural studies. We report the biochemical and functional characterization of Cys-loop receptor homologues identified in the proteome of Alvinella pompejana, an extremophilic, polychaete annelid found in hydrothermal vents at the bottom of the Pacific Ocean. Seven homologues were selected, named Alpo1-7. Five of them, Alpo2-6, were unidentified prior to this study. Two-electrode voltage clamp experiments revealed that wild type Alpo5 and Alpo6, both sharing remarkably high sequence identity with human glycine receptor α subunits, are anion-selective channels that can be activated by glycine, GABA and taurine. Furthermore, upon expression in insect cells fluorescence size-exclusion chromatography experiments indicated that four homologues, Alpo1, Alpo4, Alpo6 and Alpo7, can be extracted out of the membrane by a wide variety of detergents while maintaining their oligomeric state. Finally, large-scale purification efforts of Alpo1, Alpo4 and Alpo6 resulted in milligram amounts of biochemically stable and monodisperse protein. Overall, our results establish the evolutionary conservation of glycine receptors in annelids and pave the way for future structural studies.which depolarizes or hyperpolarizes the postsynaptic cell. Because of their pivotal role in neurotransmission, dysfunction of these receptors is linked to several neurological disorders including Alzheimer's disease [1], epilepsy [2-4], myasthenia gravis [5] and hyperekplexia [6]. Additionally, they are the target of several clinically important drugs like benzodiazepines, general anesthetics, anti-emetics and smoking cessation aids. Due to this high clinical relevance, detailed structural insights about these receptors might assist in unraveling the molecular mechanisms of diseases correlated to CLRs. Moreover, this structural information can lead to novel pathways for the development of more efficient drugs.Over the last decade, several X-ray crystal structures have been solved for this class of ion channels. Crystal structures of Acetylcholine Binding Proteins (AChBPs), water-soluble homologues of the extracellular domain of nicotinic acetylcholine receptors (nAChRs), in complex with a wide variety of known CLR ligands have provided insights concerning the molecular determinants of ligand recognition [7]. On the other hand, structures of the full-length prokaryotic channels ELIC [8] and GLIC [9][10][11] have offered a first glimpse of channel gating and ion conductance. ELIC is derived from the bacterium Erwinia chrysanthemi and can be activated by primary amines, such as GABA [12,13]. S...

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Thermal Unfolding of a Mammalian Pentameric Ligand-gated Ion Channel Proceeds at Consecutive, Distinct Steps*

Cited by 22 publications

References 72 publications

Intramembrane Aromatic Interactions Influence the Lipid Sensitivities of Pentameric Ligand-gated Ion Channels

Intramembrane Aromatic Interactions Influence the Lipid Sensitivities of Pentameric Ligand-gated Ion Channels

Expression, purification, and preliminary characterization of human presenilin-2

Functional and Biochemical Characterization of Alvinella Pompejana Cys-Loop Receptor Homologues

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