An Internally Modulated, Thermostable, pH-sensitive Cys Loop Receptor from the Hydrothermal Vent Worm Alvinella pompejana

Juneja, Puneet; Horlacher, Reinhold; Bertrand, Daniel; Krause, Ryoko; Marger, Fabrice; Welte, Wolfram

doi:10.1074/jbc.m113.525576

Cited by 7 publications

(15 citation statements)

References 46 publications

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“…Moreover, we could confirm the activation of Alpo7-wt by elevated proton concentrations as previously described by Juneja, P. et al [27]. For Alpo1-wt, Alpo2-wt and Alpo4-wt none of the tested substances (Table 1) evoked channel activation.…”

Section: Resultssupporting

confidence: 89%

“…to our nomenclature, Alv-a9 corresponds to Alpo1 and Alv-a1-pHCl corresponds to Alpo7. For Alv-a9 no activating ligand was found, whereas Alv-a1-pHCl is pH-activated [27], similar to ion channels found in other invertebrates [28]. In our study, we extended on these observations and identified five additional Alvinella pompejana CLR homologues.…”

Section: Introductionsupporting

confidence: 80%

“…The protein sequences have been deposited in the protein database of The Max Planck Institute for Developmental Biology (http://jekelylab.tuebingen.mpg.de/) under accession numbers P74014 (Alpo1), P64117 (Alpo2), P61179 (Alpo3), P60768 (Alpo4), P65805 (Alpo5), P52796 (Alpo6) and P60838 (Alpo7). Alpo1 is equal to Alv-a9 and Alpo7 is equal to Alv-a1-pHCl as reported by Juneja, P. et al [27]. The cladogram provides a visual representation of the relationship between Alpo CLR homologues, known human CLR subunits and GluCl (Fig 2A).…”

Section: Resultssupporting

confidence: 57%

“…Once suitable solubilization conditions were identified, large-scale purifications were performed for Alpo1, Alpo4 and Alpo6. For Alpo7 no purification efforts were undertaken given the recent publication of preliminary biochemical data describing the expression and purification of this protein [27]. The overall purification strategy was similar for all three constructs and consisted of a 2-step purification protocol composed of immobilized metal affinity chromatography (IMAC) and SEC.…”

Section: Resultsmentioning

confidence: 99%

“…It has been shown that proteins from extremophilic organisms display superior stability under laboratory conditions, making them ideal candidates for structural studies [23][24][25][26]. The potential application of Alvinella pompejana CLRs in structural studies was previously recognized by Juneja, P. et al [27]. They identified two CLR homologues: Alv-a9 and Alv-a1-pHCl.…”

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confidence: 99%

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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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Section: Resultssupporting

confidence: 89%

Section: Introductionsupporting

confidence: 80%

Section: Resultssupporting

confidence: 57%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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