The Interface between Extracellular and Transmembrane Domains of Homomeric Cys-Loop Receptors Governs Open-Channel Lifetime and Rate of Desensitization

Bouzat, Cecilia; Bartos, Mariana; Corradi, Jeremías; Sine, Steven M.

doi:10.1523/jneurosci.0448-08.2008

Cited by 120 publications

(252 citation statements)

References 48 publications

(83 reference statements)

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“…The observed kinetic parameters are compatible with those reported for α7 AChRs transiently expressed in oocytes [19] and in BOSC 23 cells [20] . A recent report [21] describing the functional properties of α7 AChRs heterologously expressed in CHO cells failed to observe receptor-mediated Ca 2+ fluxes when nicotinic agonists were used to activate the AChR in the absence of the positive allosteric modulator PNU-120596 or after pretreatment of cells with the tyrosine kinase inhibitor genistein.…”

Section: Discussionsupporting

confidence: 88%

“…Few studies have reported single-channel currents in oocytes from wild-type and mutant α7 AChRs [18,19] . Recently, the human α7 AChR and the Ric-3 protein were transiently transfected in BOSC 23 cells [20] . Furthermore, Roncarati et al [21] produced a CHO-derived cell line that stably expresses α7 AChRs with the aid of the Ric-3 chaperone.…”

Section: Introductionmentioning

confidence: 99%

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Ric-3 chaperone-mediated stable cell-surface expression of the neuronal α7 nicotinic acetylcholine receptor in mammalian cells

2009

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Aim: Studies of the α7-type neuronal nicotinic acetylcholine receptor (AChR), one of the receptor forms involved in many physiologically relevant processes in the central nervous system, have been hampered by the inability of this homomeric protein to assemble in most heterologous expression systems. In a recent study, it was shown that the chaperone Ric-3 is necessary for the maturation and functional expression of α7-type AChRs [1] . The current work aims at obtaining and characterizing a cell line with high functional expression of the human α7 AChR. Methods: Ric-3 cDNA was incorporated into SHE-P1-hα7 cells expressing the α7-type AChR. Functional studies were undertaken using single-channel patch-clamp recordings. Equilibrium and kinetic [ 125 I]α-bungarotoxin binding assays, as well as fluorescence microscopy using fluorescent α-bungarotoxin, anti-α7 antibody, and GFP-α7 were performed on the new clone. Results: The human α7-type AChR was stably expressed in a new cell line, which we coined SHE-P1-hα7-Ric-3, by co-expression of the chaperone Ric-3. Cell-surface AChRs exhibited [ 125 I]αBTX saturable binding with an apparent K D of about 55 nmol/L. Fluorescence microscopy revealed dispersed and micro-clustered AChR aggregates at the surface of SHE-P1-hα7-Ric-3 cells. Larger micron-sized clusters were observed in the absence of receptor-clustering proteins or upon aggregation with anti-α7 antibodies. In contrast, chaperone-less SHE-P1-hα7 cells expressed only intracellular α7 AChRs and failed to produce detectable single-channel currents. Conclusion:The production of a stable and functional cell line of neuroepithelial lineage with robust cell-surface expression of neuronal α7-type AChR, as reported here, constitutes an important advance in the study of homomeric receptors in mammalian cells.

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Section: Discussionsupporting

confidence: 88%

Section: Introductionmentioning

confidence: 99%

Ric-3 chaperone-mediated stable cell-surface expression of the neuronal α7 nicotinic acetylcholine receptor in mammalian cells

2009

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“…A study using a chimeric receptor with AChBP and channel domains of 5HT 3 R revealed that the coupling interface requires matching of three loops (loop 2, loop 7, and loop 9) from the amino-terminal domain and one loop (M2-M3 linker) from the transmembrane domain for the receptor to be functional [43] . Additionally, a region in pre-M1 and the beginning of M1 that covalently links the amino-terminal domain to the transmembrane domain, is also important in channel gating ( Figure 4C) [44] .…”

Section: Functional Domains Of the Cys-loop Receptorsmentioning

confidence: 99%

“…Coupling between amino-terminal domain and the gating machinery As mentioned above, coupling between binding and gating domains requires matching of three loops (loop 2, loop 7/cys-loop, and loop 9/loop F) from the amino-terminal domain and one loop (M2-M3 linker) from the transmembrane domain [43] and pre-M1 and the beginning of M1 [44] . Since M2-M3 linker is not conserved across the entire cys-loop receptor family, detailed coupling residues could vary depending on subfamilies, although the general mechanism is likely to be conserved.…”

Section: Activation Mechanismmentioning

confidence: 99%

Allosteric activation mechanism of the cys-loop receptors

et al. 2009

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Binding of a neurotransmitter to its ionotropic receptor opens a distantly located ion channel, a process termed allosteric activation. Here we review recent advances in the molecular mechanism by which the cys-loop receptors are activated with emphasis on the best studied nicotinic acetylcholine receptors (nAChRs). With a combination of affinity labeling, mutagenesis, electrophysiology, kinetic modeling, electron microscopy (EM), and crystal structure analysis, the allosteric activation mechanism is emerging. Specifically, the binding domain and gating domain are interconnected by an allosteric activation network. Agonist binding induces conformational changes, resulting in the rotation of a β sheet of aminoterminal domain and outward movement of loop 2, loop F, and cys-loop, which are coupled to the M2-M3 linker to pull the channel to open. However, there are still some controversies about the movement of the channel-lining domain M2. Nine angstrom resolution EM structure of a nAChR imaged in the open state suggests that channel opening is the result of rotation of the M2 domain. In contrast, recent crystal structures of bacterial homologues of the cys-loop receptor family in apparently open state have implied an M2 tilting model with pore dilation and quaternary twist of the whole pentameric receptor. An elegant study of the nAChR using protonation scanning of M2 domain supports a similar pore dilation activation mechanism with minimal rotation of M2. This remains to be validated with other approaches including high resolution structure determination of the mammalian cys-loop receptors in the open state.

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“…[6][7][8][9][10][11] Indeed, engineered chimeric LGICs in which AChBP replaces the wild-type extracellular domain (ECD) are functional (n.b. mutation of several key residues in the transmembrane interface are required for functional chimeras), [12][13][14][15][16] indicating AChBP and the ECD of LGICs may share similar conformational changes upon ligand binding.…”

Section: Introductionmentioning

confidence: 99%

Enhanced meta‐analysis of acetylcholine binding protein structures reveals conformational signatures of agonism in nicotinic receptors

Stober

Abrams

2012

Protein Science

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The soluble acetylcholine binding protein (AChBP) is the default structural proxy for pentameric ligand-gated ion channels (LGICs). Unfortunately, it is difficult to recognize conformational signatures of LGIC agonism and antagonism within the large set of AChBP crystal structures in both apo and ligand-bound states, primarily because AChBP conformations in this set are nearly superimposable (root mean square deviation < 1.5 Å ). We have undertaken a systematic, alignment-free approach to elucidate conformational differences displayed by AChBP that cleanly differentiate apo/antagonist-bound from agonist-bound states. Our approach uses statistical inference based on both crystallographic states and conformations sampled during long molecular dynamics simulations to select important inter-C a distances and map their collective values onto functional states. We observe that binding of (nAChR) agonists to AChBP elicits clockwise rotation of the inner b-sheet with respect to the outer b-sheet, causing tilting of the cys-loop away from the five-fold axis, in a manner quite similar to that speculated for a-subunits of the heteromeric nAChR structure (Unwin, J Mol Biol 2005;346:967), making this motion potentially important in transmission of the gating signal to the transmembrane domain of a LGIC. The method is also successful at discriminating partial from full agonists and supports the hypothesis that a particularly controversial ligand, lobeline, is in fact an LGIC antagonist.

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The Interface between Extracellular and Transmembrane Domains of Homomeric Cys-Loop Receptors Governs Open-Channel Lifetime and Rate of Desensitization

Cited by 120 publications

References 48 publications

Ric-3 chaperone-mediated stable cell-surface expression of the neuronal α7 nicotinic acetylcholine receptor in mammalian cells

Ric-3 chaperone-mediated stable cell-surface expression of the neuronal α7 nicotinic acetylcholine receptor in mammalian cells

Allosteric activation mechanism of the cys-loop receptors

Enhanced meta‐analysis of acetylcholine binding protein structures reveals conformational signatures of agonism in nicotinic receptors

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