Design, synthesis and functional characterization of a pentameric channel protein that mimics the presumed pore structure of the nicotinic cholinergic receptor

Montal, Myrta S.; Iwamoto, Takeo; Tomich, John M.; Montal, Mauricio

doi:10.1016/0014-5793(93)80599-p

Cited by 61 publications

(33 citation statements)

References 35 publications

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“…12°relative to the bilayer normal. Molecular modelling studies 1,12,13 show that this tilt angle is consistent with a left-handed pentameric helix bundle. However, to date modelling studies have been performed in the absence of the complex anisotropic environment provided by a lipid bilayer.…”

Section: Introductionmentioning

confidence: 53%

Structure and dynamics of the pore-lining helix of the nicotinic receptor: MD simulations in water, lipid bilayers, and transbilayer bundles

et al. 2000

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Multiple nanosecond duration molecular dynamics simulations on the pore-lining M2 helix of the nicotinic acetylcholine receptor reveal how its structure and dynamics change as a function of environment. In water, the M2 helix partially unfolds to form a molecular hinge in the vicinity of a central Leu residue that has been implicated in the mechanism of ion channel gating. In a phospholipid bilayer, either as a single transmembrane helix, or as part of a pentameric helix bundle, the M2 helix shows less flexibility, but still exhibits a kink in the vicinity of the central Leu. The single M2 helix tilts relative to the bilayer normal by 12 degrees, in agreement with recent solid state NMR data (Opella et al., Nat Struct Biol 6:374-379, 1999). The pentameric helix bundle, a model for the pore domain of the nicotinic receptor and for channels formed by M2 peptides in a bilayer, is remarkably stable over a 2-ns MD simulation in a bilayer, provided one adjusts the pK(A)s of ionizable residues to their calculated values (when taking their environment into account) before starting the simulation. The resultant transbilayer pore shows fluctuations at either mouth which transiently close the channel. Proteins 2000;39:47-55.

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Section: Introductionmentioning

confidence: 53%

Structure and dynamics of the pore-lining helix of the nicotinic receptor: MD simulations in water, lipid bilayers, and transbilayer bundles

et al. 2000

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“…The gate in the nicotinic channel and other Cys-loop LGICs undoubtedly exists in the M2 helix (75)(76)(77)(78)(79). Much evidence also suggests that the gate is in different conformations in the closed and desensitized states (80,81).…”

Section: Conclusion and Discussionmentioning

confidence: 99%

A gating mechanism proposed from a simulation of a human α7 nicotinic acetylcholine receptor

Law

Henchman

McCammon

2005

Proc. Natl. Acad. Sci. U.S.A.

134

142

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The nicotinic acetylcholine receptor is a well characterized ligandgated ion channel, yet a proper description of the mechanisms involved in gating, opening, closing, ligand binding, and desensitization does not exist. Until recently, atomic-resolution structural information on the protein was limited, but with the production of the x-ray crystal structure of the Lymnea stagnalis acetylcholine binding protein and the EM image of the transmembrane domain of the torpedo electric ray nicotinic channel, we were provided with a window to examine the mechanism by which this channel operates. A 15-ns all-atom simulation of a homology model of the homomeric human ␣7 form of the receptor was conducted in a solvated palmitoyl-2-oleoyl-sn-glycerol-phosphatidylcholine bilayer and examined in detail. The receptor was unliganded. The structure undergoes a twist-to-close motion that correlates movements of the C loop in the ligand binding domain, via the ␤10-strand that connects the two, with the 10°rotation and inward movement of two nonadjacent subunits. The Cys loop appears to act as a stator around which the ␣-helical transmembrane domain can pivot and rotate relative to the rigid ␤-sheet binding domain. The M2-M3 loop may have a role in controlling the extent or kinetics of these relative movements. All of this motion, along with essential dynamics analysis, is suggestive of the direction of larger motions involved in gating of the channel.T he nicotinic acetylcholine receptor is one of the best-studied ligand-gated ion channels (LGICs) (1). It has been found to have a role in a wide range of pathological conditions and diseases including epilepsy (2), schizophrenia (3), Alzheimer's (4), Parkinson's (5), neuromuscular diseases (6), inflammation (7), nicotine addiction (8), and addiction to other drugs such as alcohol (9) and cocaine (10), as well as a role in anesthetic action (11). The nicotinic acetylcholine receptor is a member of a superfamily of pentameric receptors that include the serotonin (excitory), GABA, and glycine (inhibitory) receptors that are all involved in the transmission and modification of electrical signals in excitory cells. The family is known as the Cys-loop LGIC family, because of a conserved pair of linked cysteines in the N-terminal domain of the receptor (12). Within the superfamily, an array of different nicotinic receptor subunits exists (13,14), making different subunit assemblies possible, for different roles, in different cell types. In all of these receptors, the ligand binds at the interface between the ␣ and other subunits of the ligand binding domain (LBD). This event begins a chain reaction of conformational changes within the receptor that transmits the message of ligand binding to the transmembrane domain (TMD), which then opens to allow the passage of ions through the channel. Although the genetics, kinetics, electrophysiology, and many topological aspects are well characterized for the nicotinic receptor (15, 16), the exact nature of the structural rearrangments involved in activ...

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“…The second most common Bcl-2 channel exhibited a conductance of 41 Ϯ 2 pS. Channels with similar conductance have been measured using synthetic peptides that form pentameric helical bundles in membranes (39), raising the possibility that these larger channels may be produced by Bcl-2 trimers or by insertion of additional amphipathic helices from Bcl-2 into the membrane (24). Moreover, the presence of three discrete channel activities with progressively greater conductances (Ϸ20 pS, Ϸ40 pS, and Ϸ90 pS) but occurring with progressively lesser frequency raises the possibility of step-wise oligomerization of Bcl-2 protein molecules in planar bilayers.…”

Section: Discussionmentioning

confidence: 99%

Channel formation by antiapoptotic protein Bcl-2

Schendel

Xie

Montal

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

568

351

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Bcl-2 is the prototypical member of a large family of apoptosis-regulating proteins, consisting of blockers and promoters of cell death. The three-dimensional structure of a Bcl-2 homologue, Bcl-X L , suggests striking similarity to the pore-forming domains of diphtheria toxin and the bacterial colicins, prompting exploration of whether Bcl-2 is capable of forming pores in lipid membranes. Using chloride efflux from KCl-loaded unilamellar lipid vesicles as an assay, purified recombinant Bcl-2 protein exhibited pore-forming activity with properties similar to those of the bacterial toxins, diphtheria toxin, and colicins, i.e., dependence on low pH and acidic lipid membranes. In contrast, a mutant of Bcl-2 lacking the two core hydrophobic ␣-helices (helices 5 and 6), predicted to be required for membrane insertion and channel formation, produced only nonspecific effects. In planar lipid bilayers, where detection of single channels is possible, Bcl-2 formed discrete ion-conducting, cation-selective channels, whereas the Bcl-2 (⌬h5, 6) mutant did not. The most frequent conductance observed (18 ؎ 2 pS in 0.5 M KCl at pH 7.4) is consistent with a four-helix bundle structure arising from Bcl-2 dimers. However, larger channel conductances (41 ؎ 2 pS and 90 ؎ 10 pS) also were detected with progressively lower occurrence, implying the step-wise formation of larger oligomers of Bcl-2 in membranes. These findings thus provide biophysical evidence that Bcl-2 forms channels in lipid membranes, suggesting a novel function for this antiapoptotic protein.

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Design, synthesis and functional characterization of a pentameric channel protein that mimics the presumed pore structure of the nicotinic cholinergic receptor

Cited by 61 publications

References 35 publications

Structure and dynamics of the pore-lining helix of the nicotinic receptor: MD simulations in water, lipid bilayers, and transbilayer bundles

Structure and dynamics of the pore-lining helix of the nicotinic receptor: MD simulations in water, lipid bilayers, and transbilayer bundles

A gating mechanism proposed from a simulation of a human α7 nicotinic acetylcholine receptor

Channel formation by antiapoptotic protein Bcl-2

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