Β-Structure in the Membrane-Spanning Part of the Nicotinic Acetylcholine Receptor (Or How Helical Are Transmembrane Helices?) (original) (raw)
Related papers
The EMBO Journal, 1994
2Corresponding author Communicated by B.Sakmann The transmembrane domain of the nicotinic acetylcholine receptor (nAChR) from Torpedo caifornica electric tissue contains both a-helical and (structures. The secondary structure was investigated by Fourier transform infrared (FTIR) spectroscopy after the extramembrane moieties of the protein from the extracellular and intracellular sides of the membrane were removed by proteolysis using proteinase K. The secondary structure composition of this membrane structure was: a-helical 50%, (structure and turns 40%, random 10%. The ahelices are shown to be oriented with respect to the membrane plane in a way allowing them to span the membrane, while no unidirectional structure for the (structures was observed. These findings contradict previous secondary structure models based on hydropathy plots alone.
2010
Aromatic-aromatic interactions are a prominent feature of the crystal structure of ELIC [Protein Data Bank (PDB) code 2VL0], a bacterial member of the nicotinic receptor superfamily of ion channels where five pore-facing phenylalanines come together to form a structure akin to a narrow iris that occludes the transmembrane pore. To identify the functional state of the channel that this structure represents, we engineered phenylalanines at various pore-facing positions of the muscle acetylcholine (ACh) receptor (one position at a time), including the position that aligns with the native phenylalanine 246 of ELIC, and assessed the consequences of such mutations using electrophysiological and toxin-binding assays. From our experiments, we conclude that the interaction among the side chains of pore-facing phenylalanines, rather than the accumulation of their independent effects, leads to the formation of a nonconductive conformation that is unresponsive to the application of ACh and is highly stable even in the absence of ligand. Moreover, electrophysiological recordings from a GLIC channel (another bacterial member of the superfamily) engineered to have a ring of phenylalanines at the corresponding pore-facing position suggest that this novel refractory state is distinct from the well-known desensitized state. It seems reasonable to propose then that it is in this peculiar nonconductive conformation that the ELIC channel was crystallized. It seems also reasonable to propose that, in the absence of rings of porefacing aromatic side chains, such stable conformation may never be attained by the ACh receptor. Incidentally, we also noticed that the response of the proton-gated wild-type GLIC channel to a fast change in pH from pH 7.4 to pH 4.5 (on the extracellular side) is only transient, with the evoked current fading completely in a matter of seconds. This raises the possibility that the crystal structures of GLIC obtained at pH 4.0 (PDB code 3EHZ) and pH 4.6 (PDB code 3EAM) correspond to the to the (well-known) desensitized state.
Medical Hypotheses, 1981
This paper presents the hypothesis, based on the amino acid sequence of the AChR proteins, that the main body of the channel is in the form of a B-barrel. This is based both on a Chou and Fasman analysis of the reported sequence, which predicts B-sheet for residues %28 to %46, of all four subunits, as well as the amphoteric nature of the B-sheet so formed, in which one side is almost entirely lipophilic and the other is hydrophilic. A means of testing my previous hypothesis, that residues 1-17 of the two a-subunits contain the actual ACh binding site, is also presented.
Toward a structural basis for the function of nicotinic acetylcholine receptors and their cousins
Neuron, 1995
The nicotinic acetylcholine (ACh) receptors and the other neurotransmitter-gated ion channels have key roles in fast synaptic transmission throughout the nervous system. These receptors have a simple functional repertoire: they bind a specific neurotransmitter, open a gate, conduct specific ions across the membrane, and desensitize. Although it is easy for us to imagine in a general way how they might carry out these steps, to determine the actual mechanism requires more detailed structural information than we now have. Nevertheless, new information about the parts of these receptors that are in the front line of function, the neurotransmitter binding sites, the ion-conducting channel, and the gate, provides intriguing clues, albeit not always consistent ones, about the mechanisms of these receptors. Most progress toward understanding function in terms of structure has been made with the ACh receptors, on which we will focus, at the same time noting what is conserved and what is variable among all of the neurotransmitter-gated ion channels. The ACh receptors are members of a family of neurotransmitter-gated ion channels, which also includes receptors for y-aminobutyric acid (GABA), glycine (Gly), and 5hydroxytryptamine (5HT; Nodaet al., 1983; Grenningloh et al., 1987; Schofieldet al., 1987; Maricqet al., 1991); also in this family is an invertebrate glutamate-gated chloride channel (Gully et al., 1994). The subunits of these receptors have similar sequences and distributions of hydrophobic, membrane-spanning segments and are homologous (Figures 1 and 2). In this family, each subunit contains, in its N-terminal extracellular half, 2 cysteine (Cys) residues separated by 13 other residues. These Cys residues are disulfide linked in the ACh receptor (Kao and Karlin, 1986) and presumably in the homologous receptors, thereby closing a 15-residue loop. Because of this unique, invariant feature, we will call this family the Cys-loop receptors. The subunits of other ligand-gated ion channels-the receptors for glutamate (cation-conducting), for ATP, and for the second messengers, CAMP and cGMP-have sequences and distributions of putative membrane-spanning segments that are dissimilar from those of the Cysloop receptors (Figures 1 and 2). Despite the differences in their structures, all of these ligand-gated ion channels carry out the same general functions. This implies that, at the level of detailed mechanisms, there are many ways of recognizing specific ligands, of transducing binding into propagated structural changes, of gating a channel, and of selecting and conducting specific ions through a membrane. It is likely, however, that among the homologous Cys-loop receptors the mechanisms are very similar and that insight into one is applicable to all.
Opening a hydrophobic gate: the nicotinic acetylcholine receptor as an example
2009
To what extent must a hydrophobic gate expand for the channel to count as open? We address this question using the nicotinic acetylcholine receptor (nAChR) as the exemplar. The nAChR is an integral membrane protein which forms a cation selective channel gated by neurotransmitter binding to its extracellular domain. A hydrophobic gating model has been proposed for the nAChR, whereby the pore is incompletely occluded in the closed state channel, with a narrow hydrophobic central gate region which presents an energetic barrier to ion permeation. The nAChR pore is lined by a parallel bundle of five M2 α-helices, with the gate formed by three rings of hydrophobic sidechains (9′, 13′, and 17′ of M2). A number of models have been proposed to describe the nature of the conformational change underlying the closed to open transition of the nAChR. These models involve different degrees of M2 helix displacement, rotation, and/or kinking. In this study, we use a simple pore expansion method (previously used to model opening of potassium channels) to generate a series of progressively wider models of the nAChR transmembrane domain. Continuum electrostatics calculations are used to assess the change in the barrier height of the hydrophobic gate as a function of pore expansion. The results
Biochemistry, 2000
Previous amino acid substitutions at the M4 domain of the Torpedo californica and mouse acetylcholine receptor suggested that the location of the substitution relative to the membrane-lipid interface and perhaps to the ion pore can be critical to the channel gating mechanism [Lasalde, J. along this postulated lipid-exposed segment M4 so that we can examine functional consequences on channel gating. The expression levels of mutants C412W, G421W, S424W, and V425W were almost the same as that of the wild type, whereas other mutants (M415W, L416W, C418W, I419W, I420W, T422W, and V423W) had relatively lower expression levels compared to that of the wild type as measured by iodinated R-bungarotoxin binding ([ 125 I]-R-BgTx). Two positions (L416W and I419W) had less than 20% of the wild type expression level. I417W gave no detectable [ 125 I]BgTx binding on the surface of oocyte, suggesting that this position might be involved in the AChR assembly, oligomerization, or transport to the cell membrane. The RV425W mutant exhibited a significant increase in the open channel probability with a moderate increase in the macroscopic response at higher ACh concentrations very likely due to channel block. The periodicity for the alteration of receptor assembly and ion channel function seems to favor a potential R-helical structure. Mutants that have lower levels of expression are clustered on one side of the postulated R-helical structure. Mutations that display normal expression and functional activity have been shown previously to face the membrane lipids by independent labeling studies. The functional analysis of these mutations will be presented and discussed in terms of possible structural models.
Pore-opening mechanism of the nicotinic acetylcholine receptor evinced by proton transfer
Nature Structural & Molecular Biology, 2008
The conformational changes underlying Cys-loop receptor channel gating remain elusive and controversial. We previously developed a single-channel electrophysiological method that allows structural inferences about the transient open-channel conformation to be made from the effect and properties of introduced charges on systematically engineered ionizable amino acids. Here, we applied this methodology to the entire M1 and M3 segments of the muscle nicotinic acetylcholine receptor, two transmembrane α-helices that pack against the pore-lining M2 α-helix. Together with our previous results on M2, these data suggest that the dilation of the pore underlying channel opening involves only a subtle rearrangement of these three transmembrane helices. Such a limited conformational change seems optimal to allow rapid closed-open interconversion rates, and hence, a fast postsynaptic response upon neurotransmitter binding. Thus, this receptor-channel seems to have evolved to take full advantage of the steep dependence of ion-and water-conduction rates on pore diameter characteristic of model hydrophobic nanopores. A variety of approaches have contributed to the development of a structural model of the closedchannel conformation of the muscle-type nicotinic acetylcholine receptor 1 (AChR), an archetypal member of the Cys-loop receptor superfamily of pentameric neurotransmitter-gated ion channels (Fig. 1). At the level of the membrane, twenty helices (four per subunit) are arranged in concentric layers around a central aqueous pore, with M2 directly lining the cationselective permeation pathway, M1 and M3 shielding M2 from the surrounding lipid bilayer, and M4 being the outermost and most lipid-exposed segment. Although some uncertainty remains as to the exact positioning of the side chains, it seems clear that the model is, otherwise, essentially correct. Less is known about the open-channel conformation, though. Since, on exposure to saturating concentrations of acetylcholine (ACh), closed AChRs open only to shut ("desensitize") again with a ∼50-ms time constant (ref. 2), the structure of the open conformation (and thus, the molecular basis of Cys-loop receptor-channel gating) has remained largely elusive. And although freeze-trapping experiments likely succeed in isolating the open state of the receptor (upon fast application of high concentrations of agonist) important limitations remain.
Biophysical Journal, 2005
A three-dimensional model of the transmembrane domain of a neuronal-type nicotinic acetylcholine receptor (nAChR), (a4) 2 (b2) 3 , was constructed from a homology structure of the muscle-type nAChR recently determined by cryoelectron microscopy. The neuronal channel model was embedded in a fully hydrated DMPC lipid bilayer, and moleculardynamics simulations were performed for 5 ns. A comparative analysis of the neuronal-versus muscle-type nAChR models revealed many conserved pore-lining residues, but an important difference was found near the periplasmic mouth of the pore. A flickering salt-bridge of a4-E266 with its adjacent b2-K260 was observed in the neuronal-type channel during the course of the molecular-dynamics simulations. The narrowest region, with a pore radius of ;2 Å formed by the salt-bridges, does not seem to be the restriction site for a continuous water passage. Instead, two hydrophobic rings, formed by a4-V259, a4-L263, and the homologous residues in the b 2-subunits, act as the gates for water flow, even though the region has a slightly larger pore radius. The model offers new insight into the water transport across the (a4) 2 (b2) 3 nAChR channel, and may lead to a better understanding of the structures, dynamics, and functions of this family of ion channels.