Sources of energy for gating by neurotransmitters in acetylcholine receptor channels (original) (raw)
Related papers
PLoS ONE, 2008
Gating of nicotinic acetylcholine receptors from a C(losed) to an O(pen) conformation is the initial event in the postsynaptic signaling cascade at the vertebrate nerve-muscle junction. Studies of receptor structure and function show that many residues in this large, five-subunit membrane protein contribute to the energy difference between C and O. Of special interest are amino acids located at the two transmitter binding sites and in the narrow region of the channel, where C«O gating motions generate a low«high change in the affinity for agonists and in the ionic conductance, respectively. We have measured the energy changes and relative timing of gating movements for residues that lie between these two locations, in the C-terminus of the pore-lining M2 helix of the a subunit ('aM2-cap'). This region contains a binding site for noncompetitive inhibitors and a charged ring that influences the conductance of the open pore. aM2-cap mutations have large effects on gating but much smaller effects on agonist binding, channel conductance, channel block and desensitization. Three aM2-cap residues (aI260, aP265 and aS268) appear to move at the outset of channel-opening, about at the same time as those at the transmitter binding site. The results suggest that the aM2-cap changes its secondary structure to link gating motions in the extracellular domain with those in the channel that regulate ionic conductance.
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.
Energetic Contributions to Channel Gating of Residues in the Muscle Nicotinic Receptor β1 Subunit
PLoS ONE, 2013
In the pentameric ligand-gated ion channel family, transmitter binds in the extracellular domain and conformational changes result in channel opening in the transmembrane domain. In the muscle nicotinic receptor and other heteromeric members of the family one subunit does not contribute to the canonical agonist binding site for transmitter. A fundamental question is whether conformational changes occur in this subunit. We used records of single channel activity and rate-equilibrium free energy relationships to examine the β1 (non-ACh-binding) subunit of the muscle nicotinic receptor. Mutations to residues in the extracellular domain have minimal effects on the gating equilibrium constant. Positions in the channel lining (M2 transmembrane) domain contribute strongly and relatively late during gating. Positions thought to be important in other subunits in coupling the transmitter-binding to the channel domains have minimal effects on gating. We conclude that the conformational changes involved in channel gating propagate from the binding-site to the channel in the ACh-binding subunits and subsequently spread to the non-binding subunit.
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.
Energetic Contributions to Channel Gating of Residues in the Muscle Nicotinic Receptor β1 Subunit
PLoS ONE, 2013
In the pentameric ligand-gated ion channel family, transmitter binds in the extracellular domain and conformational changes result in channel opening in the transmembrane domain. In the muscle nicotinic receptor and other heteromeric members of the family one subunit does not contribute to the canonical agonist binding site for transmitter. A fundamental question is whether conformational changes occur in this subunit. We used records of single channel activity and rate-equilibrium free energy relationships to examine the β1 (non-ACh-binding) subunit of the muscle nicotinic receptor. Mutations to residues in the extracellular domain have minimal effects on the gating equilibrium constant. Positions in the channel lining (M2 transmembrane) domain contribute strongly and relatively late during gating. Positions thought to be important in other subunits in coupling the transmitter-binding to the channel domains have minimal effects on gating. We conclude that the conformational changes involved in channel gating propagate from the binding-site to the channel in the ACh-binding subunits and subsequently spread to the non-binding subunit.
Journal of Biological Chemistry, 2008
The muscle nicotinic acetylcholine receptor is a large, allosteric, ligand-gated ion channel with the subunit composition ␣ 2 ␥␦. Although much is now known about the structure of the binding site, relatively little is understood about how the binding event is communicated to the channel gate, causing the pore to open. Here we identify a key hydrogen bond near the binding site that is involved in the gating pathway. Using mutant cycle analysis with the novel unnatural residue ␣-hydroxyserine, we find that the backbone N-H of ␣Ser-191 in loop C makes a hydrogen bond to an anionic side chain of the complementary subunit upon agonist binding. However, the anionic partner is not the glutamate predicted by the crystal structures of the homologous acetylcholine-binding protein. Instead, the hydrogen-bonding partner is the extensively researched aspartate ␥Asp-174/␦Asp-180, which had originally been identified as a key binding residue for cationic agonists. The Cys loop family of ligand-gated ion channels is involved in mediating fast synaptic transmission throughout the central and peripheral nervous systems (1-3). These neuroreceptors are among the molecules of learning, memory, and sensory perception, and they are implicated in numerous neurological disorders, including Alzheimer disease, Parkinson disease, and schizophrenia. The muscle nicotinic acetylcholine receptor (nAChR) 5 is arguably the best-studied member of the Cys loop family. This heteropentameric receptor is composed of homologous but functionally distinct subunits arranged symmetrically around a central ion-conducting pore with the stoichiometry ␣ 2 ␥␦. The agonist binding sites are located at the interfaces between the ␣␥ and ␣␦ subunits. The binding of two
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.
The intrinsic energy of the gating isomerization of a neuromuscular acetylcholine receptor channel
The Journal of General Physiology, 2012
Nicotinic acetylcholine receptor (AChR) channels at neuromuscular synapses rarely open in the absence of agonists, but many different mutations increase the unliganded gating equilibrium constant (E0) to generate AChRs that are active constitutively. We measured E0for two different sets of mutant combinations and by extrapolation estimated E0for wild-type AChRs. The estimates were 7.6 and 7.8 × 10−7in adult-type mouse AChRs (−100 mV at 23°C). The values are in excellent agreement with one obtained previously by using a completely different method (6.5 × 10−7, from monoliganded gating). E0decreases with depolarization to the same extent as does the diliganded gating equilibrium constant, e-fold with ∼60 mV. We estimate that at −100 mV the intrinsic energy of the unliganded gating isomerization is +8.4 kcal/mol (35 kJ/mol), and that in the absence of a membrane potential, the intrinsic chemical energy of this global conformational change is +9.4 kcal/mol (39 kJ/mol). Na+and K+in the e...