Structure of the human M2 muscarinic acetylcholine receptor bound to an antagonist

The parasympathetic branch of the autonomic nervous system regulates the activity of multiple organ systems. Muscarinic receptors are G-protein-coupled receptors that mediate the response to acetylcholine released from parasympathetic nerves. Their role in the unconscious regulation of organ and central nervous system function makes them potential therapeutic targets for a broad spectrum of diseases. The M2 muscarinic acetylcholine receptor (M2 receptor) is essential for the physiological control of cardiovascular function through activation of G-protein-coupled inwardly rectifying potassium channels, and is of particular interest because of its extensive pharmacological characterization with both orthosteric and allosteric ligands. Here we report the structure of the antagonist-bound human M2 receptor, the first human acetylcholine receptor to be characterized structurally, to our knowledge. The antagonist 3-quinuclidinyl-benzilate binds in the middle of a long aqueous channel extending approximately two-thirds through the membrane. The orthosteric binding pocket is formed by amino acids that are identical in all five muscarinic receptor subtypes, and shares structural homology with other functionally unrelated acetylcholine binding proteins from different species. A layer of tyrosine residues forms an aromatic cap restricting dissociation of the bound ligand. A binding site for allosteric ligands has been mapped to residues at the entrance to the binding pocket near this aromatic cap. The structure of the M2 receptor provides insights into the challenges of developing subtype-selective ligands for muscarinic receptors and their propensity for allosteric regulation.     

Location of a delta-subunit region determining ion transport through the acetylcholine receptor channel

The combination of complementary DNA expression and single-channel current analysis provides a powerful tool for studying the structure-function relationship of the nicotinic acetylcholine receptor (AChR) (refs 1-5). We have previously shown that AChR channels consisting of subunits from different species, expressed in the surface membrane of Xenopus oocytes, can be used to relate functional properties to individual subunits. Here we report that, in extracellular solution of low divalent cation concentration, the bovine AChR channel has a smaller conductance than the Torpedo AChR channel. Replacement of the delta-subunit of the Torpedo AChR by the bovine delta-subunit makes the channel conductance similar to that of the bovine AChR channel. To locate the region in the delta-subunit responsible for this difference, we have constructed chimaeric delta-subunit cDNAs with different combinations of the Torpedo and bovine counterparts. The conductances of AChR channels containing chimaeric delta-subunits suggest that a region comprising the putative transmembrane segment M2 and the adjacent bend portion between segments M2 and M3 is involved in determining the rate of ion transport through the open channel.     

Principal pathway coupling agonist binding to channel gating in nicotinic receptors

Synaptic receptors respond to neurotransmitters by opening an intrinsic ion channel in the final step in synaptic transmission. How binding of the neurotransmitter is conveyed over the long distance to the channel remains a central question in neurobiology. Here we delineate a principal pathway that links neurotransmitter binding to channel gating by using a structural model of the Torpedo acetylcholine receptor at 4-A resolution, recordings of currents through single receptor channels and determinations of energetic coupling between pairs of residues. We show that a pair of invariant arginine and glutamate residues in each receptor alpha-subunit electrostatically links peripheral and inner beta-sheets from the binding domain and positions them to engage with the channel. The key glutamate and flanking valine residues energetically couple to conserved proline and serine residues emerging from the top of the channel-forming alpha-helix, suggesting that this is the point at which the binding domain triggers opening of the channel. The series of interresidue couplings identified here constitutes a primary allosteric pathway that links neurotransmitter binding to channel gating.     

Mapping the conformational wave of acetylcholine receptor channel gating

Allosteric transitions allow fast regulation of protein function in living systems. Even though the end points of such conformational changes are known for many proteins, the characteristics of the paths connecting these states remain largely unexplored. Rate-equilibrium linear free-energy relationships (LFERs) provide information about such pathways by relating changes in the free energy of the transition state to those of the ground states upon systematic perturbation of the system. Here we present an LFER analysis of the gating reaction pathway of the muscle acetylcholine receptor. We studied the closed <==> open conformational change at the single-molecule level following perturbation by series of single-site mutations, agonists and membrane voltages. This method provided a snapshot of several regions of the receptor at the transition state in terms of their approximate positions along the reaction coordinate, on a scale from 0 (closed-like) to 1 (open-like). The resulting map reveals a spatial gradient of positional values, which suggests that the conformational change proceeds in a wave-like manner, with the low-to-high affinity change at the transmitter-binding sites preceding the complete opening of the pore.     

Crystal structure and mechanism of a calcium-gated potassium channel

Ion channels exhibit two essential biophysical properties; that is, selective ion conduction, and the ability to gate-open in response to an appropriate stimulus. Two general categories of ion channel gating are defined by the initiating stimulus: ligand binding (neurotransmitter- or second-messenger-gated channels) or membrane voltage (voltage-gated channels). Here we present the structural basis of ligand gating in a K(+) channel that opens in response to intracellular Ca(2+). We have cloned, expressed, analysed electrical properties, and determined the crystal structure of a K(+) channel (MthK) from Methanobacterium thermoautotrophicum in the Ca(2+)-bound, opened state. Eight RCK domains (regulators of K(+) conductance) form a gating ring at the intracellular membrane surface. The gating ring uses the free energy of Ca(2+) binding in a simple manner to perform mechanical work to open the pore.     

Quaternary structure of the acetylcholine receptor

The five membrane-spanning subunits of the acetylcholine receptor have been resolved in electron microscope images and are shown to lie at pentagonally symmetrical positions around the channel over a large fraction of their length. The channel consists of a wide synaptic portion and a narrow portion extending through the membrane into the interior of the cell.     

Cis-trans isomerization at a proline opens the pore of a neurotransmitter-gated ion channel

5-hydroxytryptamine type 3 (5-HT3) receptors are members of the Cys-loop receptor superfamily. Neurotransmitter binding in these proteins triggers the opening (gating) of an ion channel by means of an as-yet-uncharacterized conformational change. Here we show that a specific proline (Pro 8*), located at the apex of the loop between the second and third transmembrane helices (M2-M3), can link binding to gating through a cis-trans isomerization of the protein backbone. Using unnatural amino acid mutagenesis, a series of proline analogues with varying preference for the cis conformer was incorporated at the 8* position. Proline analogues that strongly favour the trans conformer produced non-functional channels. Among the functional mutants there was a strong correlation between the intrinsic cis-trans energy gap of the proline analogue and the activation of the channel, suggesting that cis-trans isomerization of this single proline provides the switch that interconverts the open and closed states of the channel. Consistent with this proposal, nuclear magnetic resonance studies on an M2-M3 loop peptide reveal two distinct, structured forms. Our results thus confirm the structure of the M2-M3 loop and the critical role of Pro 8* in the 5-HT3 receptor. In addition, they suggest that a molecular rearrangement at Pro 8* is the structural mechanism that opens the receptor pore.     

Inactivation of the sarcoplasmic reticulum calcium channel by protein kinase.

The ryanodine receptor protein of skeletal muscle sarcoplasmic reticulum (SR) membranes is a calcium ion channel which allows movement of calcium from the SR lumen into the cytoplasm during muscle activation. Gating of this channel is modulated by a number of physiologically important substances including calcium. Interestingly, calcium has both activating and inactivating effects which are concentration- and tissue-specific. In skeletal muscle, calcium-dependent inactivation of calcium release occurs at concentrations reached physiologically, suggesting that calcium may modulate the release process by a negative feedback mechanism. To determine the cellular mechanism responsible for calcium-dependent inactivation, we have investigated the ability of protein phosphorylation to affect single channel gating behaviour using the patch clamp technique. Here we demonstrate that the ryanodine receptor protein/calcium release channel of skeletal muscle SR is inactivated under conditions permissive for protein phosphorylation. This inactivation is reversed by the application of phosphatase and prevented by a peptide inhibitor specific for calcium/calmodulin-dependent protein kinase II. The results provide evidence for an endogenous protein kinase which is closely associated with the ryanodine receptor protein and regulates channel gating.     

Modulation of glycine receptor chloride channels by cAMP-dependent protein kinase in spinal trigeminal neurons

Glycine is an important inhibitory transmitter in the brainstem and spinal cord. In the trigeminal subnucleus caudalis (medullary dorsal horn) and in the spinal dorsal horn (the relaying centres for processing pain and sensory information), glycine inhibits the glutamate-evoked depolarization and depresses firing of neurons. The binding of glycine to its receptor produces a large increase in Cl- conductance, which causes membrane hyperpolarization. The selectivity and gating properties of glycine receptor channels have been well characterized; the glycine receptor molecules have also been purified. The amino-acid sequence, deduced from complementary DNA clones encoding one of the peptides (the 48K subunit), shows significant homology with gamma-aminobutyric acid A (GABAA) and nicotinic acetylcholine receptor subunits, suggesting that glycine receptors may belong to a superfamily of chemically gated channel proteins. However, very little is known about the modulation of glycine receptor channels. We have investigated the regulation of strychnine-sensitive glycine receptor channels by cyclic AMP-dependent protein kinase in neurons isolated from spinal trigeminal nucleus of rat and report here that the protein kinase A dramatically increased the glycine-induced Cl- currents by increasing the probability of the channel openings. GS protein, which is sensitive to cholera toxin, was involved in the modulation.     

Mechanism of glutamate receptor desensitization

Ligand-gated ion channels transduce chemical signals into electrical impulses by opening a transmembrane pore in response to binding one or more neurotransmitter molecules. After activation, many ligand-gated ion channels enter a desensitized state in which the neurotransmitter remains bound but the ion channel is closed. Although receptor desensitization is crucial to the functioning of many ligand-gated ion channels in vivo, the molecular basis of this important process has until now defied analysis. Using the GluR2 AMPA-sensitive glutamate receptor, we show here that the ligand-binding cores form dimers and that stabilization of the intradimer interface by either mutations or allosteric modulators reduces desensitization. Perturbations that destabilize the interface enhance desensitization. Receptor activation involves conformational changes within each subunit that result in an increase in the separation of portions of the receptor that are linked to the ion channel. Our analysis defines the dimer interface in the resting and activated state, indicates how ligand binding is coupled to gating, and suggests modes of dimer dimer interaction in the assembled tetramer. Desensitization occurs through rearrangement of the dimer interface, which disengages the agonist-induced conformational change in the ligand-binding core from the ion channel gate.     

Mutations in the channel domain alter desensitization of a neuronal nicotinic receptor.

A variety of ligand-gated ion channels undergo a fast activation process after the rapid application of agonist and also a slower transition towards desensitized or inactivated closed channel states when exposure to agonist is prolonged. Desensitization involves at least two distinct closed states in the acetylcholine receptor, each with an affinity for agonists higher than those of the resting or active conformations. Here we investigate how structural elements could be involved in the desensitization of the acetylcholine-gated ion channel from the chick brain alpha-bungarotoxin sensitive homo-oligomeric alpha 7 receptor, using site-directed mutagenesis and expression in Xenopus oocytes. Mutations of the highly conserved leucine 247 residue from the uncharged MII segment of alpha 7 suppress inhibition by the open-channel blocker QX-222, indicating that this residue, like others from MII, faces the lumen of the channel. But, unexpectedly, the same mutations decrease the rate of desensitization of the response, increase the apparent affinity for acetylcholine and abolish current rectification. Moreover, unlike wild-type alpha 7, which has channels with a single conductance level, the leucine-to-threonine mutant has an additional conducting state active at low acetylcholine concentrations. It is possible that mutation of Leu 247 renders conductive one of the high-affinity desensitized states of the receptor.     

Rings of negatively charged amino acids determine the acetylcholine receptor channel conductance

The structure-function relationship of the nicotinic acetylcholine receptor (AChR) has been effectively studied by the combination of complementary DNA manipulation and single-channel current analysis. Previous work with chimaeras between the Torpedo californica and bovine AChR delta-subunits has shown that the region comprising the hydrophobic segment M2 and its vicinity contains an important determinant of the rate of ion transport through the AChR channel. It has also been suggested that this region is responsible for the reduction in channel conductance caused by divalent cations and that segment M2 contributes to the binding site of noncompetitive antagonists. To identify those amino acid residues that interact with permeating ions, we have introduced various point mutations into the Torpedo AChR subunit cDNAs to alter the net charge of the charged or glutamine residues around the proposed transmembrane segments. The single-channel conductance properties of these AChR mutants expressed in Xenopus laevis oocytes indicate that three clusters of negatively charged and glutamine residues neighbouring segment M2 of the alpha-, beta-, gamma- and delta-subunits, probably forming three anionic rings, are major determinants of the rate of ion transport.     

5-HT3 receptors are membrane ion channels

The neurohormone 5-hydroxytryptamine (5HT or serotonin) exerts its effects by binding to several distinct receptors. One of these is the M-receptor of Gaddum and Picarelli, now called the 5-HT3 receptor, through which 5-HT acts to excite enteric neurons. Ligand-binding and functional studies have shown that the 5-HT3 receptor is widely distributed in peripheral and central nervous tissue and evidence suggests that the receptor might incorporate an ion channel permeable to cations. We now report the first recordings of currents through single ion channels activated by 5-HT3 receptors, in excised (outside-out) membrane patches from neurons of the guinea pig submucous plexus. Whereas application of acetylcholine activated predominantly a 40-pS channel, 5-HT caused unitary currents apparently through two channels of conductances of 15 and 9 pS, which were reversibly blocked by antagonists of the 5-HT3 receptor. Receptors for amine neurotransmitters, including 5-HT1 and 5-HT2, have previously been thought to transduce their effects through GTP-binding proteins: the direct demonstration that 5-HT3 receptors are ligand-gated ion channels implies a role for 5-HT, and perhaps other amines, as a 'fast' synaptic transmitter.     

The gating mechanism of the large mechanosensitive channel MscL

The mechanosensitive channel of large conductance, MscL, is a ubiquitous membrane-embedded valve involved in turgor regulation in bacteria. The crystal structure of MscL from Mycobacterium tuberculosis provides a starting point for analysing molecular mechanisms of tension-dependent channel gating. Here we develop structural models in which a cytoplasmic gate is formed by a bundle of five amino-terminal helices (S1), previously unresolved in the crystal structure. When membrane tension is applied, the transmembrane barrel expands and pulls the gate apart through the S1-M1 linker. We tested these models by substituting cysteines for residues predicted to be near each other only in either the closed or open conformation. Our results demonstrate that S1 segments form the bundle when the channel is closed, and crosslinking between S1 segments prevents opening. S1 segments interact with M2 when the channel is open, and crosslinking of S1 to M2 impedes channel closing. Gating is affected by the length of the S1-M1 linker in a manner consistent with the model, revealing critical spatial relationships between the domains that transmit force from the lipid bilayer to the channel gate.     

Gating pore current in an inherited ion channelopathy

Ion channelopathies are inherited diseases in which alterations in control of ion conductance through the central pore of ion channels impair cell function, leading to periodic paralysis, cardiac arrhythmia, renal failure, epilepsy, migraine and ataxia. Here we show that, in contrast with this well-established paradigm, three mutations in gating-charge-carrying arginine residues in an S4 segment that cause hypokalaemic periodic paralysis induce a hyperpolarization-activated cationic leak through the voltage sensor of the skeletal muscle Na(V)1.4 channel. This 'gating pore current' is active at the resting membrane potential and closed by depolarizations that activate the voltage sensor. It has similar permeability to Na+, K+ and Cs+, but the organic monovalent cations tetraethylammonium and N-methyl-D-glucamine are much less permeant. The inorganic divalent cations Ba2+, Ca2+ and Zn2+ are not detectably permeant and block the gating pore at millimolar concentrations. Our results reveal gating pore current in naturally occurring disease mutations of an ion channel and show a clear correlation between mutations that cause gating pore current and hypokalaemic periodic paralysis. This gain-of-function gating pore current would contribute in an important way to the dominantly inherited membrane depolarization, action potential failure, flaccid paralysis and cytopathology that are characteristic of hypokalaemic periodic paralysis. A survey of other ion channelopathies reveals numerous examples of mutations that would be expected to cause gating pore current, raising the possibility of a broader impact of gating pore current in ion channelopathies.     

Coupling of agonist binding to channel gating in the GABA(A) receptor

Neurotransmitters such as acetylcholine and GABA (gamma-aminobutyric acid) mediate rapid synaptic transmission by activating receptors belonging to the gene superfamily of ligand-gated ion channels (LGICs). These channels are pentameric proteins that function as signal transducers, converting chemical messages into electrical signals. Neurotransmitters activate LGICs by interacting with a ligand-binding site, triggering a conformational change in the protein that results in the opening of an ion channel. This process, which is known as 'gating', occurs rapidly and reversibly, but the molecular rearrangements involved are not well understood. Here we show that optimal gating in the GABA(A) receptor, a member of the LGIC superfamily, is dependent on electrostatic interactions between the negatively charged Asp 57 and Asp 149 residues in extracellular loops 2 and 7, and the positively charged Lys 279 residue in the transmembrane 2-3 linker region of the alpha1-subunit. During gating, Asp 149 and Lys 279 seem to move closer to one another, providing a potential mechanism for the coupling of ligand binding to opening of the ion channel.     

Regulation of NMDA receptor desensitization in mouse hippocampal neurons by glycine

Responses to the excitatory amino acid N-methyl-D-aspartate (NMDA) are markedly potentiated by nanomolar concentrations of glycine. This is due to the action of glycine at a novel strychnine-resistant binding site with an anatomical distribution identical to that for NMDA receptors, suggesting that the NMDA receptor channel complex contains at least two classes of amino-acid recognition site. Antagonists at the glycine-binding site associated with NMDA receptors act as potent non-competitive antagonists, but do not alter the mean open time or conductance, as estimated by fluctuation analysis. The mechanisms by which glycine acts on NMDA receptors are unknown, but single-channel recording experiments show an increase in opening frequency with no change in mean open time or conductance, suggesting that glycine could regulate transitions to states that are intermediate between binding of NMDA receptor agonists and ion-channel gating. It has been suggested that glycine acts as a co-agonist at the NMDA receptor, and that responses to NMDA cannot be obtained in the complete absence of glycine, but in these experiments the response to NMDA was measured at equilibrium, and it is unlikely that sufficient temporal resolution was achieved to detect rapid alterations in receptor gating. Using a fast perfusion system we find that glycine regulates desensitization at NMDA receptors; this has a major effect on the response to NMDA measured at equilibrium, as would occur with slower applications of agonist. Reduction of NMDA receptor desensitization by glycine provides an example of a novel mechanism for regulation of ion-channel activity.     

Novel mechanism of voltage-dependent gating in L-type calcium channels

Activation of voltage-dependent calcium channels by membrane depolarization triggers a variety of key cellular responses, such as contraction in heart and smooth muscle and exocytotic secretion in endocrine and nerve cells. Modulation of calcium channel gating is believed to be the mechanism by which several neurotransmitters, hormones and therapeutic agents mediate their effects on cell function. Here we describe a novel type of voltage-dependent equilibrium between different gating patterns of dihydropyridine-sensitive (L-type) cardiac Ca2+ channels. Strong depolarizations drive the channel from its normal gating pattern into a mode of gating characterized by long openings and high open probability. The rate constants for conversions between gating modes, estimated from single channel recordings, are much slower than normal channel opening and closing rates, but the equilibrium between modes is almost as steeply voltage-dependent as channel activation and deactivation at more negative potentials. This new mechanism of voltage-dependent gating can explain previous reports of activity-dependent Ca2+ channel potentiation in cardiac and other cells and forms a potent mechanism by which Ca2+ uptake into cells could be regulated.     

Study of membrane permeability changes by fluctuation analysis.

The molecular basis of the changes in membrane permeability that underlie the nerve impulse can be studied by statistical analysis of the random fluctuations in current through the nerve membrane. Such "noise analysis" has already begun to clarify the nature of the gating mechanisms that control the flow of ions across the membrane at the neuromuscular junction.