Open channel structure of MscL and the gating mechanism of mechanosensitive channels

Mechanosensitive channels act as membrane-embedded mechano-electrical switches, opening a large water-filled pore in response to lipid bilayer deformations. This process is critical to the response of living organisms to direct physical stimulation, such as in touch, hearing and osmoregulation. Here, we have determined the structural rearrangements that underlie these events in the large prokaryotic mechanosensitive channel (MscL) using electron paramagnetic resonance spectroscopy and site-directed spin labelling. MscL was trapped in both the open and in an intermediate closed state by modulating bilayer morphology. Transition to the intermediate state is characterized by small movements in the first transmembrane helix (TM1). Subsequent transitions to the open state are accompanied by massive rearrangements in both TM1 and TM2, as shown by large increases in probe dynamics, solvent accessibility and the elimination of all intersubunit spin-spin interactions. The open state is highly dynamic, supporting a water-filled pore of at least 25 A, lined mostly by TM1. These structures suggest a plausible molecular mechanism of gating in mechanosensitive channels.     

A stepwise mechanism for acetylcholine receptor channel gating

Muscle contraction is triggered by the opening of acetylcholine receptors at the vertebrate nerve-muscle synapse. The M2 helix of this allosteric membrane protein lines the channel, and contains a 'gate' that regulates the flow of ions through the pore. We used single-molecule kinetic analysis to probe the transition state of the gating conformational change and estimate the relative timing of M2 motions in the alpha-subunit of the murine acetylcholine receptor. This analysis produces a 'Phi-value' for a given residue that reflects its open-like versus closed-like character at the transition state. Here we show that most of the residues throughout the length of M2 have a Phi-value of approximately 0.64 but that some near the middle have lower Phi-values of 0.52 or 0.31, suggesting that alphaM2 moves in three discrete steps. The core of the channel serves both as a gate that regulates ion flow and as a hub that directs the propagation of the gating isomerization through the membrane domain of the acetylcholine receptor.     

机械敏感通道蛋白的研究进展

机械敏感通道蛋白是一类在生物体内广泛分布的离子通道蛋白,参与生物体内多种正常的生理过程,其结构异常及功能失调与多种疾病的发生、发展密切相关. 研究归纳已知的几类机械敏感通道蛋白及其在生理、病理过程中发挥的作用,并对相关疾病的靶向诊断、治疗的研究进展进行综述,最后展望其在生物医学领域的应用.     

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.     

Role of acetylcholine receptor subunits in gating of the channel

The Torpedo and calf acetylcholine receptors and hybrids composed of subunits from the two species have been produced in Xenopus oocytes by the use of the cloned complementary DNAs. Single-channel current measurements indicate that these receptors form channels of similar conductance but with different gating behaviour.     

Structural mechanism of plant aquaporin gating

Plants counteract fluctuations in water supply by regulating all aquaporins in the cell plasma membrane. Channel closure results either from the dephosphorylation of two conserved serine residues under conditions of drought stress, or from the protonation of a conserved histidine residue following a drop in cytoplasmic pH due to anoxia during flooding. Here we report the X-ray structure of the spinach plasma membrane aquaporin SoPIP2;1 in its closed conformation at 2.1 A resolution and in its open conformation at 3.9 A resolution, and molecular dynamics simulations of the initial events governing gating. In the closed conformation loop D caps the channel from the cytoplasm and thereby occludes the pore. In the open conformation loop D is displaced up to 16 A and this movement opens a hydrophobic gate blocking the channel entrance from the cytoplasm. These results reveal a molecular gating mechanism which appears conserved throughout all plant plasma membrane aquaporins.     

The principle of gating charge movement in a voltage-dependent K+ channel

The steep dependence of channel opening on membrane voltage allows voltage-dependent K+ channels to turn on almost like a switch. Opening is driven by the movement of gating charges that originate from arginine residues on helical S4 segments of the protein. Each S4 segment forms half of a 'voltage-sensor paddle' on the channel's outer perimeter. Here we show that the voltage-sensor paddles are positioned inside the membrane, near the intracellular surface, when the channel is closed, and that the paddles move a large distance across the membrane from inside to outside when the channel opens. KvAP channels were reconstituted into planar lipid membranes and studied using monoclonal Fab fragments, a voltage-sensor toxin, and avidin binding to tethered biotin. Our findings lead us to conclude that the voltage-sensor paddles operate somewhat like hydrophobic cations attached to levers, enabling the membrane electric field to open and close the pore.     

TRPA1 is a candidate for the mechanosensitive transduction channel of vertebrate hair cells

Mechanical deflection of the sensory hair bundles of receptor cells in the inner ear causes ion channels located at the tips of the bundle to open, thereby initiating the perception of sound. Although some protein constituents of the transduction apparatus are known, the mechanically gated transduction channels have not been identified in higher vertebrates. Here, we investigate TRP (transient receptor potential) ion channels as candidates and find one, TRPA1 (also known as ANKTM1), that meets criteria for the transduction channel. The appearance of TRPA1 messenger RNA expression in hair cell epithelia coincides developmentally with the onset of mechanosensitivity. Antibodies to TRPA1 label hair bundles, especially at their tips, and tip labelling disappears when the transduction apparatus is chemically disrupted. Inhibition of TRPA1 protein expression in zebrafish and mouse inner ears inhibits receptor cell function, as assessed with electrical recording and with accumulation of a channel-permeant fluorescent dye. TRPA1 is probably a component of the transduction channel itself.     

Structure and gating mechanism of the acetylcholine receptor pore

The nicotinic acetylcholine receptor controls electrical signalling between nerve and muscle cells by opening and closing a gated, membrane-spanning pore. Here we present an atomic model of the closed pore, obtained by electron microscopy of crystalline postsynaptic membranes. The pore is shaped by an inner ring of 5 alpha-helices, which curve radially to create a tapering path for the ions, and an outer ring of 15 alpha-helices, which coil around each other and shield the inner ring from the lipids. The gate is a constricting hydrophobic girdle at the middle of the lipid bilayer, formed by weak interactions between neighbouring inner helices. When acetylcholine enters the ligand-binding domain, it triggers rotations of the protein chains on opposite sides of the entrance to the pore. These rotations are communicated through the inner helices, and open the pore by breaking the girdle apart.     

Evidence for cooperative interactions in potassium channel gating.

Cloning and expression of voltage-activated potassium ion-channel complementary DNAs has confirmed that these channels are composed of four identical subunits, each containing a voltage sensor. It has been generally accepted that the voltage sensors must reach a permissive state through one or more conformational ('gating') transitions before the channel can open. To test whether each subunit gates independently, we have constructed cDNAs encoding four subunits on a single polypeptide chain, enabling us to specify the subunit stoichiometry. The gating of heterotetramers made up from combinations of subunits with different gating phenotypes strongly suggests that individual subunits gate cooperatively, rather than independently. Nonindependent subunit gating is consistent with measurements of the kinetics of K(+)-channel gating currents and in line with the widespread subunit cooperativity observed in other multisubunit proteins.     

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.     

Coupling of agonist binding to channel gating in an ACh-binding protein linked to an ion channel

Neurotransmitter receptors from the Cys-loop superfamily couple the binding of agonist to the opening of an intrinsic ion pore in the final step in rapid synaptic transmission. Although atomic resolution structural data have recently emerged for individual binding and pore domains, how they are linked into a functional unit remains unknown. Here we identify structural requirements for functionally coupling the two domains by combining acetylcholine (ACh)-binding protein, whose structure was determined at atomic resolution, with the pore domain from the serotonin type-3A (5-HT3A) receptor. Only when amino-acid sequences of three loops in ACh-binding protein are changed to their 5-HT3A counterparts does ACh bind with low affinity characteristic of activatable receptors, and trigger opening of the ion pore. Thus functional coupling requires structural compatibility at the interface of the binding and pore domains. Structural modelling reveals a network of interacting loops between binding and pore domains that mediates this allosteric coupling process.     

Ion permeation mechanism of the potassium channel

Ion-selective channels enable the specific permeation of ions through cell membranes and provide the basis of several important biological functions; for example, electric signalling in the nervous system. Although a large amount of electrophysiological data is available, the molecular mechanisms by which these channels can mediate ion transport remain a significant unsolved problem. With the recently determined crystal structure of the representative K+ channel (KcsA) from Streptomyces lividans, it becomes possible to examine ion conduction pathways on a microscopic level. K+ channels utilize multi-ion conduction mechanisms, and the three-dimensional structure also shows several ions present in the channel. Here we report results from molecular dynamics free energy perturbation calculations that both establish the nature of the multiple ion conduction mechanism and yield the correct ion selectivity of the channel. By evaluating the energetics of all relevant occupancy states of the selectivity filter, we find that the favoured conduction pathway involves transitions only between two main states with a free difference of about 5 kcal mol(-1). Other putative permeation pathways can be excluded because they would involve states that are too high in energy.     

A C. elegans stretch receptor neuron revealed by a mechanosensitive TRP channel homologue

The nematode Caenorhabditis elegans is commonly used as a genetic model organism for dissecting integration of the sensory and motor systems. Despite extensive genetic and behavioural analyses that have led to the identification of many genes and neural circuits involved in regulating C. elegans locomotion behaviour, it remains unclear whether and how somatosensory feedback modulates motor output during locomotion. In particular, no stretch receptors have been identified in C. elegans, raising the issue of whether stretch-receptor-mediated proprioception is used by C. elegans to regulate its locomotion behaviour. Here we have characterized TRP-4, the C. elegans homologue of the mechanosensitive TRPN channel. We show that trp-4 mutant worms bend their body abnormally, exhibiting a body posture distinct from that of wild-type worms during locomotion, suggesting that TRP-4 is involved in stretch-receptor-mediated proprioception. We show that TRP-4 acts in a single neuron, DVA, to mediate its function in proprioception, and that the activity of DVA can be stimulated by body stretch. DVA both positively and negatively modulates locomotion, providing a unique mechanism whereby a single neuron can fine-tune motor activity. Thus, DVA represents a stretch receptor neuron that regulates sensory-motor integration during C. elegans locomotion.     

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.