Focus on touch and hearing distracts attention from numerous subconscious force sensors, such as the vital control of blood pressure and systemic osmolarity, and sensors in nonanimals. Multifarious ...manifestations should not obscure invariant and fundamental physicochemical principles. We advocate that force from lipid (FFL) is one such principle. It is based on the fact that the self-assembled bilayer necessitates inherent forces that are large and anisotropic, even at life’s origin. Functional response of membrane proteins is governed by bilayer force changes. Added stress can redirect these forces, leading to geometric changes of embedded proteins such as ion channels. The FFL principle was first demonstrated when purified bacterial mechanosensitive channel of large conductance (MscL) remained mechanosensitive (MS) after reconstituting into bilayers. This key experiment has recently been unequivocally replicated with two vertebrate MS K
2p
channels. Even the canonical Kv and the
Drosophila
canonical transient receptor potentials (TRPCs) have now been shown to be MS in biophysical and in physiological contexts, supporting the universality of the FFL paradigm. We also review the deterministic role of mechanical force during stem cell differentiation as well as the cell-cell and cell-matrix tethers that provide force communications. In both the ear hair cell and the worm’s touch neuron, deleting the cadherin or microtubule tethers reduces but does not eliminate MS channel activities. We found no evidence to distinguish whether these tethers directly pull on the channel protein or a surrounding lipid platform. Regardless of the implementation, pulling tether tenses up the bilayer. Membrane tenting is directly visible at the apexes of the stereocilia.
Binding of the neurotransmitter acetylcholine to its receptors on muscle fibers depolarizes the membrane and thereby triggers muscle contraction. We sought to understand at the level of ...three-dimensional structure how agonists and antagonists alter nicotinic acetylcholine receptor conformation. We used the muscle-type receptor from the Torpedo ray to first define the structure of the receptor in a resting, activatable state. We then determined the receptor structure bound to the agonist carbachol, which stabilizes an asymmetric, closed channel desensitized state. We find conformational changes in a peripheral membrane helix are tied to recovery from desensitization. To probe mechanisms of antagonism, we obtained receptor structures with the active component of curare, a poison arrow toxin and precursor to modern muscle relaxants. d-Tubocurarine stabilizes the receptor in a desensitized-like state in the presence and absence of agonist. These findings define the transitions between resting and desensitized states and reveal divergent means by which antagonists block channel activity of the muscle-type nicotinic receptor.
Life’s origin entails enclosing a compartment to hoard material, energy, and information. The envelope necessarily comprises amphipaths, such as prebiotic fatty acids, to partition the two aqueous ...domains. The self-assembled lipid bilayer comes with a set of properties including its strong anisotropic internal forces that are chemically or physically malleable. Added bilayer stretch can alter force vectors on embedded proteins to effect conformational change. The force-from-lipid principle was demonstrated 25 y ago when stretches opened purified Escherichia coli MscL channels reconstituted into artificial bilayers. This reductionistic exercise has rigorously been recapitulated recently with two vertebrate mechanosensitive K ⁺ channels (TREK1 and TRAAK). Membrane stretches have also been known to activate various voltage-, ligand-, or Ca ²⁺-gated channels. Careful analyses showed that Kv, the canonical voltage-gated channel, is in fact exquisitely sensitive even to very small tension. In an unexpected context, the canonical transient-receptor-potential channels in the Drosophila eye, long presumed to open by ligand binding, is apparently opened by membrane force due to PIP ₂ hydrolysis-induced changes in bilayer strain. Being the intimate medium, lipids govern membrane proteins by physics as well as chemistry. This principle should not be a surprise because it parallels water’s paramount role in the structure and function of soluble proteins. Today, overt or covert mechanical forces govern cell biological processes and produce sensations. At the genesis, a bilayer’s response to osmotic force is likely among the first senses to deal with the capricious primordial sea.
The nicotinic acetylcholine receptor, a pentameric ligand-gated ion channel, converts the free energy of binding of the neurotransmitter acetylcholine into opening of its central pore. Here we ...present the first high-resolution structure of the receptor type found in muscle-endplate membrane and in the muscle-derived electric tissues of fish. The native receptor was purified from Torpedo electric tissue and functionally reconstituted in lipids optimal for cryo-electron microscopy. The receptor was stabilized in a closed state by the binding of α-bungarotoxin. The structure reveals the binding of a toxin molecule at each of two subunit interfaces in a manner that would block the binding of acetylcholine. It also reveals a closed gate in the ion-conducting pore, formed by hydrophobic amino acid side chains, located ∼60 Å from the toxin binding sites. The structure provides a framework for understanding gating in ligand-gated channels and how mutations in the acetylcholine receptor cause congenital myasthenic syndromes.
•High-resolution structure of a native muscle-type nicotinic acetylcholine receptor•Previously unresolved structural elements contribute to neurotoxin binding•Channel is stabilized in a closed conformation by α-bungarotoxin from snake venom•Transduction mechanism and myasthenic disease mutations
Rahman et al. report the high-resolution single-particle cryo-EM structure of a native muscle-type nicotinic acetylcholine receptor from the Torpedo electric ray, in complex with α-bungarotoxin from the banded krait. The structure was obtained in a lipidic environment shown to support channel function and reveals a closed, hydrophobic ion channel gate.
Abstract
γ-Aminobutyric acid type A (GABA
A
) receptors are pentameric ligand-gated ion channels abundant in the central nervous system and are prolific drug targets for treating anxiety, sleep ...disorders and epilepsy. Diverse small molecules exert a spectrum of effects on γ-aminobutyric acid type A (GABA
A
) receptors by acting at the classical benzodiazepine site. They can potentiate the response to GABA, attenuate channel activity, or counteract modulation by other ligands. Structural mechanisms underlying the actions of these drugs are not fully understood. Here we present two high-resolution structures of GABA
A
receptors in complex with zolpidem, a positive allosteric modulator and heavily prescribed hypnotic, and DMCM, a negative allosteric modulator with convulsant and anxiogenic properties. These two drugs share the extracellular benzodiazepine site at the α/γ subunit interface and two transmembrane sites at β/α interfaces. Structural analyses reveal a basis for the subtype selectivity of zolpidem that underlies its clinical success. Molecular dynamics simulations provide insight into how DMCM switches from a negative to a positive modulator as a function of binding site occupancy. Together, these findings expand our understanding of how GABA
A
receptor allosteric modulators acting through a common site can have diverging activities.
General anesthetics and neuromuscular blockers are used together during surgery to stabilize patients in an unconscious state. Anesthetics act mainly by potentiating inhibitory ion channels and ...inhibiting excitatory ion channels, with the net effect of dampening nervous system excitability. Neuromuscular blockers act by antagonizing nicotinic acetylcholine receptors at the motor endplate; these excitatory ligand-gated ion channels are also inhibited by general anesthetics. The mechanisms by which anesthetics and neuromuscular blockers inhibit nicotinic receptors are poorly understood but underlie safe and effective surgeries. Here we took a direct structural approach to define how a commonly used anesthetic and two neuromuscular blockers act on a muscle-type nicotinic receptor. We discover that the intravenous anesthetic etomidate binds at an intrasubunit site in the transmembrane domain and stabilizes a non-conducting, desensitized-like state of the channel. The depolarizing neuromuscular blocker succinylcholine also stabilizes a desensitized channel but does so through binding to the classical neurotransmitter site. Rocuronium binds in this same neurotransmitter site but locks the receptor in a resting, non-conducting state. Together, this study reveals a structural mechanism for how general anesthetics work on excitatory nicotinic receptors and further rationalizes clinical observations in how general anesthetics and neuromuscular blockers interact.
Abstract Methaqualone, a quinazolinone marketed commercially as Quaalude, is a central nervous system depressant that was used clinically as a sedative-hypnotic, then became a notorious recreational ...drug in the 1960s-80s. Due to its high abuse potential, medical use of methaqualone was eventually prohibited, yet it persists as a globally abused substance. Methaqualone principally targets GABA A receptors, which are the major inhibitory neurotransmitter-gated ion channels in the brain. The restricted status and limited accessibility of methaqualone have contributed to its pharmacology being understudied. Here, we use cryo-EM to localize the GABA A receptor binding sites of methaqualone and its more potent derivative, PPTQ, to the same intersubunit transmembrane sites targeted by the general anesthetics propofol and etomidate. Both methaqualone and PPTQ insert more deeply into subunit interfaces than the previously-characterized modulators. Binding of quinazolinones to this site results in widening of the extracellular half of the ion-conducting pore, following a trend among positive allosteric modulators in destabilizing the hydrophobic activation gate in the pore as a mechanism for receptor potentiation. These insights shed light on the underexplored pharmacology of quinazolinones and further elucidate the molecular mechanisms of allosteric GABA A receptor modulation through transmembrane binding sites.
Abstract
γ-Aminobutyric acid type A (GABA
A
) receptors mediate fast inhibitory signaling in the brain and are targets of numerous drugs and endogenous neurosteroids. A subset of neurosteroids are ...GABA
A
receptor positive allosteric modulators; one of these, allopregnanolone, is the only drug approved specifically for treating postpartum depression. There is a consensus emerging from structural, physiological and photolabeling studies as to where positive modulators bind, but how they potentiate GABA activation remains unclear. Other neurosteroids are negative modulators of GABA
A
receptors, but their binding sites remain debated. Here we present structures of a synaptic GABA
A
receptor bound to allopregnanolone and two inhibitory sulfated neurosteroids. Allopregnanolone binds at the receptor-bilayer interface, in the consensus potentiator site. In contrast, inhibitory neurosteroids bind in the pore. MD simulations and electrophysiology support a mechanism by which allopregnanolone potentiates channel activity and suggest the dominant mechanism for sulfated neurosteroid inhibition is through pore block.
Significance Transient receptor potential (TRP) families are front-line channels that transduce various physical or chemical stimuli into ion fluxes. TRP subunits have architecture similar to that of ...voltage-gated K ⁺ (Kv) channels, but most have an additional TRP-domain helix of unknown function. An invariant tryptophan in this helix forms a hydrogen bond with a residue that begins another helix, the S4–S5 linker, which is the lever that operates the channel gate in Kv channels. By electrophysiological and other examinations of TRP vanilloid subfamily, member 4 (TRPV4) channels mutated at the two bonding partners, we show that this bond is needed to stabilize the closed conformation. Destabilized channels leak ions, explaining the harmful phenotypes of mutant TRPV4 in budding yeast (growth stoppage) and in human (bone-development blockage).
Unlike other cation channels, each subunit of most transient receptor potential (TRP) channels has an additional TRP-domain helix with an invariant tryptophan immediately trailing the gate-bearing S6. Recent cryo-electron microscopy of TRP vanilloid subfamily, member 1 structures revealed that this domain is a five-turn amphipathic helix, and the invariant tryptophan forms a bond with the beginning of the four-turn S4–S5 linker helix. By homology modeling, we identified the corresponding L596–W733 bond in TRP vanilloid subfamily, member 4 (TRPV4). The L596P mutation blocks bone development in Kozlowski-type spondylometaphyseal dysplasia in human. Our previous screen also isolated W733R as a strong gain-of-function (GOF) mutation that suppresses growth when the W733R channel is expressed in yeast. We show that, when expressed in Xenopus oocytes, TRPV4 with the L596P or W733R mutation displays normal depolarization-induced activation and outward rectification. However, these mutant channels have higher basal open probabilities and limited responses to the agonist GSK1016790A, explaining their biological GOF phenotypes. In addition, W733R current fails to inactivate during depolarization. Systematic replacement of W733 with amino acids of different properties produced similar electrophysiological and yeast phenotypes. The results can be interpreted consistently in the context of the homology model of TRPV4 molecule we have developed and refined using simulations in explicit medium. We propose that this bond maintains the orientation of the S4–S5 linker to keep the S6 gate closed. Further, the two partner helices, both amphipathic and located at the polar–nonpolar interface of the inner lipid monolayer, may receive and integrate various physiological stimuli.
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