Electrically silent mutants unravel the mechanism of binding-gating coupling in Cys-loop receptors.

The transduction of extracellular chemical signals into intracellular events relies on the communication between neighboring domains of membrane receptors. In the particular case of Cys-loop receptor channels, five short stretches of amino acids, one per subunit, link the extracellular and transmembrane domains in such a way that the ion permeability of the latter and the affinity for neurotransmitters of the former become tied to each other. Here, using direct functional approaches, we set out to understand the molecular bases of this crucial interdependence through the characterization of total loss-of-current mutations at the interface between domains.

An experimental test of the nicotinic hypothesis of COVID-19.

The pathophysiological mechanisms underlying the constellation of symptoms that characterize COVID-19 are only incompletely understood. In an effort to fill these gaps, a "nicotinic hypothesis," which posits that nicotinic acetylcholine receptors (AChRs) act as additional severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptors, has recently been put forth. A key feature of the proposal (with potential clinical ramifications) is the suggested competition between the virus' spike protein and small-molecule cholinergic ligands for the receptor's orthosteric binding sites.

Structure and function at the lipid-protein interface of a pentameric ligand-gated ion channel.

Although it has long been proposed that membrane proteins may contain tightly bound lipids, their identity, the structure of their binding sites, and their functional and structural relevance have remained elusive. To some extent, this is because tightly bound lipids are often located at the periphery of proteins, where the quality of density maps is usually poorer, and because they may be outcompeted by detergent molecules used during standard purification procedures. As a step toward characterizing natively bound lipids in the superfamily of pentameric ligand-gated ion channels (pLGICs), we applied single-particle cryogenic electron microscopy to fragments of native membrane obtained in the complete absence of detergent-solubilization steps.

Signal transduction through Cys-loop receptors is mediated by the nonspecific bumping of closely apposed domains.

One of the most fundamental questions in the field of Cys-loop receptors (pentameric ligand-gated ion channels, pLGICs) is how the affinity for neurotransmitters and the conductive/nonconductive state of the transmembrane pore are correlated despite the ∼60-Å distance between the corresponding domains. Proposed mechanisms differ, but they all converge into the idea that interactions between wild-type side chains across the extracellular-transmembrane-domain (ECD-TMD) interface are crucial for this phenomenon. Indeed, the successful design of fully functional chimeras that combine intact ECD and TMD modules from different wild-type pLGICs has commonly been ascribed to the residual conservation of sequence that exists at the level of the interfacial loops even between evolutionarily distant parent channels.

Cryo-EM structures of a lipid-sensitive pentameric ligand-gated ion channel embedded in a phosphatidylcholine-only bilayer.

The lipid dependence of the nicotinic acetylcholine receptor from the electric organ has long been recognized, and one of the most consistent experimental observations is that, when reconstituted in membranes formed by zwitterionic phospholipids alone, exposure to agonist fails to elicit ion-flux activity. More recently, it has been suggested that the bacterial homolog ELIC ( ligand-gated ion channel) has a similar lipid sensitivity. As a first step toward the elucidation of the structural basis of this phenomenon, we solved the structures of ELIC embedded in palmitoyl-oleoyl-phosphatidylcholine- (POPC-) only nanodiscs in both the unliganded (4.

Chasing the open-state structure of pentameric ligand-gated ion channels.

Remarkable advances have been made toward the structural characterization of ion channels in the last two decades. However, the unambiguous assignment of well-defined functional states to the obtained structural models has proved challenging. In the case of the superfamily of nicotinic-receptor channels (also referred to as pentameric ligand-gated ion channels [pLGICs]), for example, two different types of model of the open-channel conformation have been proposed on the basis of structures solved to resolutions better than 4.

Identifying the elusive link between amino acid sequence and charge selectivity in pentameric ligand-gated ion channels.

Among neurotransmitter-gated ion channels, the superfamily of pentameric ligand-gated ion channels (pLGICs) is unique in that its members display opposite permeant-ion charge selectivities despite sharing the same structural fold. Although much effort has been devoted to the identification of the mechanism underlying the cation-versus-anion selectivity of these channels, a careful analysis of past work reveals that discrepancies exist, that different explanations for the same phenomenon have often been put forth, and that no consensus view has yet been reached. To elucidate the molecular basis of charge selectivity for the superfamily as a whole, we performed extensive mutagenesis and electrophysiological recordings on six different cation-selective and anion-selective homologs from vertebrate, invertebrate, and bacterial origin.

Engineered Ionizable Side Chains.

One of the great challenges of mechanistic ion-channel biology is to obtain structural information from well-defined functional states. In the case of neurotransmitter-gated ion channels, the open-channel conformation is particularly elusive owing to its transient nature and brief mean lifetime. In this Chapter, we show how the analysis of single-channel currents recorded from mutants engineered to contain single ionizable side chains in the transmembrane region can provide specific information about the open-channel conformation without any interference from the closed or desensitized conformations.

The unanticipated complexity of the selectivity-filter glutamates of nicotinic receptors.

In ion channels, 'rings' of ionized side chains that decorate the walls of the permeation pathway often lower the energetic barrier to ion conduction. Using single-channel electrophysiological recordings, we studied the poorly understood ring of four glutamates (and one glutamine) that dominates this catalytic effect in the muscle nicotinic acetylcholine receptor ('the intermediate ring of charge'). We show that all four wild-type glutamate side chains are deprotonated in the range of 6.

Estimating the pKa values of basic and acidic side chains in ion channels using electrophysiological recordings: a robust approach to an elusive problem.

As a step toward gaining a better understanding of the physicochemical bases of pK(a)-value shifts in ion channels, we have previously proposed a method for estimating the proton affinities of systematically engineered ionizable side chains from the kinetic analysis of single-channel current recordings. We reported that the open-channel current flowing through mutants of the (cation-selective) muscle nicotinic acetylcholine receptor (AChR) engineered to bear single basic residues in the transmembrane portion of the pore domain fluctuates between two levels of conductance. Our observations were consistent with the idea that these fluctuations track directly the alternate protonation-deprotonation of basic side chains: protonation of the introduced basic group would attenuate the single-channel conductance, whereas its deprotonation would restore the wild-type-like level.

Tunable pKa values and the basis of opposite charge selectivities in nicotinic-type receptors.

Among ion channels, only the nicotinic-receptor superfamily has evolved to generate both cation- and anion-selective members. Although other, structurally unrelated, neurotransmitter-gated cation channels exist, no other type of neurotransmitter-gated anion channel, and thus no other source of fast synaptic inhibitory signals, has been described so far. In addition to the seemingly straightforward electrostatic effect of the presence (in the cation-selective members) or absence (in the anion-selective ones) of a ring of pore-facing carboxylates, mutational studies have identified other features of the amino-acid sequence near the intracellular end of the pore-lining transmembrane segments (M2) that are also required to achieve the high charge selectivity shown by native channels.

Decremental response to high-frequency trains of acetylcholine pulses but unaltered fractional Ca2+ currents in a panel of "slow-channel syndrome" nicotinic receptor mutants.

The slow-channel congenital myasthenic syndrome (SCCMS) is a disorder of the neuromuscular junction caused by gain-of-function mutations to the muscle nicotinic acetylcholine (ACh) receptor (AChR). Although it is clear that the slower deactivation time course of the ACh-elicited currents plays a central role in the etiology of this disease, it has been suggested that other abnormal properties of these mutant receptors may also be critical in this respect. We characterized the kinetics of a panel of five SCCMS AChRs (alphaS269I, betaV266M, epsilonL221F, epsilonT264P, and epsilonL269F) at the ensemble level in rapidly perfused outside-out patches.

Pore-opening mechanism of the nicotinic acetylcholine receptor evinced by proton transfer.

The conformational changes underlying cysteine-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 have applied this methodology to the entire M1 and M3 segments of the muscle nicotinic acetylcholine receptor, two transmembrane alpha-helices that pack against the pore-lining M2 alpha-helix.

Desensitization contributes to the synaptic response of gain-of-function mutants of the muscle nicotinic receptor.

Although the muscle nicotinic receptor (AChR) desensitizes almost completely in the steady presence of high concentrations of acetylcholine (ACh), it is well established that AChRs do not accumulate in desensitized states under normal physiological conditions of neurotransmitter release and clearance. Quantitative considerations in the framework of plausible kinetic schemes, however, lead us to predict that mutations that speed up channel opening, slow down channel closure, and/or slow down the dissociation of neurotransmitter (i.e.

Probing ion-channel pores one proton at a time.

Although membrane proteins often rely on ionizable residues for structure and function, their ionization states under physiological conditions largely elude experimental estimation. To gain insight into the effect of the local microenvironment on the proton affinity of ionizable residues, we have engineered individual lysines, histidines and arginines along the alpha-helical lining of the transmembrane pore of the nicotinic acetylcholine receptor. We can detect individual proton binding-unbinding reactions electrophysiologically at the level of a single proton on a single side chain as brief blocking-unblocking events of the passing cation current.

Dynamics of the acetylcholine receptor pore at the gating transition state.

Neuromuscular acetylcholine receptors (AChRs) are ion channels that alternatively adopt stable conformations that either allow (open) or prohibit (closed) ionic conduction. We probed the dynamics of pore (M2) residues at the diliganded gating transition state by using single-channel kinetic and rate-equilibrium free energy relationship (phi-value) analyses of mutant AChRs. The mutations were at the equatorial (9') position of the alpha, beta, and epsilon subunits (n = 15) or at sites between the equator and the extracellular domain in the alpha-subunit (n = 8).

Structure of the transition state of gating in the acetylcholine receptor channel pore: a phi-value analysis.

The gating mechanism of the acetylcholine receptor channel (AChR) was investigated by using rate equilibrium linear free energy relationships (LFERs) to probe the transition state between the closed and open conformations. The properties of the transition state of gating in the second transmembrane segment (M2) of the delta subunit, one of the five homologous pore-lining segments, was measured on a residue-by-residue basis. Series of point mutations were engineered at individual positions of this domain, and the corresponding constructs were characterized electrophysiologically, at the single-channel level.