Remigijus Lape

On the nature of partial agonism in the nicotinic receptor superfamily

Partial agonists are ligands that bind to receptors but produce only a small maximum response even at concentrations where all receptors are occupied. In the case of ligand-activated ion channels, it has been supposed since 1957 that partial agonists evoke a small response because they are inefficient at eliciting the change of conformation between shut and open states of the channel. We have investigated partial agonists for two members of the nicotinic superfamily—the muscle nicotinic acetylcholine receptor and the glycine receptor—and find that the open–shut reaction is similar for both full and partial agonists, but the response to partial agonists is limited by an earlier conformation change (‘flipping’) that takes place while the channel is still shut. This has implications for the interpretation of structural studies, and in the future, for the design of partial agonists for therapeutic use.

The α1K276E Startle Disease Mutation Reveals Multiple Intermediate States in the Gating of Glycine Receptors

Loss-of-function mutations in human glycine receptors cause hyperekplexia, a rare inherited disease associated with an exaggerated startle response. We have studied a human disease mutation in the M2–M3 loop of the glycine receptor α1 subunit (K276E) using direct fitting of mechanisms to single-channel recordings with the program HJCFIT. Whole-cell recordings from HEK293 cells showed the mutation reduced the receptor glycine sensitivity. In single-channel recordings, rat homomeric α1 K276E receptors were barely active, even at 200 mm glycine. Coexpression of the β subunit partially rescued channel function. Heteromeric mutant channels opened in brief bursts at 300 μm glycine (a concentration that is near-maximal for wild type) and reached a maximum one-channel open probability of about 45% at 100 mm glycine (compared to 96% for wild type). Distributions of apparent open times contained more than one component in high glycine and, therefore, could not be described by mechanisms with only one fully liganded open state. Fits to the data were much better with mechanisms in which opening can also occur from more than one fully liganded intermediate (e.g., “primed” models). Brief pulses of glycine (∼3 ms, 30 mm) applied to mutant channels in outside-out patches activated currents with a slower rise time (1.5 ms) than those of wild-type channels (0.2 ms) and a much faster decay. These features were predicted reasonably well by the mechanisms obtained from fitting single-channel data. Our results show that, by slowing and impairing channel gating, the K276E mutation facilitates the detection of closed reaction intermediates in the activation pathway of glycine channels.

Agonist and blocking actions of choline and tetramethylammonium on human muscle acetylcholine receptors

Choline has been used widely as an agonist for the investigation of gain‐of‐function mutants of the nicotinic acetylcholine receptor. It is useful because it behaves like a partial agonist. The efficacy of choline is difficult to measure because choline blocks the channel at concentrations about four times lower than those that activate it. We have fitted activation mechanisms to single‐channel activity elicited from HEK‐expressed human recombinant muscle nicotinic receptors by choline and by tetramethylammonium (TMA). Channel block by the agonist was incorporated into the mechanisms that were fitted, and block was found not to be selective for the open state. The results also suggest that channel block is very fast and that the channel can shut almost as fast as normal when the blocker was bound. Single‐channel data are compatible with a mechanism in which choline is actually a full agonist, its maximum response being limited only by channel block. However, they are also compatible with a mechanism incorporating a pre‐opening conformation change (‘flip’) in which choline is a genuine partial agonist. The latter explanation is favoured by concentration jump experiments, and by the fact that only this mechanism fits the TMA data. We propose that choline, like TMA, is a partial agonist because it is very ineffective (approximately 600‐fold less than acetylcholine) at eliciting the initial, pre‐opening conformation change. Once flipping has occurred, all agonists, even choline, open the channel with similar efficiency.

Perspectives on: Conformational coupling in ion channels: Allosteric coupling in ligand-gated ion channels

### What does “allosteric” mean? In the context of receptors, the word allosteric is now widely used. It is, perhaps, not helpful for clarity of thought that different authors often use it to mean somewhat different things ([Colquhoun, 1998][1]). At one extreme, the term “allosteric

Agonist and Antagonist Binding in Human Glycine Receptors

The human glycine receptor (hGlyR) is an anion-permeable ligand-gated channel that is part of a larger superfamily of receptors called the Cys-loop family. hGlyRs are particularly amenable to single-channel recordings, thus making them a model experimental system for understanding the Cys-loop receptor family in general. Understanding the relationship between agonist binding and efficacy in Cys-loop receptors should improve our future prospects for making specific agonists or antagonists. However, at present, there is no high-resolution structure for the complete hGlyR, and thus, modeling is needed to provide a physical framework on which to interpret single-channel data. The structure of the glutamate-gated chloride channel from Caenorhabditis elegans shows a much higher level of sequence identity to human hGlyR than previous templates such as AChBP or the bacterial channels, GLIC and ELIC. Thus, we constructed a model of the hGlyR and validated it against previously reported mutagenesis data. We used molecular dynamics to refine the model and to explore binding of both an agonist (glycine) and an antagonist (strychnine). The model shows excellent agreement with previous data but also suggests some unique features: (i) a water molecule that forms part of the binding site and allows us to account for some previous results that were difficult to reconcile, (ii) an interaction of the glycine agonist with S129, and (iii) an effect from E211, both of which we confirmed with new site-directed mutagenesis and patch clamp recordings. Finally, examination of the simulations suggests that strychnine binding induces movement to a conformational state distinct from the glycine-bound or apo state, not only within the ligand-binding domain but also in the transmembrane domain.

The long activations of α2 glycine channels can be described by a mechanism with reaction intermediates (“flip”)

The α2 glycine receptor (GlyR) subunit, abundant in embryonic neurons, is replaced by α1 in the adult nervous system. The single-channel activity of homomeric α2 channels differs from that of α1-containing GlyRs, as even at the lowest glycine concentration (20 µM), openings occurred in long (>300-ms) groups with high open probability (Popen; 0.96; cell-attached recordings, HEK-expressed channels). Shut-time intervals within groups of openings were dominated by short shuttings of 5–10 µs. The lack of concentration dependence in the groups of openings suggests that they represent single activations, separated by very long shut times at low concentrations. Several putative mechanisms were fitted by maximizing the likelihood of the entire sequence of open and shut times, with exact missed-events allowance (program hjcfit). Records obtained at several glycine concentrations were fitted simultaneously. The adequacy of the different schemes was judged by the accuracy with which they predicted not only single-channel data but also the time course and concentration dependence of macroscopic responses elicited by rapid glycine applications to outside-out patches. The data were adequately described only with schemes incorporating a reaction intermediate in the activation, and the best was a flip mechanism with two binding sites and one open state. Fits with this mechanism showed that for α2 channels, the opening rate constant is very fast, ∼130,000 s−1, much as for α1β GlyRs (the receptor in mature synapses), but the estimated true mean open time is 20 times longer (around 3 ms). The efficacy for the flipping step and the binding affinity were lower for α2 than for α1β channels, but the overall efficacies were similar. As we previously showed for α1 homomeric receptors, in α2 glycine channels, maximum Popen is achieved when fewer than all five of the putative binding sites in the pentamer are occupied by glycine.

Mechanism of activation of the prokaryotic channel ELIC by propylamine: A single-channel study

The prokaryotic ligand-gated channel ELIC opens to two open states when maximally activated by the binding of at least two agonist molecules.

The kinetic properties of the α3 rat glycine receptor make it suitable for mediating fast synaptic inhibition

•  In the adult, most inhibitory transmission mediated by glycine uses channels containing α1 subunits, but both α1 and the related α3 subunit are present in spinal areas that process pain. •  We recorded the effect of a range of fixed glycine concentrations on the activity of individual glycine channels expressed in vitro to contain only α3 subunits. •  Glycine is very effective in opening α3 channels. At full activation, both α1 and α3 channels are open nearly 100% of the time, but α3 channels need all, or almost all, of the five glycine binding sites to be occupied, whereas α1 channels need only three. •  When channels were activated in synaptic‐like conditions (fast, 1 ms, 10 mm glycine pulses), α3 responses decayed more slowly than α1 responses. •  This difference is likely to be too small to allow α1‐ and α3‐mediated synaptic responses to be distinguishable on the basis of time course alone.

Single Ion Channels

This chapter focuses on the use of single molecule studies on single ion channels. It first reviews results obtained with single molecule fluorescence methods and compares them with single ion channel results. There are relatively few single molecule experiments on ion channels, other than single-channel recording, possibly because of the difficulty of carrying out single molecule experiments in live cells or at least in a fairly intact biological membrane preparation. A major group of studies is the application of single-particle tracking to channels, in which single channels are observed for the purpose of studying their lateral diffusion in the cell membrane and their interaction with the cytoskeleton and specific proteins such as gephyrin. The photobleaching technique has been used for the purpose of counting subunits in ion channels through imaging by total internal reflection fluorescence (TIRF) microscopy of recombinant channel molecules expressed in Xenopus oocytes at low expression density. Furthermore, the chapter describes a stochastic theory that has been developed for the interpretation of single ion channel records and discusses how it might be applied to other sorts of single molecule observations. An example is presented for an analysis of an ion channel mechanism based on single molecule observations. Single molecule techniques are increasingly being applied to ion channels, which raise the hope that they soon will contribute information to complement and enrich that obtained from electrophysiology.

The startle disease mutation E103K impairs activation of human homomeric α1 glycine receptors by disrupting an intersubunit salt bridge across the agonist binding site

Glycine receptors (GlyR) belong to the pentameric ligand-gated ion channel (pLGIC) superfamily and mediate fast inhibitory transmission in the vertebrate CNS. Disruption of glycinergic transmission by inherited mutations produces startle disease in man. Many startle mutations are in GlyRs and provide useful clues to the function of the channel domains. E103K is one of few startle mutations found in the extracellular agonist binding site of the channel, in loop A of the principal side of the subunit interface. Homology modeling shows that the side chain of Glu-103 is close to that of Arg-131, in loop E of the complementary side of the binding site, and may form a salt bridge at the back of the binding site, constraining its size. We investigated this hypothesis in recombinant human α1 GlyR by site-directed mutagenesis and functional measurements of agonist efficacy and potency by whole cell patch clamp and single channel recording. Despite its position near the binding site, E103K causes hyperekplexia by impairing the efficacy of glycine, its ability to gate the channel once bound, which is very high in wild type GlyR. Mutating Glu-103 and Arg-131 caused various degrees of loss-of-function in the action of glycine, whereas mutations in Arg-131 enhanced the efficacy of the slightly bigger partial agonist sarcosine (N-methylglycine). The effects of the single charge-swapping mutations of these two residues were largely rescued in the double mutant, supporting the possibility that they interact via a salt bridge that normally constrains the efficacy of larger agonist molecules.