Structural mobility tunes signalling of the GluA1 AMPA glutamate receptor.

AMPA glutamate receptors (AMPARs), the primary mediators of excitatory neurotransmission in the brain, are either GluA2 subunit-containing and thus Ca2+-impermeable, or GluA2-lacking and Ca2+-permeable1. Despite their prominent expression throughout interneurons and glia, their role in long-term potentiation and their involvement in a range of neuropathologies2, structural information for GluA2-lacking receptors is currently absent. Here we determine and characterize cryo-electron microscopy structures of the GluA1 homotetramer, fully occupied with TARPγ3 auxiliary subunits (GluA1/γ3). The gating core of both resting and open-state GluA1/γ3 closely resembles GluA2-containing receptors. However, the sequence-diverse N-terminal domains (NTDs) give rise to a highly mobile assembly, enabling domain swapping and subunit re-alignments in the ligand-binding domain tier that are pronounced in desensitized states. These transitions underlie the unique kinetic properties of GluA1. A GluA2 mutant (F231A) increasing NTD dynamics phenocopies this behaviour, and exhibits reduced synaptic responses, reflecting the anchoring function of the AMPAR NTD at the synapse. Together, this work underscores how the subunit-diverse NTDs determine subunit arrangement, gating properties and ultimately synaptic signalling efficiency among AMPAR subtypes.

Acidic pH reduces agonist efficacy and responses to synaptic-like glycine applications in zebrafish α1 and rat α1ß recombinant glycine receptors.

Many pentameric ligand-gated ion channels are modulated by extracellular pH. Glycine receptors (GlyRs) share this property, but it is not well understood how they are affected by pH changes. Whole cell experiments on HEK293 cells expressing zebrafish homomeric α1 GlyR confirmed previous reports that acidic pH (6.4) reduces GlyR sensitivity to glycine, whereas alkaline pH (8.4) has small or negligible effects. In addition to that, at pH 6.4 we observed a reduction in the maximum responses to the partial agonists ß-alanine and taurine relative to the full agonist glycine. In cell-attached single-channel recording, low pH reduced agonist efficacy, as the maximum open probability decreased from 0.97, 0.91 and 0.66 to 0.93, 0.57 and 0.34 for glycine, ß-alanine and taurine, respectively, reflecting a threefold decrease in efficacy equilibrium constants for all three agonists. We also tested the effect of pH 6.4 in conditions that replicate those at the native synapse, recording outside-out currents elicited by fast application of millisecond pulses of agonists on α1 and α1ß GlyR, at a range of intracellular chloride concentrations. Acidic pH reduced the area under the curve of the currents, by reducing peak amplitude, slowing activation and speeding deactivation. Our results show that acidification of the extracellular pH by one unit, as may occur in pathological conditions such as ischaemia, impairs GlyR gating and is likely to reduce the effectiveness of glycinergic synaptic inhibition. KEY POINTS: Extracellular pH in the central nervous system (CNS) is known to shift towards acidic values during pathophysiological conditions such as ischaemia and seizures. Acidic extracellular pH is known to affect GABAergic inhibitory synapses, but its effect on signals mediated by glycine receptors (GlyR) is not well characterised. Moderate acidic conditions (pH 6.4) reduce the maximum single channel open probability of recombinant homomeric GlyRs produced by the neurotransmitter glycine or other agonists, such as ß-alanine and taurine. When glycine was applied with a piezoelectric stepper to outside out patches, to simulate its fast rise and short duration at the synapse, responses became shorter and smaller at pH 6.4. The effect was also observed with physiologically low intracellular chloride and in mammalian heteromeric GlyRs. This suggests that acidic pH is likely to reduce the strength of inhibitory signalling at glycinergic synapses.

The intracellular domain of homomeric glycine receptors modulates agonist efficacy.

Like other pentameric ligand-gated channels, glycine receptors (GlyRs) contain long intracellular domains (ICDs) between transmembrane helices 3 and 4. Structurally characterized GlyRs are generally engineered to have a very short ICD. We show here that for one such construct, zebrafish GlyREM, the agonists glycine, ß-alanine, taurine, and GABA have high efficacy and produce maximum single-channel open probabilities greater than 0.9. In contrast, for full-length human α1 GlyR, taurine and GABA were clearly partial agonists, with maximum open probabilities of 0.46 and 0.09, respectively. We found that the elevated open probabilities in GlyREM are not due to the limited sequence differences between the human and zebrafish orthologs, but rather to replacement of the native ICD with a short tripeptide ICD. Consistent with this interpretation, shortening the ICD in the human GlyR increased the maximum open probability produced by taurine and GABA to 0.90 and 0.70, respectively, but further engineering it to resemble GlyREM (by introducing the zebrafish transmembrane helix 4 and C terminus) had no effect. Furthermore, reinstating the native ICD to GlyREM converted taurine and GABA to partial agonists, with maximum open probabilities of 0.66 and 0.40, respectively. Structural comparison of transmembrane helices 3 and 4 in short- and long-ICD GlyR subunits revealed that ICD shortening does not distort the orientation of these helices within each subunit. This suggests that the effects of shortening the ICD stem from removing a modulatory effect of the native ICD on GlyR gating, revealing a new role for the ICD in pentameric ligand-gated channels.

Salt Gradient Modulation of MicroRNA Translocation through a Biological Nanopore.

In resistive pulse sensing of microRNA biomarkers, selectivity is achieved with polynucleotide-extended DNA probes, with the unzipping of a miRNA-DNA duplex in the nanopore recorded as a resistive current pulse. As the assay sensitivity is determined by the pulse frequency, we investigated the effect of cis/trans electrolyte concentration gradients applied over α-hemolysin nanopores. KCl gradients were found to exponentially increase the pulse frequency, while reducing the preference for 3'-first pore entry of the duplex and accelerating duplex unzipping, all manifestations of an enhanced electrophoretic force. Unlike silicon nitride pores, a counteracting contribution from electro-osmotic flow along the pore wall was not apparent. Significantly, a gradient of 0.5/4 M KCl increased the pulse frequency ∼60-fold with respect to symmetrical 1 M KCl, while the duplex dwell time in the nanopore remained acceptable for pulse detection and could be extended by LiCl addition. Steeper gradients caused lipid bilayer destabilization and pore instability, limiting the total number of recorded pulses. The 8-fold KCl gradient enabled a linear relationship between pulse frequency and miRNA concentration for the range of 0.1-100 nM. This work highlights differences between biological and solid-state nanopore sensing and provides strategies for subnanomolar miRNA quantification with bilayer-embedded porins.

A Low-Noise Transimpedance Amplifier for BLM-Based Ion Channel Recording.

High-throughput screening (HTS) using ion channel recording is a powerful drug discovery technique in pharmacology. Ion channel recording with planar bilayer lipid membranes (BLM) is scalable and has very high sensitivity. A HTS system based on BLM ion channel recording faces three main challenges: (i) design of scalable microfluidic devices; (ii) design of compact ultra-low-noise transimpedance amplifiers able to detect currents in the pA range with bandwidth >10 kHz; (iii) design of compact, robust and scalable systems that integrate these two elements. This paper presents a low-noise transimpedance amplifier with integrated A/D conversion realized in CMOS 0.35 µm technology. The CMOS amplifier acquires currents in the range ±200 pA and ±20 nA, with 100 kHz bandwidth while dissipating 41 mW. An integrated digital offset compensation loop balances any voltage offsets from Ag/AgCl electrodes. The measured open-input input-referred noise current is as low as 4 fA/√Hz at ±200 pA range. The current amplifier is embedded in an integrated platform, together with a microfluidic device, for current recording from ion channels. Gramicidin-A, α-haemolysin and KcsA potassium channels have been used to prove both the platform and the current-to-digital converter.

Characterisation of cell membrane interaction mechanisms of antimicrobial peptides by electrical bilayer recording.

Many antimicrobial peptides (AMPs) are cationic host defence peptides (HDPs) that interact with microbial membranes. This ability may lead to implementation of AMPs as therapeutics to overcome the wide-spread antibiotic resistance problem as the affected bacteria may not be able to recover from membrane lysis types of attack. AMP interactions with lipid bilayer membranes are typically explained through three mechanisms, i.e., barrel-stave pore, toroidal pore and carpet models. Electrical bilayer recording is a relatively simple and sensitive technique that is able to capture the nanoscale perturbations caused by the AMPs in the bilayer membranes. Molecular-level understanding of the behaviour of AMPs in relation to lipid bilayers mimicking bacterial and human cell membranes is essential for their development as novel therapeutic agents that are capable of targeted action against disease causing micro-organisms. The effects of four AMPs (aurein 1.2, caerin 1.1, citropin 1.1 and maculatin 1.1 from the skin secretions of Australian tree frogs) and the toxin melittin (found in the venom of honeybees) on two different phospholipid membranes were studied using the electrical bilayer recording technique. Bilayers composed of zwitterionic (DPhPC) and anionic (DPhPC/POPG) lipids were used to mimic the charge of eukaryotic and prokaryotic cell membranes, respectively, so as to determine the corresponding interaction mechanisms for different concentrations of the peptide. Analysis of the dataset corresponding to the four frog AMPs, as well as the resulting dataset corresponding to the bee toxin, confirms the proposed peptide-bilayer interaction models in existing publications and demonstrates the importance of using appropriate bilayer compositions and peptide concentrations for AMP studies.

Aminomethanesulfonic acid illuminates the boundary between full and partial agonists of the pentameric glycine receptor.

To clarify the determinants of agonist efficacy in pentameric ligand-gated ion channels, we examined a new compound, aminomethanesulfonic acid (AMS), a molecule intermediate in structure between glycine and taurine. Despite wide availability, to date there are no reports of AMS action on glycine receptors, perhaps because AMS is unstable at physiological pH. Here, we show that at pH 5, AMS is an efficacious agonist, eliciting in zebrafish α1 glycine receptors a maximum single-channel open probability of 0.85, much greater than that of ß-alanine (0.54) or taurine (0.12), and second only to that of glycine itself (0.96). Thermodynamic cycle analysis of the efficacy of these closely related agonists shows supra-additive interaction between changes in the length of the agonist molecule and the size of the anionic moiety. Single particle cryo-electron microscopy structures of AMS-bound glycine receptors show that the AMS-bound agonist pocket is as compact as with glycine, and three-dimensional classification demonstrates that the channel populates the open and the desensitized states, like glycine, but not the closed intermediate state associated with the weaker partial agonists, ß-alanine and taurine. Because AMS is on the cusp between full and partial agonists, it provides a new tool to help us understand agonist action in the pentameric superfamily of ligand-gated ion channels.