ARTICLE Received 4 Nov 2014 | Accepted 3 Mar 2015 | Published 20 Apr 2015 DOI: 10.1038/ncomms7829 OPEN The desensitization gate of inhibitory Cys-loop receptors Marc Gielen1,w, Philip Thomas1 & Trevor G. Smart1 Cys-loop neurotransmitter-gated ion channels are vital for communication throughout the nervous system. Following activation, these receptors enter into a desensitized state in which the ion channel shuts even though the neurotransmitter molecules remain bound. To date, the molecular determinants underlying this most fundamental property of Cys-loop receptors have remained elusive. Here we present a generic mechanism for the desensitization of Cys-loop GABAA (GABAARs) and glycine receptors (GlyRs), which both mediate fast inhibitory synaptic transmission. Desensitization is regulated by interactions between the second and third transmembrane segments, which affect the ion channel lumen near its intracellular end. The GABAAR and GlyR pore blocker picrotoxin prevented desensitization, consistent with its deep channel-binding site overlapping a physical desensitization gate. 1 Department of Neuroscience, Physiology & Pharmacology, University College London, Gower Street, London WC1E 6BT, UK. w Present address: Institut Pasteur, Channel-Receptor Unit, 25 rue du Docteur Roux, 75724 Paris, France. Correspondence and requests for materials should be addressed to T.G.S. (email: [email protected]) or to M.G. (email: [email protected]). NATURE COMMUNICATIONS | 6:6829 | DOI: 10.1038/ncomms7829 | www.nature.com/naturecommunications 1 & 2015 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7829 he presynaptic release of neurotransmitters is a prelude to supported large GABA currents with limited desensitization, their diffusion across the synaptic cleft and subsequent comparable to the profile for wild-type r1 (Fig. 1a, see Table 1 for Tactivation of postsynaptic ionotropic receptors, which all time constants and extents of desensitiszation data and are responsible for fast chemical neurotransmission. Agonist Supplementary Table 1 for GABA current amplitudes), thereby binding initiates the rapid opening of ligand-gated ion channels locating the main determinants of desensitization to the permitting a selective flow of ions through the pore. The transmembrane and/or intracellular regions of the receptor. sustained presence of the neurotransmitter will cause Our primary measure of desensitization involved monitoring ligand-gated channels to transit from the active open-channel the decline of currents activated by maximal saturating agonist-bound conformation to a desensitized shut-channel, concentrations of GABA. To ensure that the mutations did not agonist-bound state, thereby limiting current flow1,2. Receptor adversely perturb the general structure, function or long-term desensitization is a fundamental property of most ligand-gated trafficking of the receptor, we monitored both the dose–response ion channels and can have profound physiological consequences. curves and the recovery phase of the currents from desensitiza- These include the progressive reduction of postsynaptic current tion for selected mutants. Overall, the concentration–response 2–5 on repetitive synaptic neurotransmitter release ; the pre- curves and EC50 values were minimally affected by the mutations, desensitization of receptors by low ambient concentrations of though certain GABA receptor constructs did generate slightly neurotransmitter due to spillover from active neighbouring lower EC50 values, in accord with an increase in affinity that is synapses6; and the slowing of synaptic current decays leading to often associated with desensitized receptors (Supplementary their prolongation5. Fig. 2). Recoveries of the maximal peak GABA currents following In the central nervous system, neuronal activity results from a desensitization were also similar for wild-type and singly mutated balance between excitation and inhibition. This is largely dictated receptors, with complete recovery from maximal desensitization by the activity of excitatory ionotropic glutamate receptors, and always achieved in mins (Supplementary Fig. 3). In addition, we 7 inhibition caused by GABA and glycine activating GABAA have reported both the rates and extent of desensitization for all (GABAARs) and glycine receptors (GlyRs). These latter receptors mutants to gain an overall view of the desensitization process belong to the superfamily of pentameric Cys-loop receptors that (Table 1). also comprises excitatory nicotinic acetylcholine (nAChRs) and 7 serotonin receptors (5HT3R) . Differential desensitization kinetics of excitatory and inhibitory receptors has the potential Internal end of M3 and M1–M2 linker control desensitization. to profoundly affect the spike firing profiles of neurons5 and thus We refined our search using homomeric r1 receptors and neural network activity. At the molecular level, desensitization of sequentially replacing individual transmembrane segments, M1 to AMPA and kainate classes of ionotropic GluRs is relatively well M3, with those from a1. These constructs yielded very small understood and involves structural rearrangements at the dimer currents, precluding analysis; however, one chimera incorporat- interface between adjacent extracellular agonist-binding ing the intracellular end of M3, the M3–M4 intracellular linker 8–11 D346 domains . However, for the Cys-loop GABAARs and GlyRs, and M4 of a1 into the r1 subunit (r1 -a1), produced robust although the structure–function studies have improved our currents, which strongly desensitized (%Des ¼ 94% and 7,12 understanding of how these receptors activate , the molecular tW ¼ 200 ms, Fig. 1b). determinants underlying the process of desensitization have yet to Surprisingly, incorporating just M4 from the a1 subunit did be defined. not affect the desensitizing phenotype of r1(r1D433-a1, Fig. 1b). Here we show that for the inhibitory neurotransmitter GABAA We therefore targeted M3 in r1 and replaced six contiguous and glycine receptors, the mechanism of agonist-induced residues at the carboxyl-terminal (C-terminal; intracellular) end desensitization is regulated by residue interactions between the of M3 with the homologous residues from a1. This produced a transmembrane domains of the receptor, resulting in a time- receptor (r1a1(D6-postM3)) that desensitized profoundly within dependent constriction of the ion channel pore that reduces the one second following activation by GABA (Fig. 1b). Within this agonist-activated membrane conductance. This constriction has six amino-acid cassette, substituting just one residue, r1T349K, the characteristics of a desensitization ‘gate’ that is discrete from was sufficient to confer a rapid desensitizing profile on r1 the ion channel gate involved in ligand-gated channel opening. receptors (Fig. 1b). Interestingly, on the r1a1(D6 À postM3) background, incorporat- ing the M1–M2 intracellular linker from the b2 subunit Results considerably reduced desensitization towards that for wild-type b2(M1–M2 link) þ a1(D6-postM3) Desensitization profiles differ between GABAA receptors.To r1 (chimera r1 , Fig. 1b). Such a locate the molecular components responsible for desensitization, reversion was not observed after incorporating the a1 M1–M2 we selected two GABAAR isoforms that differ markedly in their linker (Supplementary Fig. 4). These data indicate that the response to supersaturating GABA concentrations (10 mM) by intracellular end of M3 and the M1–M2 linker of b2 modulate either desensitizing significantly (a1b2 heteromers; percentage of desensitization, conceivably via an intersubunit (a–b) interaction, peak current desensitization (% Des) ¼ 86% and weighted decay which is consistent with the close proximity of a1 M3 C-terminal time constant (tW) ¼ 17 s) or minimally (r1 homomers, 19% Des end to the M1–M2 linker of an adjacent b2 subunit, as seen in our and 23 s, Table 1, Fig. 1a). We then constructed chimeras to first structural models (see below). explore the role of the extracellular domain (ECD) by replacing We subsequently examined this potential interaction in a1b2 D260 the ECD of a1 with the homologous section from r1(r1 -a1; GABAARs by first introducing the M1–M2 linker of r1 into b2 Supplementary Fig. 1). The desensitization profile was similar to subunits (b2r1(M1–M2 link)). Co-expressing this chimera with that observed for wild-type a1b2 receptors (Fig. 1a), consistent wild-type a1 produced receptors that, unexpectedly, desensitized with published data13. Next we built chimeras containing M1 to almost completely at a rate 50-fold faster than wild-type a1b2 M4 and the intracellular linkers (M1–M2 and M3–M4) from receptors (Fig. 1c). Moreover, exchanging eight residues from the r1, coupled to the entire extracellular region of a1orb2, intracellular end of M3 in a1 and b2 with those from r1 resulted corresponding to their ECDs and the external M2–M3 linkers in a modest three-fold increase in the desensitization rate for (chimeras a1EXT-r1TM þ INT and b2EXT-r1TM þ INT, respectively; a1r1(D8-postM3)b2r1(D8-postM3) (Fig. 1c). However, on this Supplementary Fig. 1). Co-expression of these chimeras background, reintroducing the M1–M2 linker of r1 into b2 2 NATURE COMMUNICATIONS | 6:6829 | DOI: 10.1038/ncomms7829 | www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7829 ARTICLE Table 1 | Weighted decay time constant for desensitization (tW) and extent of