The desensitization gate of inhibitory Cys-loop receptors

Abstract

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.

Figure 1. Intracellular end of M3 and the M1–M2 linker control desensitization of GABA_ARs.

(a–c) Peak-scaled membrane currents elicited by 10 mM GABA (black line), showing the desensitization phase for the indicated receptor constructs (left column). The recovery phase is omitted for clarity. See Table 1 for values of τ_w and extent of desensitization (% Des) in all figures. The subunit chimeras are depicted by a colour code: green (α1), red (β2) and grey (ρ1) (right column). Numbering refers to the position of the interface between the two subunits in the chimera. TM/M, transmembrane domain, INT, intracellular loops.

Figure 2. Mutating the intracellular end of the M2/M3 interface regulates desensitization of α1β2 GABA_ARs.

(a) Left: 3D model of an α1β2 GABA_AR based on GluCl template. Right in a–c: membrane currents induced by 10 mM GABA. (b) Left: enlarged side view of the intracellular end of the ion channel showing transmembrane domains M1–M3 for α1 (green) and β2 (red) subunits and the positions of various labelled residues. (c) Left: enlarged plan view of the ion channel. Note, in b,c, M4 segments were omitted for clarity.

Figure 3. Effects of mutations α1^L300Vβ2^L296V and α1^G258V β2^G254V on GABA sensitivity.

GABA concentration–response curves for wild-type (wt) α1β2 (EC₅₀=4.1±0.7 μM, n_H=1.08±0.07, n=6), α1^L300Vβ2^L296V (EC₅₀=7.3±0.9 μM, n_H=0.91±0.02, n=5) and α1^G258V β2^G254V receptors (EC₅₀=3.0±0.9 μM, n_H=0.89±0.19, n=5). Error bars are s.d.

Figure 4. Residues affecting desensitization in heteromeric α1β2γ2 GABA_A receptors and homomeric GlyRα1 glycine receptors.

(a) Membrane currents activated by 10 mM GABA showing desensitization of γ2 subunit-containing wild-type and mutant GABA_A receptors. (b,c) Membrane currents activated by 10 mM glycine, showing desensitization of glycine receptors, both wild type and mutants (see text for details).

Figure 5. Promoting desensitization of GABA_ARs causes fast dissociation of the pore blocker PTX.

(a) Left: side-view cutaway section through the transmembrane domains showing two (α1—green, β2—red) of the five M2 helices from our α1β2 GABA_AR model overlaid with the M2 helices of the PTX-bound GluCl structure (light blue; pdb 3RI5 (ref. 14)). Note the deep location of the PTX-binding site between locations 2′ and −2′. Right: predicted pore diameter plotted as a function of the distance along the pore axis for GluCl, GLIC (open; pdb 3EAM (ref. 18)) and ELIC (shut; pdb 2VL0 (ref. 19)). This is vertically aligned with the left panel schema for cross comparison. The dashed line reflects the hypothesis that desensitization is an extension of the activation process, leading to a desensitization gate located between positions −3′ and 4′. (b) Representative membrane currents showing slow dissociation of PTX from α1β2 GABA_ARs when activated by a low concentration of GABA (0.3 μM, n=6). (c) Membrane currents showing fast dissociation of PTX from α1β2 GABA_ARs activated by saturating concentrations of GABA (10 mM, n=5). Note the peak currents are truncated for clarity.

Figure 6. Simulation of GABA currents after block by and recovery from PTX.

(a–c) Left: kinetic models 1–3 used to predict the recovery of agonist-induced currents after wash-out of PTX (or P) in the presence of saturating concentrations of the agonist ([A]; 10 mM GABA). Receptors states are: R inactive, agonist-unbound receptor; AR inactive, agonist-bound receptor; AR* agonist-bound activated receptor; AD agonist-bound desensitized receptor, all with or without bound PTX. Right: predicted membrane currents, generated from the models, for wild-type α1β2 GABA_ARs activated by 10 mM GABA in the presence and absence of 50 μM PTX. See Supplementary Table 2 for the numerical values of the parameters. (a) Model 1: the PTX-blocked open receptor (AR*P) can desensitize, but PTX binding and unbinding cannot occur to or from either the resting (R/RP, AR/ARP) or desensitized (AD/ADP) receptor states. (b) Model 2: PTX binding and unbinding is permitted to both open (AR*/AR*P) and desensitized states. (c) Model 3: PTX binding prevents desensitization (that is, there is no ADP state).

Figure 7. GlyR current recovery after blockade by PTX under desensitizing conditions.

(a) Representative recording of the fast dissociation of PTX from GlyRα1 activated by high concentrations (10 mM) of glycine, accompanied by a clear rebound current (n=5). (b–d) Predicted membrane currents for activated wild-type GlyRα1 using the kinetic models 1–3, respectively, described in Fig. 6. See Supplementary Table 2 for the numerical values of parameters. Note, peak currents are truncated for clarity.