Protein interactions involving the γ2 large cytoplasmic loop of GABA _A receptors modulate conductance

Abstract

Native GABA(A) channels display a single-channel conductance ranging between approximately 10 and 90 pS. Diazepam increases the conductance of some of these native channels but never those of recombinant receptors, unless they are coexpressed with GABARAP. This trafficking protein clusters recombinant receptors in the membrane, suggesting that high-conductance channels arise from receptors that are at locally high concentrations. The amphipathic (MA) helix that is present in the large cytoplasmic loop of every subunit of all ligand-gated ion channels mediates protein-protein interactions. Here we report that when applied to inside-out patches, a peptide mimicking the MA helix of the gamma2 subunit (gamma(381-403)) of the GABA(A) receptor abrogates the potentiating effect of diazepam on both endogenous receptors and recombinant GABA(A) receptors coexpressed with GABARAP, by substantially reducing their conductance. The protein interaction disrupted by the peptide did not involve GABARAP, because a shorter peptide (gamma(386-403)) known to compete with the gamma2-GABARAP interaction did not affect the conductance of recombinant alphabetagamma receptors coexpressed with GABARAP. The requirement for receptor clustering and the fact that the gamma2 MA helix is able to self-associate support a mechanism whereby adjacent GABA(A) receptors interact via their gamma2-subunit MA helices, altering ion permeation through each channel. Alteration of ion-channel function arising from dynamic interactions between ion channels of the same family has not been reported previously and highlights a novel way in which inhibitory neurotransmission in the brain may be differentially modulated.

Evidence for α-Helices in the Large Intracellular Domain Mediating Modulation of the α1-Glycine Receptor by Ethanol and Gβγ

“…a-Helix Is Required for Intracellular GlyR Modulation (Tudan et al, 2002;Everitt et al, 2009;Guzman et al, 2009;San Martin et al, 2012;Zhang et al, 2014). For instance, several structural studies of protein-Gbg interaction, including complexes between Gbg and SIGK peptide (Davis et al, 2005;Lin and Smrcka, 2011), GRK2 (Tesmer et al, 2010;Thal et al, 2011) and phosducin (Loew et al, 1998;Gaudet et al, 1999), have reported participation of helical structures like those proposed in this study.…”

“…Also, a potential helical structure in the N-terminal LIL region was predicted in silico in pharmacologic studies of ethanol using small synthetic peptides derived from these regions (Guzman et al, 2009;San Martin et al, 2012). The presence of these helical structures is likely to serve for protein-protein interactions of the type previously proposed for other proteins Everitt et al, 2009;Chin et al, 2013;Shim et al, 2013;Alexander et al, 2014) and antagonized by small peptides having helical structures Representative glycine-evoked current traces (15 mM) in the absence and presence of 100 mM ethanol in a1-LIL-g2. (F) Effects of 100 mM ethanol on a1-GlyR and chimera a1-LIL-g2 in terms of percent of potentiation by 100 mM ethanol of 15 mM glycine-evoked currents.…”

Ethanol effects on glycinergic transmission: From molecular pharmacology to behavior responses

“…Interestingly, the helical structure content was slightly lower than that previously reported for the intracellular loop domain of the α3 subunit suggesting a high conservation [163,167]. The helical structure described in the intracellular loop domain of α1 GlyRs may be required for protein/protein interactions as previously described for other protein complexes [186,187] or for peptides having the helical structure [168,169,187]. In this regard, numerous studies have reported the involvement of helical structures in the interaction sites such as complexes between Gβγ and peptides SlGK, GRK2 or phosducin [89,92,188].…”

2′‐Methoxy‐6‐methylflavone: a novel anxiolytic and sedative with subtype selective activating and modulating actions at GABAA receptors

“…Patch electrodes contained (mM): 138 choline chloride, 0.3 KCl, 7 MgCl 2 , 10 TES (pH 7.3). Peptides were dissolved in DMSO (final concentration ≤ 0.01%), diluted to the required concentration with external solution and applied to the patch by exposing the pipette tip to flow tubes containing the test solutions (Everitt et al ., 2009). Currents were recorded with an Axopatch 200A or 200B current‐to‐voltage converter, filtered at 5–10 kHz and digitized at a frequency of 10–50 kHz using a Data Translation data acquisition card (DT3001‐PGL, Data Transition Inc, Marlboro, MA, USA) interfaced with an IBM compatible computer.…”