GlyRs are members of the superfamily of Cys

GlyRs are members of the superfamily of Cys-loop receptors (CLRs) [3]. They form postsynaptic pentameric receptor complexes of α1 and β subunits anchored via the scaffolding protein gephyrin [4]. GlyRs are composed of several domains with the large N-terminal extracellular domain (ECD) important for ligand-binding followed by TM1-4 connected by intra- and extracellular loop structures, and a short extracellular C-terminus. TM2 forms the ion channel pore [3]. Several posttranslational modifications have been shown for the large intracellular TM3-4 loop, e.g., Gβγ-modulation, PKA/PKC phosphorylation, ubiquitination [5], [6]. Distinct residues in the TM3-4 loop are allosterically modulated by ethanol, neurosteroids, and endocannabinoids [7]. The basic motif 318RRKRR has been described as an important domain for surface transport and correct integration of TM3 [8]. The intracellular TM3-4 loop is of highest variability among members of the Cys-loop receptor family. Previous studies on TM3-4 loop replacements of GLIC and 5HT3A have shown that functionality was preserved by exchange in both directions [9]. A replacement of the TM3-4 loop of the α1 GlyR with short polypeptides did not influence channel deactivation but had a distinct effect on the desensitization kinetics of GlyR SSR 69071 [10]. Here, we describe the exchange of the intracellular TM3-4 loop of the GlyRα1 subunit with the short linker from GLIC, a prokaryotic Cys-loop receptor isolated from G. violaceus with and without the basic stretches attached to the GLIC sequence. GlyR–GLIC chimeras were elucidated for expression and functionality with respect to desensitization kinetics. Our data exhibit considerable evidence that the interspacial residues between both basic stretches 318RRKRR and 385KKIDK within the TM3-4 loop are crucial for non-desensitization of the GlyRα1 wild type (wt).
Materials and methods
Results
Discussion Domain swapping between various types of ion channels has been successfully reported without disturbances of ion channel function [12], [13]. Although X-ray analysis provided lots of information on the ECD structure of CLRs, the overall fold of the intracellular domain is not yet solved. Even recently solved structures of Glu-Cl and Lily (GLIC–GlyR chimera) lacked the large ICD [14], [15]. Several reports described α-helical organization close to TM3 and TM4 [16], [17] with basic residues being important determinants for channel properties. Basic motifs at both ends of the GlyR ICD have been reported to determine trafficking and Gβγ protein binding [6], [8]. Our studies on GlyR–GLIC chimera with and without basic domains revealed a significant decrease in cell surface expression for all three constructs independent of the presence or absence of motifs 318RRKRR at the N-terminal and 385KKIDK at the C-terminal end of GlyR ICD. Although transport towards the cell surface and association with the outer membrane was shown for construct GlyRα1–GLIC, labeling of native epitopes at the extracellular N-terminus failed. Independent testing of two antibodies labeling the extracellular domain of GlyRα1 demonstrated that the ECD of GlyRα1–GLIC was either not exposed to the extracellular lumen or the construct might constitute a conformation non-accessible by the antibodies. A change in the overall extracellular conformation is in line with the observation of non-functional ion channel formation of GlyRα1–GLIC. Similarly, lack of the ICD in 5HT3A receptors lead to failure in functional channel formation [13]. Besides expression level, mutating charged residues in the MA stretch of GlyRα1 (residues 369–394 including 385KK), resulted in significantly decreased single channel conductance, but overall current potency was unaffected [18]. Notably, basic stretches at both ends of the GlyR ICD determine ion channel conductance. The ICD is of highest variability in length and amino acid composition among CLRs and determines subclass specific ion channel properties such as desensitization. In a recent study, we could show in chimera between GlyRα1 and α3 that desensitization properties can be transferred between subunits by domain transplantation [12]. These results argue that (i) ion channel properties are transferable between subunits and (ii) domain swapping is a powerful tool to analyze subdomain importance. Therefore we set out a chimeric study between the ICD of α1 and the short ICD peptide SQPARAA of the prokaryotic GLIC channel to investigate loop length in regard to ion channel properties. Constructs harbor either the wt α1 or the GLIC sequence plus/minus basic residues of the α1 ICD at both ends. All chimera were able to form functional ion channels, except for the construct where the GLIC peptide was inserted between TM3 and 4 of GlyRα1. Other studies on 5HT3-GLIC, GABAC–GLIC, and GlyR–GLIC chimera also revealed functional constructs when the GLIC peptide SQPARAA was flanked by sequences of at least three to four amino acid residues at each side of the GLIC loop [10], [13]. GlyRα1–GLIC(+) bm showed maximal current values indistinguishable from GlyRα1wt. If an external 5HT3-ICD was inserted into GLIC, natural GLIC residues close to TM3 and TM4 were demonstrated as being essential for activation of GLIC by external protons [9]. All studies on chimeric proteins of various CLRs illustrate that a critical loop length is required to ensure channel functionality. We hypothesize that positively charged amino acids close to TM3 and at the beginning of TM4 of the ICD are essential determinants to retain the overall ability of the receptor for correct folding. This is in line with the proposed function of these residues for cytoplasmic portal formation enabling a funnel of the incoming ions [16].