Anomalous rectifying properties of `diazepam-insensitive' GABA_A receptors

Abstract

Studies using recombinant systems indicate that `diazepam-insensitive' GABA<sub>A</sub> receptors in the central nervous system contain α4 and α6 subunits while `diazepam-sensitive' GABA_A receptors contain α1, α2, α3 and α5 subunits. Both native and recombinant diazepam-sensitive GABA_A receptors typically exhibit large, outwardly rectifying currents. For example, in patch clamp studies, Human Embryonic Kidney (HEK) 293 cells transfected with cDNAs encoding α1β2γ2 subunits exhibit a rectification ratio (I_{+60 mV}/I_{−60 mV}) of 1.95±0.21. However, anomalous rectification was observed in recombinant diazepam-insensitive GABA_A receptors composed of either α4β2γ2 (rectification ratio, 0.74±0.09) or α6β2γ2 (rectification ratio, 0.67±0.11) subunits. Based on sequence differences between diazepam-sensitive and -insensitive GABA_A receptor α subunits in the vicinity of the putative channel lining, a point mutation was introduced at His²⁷³ on the α4 subunit. The rectification ratio in cells expressing a mutated α4(Asn²⁷³)β2γ2 receptor increased to 1.92±0.17. Moreover, mutation of the homologous residue in the α1 subunit to histidine reduced the rectification ratio of α1(His²⁷⁴)β2γ2 to 1.02±0.12. The affinities of benzodiazepine site ligands at diazepam-sensitive and -insensitive GABA_A receptors were unaffected by these mutations. Thus, the electrophysiological properties of diazepam-sensitive and -insensitive GABA_A receptors may be as divergent as their pharmacological characteristics.

Introduction

GABA_A receptors are a heterogeneous family of ligand-gated ion channels that may be assembled from at least 16 structurally related subunits (6α, 3β, 3γ, δ, ε and 2ρ) (McKernan and Whiting, 1996; Davies et al., 1997). In the central nervous system, GABA_A receptors are most often assembled as ternary complexes composed of α, β, and γ subunits (Fritschy and Mohler, 1993; De Blas, 1996). While the subunit stoichiometry (i.e., the number of α, β, and γ subunits per receptor) remains controversial (Backus et al., 1993; Chang et al., 1996; Tretter et al., 1997), converging lines of evidence have shown that subunit composition determines the physiological and pharmacological properties of GABA_A receptors (Barnard, 1995; Lüddens et al., 1995).

GABA_A receptors possess multiple, allosterically coupled modulatory sites that are loci for drug action (Skolnick and Paul, 1988; Johnston, 1996). However, from the perspective of both current therapy and future drug development, the group of compounds acting at `benzodiazepine binding sites' is perhaps the most important. Thus, this structurally diverse group of compounds includes 1,4-benzodiazepines (such as diazepam and flurazepam), imidazopyridines (e.g., zolpidem), cyclopyrrolones (e.g., zopiclone), and β-carbolines (e.g., abecarnil). Studies in recombinant GABA<sub>A</sub> receptors have shown that the α subunit is the principal determinant of affinity for this group of compounds (Pritchett and Seeburg, 1990; Lüddens et al., 1990; Hadingham et al., 1993), with the γ subunit playing a smaller, albeit significant role for a subset of these compounds (Lüddens et al., 1994; Benke et al., 1996). While most therapeutically useful benzodiazepine site ligands are high affinity, positive modulators at GABAergic synapses (Haefely and Polc, 1986), Malminiemi and Korpi (1989)demonstrated that high (μM) concentrations of the prototypic 1,4-benzodiazepine diazepam are inactive at a subpopulation of GABA<sub>A</sub> receptors in primary granule cell neurons. In contrast, the imidazodiazepine Ro 15-4513 retained high affinity for this receptor subpopulation, termed `diazepam-insensitive' GABA_A receptors. Subsequent cloning studies identified two α subunits (α4 and α6) (Lüddens et al., 1990; Kato, 1990; Wisden et al., 1991) that could reproduce this pharmacological profile when expressed with the appropriate β and γ subunits.

Diazepam-insensitive sites represent only a small fraction of the total GABA_A receptor population. Nonetheless, this group of GABA_A receptors is of considerable interest because several high affinity diazepam-insensitive receptor ligands (e.g., Ro 15-4513 and Ro 19-4603) have been reported to act as amethystic agents (Suzdak et al., 1996; Lister and Durcan, 1989; June et al., 1996). Furthermore, a point mutation in the α6 subunit has been identified in the Alcohol Non-Tolerant (ANT) rat, a strain selected for sensitivity to alcohol (Korpi et al., 1993). These observations prompted efforts to develop benzodiazepine-site ligands that are selective for diazepam-insensitive GABA_A receptors (Ananthan et al., 1993; Wong et al., 1993). While examining the structural requirements for ligand binding to recombinant receptors containing α4 and α6 subunits (Gunnersen et al., 1996), we observed that the large, outwardly rectifying currents that have been characterized in both native and recombinant GABA_A receptors (Verdoorn et al., 1990; Adodra and Hales, 1995) were absent in these diazepam-insensitive isoforms. We now report that a histidine residue located between the second and third transmembrane spanning regions imparts this low rectification to recombinant diazepam-insensitive GABA_A receptors. Mutation of this residue to an asparagine (the amino acid found in the homologous position in α1, α2, α3, and α5 subunits) restores the large, outwardly rectifying properties that are observed in diazepam-sensitive GABA_A receptors composed of α1β2γ2 subunits. Moreover, substitution of a histidine residue in the homologous position of the α1 subunit reduces the large, outward rectifying currents characteristic of wild-type receptors composed of α1β2γ2 subunits. These dramatic effects of point mutations on the rectifying properties of GABA_A receptors are independent of the ligand binding properties that have been employed to discriminate between diazepam-sensitive and -insensitive receptor isoforms.