Current awareness

The transmitter-gated channels: a range of receptor types and structures

A series of novel enaminone amides with improved side effect were synthesized by Hogenkamp et al. To explore the action mechanisms of enaminone amides, the homology model of rat α1β2γ2 GABAR was generated using the cryo-electron microscopy structure of the nAChR of Torpedo marmorata and the AChBP of Lymnaea stagnalis as the templates. Molecular docking and pharmacophore analyses allowed us to speculate the critical residues involving to the recognition of the ligands. The docking results indicated His128, Tyr186 and Tyr236 of α subunit were essential to form H-bond interactions contacts with the ligands. Specially, the N-substituents of enaminone amides as the sterically favored areas could form the important hydrophobic interactions with the residue Tyr186.

γ-Aminobutyric-acid (GABA) and ATP ionotropic receptors represent two structurally and functionally different classes of neurotransmitter-gated channels involved in fast synaptic transmission. We demonstrate here that, when the inhibitory ρ1/GABA and the excitatory P2X₂ receptor channels are co-expressed in Xenopus oocytes, activation of one channel reduces the currents mediated by the other one. This reciprocal inhibitory cross-talk is a receptor-mediated phenomenon independent of agonist cross-modulation, membrane potential, direction of ionic flux, or channel densities. Functional interaction is disrupted when the cytoplasmic C-terminal domain of P2X₂ is deleted or in competition experiments with minigenes coding for the C-terminal domain of P2X₂ or the main intracellular loop of ρ1 subunits. We also show a physical interaction between P2X₂ and ρ1 receptors expressed in oocytes and the co-clustering of these receptors in transfected hippocampal neurons. Co-expression with P2X₂ induces retargeting and recruitment of mainly intracellular ρ1/GABA receptors to surface clusters. Therefore, molecular and functional cross-talk between inhibitory and excitatory ligand-gated channels may regulate synaptic strength both by activity-dependent current occlusion and synaptic receptors co-trafficking.

The aim of this study was to better understand the mechanisms that underlie adaptive changes in GABA_A receptors following their prolonged exposure to drugs. Exposure (48 and/or 96 h) of human embryonic kidney (HEK 293) cells stably expressing recombinant α1β2γ2s GABA_A receptors for γ-aminobutyric (GABA, 1 mM) and muscimol (100 μM), but not for diazepam (1 μM), enhanced the maximum number (B_max) of [³H]flunitrazepam binding sites without affecting their affinity (K_d). The GABA-induced enhancement in B_max was reduced by the GABA receptor antagonist, bicuculline (100 μM), and by cycloheximide (10 μl/ml), a protein synthesis inhibitor. GABA (100 μM) enhanced the affinity of [³H]flunitrazepam binding to vehicle- and GABA-pretreated, but not to diazepam-pretreated, HEK 293 cells. The results suggest that chronic GABA treatment up-regulates stably expressed GABA_A receptors, presumably by stimulating their synthesis. Unlike chronic diazepam, which produced functional uncoupling of GABA and benzodiazepine binding sites, chronic GABA failed to produce this effect.

Human α₁β₃ε GABA_A receptors were expressed in Xenopus oocytes and examined using the conventional two-electrode voltage-clamp technique and compared to α₁β₃γ₂ receptors. The effects of several GABA_A agonists were studied, and the allosteric modulation of the channel by a number of GABAergic modulators investigated. The presence of the ε subunit increased the potency and efficacy of direct activation by partial GABA_A agonists (piperidine-4-sulphonic acid and thio-4-PIOL), pentobarbital and neuro-steroids. Direct activation by 3-hydroxylated neurosteroids was restricted to 3α epimers, while chirality at C5 was indifferent. The 3β-sulfate esters of pregnenolone and dehydroepiandrosterone inhibited the spontaneous currents with efficacies higher, while bicuculline methiodide and SR 95531 did so lower than picrotoxin and TBPS. Furosemide, fipronil, triphenylcyanoborate and Zn²⁺ blocked the spontaneous currents of α₁β₃ε receptors with different efficacies. Flunitrazepam and 4′-chlorodiazepam inhibited the spontaneous currents with micromolar potencies. In conclusion, spontaneously active α₁β₃ε GABA_A receptors can be potentiated and blocked by GABAergic agents within a broad range of efficacy.

Upon receipt in the dorsal horn (DH) of the spinal cord, nociceptive (pain-signalling) information from the viscera, skin and other organs is subject to extensive processing by a diversity of mechanisms, certain of which enhance, and certain of which inhibit, its transfer to higher centres. In this regard, a network of descending pathways projecting from cerebral structures to the DH plays a complex and crucial role. Specific centrifugal pathways either suppress (descending inhibition) or potentiate (descending facilitation) passage of nociceptive messages to the brain. Engagement of descending inhibition by the opioid analgesic, morphine, fulfils an important role in its pain-relieving properties, while induction of analgesia by the adrenergic agonist, clonidine, reflects actions at α₂-adrenoceptors (α₂-ARs) in the DH normally recruited by descending pathways. However, opioids and adrenergic agents exploit but a tiny fraction of the vast panoply of mechanisms now known to be involved in the induction and/or expression of descending controls. For example, no drug interfering with descending facilitation is currently available for clinical use. The present review focuses on: (1) the organisation of descending pathways and their pathophysiological significance; (2) the role of individual transmitters and specific receptor types in the modulation and expression of mechanisms of descending inhibition and facilitation and (3) the advantages and limitations of established and innovative analgesic strategies which act by manipulation of descending controls. Knowledge of descending pathways has increased exponentially in recent years, so this is an opportune moment to survey their operation and therapeutic relevance to the improved management of pain.

All GABA_A receptor (GABAR) subunits include an invariant proline in a consensus motif in the first transmembrane segment (M1). In receptors containing bovine α1, β1 and γ2 subunits, we analyzed the effect of mutating this M1 proline to alanine in the α1 or β1 subunit using 3 different expression systems. The β1 subunit mutant, β1(P228A), reduced the EC₅₀ for GABA about 10-fold in whole cell recordings in HEK293 cells and L929 fibroblasts. The corresponding α1 subunit mutant (α1(P233A)) also reduced the GABA EC₅₀ when expressed in Xenopus oocytes; α1(P233A)β1γ2S receptors failed to assemble in HEK293 cells. Binding of [³H]flumazenil and [³H]muscimol to transfected HEK293 cell membranes showed similar levels of receptor expression with GABARs containing β1 or β1(P228A) subunits and no change in the affinity for [³H]flumazenil; however, the affinity for [³H]muscimol was increased 6-fold in GABARs containing β1(P228A) subunits. In L929 cells, presence of the β1(P228A) subunit reduced enhancement by barbiturates without affecting enhancement by diazepam or alfaxalone. Single channel recordings from α1β1γ2S and α1β1(P228A)γ2L GABARs showed similar channel kinetics, but β-mutant containing receptors opened at lower GABA concentrations. We conclude that the β1 subunit M1 segment proline affects the linkage between GABA binding and channel gating and is critical for barbiturate enhancement. Mutation of the M1 proline in the α1 subunit also inhibited receptor assembly.