Nathan Absalom

Alpha9 nicotinic acetylcholine receptors and the treatment of pain.

Chronic pain is a vexing worldwide problem that causes substantial disability and consumes significant medical resources. Although there are numerous analgesic medications, these work through a small set of molecular mechanisms. Even when these medications are used in combination, substantial amounts of pain often remain. It is therefore highly desirable to develop treatments that work through distinct mechanisms of action. While agonists of nicotinic acetylcholine receptors (nAChRs) have been intensively studied, new data suggest a role for selective antagonists of nAChRs. alpha-Conotoxins are small peptides used offensively by carnivorous marine snails known as Conus. A subset of these peptides known as alpha-conotoxins RgIA and Vc1.1 produces both acute and long lasting analgesia. In addition, these peptides appear to accelerate the recovery of function after nerve injury, possibly through immune mediated mechanisms. Pharmacological analysis indicates that RgIA and Vc1.1 are selective antagonists of alpha9alpha10 nAChRs. A recent study also reported that these alpha9alpha10 antagonists are also potent GABA-B agonists. In the current study, we were unable to detect RgIA or Vc1.1 binding to or action on cloned GABA-B receptors expressed in HEK cells or Xenopus oocytes. We review the background, findings and implications of use of compounds that act on alpha9* nAChRs.(1).

Role of Charged Residues in Coupling Ligand Binding and Channel Activation in the Extracellular Domain of the Glycine Receptor

The glycine receptor is a member of the ligand-gated ion channel receptor superfamily that mediates fast synaptic transmission in the brainstem and spinal cord. Following ligand binding, the receptor undergoes a conformational change that is conveyed to the transmembrane regions of the receptor resulting in the opening of the channel pore. Using the acetylcholine-binding protein structure as a template, we modeled the extracellular domain of the glycine receptor α1-subunit and identified the location of charged residues within loops 2 and 7 (the conserved Cys-loop). These loops have been postulated to interact with the M2-M3 linker region between the transmembrane domains 2 and 3 as part of the receptor activation mechanism. Charged residues were substituted with cysteine, resulting in a shift in the concentration-response curves to the right in each case. Covalent modification with 2-(trimethylammonium) ethyl methanethiosulfonate was demonstrated only for K143C, which was more accessible in the open state than the closed state, and resulted in a shift in the EC50 toward wild-type values. Charge reversal mutations (E53K, D57K, and D148K) also impaired channel activation, as inferred from increases in EC50 values and the conversion of taurine from an agonist to an antagonist in E53K and D57K. Thus, each of the residues Glu-53, Asp-57, Lys-143, and Asp-148 are implicated in channel gating. However, the double reverse charge mutations E53K:K276E, D57K:K276E, and D148K:K276E did not restore glycine receptor function. These results indicate that loops 2 and 7 in the extracellular domain play an important role in the mechanism of activation of the glycine receptor although not by a direct electrostatic mechanism.

α4βδ GABA(A) receptors are high-affinity targets for γ-hydroxybutyric acid (GHB).

γ-Hydroxybutyric acid (GHB) binding to brain-specific high-affinity sites is well-established and proposed to explain both physiological and pharmacological actions. However, the mechanistic links between these lines of data are unknown. To identify molecular targets for specific GHB high-affinity binding, we undertook photolinking studies combined with proteomic analyses and identified several GABAA receptor subunits as possible candidates. A subsequent functional screening of various recombinant GABAA receptors in Xenopus laevis oocytes using the two-electrode voltage clamp technique showed GHB to be a partial agonist at αβδ- but not αβγ-receptors, proving that the δ-subunit is essential for potency and efficacy. GHB showed preference for α4 over α(1,2,6)-subunits and preferably activated α4β1δ (EC50 = 140 nM) over α4β(2/3)δ (EC50 = 8.41/1.03 mM). Introduction of a mutation, α4F71L, in α4β1(δ)-receptors completely abolished GHB but not GABA function, indicating nonidentical binding sites. Radioligand binding studies using the specific GHB radioligand [3H](E,RS)-(6,7,8,9-tetrahydro-5-hydroxy-5H-benzocyclohept-6-ylidene)acetic acid showed a 39% reduction (P = 0.0056) in the number of binding sites in α4 KO brain tissue compared with WT controls, corroborating the direct involvement of the α4-subunit in high-affinity GHB binding. Our data link specific GHB forebrain binding sites with α4-containing GABAA receptors and postulate a role for extrasynaptic α4δ-containing GABAA receptors in GHB pharmacology and physiology. This finding will aid in elucidating the molecular mechanisms behind the proposed function of GHB as a neurotransmitter and its unique therapeutic effects in narcolepsy and alcoholism.

Mechanisms of channel gating of the ligand‐gated ion channel superfamily inferred from protein structure

The nicotinic‐like ligand‐gated ion channel superfamily consists of a group of structurally related receptors that activate an ion channel after the binding of extracellular ligand. The recent publications of the crystal structure of an acetylcholine binding protein and a refined electron micrograph structure of the membrane‐bound segment of an acetylcholine receptor have led to insights into the molecular determinants of receptor function. Although the structures confirmed much biochemical and electrophysiological data obtained about the receptors, they also provide opportunities to study further the mechanisms that allow channel activation stimulated by ligand‐binding. Here we review the mechanisms of channel gating that have been elucidated by information gained from the structures of the acetylcholine binding protein and membrane‐bound segment of the acetylcholine receptor.

Oxytocin prevents ethanol actions at δ subunit-containing GABAA receptors and attenuates ethanol-induced motor impairment in rats

Significance Even moderate doses of alcohol can cause considerable motor impairment. This effect has been linked to ethanol-induced potentiation of GABA actions at δ subunit-containing GABAA receptors (δ-GABAARs). Here, we demonstrate that the neuropeptide oxytocin selectively attenuates ethanol-induced motor impairment in rats as well as ethanol-induced potentiation of GABAergic activity at δ-GABAARs. This effect of oxytocin is shown to be independent of the oxytocin receptor (OTR) and involves a direct action at δ-GABAARs. To our knowledge, this study provides the first evidence of oxytocin having a direct, non-OTR–mediated effect on GABA–ethanol interactions. Recent preclinical and clinical studies indicate that oxytocin may also attenuate alcohol consumption, craving, and withdrawal, and the present study shows a previously unidentified mechanism through which some of these effects may occur. Even moderate doses of alcohol cause considerable impairment of motor coordination, an effect that substantially involves potentiation of GABAergic activity at δ subunit-containing GABAA receptors (δ-GABAARs). Here, we demonstrate that oxytocin selectively attenuates ethanol-induced motor impairment and ethanol-induced increases in GABAergic activity at δ-GABAARs and that this effect does not involve the oxytocin receptor. Specifically, oxytocin (1 µg i.c.v.) given before ethanol (1.5 g/kg i.p.) attenuated the sedation and ataxia induced by ethanol in the open-field locomotor test, wire-hanging test, and righting-reflex test in male rats. Using two-electrode voltage-clamp electrophysiology in Xenopus oocytes, oxytocin was found to completely block ethanol-enhanced activity at α4β1δ and α4β3δ recombinant GABAARs. Conversely, ethanol had no effect when applied to α4β1 or α4β3 cells, demonstrating the critical presence of the δ subunit in this effect. Oxytocin had no effect on the motor impairment or in vitro effects induced by the δ-selective GABAAR agonist 4,5,6,7-tetrahydroisoxazolo(5,4-c)pyridin-3-ol, which binds at a different site on δ-GABAARs than ethanol. Vasopressin, which is a nonapeptide with substantial structural similarity to oxytocin, did not alter ethanol effects at δ-GABAARs. This pattern of results confirms the specificity of the interaction between oxytocin and ethanol at δ-GABAARs. Finally, our in vitro constructs did not express any oxytocin receptors, meaning that the observed interactions occur directly at δ-GABAARs. The profound and direct interaction observed between oxytocin and ethanol at the behavioral and cellular level may have relevance for the development of novel therapeutics for alcohol intoxication and dependence.

Functional analysis of six Kir6.2 (KCNJ11) mutations causing neonatal diabetes

ATP-sensitive potassium (KATP) channels, composed of pore-forming Kir6.2 and regulatory sulphonylurea receptor (SUR) subunits, play an essential role in insulin secretion from pancreatic beta cells. Binding of ATP to Kir6.2 inhibits, whereas interaction of Mg-nucleotides with SUR, activates the channel. Heterozygous activating mutations in Kir6.2 (KCNJ11) are a common cause of neonatal diabetes (ND). We assessed the functional effects of six novel Kir6.2 mutations associated with ND: H46Y, N48D, E227K, E229K, E292G, and V252A. KATP channels were expressed in Xenopus oocytes and the heterozygous state was simulated by coexpression of wild-type and mutant Kir6.2 with SUR1 (the beta cell type of SUR). All mutations reduced the sensitivity of the KATP channel to inhibition by MgATP, and enhanced whole-cell KATP currents. Two mutations (E227K, E229K) also enhanced the intrinsic open probability of the channel, thereby indirectly reducing the channel ATP sensitivity. The other four mutations lie close to the predicted ATP-binding site and thus may affect ATP binding. In pancreatic beta cells, an increase in the KATP current is expected to reduce insulin secretion and thereby cause diabetes. None of the mutations substantially affected the sensitivity of the channel to inhibition by the sulphonylurea tolbutamide, suggesting patients carrying these mutations may respond to these drugs.

Naringin directly activates inwardly rectifying potassium channels at an overlapping binding site to tertiapin-Q.

BACKGROUND G protein‐coupled inwardly rectifying potassium (KIR3) channels are important proteins that regulate numerous physiological processes including excitatory responses in the CNS and the control of heart rate. Flavonoids have been shown to have significant health benefits and are a diverse source of compounds for identifying agents with novel mechanisms of action.

Phenotyping murine models of non-alcoholic fatty liver disease through metabolic profiling of intact liver tissue

NAFLD (non-alcoholic fatty liver disease) is a common cause of chronic liver disease associated with the metabolic syndrome. Effective techniques are needed to investigate the potential of animal models of NAFLD. The present study aimed to characterize murine models of NAFLD by metabolic profiling of intact liver tissue. Mice of three strains (BALB/c, C3H and the novel mutant, Gena/263) were fed a control or high-fat diet. Biometric, biochemical and histological analysis demonstrated a spectrum of NAFLD from normal liver to steatohepatitis. Metabolic profiling of intact liver tissue, using (1)H MAS (proton magic angle spinning) MRS (magnetic resonance spectroscopy), showed an increase in the total lipid-to-water ratio, a decrease in polyunsaturation indices and a decrease in total choline with increasing disease severity. Principal components analysis and partial least-squares discriminant analysis showed separation of each model from its control and of each model from the total dataset. Class membership from the whole dataset was predicted with 100% accuracy in six out of eight models. Those models with steatosis discriminated from those with steatohepatitis with 100% accuracy. The separation of histologically defined steatohepatitis from simple steatosis is clinically important. Indices derived from (1)H MAS MRS studies may inform subsequent in vivo MRS studies at lower field strengths.

Low nanomolar GABA effects at extrasynaptic α4β1/β3δ GABAA receptor subtypes indicate a different binding mode for GABA at these receptors

Ionotropic GABA(A) receptors are a highly heterogenous population of receptors assembled from a combination of multiple subunits. The aims of this study were to characterize the potency of GABA at human recombinant δ-containing extrasynaptic GABA(A) receptors expressed in Xenopus oocytes using the two-electrode voltage clamp technique, and to investigate, using site-directed mutagenesis, the molecular determinants for GABA potency at α4β3δ GABA(A) receptors. α4/δ-Containing GABA(A) receptors displayed high sensitivity to GABA, with mid-nanomolar concentrations activating α4β1δ (EC₅₀=24 nM) and α4β3δ (EC₅₀=12 nM) receptors. In the majority of oocytes expressing α4β3δ subtypes, GABA produced a biphasic concentration-response curve, and activated the receptor with low and high concentrations (EC₅₀(1)=16 nM; EC₅₀(2)=1.2 μM). At α4β2δ, GABA had low micromolar activity (EC₅₀=1 μM). An analysis of 10 N-terminal singly mutated α4β3δ receptors shows that GABA interacts with amino acids different to those reported for α1β2γ2 GABA(A) receptors. Residues Y205 and R207 of the β3-subunit significantly affected GABA potency, while the residue F71 of the α4- and the residue Y97 of the β3-subunit did not significantly affect GABA potency. Mutating the residue R218 of the δ-subunit, equivalent to the GABA binding residue R207 of the β2-subunit, reduced the potency of GABA by 670-fold, suggesting a novel GABA binding site at the δ-subunit interface. Taken together, GABA may have different binding modes for extrasynaptic δ-containing GABA(A) receptors compared to their synaptic counterparts.

Pore Structure of the Cys-loop Ligand-gated Ion Channels

The Cys-loop receptor family of ligand-gated ion channels (LGICs) play a key role in synaptic transmission in the central nervous system of animals. Recent advances have led to the elucidation of two crystal structures of related prokaryotic LGICs and the electron micrograph derived structure of the acetylcholine receptor from Torpedo marmorata. Here, we review the structural and biochemical data that form our understanding of the structure of the channel pore. We introduce original data from the glycine receptor using the substituted-cysteine accessibility technique and show that while the helical structure of the segment that surrounds the channel pore is generally agreed, the location of the channel gate, the pore diameter and the structure that forms the entry to the channel pore are likely to differ between receptors. The fundamental structural differences between anion and cation selective receptors and how these differences are related to the pore structure are also considered.