Cocaine is a widely abused substance with psychostimulant effects that are attributed to inhibition of the dopamine transporter (DAT). We present molecular models for DAT binding of cocaine and cocaine analogs constructed from the high-resolution structure of the bacterial transporter homolog LeuT. Our models suggest that the binding site for cocaine and cocaine analogs is deeply buried between transmembrane segments 1, 3, 6 and 8, and overlaps with the binding sites for the substrates dopamine and amphetamine, as well as for benztropine-like DAT inhibitors. We validated our models by detailed mutagenesis and by trapping the radiolabeled cocaine analog [ 3 H]CFT in the transporter, either by cross-linking engineered cysteines or with an engineered Zn 2+ -binding site that was situated extracellularly to the predicted common binding pocket. Our data demonstrate the molecular basis for the competitive inhibition of dopamine transport by cocaine.Correspondence should be addressed to U.G. (E-mail: gether@sund.ku.dk). Note: Supplementary information is available on the Nature Neuroscience website. AUTHOR CONTRIBUTIONST.B. designed and performed the computational experiments, analyzed the data and wrote the manuscript draft together with C.J.L. J.K. generated mutants, carried out pharmacological analyses and contributed to the data analysis. M.L.B. and K.R. generated mutants and carried out pharmacological analyses. L.S. contributed to the computational experiments and manuscript refinement. L.G. participated in the design and performance of the computational experiments. A.H.N. contributed with ideas, benztropine analogues and provided expertise in the pharmacology and medicinal chemistry of DAT inhibitors. J.A.J. contributed with ideas and to the design of experiments and writing of the manuscript. H.W. directed the design and performance of the modeling and computational experiments, participated in data analysis and contributed to writing the manuscript. U.G. supervised the project together with C.J.L., designed experiments, analyzed data and wrote the final manuscript. C.J.L. supervised the project together with U.G., designed experiments, generated mutants, performed pharmacological experiments, analyzed data and wrote the manuscript draft together with T.B.Reprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions/ NIH Public Access Author ManuscriptNat Neurosci. Author manuscript; available in PMC 2009 July 1. Published in final edited form as:Nat Neurosci. 2008 July ; 11(7): 780-789. doi:10.1038/nn.2146. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptCocaine is an alkaloid derived from the Peruvian Erythroxylon coca plant and has been used as a stimulant for centuries 1 . Today, cocaine is widely abused, especially in the western hemisphere, causing major socioeconomic burdens through increased medical expenses, lost earnings and increased crime 2 . Nonetheless, the molecular mechanisms underlying cocaine's pharmacology and abuse ...

A series of substituted 1-hydroxypyrazole analogues of the GABA(A) receptor partial agonist 5-(4-piperidyl)-3-isoxazolol (4-PIOL) have been synthesized and pharmacologically characterized. Several of the analogues displayed K(i) in the low nanomolar range at the native GABA(A) receptors and potent antagonism of the alpha(1)beta(2)gamma(2) receptor. It appears that several regions situated in proximity to the core of the orthosteric binding site of the GABA(A) receptor are able to accommodate large hydrophobic substituents.

The novel ginkgolide analog ginkgolide X was characterized functionally at human glycine and ␥-aminobutyric acid type A receptors (GlyRs and GABA A Rs, respectively) in the fluorescence-based FLIPR TM Membrane Potential assay. The compound inhibited the signaling of all GABA A R subtypes included in the study with high nanomolar/low micromolar IC 50 values, except the 1 receptor at which it was a significantly weaker antagonist. Ginkgolide X also displayed high nanomolar/low micromolar IC 50 values at the homomeric ␣1 and ␣2 GlyRs, whereas it was inactive at the heteromeric ␣1␤ and ␣2␤ subtypes at concentrations up to 300 M. Thus, the functional properties of the compound were significantly different from those of the naturally occurring ginkgolides A, B, C, J, and M but similar to those of picrotoxin. In a mutagenesis study the 6 M2 residues in the GlyR ion channel were identified as the primary molecular determinant of the selectivity profile of ginkgolide X, and a 6 M2 ring consisting of five Thr residues was found to be of key importance for its activity at the GABA A R. Conformational analysis and docking of low-energy conformations of the native ginkgolide A and ginkgolide X into a ␣1 GlyR homology model revealed two distinct putative binding sites formed by the 6 M2 residues together with the 2 residues and the 10 and 13 residues, respectively. Thus, we propose that the distinct functionalities of ginkgolide X compared with the other ginkgolides could arise from different flexibility and thus different binding modes to the ion channel of the anionic Cys-loop receptor. ␥-Aminobutyric acid (GABA)2 and glycine are the predominant inhibitory neurotransmitters in the central nervous system and also maintain important functions in several peripheral tissues (1-5). The ionotropic GABA A and glycine receptors (GABA A Rs and GlyRs) belong to the Cys-loop receptor superfamily, which also comprises nicotinic acetylcholine receptors (nAChRs) and 5-HT 3 receptors (5-HT 3 Rs) (3-9). The Cys-loop receptors are homomeric or heteromeric assemblies of five subunits, and the pentameric receptor complex consists of three domains: an extracellular domain composed of the N-terminal domains of the five subunits, a transmembrane domain formed by the M1-M4 ␣-helices of the five subunits (including an ion channel predominantly formed by the five M2 helices), and an intracellular domain composed primarily of the large second intracellular loops of the five subunits (4, 7). Signal transduction through the Cys-loop receptor is initiated by binding of the agonist to orthosteric sites situated at the interfaces between the N-terminal domains of the subunits, and this elicits a conformation change in the pentameric complex leading to flux of ions through the ion channel. Whereas nAChRs and 5-HT 3

Aims/hypothesis Previous studies have demonstrated a relationship between cognitive impairment and hypoglycaemia (<3 mmol/l). This study hypothesised that non-severe insulin-induced hypoglycaemia reduces cognitive function in individuals with type 2 diabetes. Methods In this randomised crossover study, 25 participants with type 2 diabetes attended two experimental visits with hyperinsulinaemic glucose clamping: one hypoglycaemic clamp (plasma glucose 3.0 ± 0.2 mmol/l) and one euglycaemic clamp (plasma glucose 6.0 ± 0.2 mmol/l). Participants were eligible if their diabetes was treated with diet or glucose-lowering medications (except sulfonylureas or insulin), age was 35-70 years, BMI was 23-35 kg/m 2 and HbA 1c was below 75 mmol/mol (9%). Cognitive function was assessed with a neurocognitive test battery measuring verbal memory, executive function, sustained attention and psychomotor speed. From the examined cognitive domains, a global cognition score was constructed estimating global cognition. A measurement for psychomotor speed was selected as the primary outcome. Participants and people assessing the outcomes were blinded to group assignment. Results Cognitive performance was impaired during hypoglycaemia with a mean score in the primary outcome test, Symbol Digit Modalities Test measuring psychomotor speed, of 48.7 ± 9.8 (hypoglycaemia) vs 56.6 ± 12.0 (euglycaemia); i.e. a change of −7.9 points (95% CI −10.9, −4.9; p < 0.0001). In addition, hypoglycaemia reduced global cognitive score by −0.7 (95% CI −0.9, −0.6; p < 0.0001). A stable glucose plateau was achieved during both experimental visits. For the hypoglycaemic clamp, mean plasma glucose concentration (± SD) during neurocognitive testing was 3.1 (± 0.3) mmol/l. Age, sex, fasting C-peptide, counter-regulatory hormones and the severity of hypoglycaemic symptoms did not influence cognitive function. Conclusions/interpretation Acute non-severe hypoglycaemia (mean plasma glucose 3.1 mmol/l) has a substantial negative impact on cognitive function in individuals with type 2 diabetes. Trial registration ClinicalTrials.gov NCT03014011.

BACKGROUND AND PURPOSEExplorations into the heterogeneous population of native GABA type A receptors (GABAARs) and the physiological functions governed by the multiple GABAAR subtypes have for decades been hampered by the lack of subtype-selective ligands. EXPERIMENTAL APPROACHThe functional properties of the orthosteric GABAA receptor ligand 5-(4-piperidyl)-3-isothiazolol (Thio-4-PIOL) have been investigated in vitro, ex vivo and in vivo. KEY RESULTSThio-4-PIOL displayed substantial partial agonist activity at the human extrasynaptic GABAAR subtypes expressed in Xenopus oocytes, eliciting maximal responses of up to ∼30% of that of GABA at α5β3γ2S, α4β3δ and α6β3δ and somewhat lower efficacies at the corresponding α5β2γ2S, α4β2δ and α6β2δ subtypes (maximal responses of 4-12%). In contrast, it was an extremely low efficacious agonist at the α1β3γ2S, α1β2γ2S, α2β2γ2S, α2β3γ2S, α3β2γ2S and α3β3γ2S GABAARs (maximal responses of 0-4%). In concordance with its agonism at extrasynaptic GABAARs and its de facto antagonism at the synaptic receptors, Thio-4-PIOL elicited robust tonic currents in electrophysiological recordings on slices from rat CA1 hippocampus and ventrobasal thalamus and antagonized phasic currents in hippocampal neurons. Finally, the observed effects of Thio-4-PIOL in rat tests of anxiety, locomotion, nociception and spatial memory were overall in good agreement with its in vitro and ex vivo properties. CONCLUSION AND IMPLICATIONSThe diverse signalling characteristics of Thio-4-PIOL at GABAARs represent one of the few examples of a functionally subtype-selective orthosteric GABAAR ligand reported to date. We propose that Thio-4-PIOL could be a useful pharmacological tool in future studies exploring the physiological roles of native synaptic and extrasynaptic GABAARs.

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