Pharmacological characterisation of murine α4β1δ GABAA receptors expressed in Xenopus oocytes

BACKGROUND: GABAA receptor subunit composition has a profound effect on the receptor's physiological and pharmacological properties. The receptor β subunit is widely recognised for its importance in receptor assembly, trafficking and post-translational modifications, but its influence on extrasynaptic GABAA receptor function is less well understood. Here, we examine the pharmacological properties of a potentially native extrasynaptic GABAA receptor that incorporates the β1 subunit, specifically composed of α4β1δ and α4β1 subunits. RESULTS: GABA activated concentration-dependent responses at α4β1δ and α4β1 receptors with EC50 values in the nanomolar to micromolar range, respectively. The divalent cations Zn(2+) and Cu(2+), and the β1-selective inhibitor salicylidine salicylhydrazide (SCS), inhibited GABA-activated currents at α4β1δ receptors. Surprisingly the α4β1 receptor demonstrated biphasic sensitivity to Zn(2+) inhibition that may reflect variable subunit stoichiometries with differing sensitivity to Zn(2+). The neurosteroid tetrahydro-deoxycorticosterone (THDOC) significantly increased GABA-initiated responses in concentrations above 30 nM for α4β1δ receptors. CONCLUSIONS: With this study we report the first pharmacological characterisation of various GABAA receptor ligands acting at murine α4β1δ GABAA receptors, thereby improving our understanding of the molecular pharmacology of this receptor isoform. This study highlights some notable differences in the pharmacology of murine and human α4β1δ receptors. We consider the likelihood that the α4β1δ receptor may play a role as an extrasynaptic GABAA receptor in the nervous system

Block of nicotinic acetylcholine receptors by philanthotoxins is strongly dependent on their subunit composition

Philanthotoxin-433 (PhTX-433) is an active component of the venom from the Egyptian digger wasp, Philanthus triangulum. PhTX-433 inhibits several excitatory ligand-gated ion channels, and to improve selectivity two synthetic analogues, PhTX-343 and PhTX-12, were developed. Previous work showed a 22-fold selectivity of PhTX-12 over PhTX-343 for embryonic muscle-type nicotinic acetylcholine receptors (nAChRs) in TE671 cells. We investigated their inhibition of different neuronal nAChR subunit combinations as well as of embryonic muscle receptors expressed in Xenopus oocytes. Whole-cell currents in response to application of acetylcholine alone or co-applied with PhTX analogue were studied by using two-electrode voltage-clamp. α3β4 nAChRs were most sensitive to PhTX-343 (IC50=12 nM at −80 mV) with α4β4, α4β2, α3β2, α7 and α1β1γδ being 5, 26, 114, 422 and 992 times less sensitive. In contrast α1β1γδ was most sensitive to PhTX-12 along with α3β4 (IC50values of 100 nM) with α4β4, α4β2, α3β2 and α7 being 3, 3, 26 and 49 times less sensitive. PhTX-343 inhibition was strongly voltage-dependent for all subunit combinations except α7, whereas this was not the case for PhTX-12 for which weak voltage dependence was observed. We conclude that PhTX-343 mainly acts as an open-channel blocker of nAChRs with strong subtype selectivity

Replication and Fine Mapping for Association of the C2orf43, FOXP4, GPRC6A and RFX6 Genes with Prostate Cancer in the Chinese Population

Prostate cancer represents the leading cause of male death across the world. A recent genome-wide association study (GWAS) identified five novel susceptibility loci for prostate cancer in the Japanese population. This study is to replicate and fine map the potential association of these five loci with prostate cancer in the Chinese Han population.In Phase I of the study, we tested the five single nucleotide polymorphisms (SNPs) which showed the strongest association evidence in the original GWAS in Japanese. The study sample consists of 1,169 Chinese Hans, comprising 483 patients and 686 healthy controls. Then in phase II, flanking SNPs of the successfully replicated SNPs in Phase I were genotyped and tested for association with prostate cancer to fine map those significant association signals.We successfully replicated the association of rs13385191 (located in the C2orf43 gene, P = 8.60×10(-5)), rs12653946 (P = 1.33×10(-6)), rs1983891 (FOXP4, P = 6.22×10(-5)), and rs339331 (GPRC6A/RFX6, P = 1.42×10(-5)) with prostate cancer. The most significant odds ratio (OR) was recorded as 1.41 (95% confidence interval 1.18-1.68) for rs12653946. Rs9600079 did not show significant association (P = 8.07×10(-2)) with prostate cancer in this study. The Phase II study refined these association signals, and identified several SNPs showing more significant association with prostate cancer than the very SNPs tested in Phase I.Our results provide further support for association of the C2orf43, FOXP4, GPRC6A and RFX6 genes with prostate cancer in Eastern Asian populations. This study also characterized the novel loci reported in the original GWAS with more details. Further work is still required to determine the functional variations and finally clarify the underlying biological mechanisms

Characterization of the Modes of Binding between Human Sweet Taste Receptor and Low-Molecular-Weight Sweet Compounds

One of the most distinctive features of human sweet taste perception is its broad tuning to chemically diverse compounds ranging from low-molecular-weight sweeteners to sweet-tasting proteins. Many reports suggest that the human sweet taste receptor (hT1R2–hT1R3), a heteromeric complex composed of T1R2 and T1R3 subunits belonging to the class C G protein–coupled receptor family, has multiple binding sites for these sweeteners. However, it remains unclear how the same receptor recognizes such diverse structures. Here we aim to characterize the modes of binding between hT1R2–hT1R3 and low-molecular-weight sweet compounds by functional analysis of a series of site-directed mutants and by molecular modeling–based docking simulation at the binding pocket formed on the large extracellular amino-terminal domain (ATD) of hT1R2. We successfully determined the amino acid residues responsible for binding to sweeteners in the cleft of hT1R2 ATD. Our results suggest that individual ligands have sets of specific residues for binding in correspondence with the chemical structures and other residues responsible for interacting with multiple ligands

Expression of Tas1 Taste Receptors in Mammalian Spermatozoa: Functional Role of Tas1r1 in Regulating Basal Ca2+ and cAMP Concentrations in Spermatozoa

Background: During their transit through the female genital tract, sperm have to recognize and discriminate numerous chemical compounds. However, our current knowledge of the molecular identity of appropriate chemosensory receptor proteins in sperm is still rudimentary. Considering that members of the Tas1r family of taste receptors are able to discriminate between a broad diversity of hydrophilic chemosensory substances, the expression of taste receptors in mammalian spermatozoa was examined. Methodology/Principal Findings: The present manuscript documents that Tas1r1 and Tas1r3, which form the functional receptor for monosodium glutamate (umami) in taste buds on the tongue, are expressed in murine and human spermatozoa, where their localization is restricted to distinct segments of the flagellum and the acrosomal cap of the sperm head. Employing a Tas1r1-deficient mCherry reporter mouse strain, we found that Tas1r1 gene deletion resulted in spermatogenic abnormalities. In addition, a significant increase in spontaneous acrosomal reaction was observed in Tas1r1 null mutant sperm whereas acrosomal secretion triggered by isolated zona pellucida or the Ca2+ ionophore A23187 was not different from wild-type spermatozoa. Remarkably, cytosolic Ca2+ levels in freshly isolated Tas1r1-deficient sperm were significantly higher compared to wild-type cells. Moreover, a significantly higher basal cAMP concentration was detected in freshly isolated Tas1r1-deficient epididymal spermatozoa, whereas upon inhibition of phosphodiesterase or sperm capacitation, the amount of cAMP was not different between both genotypes. Conclusions/Significance: Since Ca2+ and cAMP control fundamental processes during the sequential process of fertilization, we propose that the identified taste receptors and coupled signaling cascades keep sperm in a chronically quiescent state until they arrive in the vicinity of the egg - either by constitutive receptor activity and/or by tonic receptor activation by gradients of diverse chemical compounds in different compartments of the female reproductive tract

Presynaptic External Calcium Signaling Involves the Calcium-Sensing Receptor in Neocortical Nerve Terminals

Nerve terminal invasion by an axonal spike activates voltage-gated channels, triggering calcium entry, vesicle fusion, and release of neurotransmitter. Ion channels activated at the terminal shape the presynaptic spike and so regulate the magnitude and duration of calcium entry. Consequently characterization of the functional properties of ion channels at nerve terminals is crucial to understand the regulation of transmitter release. Direct recordings from small neocortical nerve terminals have revealed that external [Ca(2+)] ([Ca(2+)](o)) indirectly regulates a non-selective cation channel (NSCC) in neocortical nerve terminals via an unknown [Ca(2+)](o) sensor. Here, we identify the first component in a presynaptic calcium signaling pathway.By combining genetic and pharmacological approaches with direct patch-clamp recordings from small acutely isolated neocortical nerve terminals we identify the extracellular calcium sensor. Our results show that the calcium-sensing receptor (CaSR), a previously identified G-protein coupled receptor that is the mainstay in serum calcium homeostasis, is the extracellular calcium sensor in these acutely dissociated nerve terminals. The NSCC currents from reduced function mutant CaSR mice were less sensitive to changes in [Ca(2+)](o) than wild-type. Calindol, an allosteric CaSR agonist, reduced NSCC currents in direct terminal recordings in a dose-dependent and reversible manner. In contrast, glutamate and GABA did not affect the NSCC currents.Our experiments identify CaSR as the first component in the [Ca(2+)](o) sensor-NSCC signaling pathway in neocortical terminals. Decreases in [Ca(2+)](o) will depress synaptic transmission because of the exquisite sensitivity of transmitter release to [Ca(2+)](o) following its entry via voltage-activated Ca(2+) channels. CaSR may detects such falls in [Ca(2+)](o) and increase action potential duration by increasing NSCC activity, thereby attenuating the impact of decreases in [Ca(2+)](o) on release probability. CaSR is positioned to detect the dynamic changes of [Ca(2+)](o) and provide presynaptic feedback that will alter brain excitability

The evolving place of incretin-based therapies in type 2 diabetes

Treatment options for type 2 diabetes based on the action of the incretin hormone glucagon-like peptide-1 (GLP-1) were first introduced in 2005. These comprise the injectable GLP-1 receptor agonists solely acting on the GLP-1 receptor on the one hand and orally active dipeptidyl-peptidase inhibitors (DPP-4 inhibitors) raising endogenous GLP-1 and other hormone levels by inhibiting the degrading enzyme DPP-4. In adult medicine, both treatment options are attractive and more commonly used because of their action and safety profile. The incretin-based therapies stimulate insulin secretion and inhibit glucagon secretion in a glucose-dependent manner and carry no intrinsic risk of hypoglycaemia. GLP-1 receptor agonists allow weight loss, whereas DPP-4 inhibitors are weight neutral. This review gives an overview of the mechanism of action and the substances and clinical data available