Mao Xiang Chen

GlaxoSmithKline plc., Londinium, England, Belgium

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Publications (10)30.7 Total impact

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    ABSTRACT: Abstract Gap junctions (GJs) are intercellular channels which are composed of the connexin family of proteins that allow electrical and chemical communications and synchronization in tissue ensembles. Evidence suggests that pharmaceutical modulators of these channels may have therapeutic potential or carry undesired liability. In this report, we exogenously expressed human connexin 43 (Cx43, GJA1) and demonstrated functionality in a 96-well flow cytometry assay detecting intercellular transfer of the calcein dye. We have designed a 384-well high-throughput method for detecting the transfer of calcium between HeLa cells expressing Cx43. In this assay, donor cells coexpress Cx43 and the α1A adrenergic Gα-coupled receptor, while recipient cells coexpress Cx43 and the cytoplasmic version of the calcium-sensitive luminescent protein aequorin enhanced by codon optimization (cytoAeq). The two cell populations were mixed, dispensed to 384-well plates, and incubated for 3 h to allow the formation of GJs. Activation of α1A by epinephrine in donor cells led to dose-dependent calcium increases in recipient cells, which were detected by measuring the intensity of aequorin luminescence. The response was dependent on the expression of Cx43 and inhibited by the GJ blocker 18α-glycyrrhetinic acid, suggesting Cx43 GJ-mediated activity. In a parallel experiment with capsaicin and the TrpV1 ion channel in place of phenylephrine and α1A, a similar magnitude of difference in the maximal calcium response was detected in both donor and recipient cells, suggesting that calcium is likely the permeant ion through the GJ. This assay may pave the way for high-throughput screening of GJ modulators for drug discovery.
    Assay and Drug Development Technologies 10/2012; · 1.90 Impact Factor
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    ABSTRACT: The tractability of ion channels as drug targets has been significantly improved by the advent of planar array electrophysiology platforms which have dramatically increased the capacity for electrophysiological profiling of lead series compounds. However, the data quality and through-put obtained with these platforms is critically dependent on the robustness of the expression reagent being used. The generation of high quality, recombinant cell lines is therefore a key step in the early phase of ion channel drug discovery and this can present significant challenges due to the diversity and organisational complexity of many channel types. This article focuses on several complex and difficult to express ion channels and illustrates how improved stable cell lines can be obtained by integration of planar array electrophysiology systems into the cell line generation process per se. By embedding this approach at multiple stages (e.g., during development of the expression strategy, during screening and validation of clonal lines, and during characterisation of the final cell line), the cycle time and success rate in obtaining robust expression of complex multi-subunit channels can be significantly improved. We also review how recent advances in this technology (e.g., population patch clamp) have further widened the versatility and applicability of this approach.
    Combinatorial chemistry & high throughput screening 12/2009; 12(1):96-106. · 2.46 Impact Factor
  • Mao Xiang Chen, Ray M Helliwell, Jeff J Clare
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    ABSTRACT: Acquired long QT syndrome is a major cause of drug withdrawals and the failure of compounds during development. Traditionally, in vitro cardiac liability screens have been low throughput and expensive, and have used primary animal cells or tissues that are not necessarily predictive of the human heart. Recent advances in ion channel assay technologies, particularly automated patch clamp, have enabled the early liability screening of individual ion channels at medium-to-high throughput and with acceptable fidelity. The primary aim of this first-tier screening is to aid prioritization of early-stage compounds for progression along the screening cascade. Potential liabilities can be flagged to enable follow-up studies to be initiated if and when compounds approach the costlier stages of development. To date, cardiac safety screening has been focused on hERG channel. This article examines the rationale for the early screening of cardiac channels beyond hERG as part of an integrated strategy for in vitro evaluation of cardiac risk, and reviews recent developments in the relevant technologies.
    Current opinion in molecular therapeutics 07/2009; 11(3):269-81. · 3.42 Impact Factor
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    ABSTRACT: Planar array electrophysiology techniques were applied to assays for modulators of recombinant hIK and hSK3 Ca2+-activated K+ channels. In CHO-hIK-expressing cells, under asymmetric K+ gradients, small-molecule channel activators evoked time- and voltage-independent currents characteristic of those previously described by classical patch clamp electrophysiology methods. In single-hole (cell) experiments, the large cell-to-cell heterogeneity in channel expression rendered it difficult to generate activator concentration-response curves. However, in population patch clamp mode, in which signals are averaged from up to 64 cells, well-to-well variation was substantially reduced such that concentration-response curves could be easily constructed. The absolute EC50 values and rank order of potency for a range of activators, including 1-EBIO and DC-EBIO, corresponded well with conventional patch clamp data. Activator responses of hIK and hSK3 channels could be fully and specifically blocked by the selective inhibitors TRAM-34 and apamin, with IC50 values of 0.31 microM and 3 nM, respectively. To demonstrate assay precision and robustness, a test set of 704 compounds was screened in a 384-well format of the hIK assay. All plates had Z' values greater than 0.6, and the statistical cutoff for activity was 8%. Eleven hits (1.6%) were identified from this set, in addition to the randomly spiked wells with known activators. Overall, our findings demonstrate that population patch clamp is a powerful and enabling method for screening Ca2+-activated K+ channels and provides significant advantages over single-cell electrophysiology (IonWorks(HT)) and other previously published approaches. Moreover, this work demonstrates for the 1st time the utility of population patch clamp for ion channel activator assays and for non-voltage-gated ion channels.
    Journal of Biomolecular Screening 03/2007; 12(1):50-60. · 2.21 Impact Factor
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    ABSTRACT: HERG potassium channel blockade is the major cause for drug-induced long QT syndrome, which sometimes cause cardiac disrhythmias and sudden death. There is a strong interest in the pharmaceutical industry to develop high quality medium to high-throughput assays for detecting compounds with potential cardiac liability at the earliest stages of drug development. Cultivation of cells at lower temperature has been used to improve the folding and membrane localization of trafficking defective hERG mutant proteins. The objective of this study was to investigate the effect of lower temperature maintenance on wild type hERG expression and assay performance. Wild type hERG was stably expressed in CHO-K1 cells, with the majority of channel protein being located in the cytoplasm, but relatively little on the cell surface. Expression at both locations was increased several-fold by cultivation at lower growth temperatures. Intracellular hERG protein levels were highest at 27 degrees C and this correlated with maximal 3H-dofetilide binding activity. In contrast, the expression of functionally active cell surface-associated hERG measured by patch clamp electrophysiology was optimal at 30 degrees C. The majority of the cytoplasmic hERG protein was associated with the membranes of cytoplasmic vesicles, which markedly increased in quantity and size at lower temperatures or in the presence of the Ca2+-ATPase inhibitor, thapsigargin. Incubation with the endocytic trafficking blocker, nocodazole, led to an increase in hERG activity at 37 degrees C, but not at 30 degrees C. Our results are consistent with the concept that maintenance of cells at reduced temperature can be used to boost the functional expression of difficult-to-express membrane proteins and improve the quality of assays for medium to high-throughput compound screening. In addition, these results shed some light on the trafficking of hERG protein under these growth conditions.
    BMC Biotechnology 02/2007; 7:93. · 2.17 Impact Factor
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    ABSTRACT: We investigated the role of small-conductance calcium-activated potassium (SK) and intermediate-conductance calcium-activated potassium channels in modulating sensory transmission from peripheral afferents into the rat spinal cord. Subunit-specific antibodies reveal high levels of SK3 immunoreactivity in laminas I, II, and III of the spinal cord. Among dorsal root ganglion neurons, both peripherin-positive (C-type) and peripherin-negative (A-type) cells show intense SK3 immunoreactivity. Furthermore, dorsal root-stimulated sensory responses recorded in vitro are inhibited when SK channel activity is increased with 1-ethyl-2-benzimidazolinone (1-EBIO). In vivo electrophysiological recordings show that neuronal responses to naturally evoked nociceptive and nonnociceptive stimuli increase after application of the selective SK channel blocker 8,14-diaza-1,7(1,4)-diquinolinacyclotetradecaphanedium di-trifluoroacetate (UCL 1848), indicating that SK channels are normally active in moderating afferent input. Conversely, neuronal responses evoked by mechanical stimuli are inhibited when SK channel activity is increased with 1-EBIO. These effects are reversed by the subsequent application of UCL 1848. Our data demonstrate that SK channels have an important role in controlling sensory input into the spinal cord.
    Journal of Neuroscience 05/2005; 25(14):3489-98. · 6.91 Impact Factor
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    ABSTRACT: Calcium-activated potassium channels are critically important in modulating neuronal cell excitability. One member of the family, the intermediate-conductance potassium (IK) channel, is not thought to play a role in neurones because of its predominant expression in non-excitable cells such as erythrocytes and lymphocytes, in smooth muscle tissues, and its lack of apparent expression in brain. In the present study, we demonstrate that IK channels are localized on specific neurones in the mouse enteric nervous system where they mediate the slow afterhyperpolarization following an action potential. IK channels were localized by immunohistochemistry on intrinsic primary afferent neurones, identified by their characteristic Dogiel type II morphology. The slow afterhyperpolarization recorded from these cells was abolished by the IK channel blocker clotrimazole. RT-PCR and western analysis of extracts from the colon revealed an IK channel transcript and protein identical to the IK channel expressed in other cell types. These results indicate that IK channels are expressed in neurones where they play an important role in modulating firing properties.
    Journal of Neurochemistry 10/2004; 90(6):1414-22. · 3.97 Impact Factor
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    ABSTRACT: The SK/IK family of small and intermediate conductance calcium-activated potassium channels contains four members, SK1, SK2, SK3 and IK1, and is important for the regulation of a variety of neuronal and non-neuronal functions. In this study we have analysed the distribution of these channels in human tissues and their cellular localisation in samples of colon and corpus cavernosum. SK1 mRNA was detected almost exclusively in neuronal tissues. SK2 mRNA distribution was restricted but more widespread than SK1, and was detected in adrenal gland, brain, prostate, bladder, liver and heart. SK3 mRNA was detected in almost every tissue examined. It was highly expressed in brain and in smooth muscle-rich tissues including the clitoris and the corpus cavernosum, and expression in the corpus cavernosum was upregulated up to 5-fold in patients undergoing sex-change operations. IK1 mRNA was present in surface-rich, secretory and inflammatory cell-rich tissues, highest in the trachea, prostate, placenta and salivary glands. In detailed immunohistochemical studies of the colon and the corpus cavernosum, SK1-like immunoreactivity was observed in the enteric neurons. SK3-like immunoreactivity was observed strongly in smooth muscle and vascular endothelium. IK1-like immunoreactivity was mainly observed in inflammatory cells and enteric neurons of the colon, but absent in corpus cavernosum. These distinctive patterns of distribution suggest that these channels are likely to have different biological functions and could be specifically targeted for a number of human diseases, such as irritable bowel syndrome, hypertension and erectile dysfunction.
    Archiv für Experimentelle Pathologie und Pharmakologie 07/2004; 369(6):602-15. · 2.15 Impact Factor
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    ABSTRACT: IK channels, which had been previously found in hemopoetically derived cells (including erythrocytes and lymphocytes) and epithelial cells, where they regulate proliferation, cell volume regulation and secretion, have only recently been discovered in neurons, where they had previously been claimed not to occur. Based on immunohistochemical detection of IK channel-like immunoreactivity, it has been reported that IK channel expression in enteric neurons is suppressed in Crohn's disease. In the present work we have investigated whether authentic IK channels are expressed by enteric neurons. Human and mouse tissue was investigated by immunohistochemistry, Western blot and RT-PCR. Immunohistochemical studies revealed IK channel-like immunoreactivity in large myenteric neurons, but not in other cell types in the external muscle layers. Many of these nerve cells had calbindin immunoreactivity. Western blots from the external muscle revealed an immunoreactive band at the molecular weight of the IK channel. Using RT-PCR, we detected a transcript corresponding to the IK channel gene in extracts from the ganglion containing layer. The sequence obtained from the RT-PCR product was identical to that previously published for the IK channel. We conclude that IK channels are expressed by human enteric neurons, including large smooth surfaced neurons that are possibly the human equivalent of the Dogiel type II neurons that express these channels in small mammals.
    Autonomic Neuroscience 06/2004; 112(1-2):93-7. · 1.85 Impact Factor
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    ABSTRACT: Recent functional evidence suggests that intermediate conductance calcium-activated potassium channels (IK channels) occur in neurons in the small intestine and in mucosal epithelial cells in the colon. This study was undertaken to investigate whether IK channel immunoreactivity occurs at these and at other sites in the gastrointestinal tract of the rat. IK channel immunoreactivity was found in nerve cell bodies throughout the gastrointestinal tract, from the esophagus to the rectum. It was revealed in the initial segments of the axons, but not in axon terminals. The majority of immunoreactive neurons had Dogiel type II morphology and in the myenteric plexus of the ileum all immunoreactive neurons were of this shape. Intrinsic primary afferent neurons in the rat small intestine are Dogiel type II neurons that are immunoreactive for calretinin, and it was found that almost all the IK channel immunoreactive neurons were also calretinin immunoreactive. IK channel immunoreactivity also occurred in calretinin-immunoreactive, Dogiel type II neurons in the caecum. Epithelial cells of the mucosal lining were immunoreactive in the esophagus, stomach, small and large intestines. In the intestines, the immunoreactivity occurred in transporting enterocytes, but not in mucous cells. Immunoreactivity was at both the apical and basolateral surfaces. A small proportion of mucosal endocrine cells was immunoreactive in the duodenum, ileum and caecum, but not in the stomach, proximal colon, distal colon or rectum. There was immunoreactivity of vascular endothelial cells. It is concluded that IK channels are located on cell bodies and proximal parts of axons of intrinsic primary afferent neurons, where, from functional studies, they would be predicted to lower neuronal excitability when opened in response to calcium entry. In the mucosa of the small and large intestine, IK channels are probably involved in control of potassium exchange, and in the esophageal and gastric mucosa they are possibly involved in control of cell volume in response to osmotic challenge.
    Cell and Tissue Research 12/2003; 314(2):179-89. · 3.68 Impact Factor