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Nicotinic acetylcholine receptors (nAChR's) containing the α6 subunit (α6) are putative drug targets of relevance to Parkinson's disease and nicotine addiction. However, heterologous expression of α6 receptors has proven challenging which has stifled drug discovery efforts. Here, we investigate potential new avenues for achieving functional α6 receptor expression. Combinations of chimeric and mutated α6, β2 and β3 subunits were co-expressed in the human HEK293 cell line and receptor expression was assessed using Ca(2+)-imaging (FLIPR™) and whole-cell patch-clamp electrophysiology. Transient transfections of a chimeric α6/α3 subunit construct in combination with β2 and β3(V9'S) gave rise to significant acetylcholine-evoked whole-cell currents. Increasing the β3(V9'S):β2:α6/α3 cDNA ratio, resulted in a significantly higher fraction of cells with robust current levels. Using an excess of wild-type β3, significant functional expression of α6/α3β2β3 was also demonstrated. Comparing the acetylcholine concentration-response relationship of α6/α3β2β3(V9'S) to that of α6/α3β2β3 revealed the β3 point mutation to result in decreased current decay rate and increased ACh agonist potency. Ca(2+)-imaging experiments showed preservation of basic α6 receptor pharmacology. Our results establish that α6/α3β2β3(V9'S) replicate several basic features of native α6 receptors but also highlight several caveats associated with using this construct and may therefore provide guidance for future drug hunting efforts., (© 2013 Published by Elsevier B.V.)

Microglial cells play an important role during neuroinflammation in the central nervous system. Among other factors, activated microglia produce nitric oxide (NO), which is toxic to neurons and excessive microglial activation and NO production contribute to the pathology of neurodegenerative diseases. Chloride channels have previously been shown to participate in microglial activation. Here we investigate the effects of established chloride channel blockers with different chemical structures on interferon-gamma (IFNgamma)-induced activation of the murine microglial cell line, BV2. IFNgamma-induced NO production was effectively reduced by NPPB, IAA-94, tamoxifen, NS3728 and NS1652, with NS1652 being the most potent. In contrast, DIDS reduced NO production only at cytotoxic concentrations. Furthermore, NS1652 reduced the protein and mRNA levels of inducible nitric oxide synthase (iNOS), without altering STAT1 phosphorylation. These observations suggest a microglial chloride conductance as a critical permissive factor downstream in the IFNgamma-induced iNOS cascade. The nature of the underlying channel is unknown, but the pharmacological profile appears incompatible with the involvement of the volume activated anion conductance (VRAC).