Ion Channel Regulation By Signal Transduction Pathways

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We have shown previously that Rho family GTPases regulate potassium channel activity in pituitary cells, so we tested this idea in microglia that are believed to be responsive for much of the inflammatory response in the brain to neurodegeneration. As we reported in a paper in the journal Glia, chemokines that regulate microglial activation and motility also regulate inwardly rectifying potassium channels in microglia (Muessel et al., 2013). We are now using a microRNA that allows selective expression of transgenes in microglia to test whether the channels themselves are involved in the regulation of activation and motility, which would make them important new drug targets for neurodegenerative disease. To identify toxicants targeting oxytocin signaling, we engineered a human cell line (HEK293) to express human OXTR and a new generation, low affinity, genetically encoded calcium indicator, GCAMP3 (Tian, L. et al., 2009 Nature Methods 6:875-881). We screened the cells in a 96-well plate fluorescence reader. Calcium signaling was stimulated half maximally by 3 nM oxytocin and blocked half maximally by 60 nM atosiban, an established Oxtr antagonist. We identified two flame retardants, tris (2,3-dibromo-2-propyl) phosphate (TDBPP) and tris (1,3-dichloro-2-propyl) phosphate (TDCPP) that reduced the calcium signal when they were applied at concentrations between 20-100 uM. At the single cell level, however, atosiban and the flame retardants had very different effects on the calcium signals induced by oxytocin. The origin of the difference was revealed when we stimulated calcium release and entry independently of oxytocin by inhibiting the calcium pump in the endoplasmic reticulum (ER) with thapsigargin. As expected for an oxytocin receptor antagonist, atosiban had no effect on the calcium signals produced by thapsigargin. In contrast, both flame retardants selectively suppressed calcium entry after the ER calcium was depleted. The same concentrations of the flame retardants also inhibit the calcium-dependent synaptic potentiation induced by oxytocin on CA1 pyramidal neurons in hippocampal slices from PD 12-17 mouse brains. We have subsequently shown that the flame retardants block TRPC5 channels, which were thought to be regulated by Gq signaling through calcium and PIP2 and/or its metabolites. However, the flame retardants do not interfere with Gq signaling or calcium release. We discovered that they block TRPC5 channels completely, and not by blocking the pore directly. We also showed that TRPC5 is required for oxytocin-dependent potentiation because it is missing in a TRPC5 knockout mouse strain. We are now examining the mechanism of TRPC5 stimulation by Gq signaling with recombinant channels in a heterologous system mammalian system. We have also established that somatostatin stops spiking through a diffusible, cAMP-dependent mechanism that targets two pore potassium channels involving protein phosphatases and we have reconstituted this signaling in a heterologous system. We have confirmed this mechanism in rodent hippocampal neurons, both in acute slices and in dissociated cultures. In fact there are two mechanisms in the neurons, the appearance of one of which is also regulated by protein phosphatases.