Ion Channel Regulation By Signal Transduction Pathways

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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. Thus, we conclude that synaptic potentiation by Gq-coupled hormone receptors requires calcium entry through store-operated calcium channels, such as ORAI2, which is highly expressed in hippocampal pyramidal neurons. Subsequent work on recombinant store-operated signaling proteins has confirmed that the flame retardants which target STIM2/ORAi2 are the ones that disrupt oxytocin signaling. 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.