Gating mechanisms of TRPC1 channel in hippocampal neurons.

• ¹ Université Catholique de Louvain, Belgium

The canonical family of Transient Receptor Potential (TRPC) proteins owns seven members (TRPC1 to TRPC7) that form homo- and/or heterotetrameric Ca2+-permeable, nonselective cation membrane channels (Owsianik et al., 2006). TRPC channels are widely expressed in the central nervous system but their functions and their gating mechanisms remain unclear. They are able to modulate neuronal excitability and could promote excitotoxicity (Phelan et al., 2012; Phelan et al., 2013; Narayanan et al., 2014). In particular, heterotetrameric complexes made of TRPC1, TRPC4 and or TRPC5 seem involved presynaptically in the efficiency of synaptic transmission in the hippocampus (Broker-Lai et al., 2017). Recently, we reported that mice made deficient in TRPC1 expression (Trpc1-/- mice) showed alterations in spatial working memory and fear conditioning (Lepannetier et al., 2017). On brain slices, we also observed that LTP triggered by a theta burst could not maintain over time in Trpc1-/- mice. Interestingly, Li et al. recently found that TRPC1 deletion aggravated learning and memory deficits induced by amyloid-β, the major component of senile plaques observed in the brains of Alzheimer disease patients (Li et al., 2018). The present study was therefore focused on the gating mechanisms of TRPC1. We observed that stimulation of neurons with the selective agonist of group I mGluR dihydroxyphenylglycine (DHPG, 50 µM) induced an intracellular Ca2+ transient that was slightly reduced by pre-treatment with 10µM LY367385, a specific inhibitor of mGluR1 but completely abolished by 50 µM MPEP, a specific inhibitor of mGluR5. The response to DHPG was significantly reduced in neurons from Trpc1-/- mice. Emptying the stores of Ca2+ with the SERCA inhibitor thapsigargin triggered an influx of Ca2+ that was slightly reduced in neurons from Trpc1-/- mice, suggesting that TRPC1 acts at least partially as a store-operated channel, as shown in other cell types (Ong et al., 2017; Tajeddine et al., 2012). However, after inhibition of the mGluR-induced phospholipase C pathway by 10 µM U73122, DHPG still induced a Ca2+ response that depended on the presence of external Ca2+ and that was not observed in neurons from Trpc1-/- mice suggesting that DHPG also induced an entry of Ca2+ through TRPC1 that was independent of ER store depletion. As expected, DHPG was still able to induce an entry of Ca2+ after emptying the stores with thapsigargin in neurons from WT animals. This store-independent entry of Ca2+ induced by DHPG was not observed in neurons from Trpc1-/- mice. Electrophysiological experiments were then performed to characterize DHPG-induced response. Isolated hippocampal neurons from WT and Trpc1-/- mice were voltage-clamped at -60mV. To prevent neuronal activity, experiments were performed in the presence of inhibitors of Na+ voltage-dependent channels, AMPA, NMDA and GABA receptors. Moreover, to prevent the activation of store-dependent entry of Ca2+, the PLC inhibitor U-73122 (5 µM) was also added in the extracellular medium. In these conditions, stimulation of the wild type cells with 50 µM DHPG induced an inward current that was significantly diminished in neurons isolated from Trpc1-/- mice (1.13 ± 0.22 pA/pF, n=5 in WT vs 0.23 ± 0.06 pA/pF, n= 5 in Trpc1-/- neurons). In current clamp mode, stimulation of WT neurons with 50 µM DHPG depolarized the cells by about 11 mV. This depolarization was significantly decreased although not abolished in Trpc1-/- neurons. To examine the consequences on neuronal excitability, we measured spontaneous excitatory postsynaptic current of neurons kept in culture and treated with inhibitors of GABA receptors. We observed that stimulation of the cells with 50 µM DHPG increased by a factor of 3 the firing frequency. In conclusion, our data suggest that, in hippocampal neurons, stimulation of mGluR5 receptor activates both a store-operated and a store-independent entry of cations through TRPC1 channel, resulting in a depolarization of the membrane potential and in an increased synaptic excitability.