PGB acutely affects only large P/Q mediated currents and P/Q channel´s cellular distribution

WEISSMANN C; URBANO FJ; GONZÁLEZ INCHAUSPE C; DI GUILMI, MN; UCHITEL OD

Cancun

Congreso; I FALAN Congress; 2012

IBRO

We set out to investigate the mechanism of action of pregabalin (PGB) -an anticonvulsant and analgesic drug used in the treatment of epilepsy and neuropathic pain- on P/Q-calcium channels. We previously reported that slices from calyx of Held of WT-mice incubated acutely with PGB (500 _M) show slightly reduced P/Q-calcium (CaV2.1) currents compared to untreated ones (Di Guilmi et al, 2010). In this work, replacing calcium by barium, we showed greater differences between PGB-treated and untreated slices (17±1.2% reduction), without any changes in voltage activating properties. We also analyzed HEK293t cells transfected with subunits of CaV2.1 channels, and detected by an eGFP-tagged _1 subunit. Fluorescent cells yielded robust barium-mediated currents that decreased (56±12%) after PGB (500 _M) treatment. Thus, at a real synapse as well as in an heterologous system, PGB applied acutely determined the reduction of large CaV2.1-mediated barium currents. To test whether the effect of PGB was dependent on the magnitude of CaV2.1-mediated currents, we analyzed the effect of PGB in transgenic R192Q KI mice. These mice express a familial hemiplegic migraine type-1 mutation and show greater current amplitude during long lasting action potentials (APs) stimulation compared to WT animals (Inchauspe et al, 2010). KI slices incubated with PGB decreased the currents to WT-levels when stimulated with long duration APs (larger currents). This effect was absent with short APs, Using time-lapse fluorescence microscopy of HEK293t transfected cells. We were able to follow the distribution of the channels after acute incubation of PGB showing decreased (~30%) membrane fluorescence and suggesting the internalization of through acute PGB-mediated mechanism. These results not only provide the mechanism of action of PGB, but also highlight the importance of the drug as a potential candidate to use in other calcium channelopathies to drive currents to steady state levels.