Acute effects of pregabalin on the function and cellular distribution of Cav2.1 in HEK293t cells

We established a cell model to study the acute effects of pregabalin (PGB), a drug widely used in epilepsy and neuropathic pain, on voltage gated Ca(v)2.1 (P/Q-type) calcium channels function and distribution at the membrane level. HEK293t cells were transfected with plasmids coding for all subunits of the Ca(v)2.1 channel. We used a al fused to an eGFP tag to follow its distribution in time and at different experimental conditions. The expressed channel was functional as shown by the presence of barium-mediated, calcium currents of transfected cells measured by 'whole-cell voltage-clamp' recordings, showing a maximum current peak in the I-V curve at +20 mV. The GFP fluorescent signal was confined to the periphery of the cells. Incubation with 500 mu M PGB, that binds alpha 2 delta subunits, for 30 min induced changes in localization of the fluorescent subunits as measured by fluorescent time lapse microscopy. These changes correlated with a reversible reduction of barium currents through Ca(v)2.1 calcium channels under the same conditions. However, no changes in the cellular distribution of the subunits were visualized for cells either expressing another membrane associated protein or after exposure of the Ca(v)2.1 channels to isoleucine, another alpha 2 delta ligand. Together these results show strong evidence for an acute effect of PGB on Ca(v)2.1 calcium channels' currents and distribution and suggest that internalization of Ca(v)2.1 channels might be a mechanism of PGB action. (C) 2012 Elsevier Inc. All rights reserved.