Acute effects of pregabalin on the function and cellular distribution of CaV 2.1 in HEK293t cells

WEISSMANN C; DI GUILMI MN; URBANO FJ; UCHITEL OD

BRAIN RESEARCH BULLETIN

PERGAMON-ELSEVIER SCIENCE LTD

Lugar: Amsterdam; Año: 2013 vol. 90 p. 107 - 113

0361-9230

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 CaV2.1 (P/Q-type) calcium channels function and distribution at the membrane level. HEK293t cells were transfected with plasmids coding for all subunits of the CaV2.1 channel. We used a α1 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 microM PGB, that binds alpha2delta subunits, for 30 minutes 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 CaV2.1 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 CaV2.1 channels to Isoleucine, another alpha2delta ligand. Together these results show strong evidence for an acute effect of PGB on CaV2.1 calcium channelś currents and distribution and suggest that internalization of CaV2.1 channels might be a mechanism of PGB action.PGB induced acute changes in localization of CaV2.1 channels in a HEK cell model. PGB-induced CaV2.1 channels internalization correlates with a reversible reduction of barium-mediated calcium currents. PGB and not other alpha2delta ligands tested induced the changes in CaV2.1 channels distribution. PGB changed the distribution of CaV2.1 channels and not of other membrane receptors tested (P2X7). PGB mediated the internalization of CaV2.1 channels in a clathrin dependent manner.