Modulation of CaV2.1 channels by cholesterol levels

CARINA WEISSMANN; BETTINA BOSCO ACKERMAN; FRANCISCO J. URBANO ; OSVALDO D. UCHITEL

Huerta Grande, Córdoba

Congreso; XXVIII Congreso Anual de la Sociedad Argentina de Investigación en Neurociencia; 2013

SAN

Calcium channels show different compartmentalization on neuronal plasma membranes where they form clusters, involving both cytoskeletal elements and microdomains within the lipid bilayer. CaV2.1 channels are distributed in lipid microdomains.We showed an acute effect of pregabalin (PGB, a α2δ-binding drug) on the cellular function and distribution of CaV2.1 channels transfected in HEK293t. The system allowed us to visualize the internalization of subunits within cells after PGB treatment by means of fluorescence microscopy, while recording barium-mediated currents (IBa). We studied how the cytoskeleton and the lipid rafts organization might modulate the calcium channels. For this purpose, we treated transfected cells with methyl-cyclodextrin (MβCD, 5-10 min.), a cholesterol sequestering drug, and determined the internalization of the fluorescent subunits, and the distribution of microtubules. MβCD (10 mM, 10-20min) increased α1 internalization (with a membrane/interior ratio 10% lower compared to untreated cells), similarly as after PGB treatment. However, it did not increase α2δ internalization.  Acute MβCD reduced IBa. MβCD and PGB exerts an effect on the microtubule cytoarchitecture evidenced by a diffuse tubulin staining. The implications on the importance of this modulation lies on the fact that lipids change in the aged brain, and the fact that the different subunits might be internalised through different mechanisms (clathrin vs.caveolae-mediated mechanisms).