Rescue of ganglioside-deficient CHO-K1 cells and acetylcholine receptor domains.

IDI BONINI; M. V. PICARDI; VICENTE BERMÚDEZ; B. DE LOS SANTOS; ANA MARIA ROCCAMO; FRANCISCO JOSE BARRANTES

Bariloche

Simposio; ARGENTINE SOCIETY FOR BIOCHEMISTRY AND MOLECULAR BIOLOGY XXXIX ANNUAL MEETING. BIOPHYSICAL SOCIETY OF ARGENTINA XXXII ANNUAL MEETING. BARILOCHE PROTEIN SYMPOSIUM.; 2003

ARGENTINE SOCIETY FOR BIOCHEMISTRY AND MOLECULAR BIOLOGY y BIOPHYSICAL SOCIETY OF ARGENTINA

In orderto investigate the possible association of the nicotinic acetylcholine receptor(AChR) with lipid microdomains (?rafts?) we have exploited the intrinsic GM2synthase intrinsic deficiency of the CHO-K1/A5 cell line that stably expressesadult muscle-type AChR, to produce a double-transfectant new clone (coined CHO-K1/GM2)rescued from such deficiency by expression of UDP-GalNAc:lactosylceramide/GM3/GD3 β-1,4-N-acetylgalactosaminyltransferase, a med-Golgi localized enzyme. The resistance to Triton X-100 andother mild detergent (CHAPS, sulfobetain and octyl-POE) solubilization was usedas one of the assaysfor microdomain occurrence. Similar solubilization profiles were observed forboth cell lines. Density gradients and Western blots of detergent-treated cellslabelled with [125I]α-bungarotoxin showed the preferential AChRdistribution in high density fractions. In view of the importance ofcholesterol in AChR function, we modified its content in both cell lines using cyclodextrins.CHO-K1/A5 cells treated with 15 mM methyl-β-cyclodextrin (MβCD)showed a decrease in [125I]α-bungarotoxin label of up to 50%.A similar decrease in label occurred in Triton X-100 soluble fractions of bothcell lines treated with 0-15 mM MβCD.                      Work supported in part by grants from UNS, FONCYT, and FIRCA 1-RO3-TWO 1225-01 NIH.