Reconstitution in liposomes of the functionally active human OCTN1 (SLC22A4) transporter over-expressed in E. coli.

Lorena Pochini , Mariafrancesca Scalise , Michele Galluccio , Linda Amelio , Cesare Indiveri (1)

1 Biologia Cellulare

The hOCTN1 transporter over-expressed in E. coli and purified by Ni-chelating chromatography has been reconstituted in liposomes by detergent removal with a batch-wise procedure. The reconstitution was optimised with respect to the protein concentration, the detergent/phospholipid ratio and the time of incubation with Amberlite XAD-4 resin. Time dependent [14C]tetraethylammonium, [3H]carnitine or [3H]ergothioneine uptake was measured in proteoliposomes with activities ratio of 8.0 : 1.3 : 1.0, respectively. Optimal activity was found at pH 8.0. The transport depended on intraliposomal ATP. [14C]tetraethylammonium transport was inhibited by several compounds; the most effective were acetylcholine and γ-butyrobetaine, followed by acetylcarnitine and tetramethylammonium. Reagents such as pyridoxal 5-phosphate, MTSES and mercurials strongly inhibited the transport. From kinetic analysis of tetraethylammonium transport a Km of 0.77 mM was calculated. Acetylcholine and γ-butyrobetaine behaved as competitive inhibitors of tetraethylammonium transport with Ki values of 0.44 and 0.63 mM, respectively.

Mots clés: Life Sciences

Lorena Pochini, Mariafrancesca Scalise, Michele Galluccio, Linda Amelio, Cesare Indiveri. Reconstitution in liposomes of the functionally active human OCTN1 (SLC22A4) transporter over-expressed in E. coli.. Biochemical Journal, 2011, 439 (2), pp.227-233. ⟨10.1042/BJ20110544⟩. ⟨hal-00628665⟩