(M0930-04-24) Role of FLVCR2 in the Intestinal Choline Uptake in the Rat Model

Purpose: Choline is a nutritional compound that needs to be supplied dietarily and absorbed from the small intestine. Carrier-mediated transport has been suggested to be involved in the intestinal uptake of this compound, which is otherwise only poorly permeable across the cellular membrane by simple diffusion because of its high hydrophilicity, to facilitate the process. However, the carrier-mediated mechanism has not been fully clarified. Particularly, the molecular aspects including the transporter responsible for that remain to be elucidated. To address the issue, we examined the possibility that feline leukemia virus subgroup C receptor 2 (FLVCR2/SLC49A2), which is a newly identified choline transporter and highly expressed in the small intestine, might be involved in intestinal choline uptake, using the rat model. Clarifying the carrier-mediated mechanism should be of help in further exploring the potential role of the transporter in the intestinal absorption of drugs analogous to choline and also its exploitation for oral drug delivery.

Methods: The uptake of [³H]choline (2 nM) was evaluated for 1 min at pH 7.4 and 37°C, unless otherwise indicated, in MDCKII cells stably expressing rat FLVCR2 for the functional analysis. The subcellular localization of FLVCR2 was assessed in polarized FLVCR2-expressing MDCKII cells by immunofluorescent analysis. Experiments were also conducted using the everted sacs prepared from the small intestine of male Wistar rats. The uptake of [³H]choline (1 nM) into the everted sacs was evaluated for 2 min at pH 7.4 and 37°C, unless otherwise indicated.

Results: The uptake of choline was greater in FLVCR2-expressing MDCKII cells than in mock cells, indicating that rat FLVCR2 is capable of transporting choline. The specific uptake of choline by FLVCR2 was reduced, although moderately, by the removal of Na⁺ in the medium and with a decrease in pH. The rather moderate effects suggested that neither Na⁺ nor H⁺ is involved in FLVCR2-mediated choline transport as the driving ion. Therefore, it is likely that FLVCR2 operates in a facilitative manner for choline transport. Kinetically, the concentration-dependent profile of the specific uptake was mostly accounted for by a saturable component with the Michaelis constant (K_m) of 1.36 mM. Although it was accompanied by another component with a smaller K_m of 0.051 mM, suggesting the presence of two distinct choline recognition sites, the former component with the greater K_m (lower affinity) was prevailing. The specific uptake was also found to be specifically inhibited by ethanolamine and hemicholinium-3. In addition, FLVCR2 was found to be localized to the apical membrane in polarized FLVCR2-expressing MDCKII cells, suggesting its role as an apical uptake transporter. Finally, choline uptake in the everted sacs of the rat small intestine was almost fully accounted for by a single saturable transport component with the K_m of 1.95 mM, which was comparable to that of the prevailing lower affinity component of the FLVCR2-mediated choline transport. Also, choline uptake was moderately dependent on Na⁺ and pH, and specifically inhibited by ethanolamine and hemicholinium-3, in agreement with the characteristics observed for FLVCR2. Based on all these, FLVCR2 is likely to be responsible for intestinal choline uptake. It seems that its function for the prevailing lower affinity component is mainly involved in that, while the function for another accompanying component does not have a detectable contribution at the tissue level.

Conclusion: Functional characterization of rat FLVCR2 as a choline transporter was achieved using MDCKII cells stably expressing FLVCR2. Furthermore, FLVCR2 was suggested to be responsible for intestinal choline uptake at the apical membrane in the rat model. Future studies should be warranted to clarify the role of FLVCR2 in intestinal choline uptake in humans.