Mapping the urea channel through the rabbit Na+-glucose cotransporter SGLT1

1. The rabbit Na+-glucose cotransporter rbSGLT1 and its carboxy-terminal pa rt, C5, which contains transmembrane helices 10-14 of SGLT1 and functions a s a low affinity glucose uniporter, were expressed as individual proteins i n Xenopus oocytes. Transport of 55 muM urea, ethylene glycol, mannitol and alpha -methyl-D-glueopyranoside (alpha KDG) by control oocytes and by oocyt es expressing SGLT1 and C5 was studied by uptake measurements of the C-14-l abelled substrates. 2. There was a 5- to 6-fold increase in urea transport mediated by C5, comp ared with control oocytes. Similar to SGLT1, the C5-urea uptake was cation independent, linear in time and with increasing urea concentration, and blo cked with the same sensitivity by the inhibitor phloretin (K-i approximate to 1 mM). Like SGLT1 in choline buffer, the C5-mediated uptake was insensit ive to phlorizin. 3. Mannitol was transported by C5 but not by SGLT1 or control oocytes. 4. The activation energy (E-a) for urea transport through C5 was low (5 +/- 3 kcal mol(-1)) compared with that of non-injected oocytes (16 +/-0.5 kcal mol(-1)) and comparable with the E-a of passive urea or water transport thr ough intact SGLT1. 5. The urea influx through C5 increased in the presence of alpha MDG, but n ot in the presence of the same concentration of mannitol. 6. We conclude that the five carboxy-terminal transmembrane helices of SGLT 1 form a channel for the permeation of small molecules such as urea and wat er.