Mapping the urea channel through the rabbit Na(+)-glucose cotransporter SGLT1.
1. The rabbit Na(+)-glucose cotransporter rbSGLT1 and its carboxy-terminal part, C5, which contains transmembrane helices 10-14 of SGLT1 and functions as a low affinity glucose uniporter, were expressed as individual proteins in Xenopus oocytes. Transport of 55 microM urea, ethylene glycol, mannitol and alpha-methyl-D-glucopyranoside (alphaMDG) by control oocytes and by oocytes expressing SGLT1 and C5 was studied by uptake measurements of the 14C-labelled substrates. 2. There was a 5- to 6-fold increase in urea transport mediated by C5, compared with control oocytes. Similar to SGLT1, the C5-urea uptake was cation independent, linear in time and with increasing urea concentration, and blocked with the same sensitivity by the inhibitor phloretin (K(i) approximately 1 mM). Like SGLT1 in choline buffer, the C5-mediated uptake was insensitive 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 through intact SGLT1. 5. The urea influx through C5 increased in the presence of alphaMDG, but not in the presence of the same concentration of mannitol. 6. We conclude that the five carboxy-terminal transmembrane helices of SGLT1 form a channel for the permeation of small molecules such as urea and water.
Panayotova-Heiermann
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Department of Physiology, UCLA Medical Center, Los Angeles, CA 90095-1751, USA. mheiermann@mednet.ucla.edu
Wright
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EM
D000818:Animals; D002250:Carbon Radioisotopes / Q000493:pharmacokinetics; D004234:Diuretics, Osmotic / Q000493:pharmacokinetics; D019855:Ethylene Glycol / Q000493:pharmacokinetics; D005260:Female; D005947:Glucose / Q000378:metabolism; D008353:Mannitol / Q000493:pharmacokinetics; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008757:Methylglucosides / Q000493:pharmacokinetics; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D009865:Oocytes / Q000502:physiology; D011817:Rabbits; D012964:Sodium / Q000378:metabolism; D051269:Sodium-Glucose Transporter 1; D014508:Urea / Q000378:metabolism*; D014982:Xenopus laevis
D002250:Carbon Radioisotopes; D004234:Diuretics, Osmotic; D008562:Membrane Glycoproteins; D008757:Methylglucosides; D009004:Monosaccharide Transport Proteins; D051269:Sodium-Glucose Transporter 1; D008353:Mannitol; C027020:methylglucoside; D014508:Urea; D012964:Sodium; D019855:Ethylene Glycol; D005947:Glucose