Renal Na+-K+-Cl- cotransporter activity and vasopressin-induced trafficking are lipid raft-dependent.
Apical bumetanide-sensitive Na(+)-K(+)-2Cl(-) cotransporter (NKCC2), the kidney-specific member of a cation-chloride cotransporter superfamily, is an integral membrane protein responsible for the transepithelial reabsorption of NaCl. The role of NKCC2 is essential for renal volume regulation. Vasopressin (AVP) controls NKCC2 surface expression in cells of the thick ascending limb of the loop of Henle (TAL). We found that 40-70% of Triton X-100-insoluble NKCC2 was present in cholesterol-enriched lipid rafts (LR) in rat kidney and cultured TAL cells. The related Na(+)-Cl(-) cotransporter (NCC) from rat kidney was distributed in LR as well. NKCC2-containing LR were detected both intracellularly and in the plasma membrane. Bumetanide-sensitive transport of NKCC2 as analyzed by (86)Rb(+) influx in Xenopus laevis oocytes was markedly reduced by methyl-beta-cyclodextrin (MbetaCD)-induced cholesterol depletion. In TAL, short-term AVP application induced apical vesicular trafficking along with a shift of NKCC2 from non-raft to LR fractions. In parallel, increased colocalization of NKCC2 with the LR ganglioside GM1 and their polar translocation were assessed by confocal analysis. Apical biotinylation showed twofold increases in NKCC2 surface expression. These effects were blunted by mevalonate-lovastatin/MbetaCD-induced cholesterol deprivation. Collectively, these findings demonstrate that a pool of NKCC2 distributes in rafts. Results are consistent with a model in which LR mediate polar insertion, activity, and AVP-induced trafficking of NKCC2 in the control of transepithelial NaCl transport.
Welker
Pia
P
Institute of Anatomy, Charité-Universitätsmedizin Berlin, Philippstrasse 12, Berlin, Germany.
Böhlick
Alexandra
A
Mutig
Kerim
K
Salanova
Michele
M
Kahl
Thomas
T
Schlüter
Hartmut
H
Blottner
Dieter
D
Ponce-Coria
Jose
J
Gamba
Gerardo
G
Bachmann
Sebastian
S
D000818:Animals; D001127:Arginine Vasopressin / Q000378:metabolism*; D019921:Biotinylation; D016764:Cell Polarity; D002478:Cells, Cultured; D002784:Cholesterol / Q000378:metabolism; D002853:Chromatography, Liquid; D020790:Diabetes Insipidus, Neurogenic / Q000378:metabolism*; D007668:Kidney / Q000378:metabolism*; D008138:Loop of Henle / Q000166:cytology; D008297:Male; D013058:Mass Spectrometry; D021962:Membrane Microdomains / Q000378:metabolism* / Q000648:ultrastructure; D009865:Oocytes; D011817:Rabbits; D051381:Rats; D020318:Rats, Long-Evans; D017207:Rats, Sprague-Dawley; D017483:Receptors, Vasopressin / Q000378:metabolism; D028021:Sodium-Potassium-Chloride Symporters / Q000378:metabolism*; D064466:Solute Carrier Family 12, Member 1; D014981:Xenopus
D017483:Receptors, Vasopressin; C578716:Slc12a1 protein, rat; D028021:Sodium-Potassium-Chloride Symporters; D064466:Solute Carrier Family 12, Member 1; D001127:Arginine Vasopressin; D002784:Cholesterol