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A small fraction of total NKCC2 is located in the apical membrane of thick ascending limbs under basal conditions
Author(s) -
Ortiz Pablo A
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.a1223-b
Subject(s) - apical membrane , chemistry , apical cell , membrane , basal (medicine) , absorption (acoustics) , biochemistry , biophysics , biology , endocrinology , insulin , physics , acoustics
We previously showed that trafficking of the apical Na/K/2Cl cotransporter NKCC2 is involved in the regulation of NaCl absorption by thick ascending limbs (TAL). It is not known which fraction of the total pool of NKCC2 is located in the apical membrane under basal conditions. We hypothesized that a small fraction of total NKCC2 in the apical membrane mediates NaCl absorption by TALs. We measured surface and total NKCC2 in TALs by biotinylation and Western blot. In TAL suspensions surface NKCC2 accounted for 2.6 ± 0.5% of total NKCC2 (n = 9). We also measured NKCC2 levels in the apical membrane of isolated perfused TALs with confocal microscopy. To resolve apical NKCC2, the apical membrane was labeled with a fluorescent lectin. NKCC2 immunofluorescence was quantified in the apical membrane (labeled by the lectin) and in the whole cell. NKCC2 intensity in the apical membrane accounted for 5.2 ± 0.6 % of total NKCC2 fluorescence (n = 4). Finally, we tested whether decreasing surface NKCC2 levels could inhibit net Cl absorption (J Cl ) in TALs. Incubation of TALs with tetanus toxin (20 min) decreased surface NKCC2 levels by 59.2 ± 6.5 % (n = 4; p <0.04). In isolated TALs, tetanus toxin (20 min) decreased J Cl by 59.5 ± 9.5 % (n = 4, p<0.05). We concluded that a small fraction of the total pool of NKCC2 is located in the apical membrane and mediates NaCl absorption under basal conditions.