Figure 4.

Water-flux is proportional to SLC4A11 expression level and is inhibited by stilbene disulfonates. (A and B) HEK293 cells were co-transfected with eGFP cDNA and indicated amount of cDNA encoding HA-tagged wild-type (WT) SLC4A11. (A) SLC4A11 expression was quantified on immunoblots of cell lysates probed with anti-HA antibody. (B) Cell swelling of HEK293 cells, expressing various amounts of SLC4A11, was assessed by the eGFP dilution assay (as in Fig. 2C). The rate of swelling observed for vector-alone transfected cells was subtracted from the values observed for cells expressing SLC4A11. (C) Chemical structures of SLC4A11 inhibitors used here. (D and E) HEK293 cells were co-transfected with eGFP and either SLC4A11, AQP1 or vector cDNA. Water-flux following shift to hypo-osmotic medium, assessed by measuring the rate of eGFP fluorescence change (as in Fig. 2C), was corrected for rate in vector-transfected cells. (D) Water-flux assays were performed in the presence of varied concentrations of DNDS, a non-covalently acting compound classically used to inhibit proteins of the SLC4 family (48). Half-maximal effective concentration determined from the curve is 24 ± 1 µM DNDS, n = 3. (E) eGFP dilution water-flux assays were performed in the absence or presence of 100 µM H₂DIDS, a covalently acting stilbene disulfonate. Rates of fluorescence change were corrected for the value found for vector-transfected cells. *P < 0.05. N.S., not significant. n = 3.