Volume-sensitive Conformational Changes and Multimerization of theNa-K-Cl Cotransporter Tagged with CFP and YFP

1.2 Summary The Na-K-Cl cotransporter, NKCC1, is involved in the regulation of intracellular chloride concentration and cell volume. We have tagged both the N terminus and a non-conserved loop in the C terminus of shark NKCC1 with ECFP and EYFP and expressed these constructs in HEK cells. The constructs are fluorescent and correctly trafficked to the cell membrane. We show that NKCC1 with either the N-terminal or the C-terminal tag, as well as NKCC1 with both tags, are active and regulated by intracellular chloride. There is a significant amount of FRET between the two dyes in all cell lines, including cell lines coexpressing two NKCC1s, one fused with a single CFP, and the other one with YFP. The latter result indicates cotransporter multimerization. There is an increase in FRET upon cell shrinkage. In order to assess the mechanism of the observed FRET changes we have cotransfected NKCC1 with two tags and untagged 'wt NKCC1'. We can show that the amount of FRET is dependent on the ratio of wt NKCC1 to tagged NKCC1. This confirms that part of the FRET signal is due to multimerization of NKCC1 rather than dimerization of GFP. The FRET changes are of the same magnitude in cell lines cotransfected with untagged NKCC1, suggesting that they reflect a conformational change rather than an increase in the tendency to form multimers. The FRET studies have been carried out with the Q69M mutant of YFP, which has a low chloride sensitivity (Griesbeck et al., 2001). The constructs using regular YFP are quenched by chloride in the 0 to 100 mM range, CFP is not chloride sensitive. The ratio of YFP to CFP fluorescence in cell lines transfected with double-tagged NKCC1 is a dye concentration independent measure of the intracellular chloride concentration in HEK cells.