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| TR |
TITLE |
AUTHORS |
KEYWORDS |
MATERIALS & METHODS
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MICROSCOPIC
TECHNIQUES |
SPECIES |
MORPHOLOGY |
CELL
LINE |
| 607 |
Volume-sensitive
Conformational Changes
and Multimerization of the Na-K-Cl
Cotransporter Tagged with CFP and YFP |
Vorgelegt
von Meike Pedersen |
NKCC1,
intracellular chloride concentration, cell volume, FRET, cell shrinkage |
Cells used for FRET-imaging were
grown on 6 cm culture dishes with a coverslip on the bottom (Mattek Corp.).
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Fluorescence
Microscopy |
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HEK-293 |
| Abstract |
| |
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. |
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