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J Biol Chem. 2012 Jan 13;287(3):2210-20. doi: 10.1074/jbc.M111.309211. Epub 2011 Nov 25.

Regulatory activation is accompanied by movement in the C terminus of the Na-K-Cl cotransporter (NKCC1).

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1
Department of Cellular and Molecular Physiology, Yale School of Medicine, New Haven, Connecticut 06520, USA. michelle.monette@yale.edu

Abstract

The Na-K-Cl cotransporter (NKCC1) is expressed in most vertebrate cells and is crucial in the regulation of cell volume and intracellular chloride concentration. To study the structure and function of NKCC1, we tagged the transporter with cyan (CFP) and yellow (YFP) fluorescent proteins at two sites within the C terminus and measured fluorescence resonance energy transfer (FRET) in stably expressing human embryonic kidney cell lines. Both singly and doubly tagged NKCC1s were appropriately produced, trafficked to the plasma membrane, and exhibited (86)Rb transport activity. When both fluorescent probes were placed within the same C terminus of an NKCC1 transporter, we recorded an 11% FRET decrease upon activation of the transporter. This result clearly demonstrates movement of the C terminus during the regulatory response to phosphorylation of the N terminus. When we introduced CFP and YFP separately in different NKCC1 constructs and cotransfected these in HEK cells, we observed FRET between dimer pairs, and the fractional FRET decrease upon transporter activation was 46%. Quantitatively, this indicates that the largest FRET-signaled movement is between dimer pairs, an observation supported by further experiments in which the doubly tagged construct was cotransfectionally diluted with untagged NKCC1. Our results demonstrate that regulation of NKCC1 is accompanied by a large movement between two positions in the C termini of a dimeric cotransporter. We suggest that the NKCC1 C terminus is involved in transport regulation and that dimerization may play a key structural role in the regulatory process. It is anticipated that when combined with structural information, our findings will provide a model for understanding the conformational changes that bring about NKCC1 regulation.

PMID:
22121194
PMCID:
PMC3265899
DOI:
10.1074/jbc.M111.309211
[Indexed for MEDLINE]
Free PMC Article
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