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Abstract: SA-PO1053

A Truncation Mutant Targets Wild-Type NKCC1 to the Apical Membrane of Epithelia

Session Information

  • Na+, K+, Cl-
    November 04, 2017 | Location: Hall H, Morial Convention Center
    Abstract Time: 10:00 AM - 10:00 AM

Category: Fluid, Electrolytes, and Acid-Base

  • 703 Na+, K+, Cl- Basic


  • Koumangoye, Rainelli, Vanderbilt University Medical Center, Nashville, Tennessee, United States
  • Omer, Salma, Vanderbilt University Medical Center, Nashville, Tennessee, United States
  • Delpire, Eric J., Vanderbilt University Medical Center, Nashville, Tennessee, United States

We recently reported the case of a 13-year old patient with complete gastrointestinal and bladder shut-downs; thyroid-, parathyroid-, and pancreatic-insufficiencies; and orthostatic intolerance. The patient carries a de novo mutation in SLC12A2, the gene encoding the Na-K-2Cl cotransporter-1 or NKCC1. The 11 bp deletion resulted in a non-functional transporter with a shorter cytosolic COOH-terminal tail. Presence of the mutant transporter was shown to increase the amount of dimer, indicating the possibility that the mutant transporter exerts dominant-negative effects on wild-type transporter. No dominant-negative effects were observed in Xenopus laevis oocytes.


In this study, we used fluorescent-tagged wild-type and mutant NKCC1 transporter cDNAs transfected in HEK293 and MDCK cells to examine cell membrane trafficking. HEK293 cells were grown on glass coverslips, whereas MDCK cells were grown on glass coverslips, permeabilized support, and matrigel.


Cells transfected with mutant NKCC1 showed rounding and disruption of well-organized honeycomb-like structures. The overall NKCC1 signal was markedly reduced, indicating that the mutant transporter significantly affected expression of the wild-type transporter. In polarized MDCK cells, most of the mutant transporter signal was observed on the apical membrane. The mutant transporter also affected the formation of MDCK cysts in 3-D cultures, as we observed the presence of multiple lumens per cyst. To test whether mutant NKCC1 affected the trafficking of the wild-type cotransporter, co-transfection experiments with tdTomato- and EGFP-tagged transporters were performed. We demonstrated that the mutant transporter caused trafficking of wild-type cotransporter to the apical membrane. The NKCC1 mutant transporter, however, did not affect the overall polarity of the cells, as the alpha subunit of the Na+/K+-ATPase was still observed on the basolateral membrane.


Our data demonstrate that expression of a truncated Na-K-2Cl cotransporter in epithelia causes targeting of the wild-type cotransporters to the wrong membrane and also affects proper lumen formation in MDCK cysts. These observations may explain the multisystem dysfunction that is observed in the patient.


  • NIDDK Support