Kidney Week

Abstract: SA-PO1030

Disruption of a C-Terminal Di-Leucine Motif Directs NKCC1 to the Apical Membrane and Lysosomes in Polarized Epithelia

Session Information

• Fluid and Electrolytes: Basic - II
  October 27, 2018 | Location: Exhibit Hall, San Diego Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Fluid and Electrolytes

• 901 Fluid and Electrolytes: Basic

Authors

• Koumangoye, Rainelli, Vanderbilt University Medical Center, Nashville, Tennessee, United States

Rainelli Koumangoye,

• Delpire, Eric J., Vanderbilt University Medical Center, Nashville, Tennessee, United States

Eric J. Delpire,

Background

Delivery of NKCC1 cotransporter to the basolateral membranes play in crucial rule in ions homeostasis in epithelia. We recently reported the first known SLC12A2 (NKCC1) mutation that causes epithelial dysfunction in an undiagnosed clinical case. The 11 bp deletion resulted in a non-functional transporter with a shorter cytosolic COOH-terminal tail. NKCC1-DFX maintains its ability to interact with wild-type NKCC1, leads to increase dimerization of the cotransporter, and causes mis-tafficking of the NKCC1-DFX mutant and wild-type transporters to the apical/subapical region of polarized epithelia.

Methods

In this study, we used sequential truncation and site-directed mutagenesis within NKCC1 COOH domain and immunofluorescence to examine the trafficking of wild-type and mutants NKCC1 cotransporters in polarized MDCK cells.

Results

Our results show that truncation of NKCC1 COOH domain uncouple the co-transporter from the lateral membrane. We also targeted a di-leucine motif (DxxxLL^1193-1194) part of a previously described tetrad in the extreme C-terminus of NKCC1 as well as a di-leucine motif (DxxxLL^1036-1037) located upstream. Disruption of the DxxxLL^1036-1037 motif led to some increase localization in the cytoplasm, although most the cotransporter remained at the plasma membrane. In contrast, mutation of the terminal DxxxLL^1193-1194 motif led to cotransporter accumulation in the cytoplasm and mis-trafficking to the apical/sub-apical region of polarized epithelial; recapitulating the phenotype observed in NKCC1-DFX mutant. This observation indicates that the DxxxLL^1193-1194 motif is important for trafficking and maintenance of NKCC1 to the basolateral membrane. Truncation deletion and LL substitution mutants are trafficked out of the Trans-Golgi network and ER but accumulates in early endosomes and late endosomes where they are degraded. Whether they reach the endosomes by trafficking transiently through the plasma membrane is currently under investigation.

Conclusion

Our data demonstrate that NKCC1 basolateral trafficking is regulated by a conserved di-leucine motif in its C-terminal domain. Loss of DxxxLL^1193-1194 leads to mis-trafficking of NKCC1 to the apical membrane and accumulation in late endosomes in the subapical region of the polarized epithelia.

Funding

• Other NIH Support