Table of Contents
Physiology Journal
Volume 2013, Article ID 598321, 9 pages
Research Article

A Single Amino Acid Substitution in the Renal Betaine/GABA Transporter Prevents Trafficking to the Plasma Membrane

Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Medical Science Building 385, 635 Barnhill Drive, Indianapolis, IN 46202-5120, USA

Received 29 November 2012; Revised 22 March 2013; Accepted 29 March 2013

Academic Editor: Andre Van Wijnen

Copyright © 2013 Christopher R. Day et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


One response to hypertonic stress in the renal medulla and MDCK cells is the upregulation of betaine transporter (BGT1) synthesis, followed by trafficking to the plasma membrane (PM) and an increase in betaine transport. Upregulation of BGT1 was enhanced by inhibitors of phosphatases PP1 and PP2A and was attenuated by inhibitors of protein kinase C, suggesting an important role for phosphorylation reactions. This was tested using mutants of BGT1 tagged with EGFP. The PM trafficking motifs of BGT1 reside near the C terminus, and truncation at lysine560 resulted in a protein that remained intracellular during hypertonic stress. This K560 mutant colocalized with endoplasmic reticulum (ER). Substitution of alanine at Thr40, a putative phosphorylation site, also prevented trafficking to the PM during hypertonic stress. Live-cell imaging showed that T40A was not retained in the ER and colocalized with markers for Golgi and endosomes. In contrast, substitution of aspartate or glutamate at Thr40, to mimic phosphorylation, restored normal trafficking to the PM. HEK293 cells transfected with K560 or T40A mutants had 10% of the GABA transport activity of native BGT1, but normal transport activity was restored in cells expressing T40E. Normal BGT1 trafficking likely requires phosphorylation at Thr40 in addition to C-terminal motifs.