MetaTOC stay on top of your field, easily

Mutation of a single threonine in the cytoplasmic N-terminus disrupts trafficking of the renal betaine-GABA transporter 1 during hypertonic stress

, , ,

Renal Physiology

Published online on

Abstract

Betaine is an important osmolyte and is, compared to other organs, much more abundant in the kidneys where it enters cells in the medulla by the Betaine-GABA-Transporter 1 (BGT1) to balance osmoregulation in the counter current system. In wildtype BGT1 (wt-BGT1)-expressing oocytes, GABA-mediated currents were diminished by preincubation of the oocytes with 100 nM PMA or 5 µM DOG, activators of the protein kinase C (PKC), while application of staurosporine prior to application of DOG restored the response to GABA. Four potential phosphorylation sites on BGT1 were mutated to alanine by site-directed mutagenesis. Three mutants (T235A, S428A, and S564A) evoked GABA currents comparable in magnitude to the currents observed in wt-BGT1-expressing oocytes, whereas GABA currents in T40A were barely detectable. Uptake of [3H]GABA was also determined in HEK293 cells expressing EGFP-tagged BGT1 with the same mutations. T235A, S428A, and S564A showed up-regulation of GABA uptake after hypertonic stress and down-regulation by PMA similar to EGFP-wt-BGT1. In contrast T40A did not respond to either hypertonicity or PMA. Confocal microscopy of the EGFP-BGT1 mutants expressed in MDCK cells revealed that T40A was present in cytoplasm after 24 hours of hypertonic stress while the other mutants and EGFP-wt-BGT1 were in the plasma membrane. All mutants, including T40A, co-migrated with wt-BGT1 on Western blots suggesting that they are full length proteins. T40A, however, cannot be phosphorylated as revealed using a specific anti-phospho antibody and therefore T40 may be important for trafficking and insertion of BGT1 in the plasma membrane.