Mutation of a single threonine in the cytoplasmic NH2 terminus disrupts trafficking of renal betaine-GABA transporter 1 during hypertonic stress.
Betaine is an important osmolyte and is, compared with other organs, much more abundant in the kidneys, where it enters cells in the medulla by betaine-GABA transporter 1 (BGT1) to balance osmoregulation in the countercurrent system. In wild-type (wt-)BGT1-expressing oocytes, GABA-mediated currents were diminished by preincubation of oocytes with 100 nM PMA or 5 μM dioctanoyl-sn-glycerol, activators of PKC, whereas the application of staurosporine before the application of dioctanoyl-sn-glycerol 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 currents observed in wt-BGT1-expressing oocytes, whereas GABA currents in T40A were barely detectable. Uptake of [(3)H]GABA was also determined in human embryonic kidney-293 cells expressing enhanced green fluorescent protein (EGFP)-tagged BGT1 with the same mutations. T235A, S428A, and S564A showed upregulation of GABA uptake after hypertonic stress and downregulation 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 Madin-Darby canine kidney cells revealed that T40A was present in the cytoplasm after 24 h of hypertonic stress. whereas the other mutants and EGFP-wt-BGT1 were in the plasma membrane. All mutants, including T40A, comigrated with wt-BGT1 on Western blots, suggesting that they are full-length proteins. T40A, however, cannot be phosphorylated, as revealed using a specific anti-phosphoantibody, and, therefore, T40 may be important for the trafficking and insertion of BGT1 in the plasma membrane.
Structural Biology Department, Max-Planck-Institute of Biophysics, Frankfurt am Main, Germany; and.
Eva S
ES
Schweikhard
Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Indianapolis, Indiana.
Stephen A
SA
Kempson
Structural Biology Department, Max-Planck-Institute of Biophysics, Frankfurt am Main, Germany; and.
Christine
C
Ziegler
Institute of Systemic Physiology and Pathophysiology, University Medical Center Göttingen, Göttingen, Germany; Birgitta.Burckhardt@med.uni-goettingen.de.
Birgitta C
BC
Burckhardt
D000818:Animals; D001622:Betaine / Q000494:pharmacology*; D002460:Cell Line; D050485:GABA Plasma Membrane Transport Proteins / Q000235:genetics*; D006801:Humans; D007668:Kidney / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed / Q000379:methods; D009154:Mutation / Q000235:genetics*; D009997:Osmotic Pressure / Q000187:drug effects* / Q000502:physiology; D021381:Protein Transport / Q000502:physiology; D013912:Threonine / Q000235:genetics* / Q000378:metabolism; D015533:Transcriptional Activation / Q000187:drug effects / Q000235:genetics; D014981:Xenopus
D050485:GABA Plasma Membrane Transport Proteins; D013912:Threonine; D001622:Betaine
endocytosis; membrane trafficking; osmotic stress; protein kinase C