Inherited Defects of Sodium-dependent Glutamate Transport Mediated by Glutamate/Aspartate Transporter in Canine Red Cells Due to a Decreased Level of Transporter Protein Expression

Abstract

Canine red cells have a high affinity Na؉ /K ؉ -dependent glutamate transporter. We herein demonstrate that this transport is mediated by the canine homologue of glutamate/aspartate transporter (GLAST), one of the glutamate transporter subtypes abundant in the central nervous system. We also demonstrate that GLAST is the most ubiquitous glutamate transporter among the transporter subtypes that have been cloned to date. The GLAST protein content was extremely reduced in variant red cells, low glutamate transport (LGlut) red cells characterized by an inherited remarkable decrease in glutamate transport activity. All LGluT dogs carried a missense mutation of Gly 492 to Ser (G492S) in either the heterozygous or homozygous state. The GLAST protein with G492S mutation was fully functional in glutamate transport in Xenopus oocytes. However, G492S GLAST exhibited a marked decrease in activity after the addition of cycloheximide, while the wild type showed no significant change, indicating that G492S GLAST was unstable compared with the wild-type transporter. Moreover, LGluT dogs, but not normal dogs, heterozygous for the G492S mutation showed a selective decrease in the accumulation of GLAST mRNA from the normal allele. Based on these findings, we conclude that a complicated heterologous combination of G492S mutation and some transcriptional defect contributes to the pathogenesis of the LGluT red cell phenotype.

Molecular Cloning of Canine Excitatory Amino Acid Transporter 5 and Its Detection in Primary Lens Epithelial Cells

“…After isolation of total RNA from a primary culture of LEC, RT-PCR amplification was performed with primer set, s-1 and a-2, employing a SuperScript first cDNA system kit (Invitrogen) and Hot start Ex Taq DNA polymerase (Takara) under the following conditions: 25 cycles of at 94°C 10 s, 58°C 15 s, and 72°C 60 s. Primers used for the amplification of canine EAAT1-EAAT4 and GAPDH were prepared as described by Sato et al [16] (Table 1). …”

Ubiquitylation-independent ER-associated degradation of an AE1 mutant associated with dominant hereditary spherocytosis in cattle

“…In vitro transcription of RNA and microinjection into Xenopus oocytes were performed essentially as described (Sato et al, 2000). Groups of five to ten oocytes were incubated in ND-96 medium (15 l/oocyte) containing 7-10 Ci/ml Na 36 Cl (Amersham) for 1 hour at 19°C in the presence or absence of 10 M DIDS (Dojin Chemical Laboratories, Kumamoto, Japan).…”

“…Preparation of red-cell ghosts, determination of membrane protein contents, deglycosylation of AE1 in red-cell membranes, and in vitro translation were carried out as described previously (Inaba and Maede, 1988;Inaba et al, 1996;Sato et al, 2000).…”

Changes in Extracellular Neurotransmitters in the Cerebrum of Familial Idiopathic Epileptic Shetland Sheepdogs Using an Intracerebral Microdialysis Technique and Immunohistochemical Study for Glutamate Metabolism

“…Sections were stained with the hematoxylin and eosin (HE) and Klüver-Barrera (KB) stains. Three polyclonal antibodies, one for GLT-1 (Wako, Japan diluted 1:100), one for GLAST [23], and one for Glu (Chemicon, U.S.A., diluted 1:5), and one monoclonal antibody for GS (Chemicon, U.S.A., diluted 1: 300) were used for immunohistochemistry. Sections were employed for immunohistochemical analysis using the labeled streptavidin procedure (DAKO, Glostrup, Denmark).…”