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Cloning, immunolocalization, and functional expression of a GABA transporter from the retina of the skate

Published online by Cambridge University Press:  02 June 2005

ANDREA D. BIRNBAUM ,
SUSAN K. ROHDE ,
HAOHUA QIAN ,
MUAYYAD R. AL-UBAIDI ,
JOHN H. CALDWELL  and
ROBERT P. MALCHOW
ANDREA D. BIRNBAUM
Affiliation:
Department of Biological Sciences, University of Illinois at Chicago, Chicago
SUSAN K. ROHDE
Affiliation:
Department of Biological Sciences, University of Illinois at Chicago, Chicago
HAOHUA QIAN
Affiliation:
Department of Biological Sciences, University of Illinois at Chicago, Chicago Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago
MUAYYAD R. AL-UBAIDI
Affiliation:
Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City
JOHN H. CALDWELL
Affiliation:
Department of Cellular and Developmental Biology, University of Colorado Health Sciences Center, Denver
ROBERT P. MALCHOW
Affiliation:
Department of Biological Sciences, University of Illinois at Chicago, Chicago Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago
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Abstract

Termination of GABA signals within the retina occurs through high-affinity reuptake of the released neurotransmitter by GABA transporters (GATs) present in neurons and glia surrounding the release site. In the present work, we have cloned a novel GAT from the retina of the skate (Raja erinacea). The clone codes for a 622 amino acid protein whose sequence has highest similarity to the GABA/β-alanine transporter of the electric ray (Torpedo marmorata) (88% identity) and the GAT-3 isolated from rat brain (75% identity). The protein was expressed in Xenopus oocytes and characterized using the two-electrode voltage-clamp technique. Application of GABA induced a dose-dependent inward current, with 8 μM GABA producing a half-maximal response. The current required the presence of extracellular sodium and was unaffected by the GABA receptor blocker picrotoxin or the GAT-1 specific antagonist NO-711. The high homology between the cloned skate GABA transporter and the GAT-3 equivalents of other species, coupled with the strikingly similar pharmacological profile to GAT-3s of other species, lead us to conclude that we had cloned the GAT-3 homologue for the skate. Polyclonal antibodies specific to GAT-3 and the previously cloned skate GAT-1 transporter were used to examine the distribution of GAT-3 and GAT-1 immunoreactivity in the retina and in isolated cells of the skate. Antibodies for both transporters showed labeling in the outer and inner plexiform layers, and staining extended from the outer to inner limiting membranes. Both GAT-1 and GAT-3 antibodies labeled enzymatically isolated Müller cells, while bipolar cells and horizontal cells did not appear to express either transporter. These results imply that GAT-1 and GAT-3 are both present in Müller cells of the skate retina where they are likely involved in regulating extracellular concentrations of GABA.

Keywords

Xenopus oocyteImmunocytochemistryMüller cell
Type
Research Article
Information
Visual Neuroscience , Volume 22 , Issue 2 , March 2005 , pp. 211 - 223
Copyright
© 2005 Cambridge University Press

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