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.