Hostname: page-component-cd9895bd7-jn8rn Total loading time: 0 Render date: 2024-12-23T17:44:36.788Z Has data issue: false hasContentIssue false
  • English
  • Français

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
Get access Rights & Permissions [Opens in a new window]

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

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

REFERENCES

Agardh, E., Bruun, A., Ehinger, B., Ekstrom, P., van Veen, T., & Wu, J.Y. (1987). Gamma-aminobutyric acid- and glutamic acid decarboxylase-immunoreactive neurons in the retina of different vertebrates. Journal of Comparative Neurology 258, 622–630.CrossRefGoogle Scholar
Ariel, M. & Daw, N.W. (1982). Pharmacological analysis of directionally sensitive rabbit retinal ganglion cells. Journal of Physiology 324, 161–185.CrossRefGoogle Scholar
Ayoub, G.S. & Lam, D.M. (1984). The release of gamma-aminobutyric acid from horizontal cells of the goldfish (Carassius auratus) retina. Journal of Physiology 355, 191–214.CrossRefGoogle Scholar
Bennett, E.R. & Kanner, B.I. (1997). The membrane topology of GAT-1, a (Na+/Cl−) coupled γ-aminobutyric acid transporter from rat brain. Journal of Biological Chemistry 272, 1203–1210.CrossRefGoogle Scholar
Bennett, E.R., Su, H., & Kanner, B.I. (2000). Mutation of Arginine 44 of GAT-1, a (Na(+) + Cl(−))-coupled γ-aminobutyric acid transporter from rat brain, impairs net flux but not exchange. Journal of Biological Chemistry 275, 34106–34113.CrossRefGoogle Scholar
Biedermann, B., Wolf, S., Kohen, L., Wiedemann, P., Buse, E., Reichenbach, A., & Pannicke, T. (2002). Patch-clamp recording of Müller glial cells after cryopreservation. Journal of Neuroscience Methods 120, 173–178.CrossRefGoogle Scholar
Borden, L.A., Smith, K.E., Hartig, P.R., Branchek, T.A., & Weinshank, R.L. (1992). Molecular heterogeneity of the gamma-aminobutyric acid (GABA) transport system. Cloning of two novel high affinity GABA transporters from rat brain. Journal of Biological Chemistry 267, 21098–21104.Google Scholar
Borden, L.A., Murali Dhar, T.G., Smith, K.E., Weinshank, R.L., Branchek, T.A., & Gluchowski, C. (1994a). Tiagabine, SK&F 89976-A, CI-966, and NNC-711 are selective for the cloned GABA transporter GAT-1. European Journal of Pharmacology 269, 219–224.Google Scholar
Borden, L.A., Dhar, T.G., Smith, K.E., Branchek, T.A., Gluchowski, C., & Weinshank, R.L. (1994b). Cloning of the human homologue of the GABA transporter GAT-3 and identification of a novel inhibitor with selectivity for this site. Receptors Channels 2, 207–213.Google Scholar
Brecha, N.C. & Weigmann, C. (1994). Expression of GAT-1, a high-affinity gamma-aminobutyric acid plasma membrane transporter in rat retina. Journal of Comparative Neurology 345, 602–611.CrossRefGoogle Scholar
Brunken, W.J., Witkovsky, P., & Karten, H.J. (1986). Retinal neurochemistry of three elasmobranch species: An immunohistochemical approach. Journal of Comparative Neurology 243, 1–12.CrossRefGoogle Scholar
Bruun, A., Ehinger, B., & Sytsma, V.M. (1984). Neurotransmitter localization in the skate retina. Brain Research 295, 233–248.CrossRefGoogle Scholar
Caldwell, J.H., Daw, N.W., & Wyatt, H.J. (1978). Effects of picrotoxin and strychnine on rabbit retinal ganglion cells: Lateral interactions for cells with more complex receptive fields. Journal of Physiology 276, 277–298.CrossRefGoogle Scholar
Cammack, J.N. & Schwartz, E.A. (1993). Ions required for the electrogenic transport of GABA by horizontal cells of the catfish retina. Journal of Physiology 472, 81–102.CrossRefGoogle Scholar
Daw, N.W. & Wyatt, H.J. (1976). Kittens reared in a unidirectional environment: Evidence for a critical period. Journal of Physiology 257, 155–170.CrossRefGoogle Scholar
Ekstrom, P. & Anzelius, M. (1998). GABA and GABA-transporter (GAT-1) Immunoreactivities in the retina of salmon (Salmo salar L.) Brain Research 812, 179–185.Google Scholar
Guastella, J., Nelson, N., Nelson, H., Czyzyk, L., Keynan, S., Miedel, M.C., Davidson, N., Lester, H.A., & Kanner, B.I. (1990). Cloning and expression of a rat brain GABA transporter. Science 249, 1303–1306.CrossRefGoogle Scholar
Guimbal, C., Klostermann, A., & Kilimann, M.W. (1995). Phylogenetic conservation of 4-aminobutyric acid (GABA) transporter isoforms. Cloning and pharmacological characterization of a GABA/β-alanine transporter from Torpedo. European Journal of Biochemistry 234, 794–800.Google Scholar
Hofmann, K. & Stoffel, W. (1993). TMBase—a database of membrane spanning segments. Biological Chemistry 374, 166.Google Scholar
Honda, S., Yamamoto, M., & Saito, N. (1995). Immunocytochemical localization of three subtypes of GABA transporter in rat retina. Brain Research Molecular Brain Research 33, 319–325.CrossRefGoogle Scholar
Hu, M., Bruun, A., & Ehinger, B. (1999). Expression of GABA transporter subtypes (GAT1, GAT3) in the adult rabbit retina. Acta Ophthalmologica Scandinavica 77(3), 255–260.CrossRefGoogle Scholar
Johnson, J., Chen, T.K., Rickman, D.W., Evans, C., & Brecha, N.C. (1996). Multiple gamma-aminobutyric acid plasma membrane transporters (GAT-1, GAT-2, GAT-3) in the rat retina. Journal of Comparative Neurology 345, 212–224.3.0.CO;2-5>CrossRefGoogle Scholar
Keshet, F.I., Bendahan, A., Su, H., Mager, S., Lester, H.A., & Kanner, B.I. (1995). Glutamate-101 is critical for the function of the sodium and chloride-coupled GABA transporter GAT-1. FEBS Letters 371, 39–42.CrossRefGoogle Scholar
Kreitzer, M.A., Andersen, K.A., & Malchow, R.P. (2003). Glutamate modulation of GABA transport in retinal horizontal cells of the skate. Journal of Physiology 546, 717–731.CrossRefGoogle Scholar
Lam, D.M. (1975). Synaptic chemistry of identified cells in the vertebrate retina. Cold Spring Harbor Symposium on Quantitative Biology, Volume XL, pp. 571–579.
Linser, P.J., Sorrentino, M., & Moscona, A.A. (1984). Cellular compartmentalization of carbonic anhydrase-C and glutamine synthetase in developing and mature neural retina. Developmental Brain Research 13, 67–71.Google Scholar
Liu, Q.R., Lopez-Corcuera, B., Mandiyan, S., Nelson, H., & Nelson, N. (1993). Molecular characterization of four pharmacologically distinct gamma-aminobutyric acid transporters in mouse brain. Journal of Biological Chemistry 268, 2106–2112.Google Scholar
Lopez-Corcuera, B., Liu, Q.R., Mandiyan, S., Nelson, H., & Nelson, N. (1992). Expression of a mouse brain cDNA encoding novel gamma-aminobutyric acid transporter. Journal of Biological Chemistry 267, 17491–17493.Google Scholar
Mabjeesh, N.J. & Kanner, B.I. (1992). Neither amino nor carboxyl termini are required for function for the sodium- and chloride-coupled γ-aminobutyric acid transporter from rat brain. Journal of Biological Chemistry 267, 2563–2568.Google Scholar
MacKinnon, R. (1995). Pore loops: An emerging theme in ion channel structure. Neuron 14, 889–892.CrossRefGoogle Scholar
Malchow, R.P., Qian, H.H., & Ripps, H. (1989). γ-aminobutyric acid (GABA)-induced currents of skate Muller (glial) cells are mediated by neuronal-like GABAA receptors. Proceedings of the National Academy of Sciences of the U.S.A. 86, 4226–4230.Google Scholar
Malchow, R.P. & Ripps, H. (1990). Effects of gamma-aminobutyric acid on skate retinal horizontal cells: Evidence for an electrogenic uptake mechanism. Proceedings of the National Academy of Sciences of the U.S.A. 87, 8945–8949.CrossRefGoogle Scholar
Malchow, R.P., Qian, H.H., Ripps, H., & Dowling, J.E. (1990). Structural and functional properties of two types of horizontal cell in the skate retina. Journal of General Physiology 95, 177–198.CrossRefGoogle Scholar
Malchow, R.P. & Andersen, K.A. (2001). GABA transporter function in the horizontal cells of the skate. Progress in Brain Research 131, 267–275.CrossRefGoogle Scholar
Massey, S.C. & Redburn, D.A. (1987). Transmitter circuits in the vertebrate retina. Progress in Neurobiology 28, 55–96.CrossRefGoogle Scholar
Muth, T.R., Ahn, J., & Caplan, M.J. (1998). Identification of sorting determinants in the C-terminal cytoplasmic tails of the gamma-aminobutyric acid transporters GAT-2 and GAT-3. Journal of Biological Chemistry 273, 25616–25627.CrossRefGoogle Scholar
Nelson, H., Mandiyan, S., & Nelson, N. (1990). Cloning of the human brain GABA transporter. FEBS Letters 269, 181–184.CrossRefGoogle Scholar
Nelson, N. (1998). The family of Na+/Cl− neurotransmitter transporters. Journal of Neurochemistry 71, 1785–1803.CrossRefGoogle Scholar
Page, R.D.M. (1996). TREEVIEW: An application to display phylogenetic trees on personal computers. Computer Applications in the Biosciences 12, 357–358.Google Scholar
Palacin, M., Estevez, R., Bertran, J., & Zorzano, A. (1998). Molecular biology of mammalian plasma membrane amino acid transporters. Physiological Reviews 78, 969–1054.Google Scholar
Pantanowitz, S., Bendahan, A., & Kanner, B.I. (1993). Only one of the charged amino acids located in the transmembrane α-helices of the γ-aminobutryic acid transporter (subtype A) is essential for its activity. Journal of Biological Chemistry 268, 3222–3225.Google Scholar
Pourch, R.G., Goebel, D.J., & McReynolds, J.S. (1984). Autoradiographic studies of [3H] glycine, [3H]-GABA, and [3H]-muscimol uptake in the mudpuppy retina. Experimental Eye Research 39, 69–81.CrossRefGoogle Scholar
Qian, H., Malchow, R.P., & Ripps, H. (1993). The effects of lowered extracellular sodium on gamma-aminobutyric acid (GABA)-induced currents of Muller (glial) cells of the skate retina. Cellular and Molecular Neurobiology 13, 147–158.CrossRefGoogle Scholar
Qian, X., Malchow, R.P., O'Brien, J., & Al-Ubaidi, M.R. (1998). Isolation and characterization of a skate retinal GABA transporter cDNA. Molecular Vision 4, 6.Google Scholar
Risso, S., DeFelice, L.J., & Blakely, R.D. (1996). Sodium-dependent GABA-induced currents in GAT1-transfected HeLa cells. Journal of Physiology 490, 691–702.CrossRefGoogle Scholar
Ruiz, M., Egal, H., Sarthy, V., Qian, X., & Sarkar, H.K. (1994). Cloning, expression, and localization of a mouse retinal γ-aminobutryic acid transporter. Investigative Ophthalmology and Visual Science 35, 4039–4048.Google Scholar
Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., & Higgins, D.G. (1997). The CLUSTAL_X windows interface: Flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research 25, 4876–4882.CrossRefGoogle Scholar
Vaney, D.I. & Young, H.M. (1988). GABA-like immunoreactivity in cholinergic amacrine cells of the rabbit retina. Brain Research 438, 369–373.CrossRefGoogle Scholar
Werblin, F. (1991). Synaptic connections, receptive fields, and patterns of higher activity in the tiger salamander retina. Investigative Ophthalmology and Visual Science 32, 459–483.Google Scholar
Wu, S. (1992). Opponent-processing effects on the field spectral sensitivity of pattern-elicited electroretinograms. Vision Research 32, 2031–2041.Google Scholar
Yang, C.Y., Brecha, N.C., & Tsao, E. (1997). Immunocytochemical localization of gamma-aminobutyric acid plasma membrane transporters in the tiger salamander retina. Journal of Comparative Neurology 389, 117–126.3.0.CO;2-5>CrossRefGoogle Scholar
Yang, C.Y. & Yazulla, S. (1988). Light microscopic localization of putative glycinergic neurons in the larval tiger salamander retina by immunocytochemical and autoradiographical methods. Journal of Comparative Neurology 272, 343–357.CrossRefGoogle Scholar
Yazulla, S. (1985). Factors controlling the release of GABA from goldfish retina horizontal cells. Neuroscience Research Supplement 2, 147–165.CrossRefGoogle Scholar
Zhao, J.W., Du, J.L., Li, Y.S., & Yang, X.L. (2000). Expression of GABA transporters on bullfrog retinal Muller cells. Glia 31, 104–117.3.0.CO;2-E>CrossRefGoogle Scholar