Endogenous l-glutamate transport in oocytes of Xenopus laevis

doi:10.1016/0005-2736(91)90244-3

Biochimica et Biophysica Acta (BBA) - Biomembranes

Volume 1066, Issue 1, 1 July 1991, Pages 14-20

https://doi.org/10.1016/0005-2736(91)90244-3 Get rights and content

Abstract

The existence of an endogenous Na⁺-glutamate cotransporter in the oocytes of Xenopus laevis is demonstrated. The transporter does not accept d-glutamate as substrate. The dependence on substrate displays two saturating components with low (K_1/2 = 9mM) and high (K_1/2 = 0.35 μM) affinities for l-glutamate. The dependence on external Na⁺ exhibits a saturating component with a K_1/2 value of about 5 mM and a component that has not saturated up to 110 mM Na⁺. In voltage-clamped oocytes, it is possible to demonstrate that Na⁺-dependent l-glutamate transport is directly coupled to countertransport of Rb⁺. The analysis of the voltage dependence of the Na⁺, K⁺-dependent l-glutamate uptake suggests that positive charges are moved inwardly during the transport cycle.

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Oocytes of the South African clawed frog Xenopus laevis are widely used as a heterologous expression system for the characterization of transport systems such as passive and active membrane transporters, receptors and a whole plethora of other membrane proteins originally derived from animal or plant tissues. The large size of the oocytes and the high degree of expression of exogenous mRNA or cDNA makes them an optimal tool, when compared with other expression systems such as yeast, Escherichia coli or eukaryotic cell lines, for the expression and functional characterization of membrane proteins. This easy to handle expression system is becoming increasingly attractive for pharmacological research. Commercially available automated systems that microinject mRNA into the oocytes and perform electrophysiological measurements fully automatically allow for a mass screening of new computer designed drugs to target membrane transport proteins. Yet, the oocytes possess a large variety of endogenous membrane transporters and it is absolutely mandatory to distinguish the endogenous transporters from the heterologous, expressed transport systems. Here, we review briefly the endogenous membrane transport systems of the oocytes.
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The excitatory amino acid transporter EAAC1 is an electrogenic Na⁺- and K⁺-gradient-driven transporter. In addition, the transporter mediates in the presence of Na⁺ and glutamate an anion conductance uncoupled from the transport of the glutamate. The first two N-terminal domains, important for forming the conductance mode, are extracellularly bordered by positively charged arginine residues, R39 and R61, being completely conserved throughout the transporter family. Also the conserved tyrosine residue Y98 could be important for Cl⁻ conductance. We have investigated, by measurements of glutamate uptake and glutamate-induced currents, the effects of mutation of the arginines and the tyrosine to alanine. The mutation R39A hardly affects transport and channel mode. The mutation R61A, on the other hand, reduces the activity of transport but stimulates the channel conductance. In addition, the apparent K_m values for glutamate uptake and for the glutamate-activated current are reduced. Glutamate stimulation of current seems to be associated with a voltage-dependent step, and the apparent valence of charge moved during binding is reduced in the R61A mutant. The mutation Y98A leads to reduced function with reduced apparent K_m value for glutamate, and with strong reduction of the selectivity ration between NO₃⁻ and Cl⁻ of the conductance mode.
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^*: Present address: Zentrum für Innere Medizin, Univ. Ulm, W-7900 Ulm, F.R.G.

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Endogenous l-glutamate transport in oocytes of Xenopus laevis

Abstract

Biochim. Biophys. Acta

J. Biol. Chem.

J. Biol. Chem.

Biochim. Biophys. Acta

Biochim. Biophys. Acta

Arch. Biochem. Biophys.

Biochim. Biophys. Acta

Cell Biol. Int. Rep.

J. Biol. Chem.

Comp. Biochem. Physiol.

Rev. Physiol. Biochem. Phar.

Biochemistry

Nature

Eur. J. Biochem.

Am. J. Physiol.

Am. J. Physiol.

Biochemistry

Pflügers Arch.

Jap. J. Physiol.