Glutamate is taken up avidly by glial cells in the central nervous system. Glutamate uptake may terminate the transmitter action of glutamate released from neurons, and keep extracellular glutamate at concentrations below those which are neurotoxic. We report here that glutamate evokes a large inward current in retinal glial cells which have their membrane potential and intracellular ion concentrations controlled by the whole-cell patch-clamp technique. This current seems to be due to an electrogenic glutamate uptake carrier, which transports at least two sodium ions with every glutamate anion carried into the cell. Glutamate uptake is strongly voltage-dependent, decreasing at depolarized potentials: when fully activated, it contributes almost half of the conductance in the part of the glial cell membrane facing the retinal neurons. The spatial localization, glutamate affinity and magnitude of the uptake are appropriate for terminating the synaptic action of glutamate released from photoreceptors and bipolar cells. These data challenge present explanations of how the b-wave of the electroretinogram is generated, and suggest a mechanism for non-vesicular voltage-dependent release of glutamate from neurons.

Uptake of glutamate into glial cells in the CNS maintains the extracellular glutamate concentration below neurotoxic levels and helps terminate its action as a neurotransmitter. The co-transport of two sodium ions on the glutamate carrier is thought to provide the energy needed to transport glutamate into cells. We have shown recently that glutamate uptake can be detected electrically because the excess of Na+ ions transported with each glutamate anion results in a net current flow into the cell. We took advantage of the control of the environment, both inside and outside the cell, provided by whole-cell patch-clamping and now report that glutamate uptake is activated by intracellular potassium and inhibited by extracellular potassium. Our results indicate that one K+ ion is transported out of the cell each time a glutamate anion and three Na+ ions are transported in. A carrier with this stoichiometry can accumulate glutamate against a much greater concentration gradient than a carrier co-transporting one glutamate anion and two Na+ ions. Pathological rises in extracellular potassium concentration will inhibit glutamate uptake by depolarizing glial cells and by preventing the loss of K+ from the glutamate carrier. This will facilitate a rise in the extracellular glutamate concentration to neurotoxic levels and contribute to the neuronal death occurring in brain anoxia and ischaemia.

SUMMARY1. The effects of excitatory amino acids on the membrane current of isolated retinal glial cells (Muller cells) were investigated using whole-cell patch clamping.2. L-Glutamate evoked an inward current at membrane potentials between -140 and +50 mV. The current was larger at more negative potentials.3. The glutamate-evoked current was activated by external cations with relative efficacies: Na+ > Li+> K+ > Cs+, choline. It was activated by internal cations with relative efficacies K+ > Rb+ > Cs+ > choline. Chloride and divalent cations did not affect the glutamate-evoked current.4. Raising the intracellular sodium or glutamate concentrations, or raising the extracellular potassium concentration, reduced the current evoked by external glutamate. The suppressive effect of internal glutamate was larger when the internal sodium concentration was high.5. Some analogues of glutamate also evoked an inward current. Responses to Laspartate resembled those to glutamate, but for aspartate the apparent affinity was higher and the voltage dependence of the current was steeper. In the physiological potential range the current evoked by a saturating dose of aspartate was less than that evoked by a saturating dose of glutamate.6. The uptake blocker threo-3-hydroxy-DL-aspartate (30,tM) reduced the glutamate-evoked current, but also generated a current itself. Dihydrokainate (510 /LM) weakly inhibited the glutamate-evoked current without generating a current itself.7. The commonly used blockers of glutamate-gated ion channels, 2-amino-5-phosphonovalerate (APV; 100 ,M), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 20 /bM), and kynurenate (1 mM) had no effect on the glutamate-evoked current. 8. The voltage dependence, cation dependence and pharmacological profile of the current evoked by excitatory amino acids indicate that it is caused by activation of the high-affinity glutamate uptake carrier. This carrier appears to transport one glutamate anion into the cell, one K+ ion out of the cell, and two or more Na+ ions into the cell, on each carrier cycle. At the inner membrane surface some or all of the transported Na+ dissociates from the carrier after the transported glutamate has dissociated.MS 8441

Genes Kcna1 and Kcna2 code for the voltage-dependent potassium channel subunits Kv1.1 and Kv1.2, which are coexpressed in large axons and commonly present within the same tetramers. Both contribute to the low-voltage-activated potassium current I Kv1, which powerfully limits excitability and facilitates temporally precise transmission of information, e.g., in auditory neurons of the medial nucleus of the trapezoid body (MNTB). Kcna1-null mice lacking Kv1.1 exhibited seizure susceptibility and hyperexcitability in axons and MNTB neurons, which also had reduced I Kv1. To explore whether a lack of Kv1.2 would cause a similar phenotype, we created and characterized Kcna2-null mice (-/-). The -/- mice exhibited increased seizure susceptibility compared with their +/+ and +/- littermates, as early as P14. The mRNA for Kv1.1 and Kv1.2 increased strongly in +/+ brain stems between P7 and P14, suggesting the increasing importance of these subunits for limiting excitability. Surprisingly, MNTB neurons in brain stem slices from -/- and +/- mice were hypoexcitable despite their Kcna2 deficit, and voltage-clamped -/- MNTB neurons had enlarged I Kv1. This contrasts strikingly with the Kcna1-null MNTB phenotype. Toxin block experiments on MNTB neurons suggested Kv1.2 was present in every +/+ Kv1 channel, about 60% of +/- Kv1 channels, and no -/- Kv1 channels. Kv1 channels lacking Kv1.2 activated at abnormally negative potentials, which may explain why MNTB neurons with larger proportions of such channels had larger I Kv1. If channel voltage dependence is determined by how many Kv1.2 subunits each contains, neurons might be able to fine-tune their excitability by adjusting the Kv1.1:Kv1.2 balance rather than altering Kv1 channel density.

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