Electrophysiology of glutamate and sodium co‐transport in a glial cell of the salamander retina.

Publisher Website
Google Scholar
Abstract
1. Muller cells were isolated from salamander retinas and their membrane voltage was controlled with a whole-cell voltage clamp. External D-aspartate, L-aspartate and L-glutamate each induced a membrane current. D-Glutamate, kainate, quisqualate and N-methyl-D-aspartate wre more than 100.times. less effective than L-aspartate. Kynurenic acid had no effect on the current produced by L-glutamate, L-aspartate or D-aspartate. 2. The current induced by an acidic amino acid (AAA) was completely dependent on the presence of external Na+. Neither Li+, Cs+, choline nor TEA+ were able to substitute for Na+. The relationship between external Na+ concentration and current amplitude can be explained if the binding of three Na+ ions enabled transport. The apparent affinity constant for Na+ binding was 41 mM. Altering K+, H+ and Cl- concentrations demonstrated that these ions are not required for transport. 3. The shape of the current-voltage relation did not depend on the external amino acid concentration. The relationship between D-aspartate concentration and current amplitude can be described by the binding of D-aspartate to a single site with an apparent affinity constant of 20 .mu.M. 4. Influx and efflux of AAA were not symmetric. Although influx was electrogenic, efflux did not produce a current. Moreover, influx stimulated efflux; but efflux inhibited influx. 5. Removing external Na+ demonstrated that Na+ carried a current in the absence of an AAA. Li+ was a very poor substitute for Na+. This current may be due to the uncoupled movement of Na+ through the transporter. The relationship between the external Na+ concentration and the amplitude of the uncoupled current can be explained if the binding of two or three Na+ ions enabled the translocation of Na+ in the absence of an AAA. The apparent affinity constant for Na+ binding was approximately 90 mM. 6. The temperature dependence of the AAA-induced current had a Q10 between 8 and 18.degree. C of 1.95. The Q10 is consistent with a rate constant for influx of 104 s-1 (at -70 mV and 20.degree. C). The maximum rate of influx was measured following a concentration jump produced by the photolysis of ''caged'' L-glutamate. The onset of the observed current was limited by the 1.3 ms resolution of the recording system. Hence, the rate constant for influx must be faster than 103 s-1. 7. These results can be explained by a model in which Na+ and an AAA bind at sites outside the membrane''s voltage filed and move as a multi-ion complex through a pore-like structure. The model has three binding sites for Na+ at the external surface and only one binding site for Na+ at the internal surface. The asymmetry in Na+ binding produces an electrogenic influx and an electroneutral efflux. Inward and outward components of the current induced by an external AAA can be explained if co-transport and uncoupled Na+ flux are both mediated by the same transporter. 8. Muller cells had large K+ conductances which determined the resting potential and input resistance. Because the input resistance was low, activation of the AAA transporter produced only a few millivolts of depolarization. 9. The concentration of an AAA in the restricted extracellular space of a tissue should be a steep function of membrane voltage. Muller cells normally maintain a relatively constant hyperpolarization and continuously accumulate AAA. During pathological conditions, when Muller cells are depolarized, extracellular AAA concentration would increase.