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4.2.2. Anion Conductance and Glutamate Transport

An unusual property of the glutamate transporter is its association with an anionic conductance carried by CI- (Fairman et al., 1995). This glutamate-activated chloride current has been recorded from Müller cells (Billups et al., 1996; Eliasof and Jahr, 1996), from oocytes expressing several subtypes of excitatory amino acid transporters (Arriza et al., 1997; Eliasof et al., 1998), as well as from retinal neurons (e.g. cone photoreceptors, ON bipolar cells) that exhibit glutamate transport activity (Eliasof and Werblin, 1993; Grant and Dowling, 1995). In each case, the anion conductance, activated when the transporter binds glutamate and Na+, accounts for a significant fraction of the glutamate-elicited current.

Patch-clamp recordings from isolated salamander Müller cells (Fig. 4.7) show that in the presence of normal extracellular chloride, exposure to 100 ^M L-glutamate evokes a large inwardly rectifying current. The current is inward at hyperpolarizing potentials and reverses to an outward current at potentials more positive than the chloride equilibrium potential (ECI ~ 0 mV). Removing chloride from the bath (replacement with gluconate) eliminates the outward current, but has no effect on the transporter-activated inward current. Thus, transport activity is not dependent upon activation of the anionic conductance. Moreover, the outward current occurs at depolarizing potentials for which the transporter is inactive, indicating the an-ionic conductance can occur independently of net glutamate transport. Nevertheless, both response components require extracellular sodium.

The cloning of excitatory amino acid transporters and their functional characterization in the oocyte expression system has enabled extension of these observations to the rapidly expanding subfamily of glutamate transporters. cDNAs encoding a variety of excitatory amino acid transporter (EAAT) subtypes were identified and shown by immunocytochemistry to be present in amphibian and mammalian retina (cf. Rauen et al., 1996; Arriza et al., 1997; Eliasof et al., 1998). Five distinct subtypes were isolated from a

Figure 4.6. The potassium dependence of glutamate transport. A. Removing intracellular K+ by perfusion through the whole-cell patch pipette reduces glutamate-induced currents in a concentrationdependent fashion (bracketed values are the number of experiments for each data point). Inset shows actual recordings of the responses to 100 ^M glutamate obtained at different values of [K+]j. Glutamate does not produce an inward current in the absence of intracellular K+ (Barbour et al., 1988). B. Stoichiometry of electrogenic glutamate transport in Müller cells. A net transfer of positive charge results from the inward movement of one glutamate anion, two sodium ions, and one proton, coupled to the outward movement of one potassium ion (Barbour et al., 1988). (Copyright 1988 Macmillan Magazines, Ltd., reprinted with permission.)

Figure 4.7. The association of the glutamate transporter with an anionic conductance is evident in current-voltage data from tiger salamander Müller cells. Steady-state responses (Iss) were elicited by 100 ^M L-glutamate in normal (high) chloride (■) and in chloride-free (•) solutions; the internal (pipette) solution also contained high chloride. The removal of [CI-]o eliminates the outward current carried predominantly by the influx of external chloride ions, but has no effect on the inward (transport) current (Eliasof and Jahr, 1996.) (Copyright 1996 National Academy of Sciences, U.S.A, reprinted with permission.)

Figure 4.7. The association of the glutamate transporter with an anionic conductance is evident in current-voltage data from tiger salamander Müller cells. Steady-state responses (Iss) were elicited by 100 ^M L-glutamate in normal (high) chloride (■) and in chloride-free (•) solutions; the internal (pipette) solution also contained high chloride. The removal of [CI-]o eliminates the outward current carried predominantly by the influx of external chloride ions, but has no effect on the inward (transport) current (Eliasof and Jahr, 1996.) (Copyright 1996 National Academy of Sciences, U.S.A, reprinted with permission.)

cDNA library prepared from the tiger salamander retina, and at least four of these (sEAATl, sEAAT2A, sEAAT5A, and sEAAT5B) were shown to be present in Müller cells (Eliasof et al., 1998). It is difficult to explain why so many different glutamate transporters exist in the Müller cell of a single species. Although the various subtypes exhibit somewhat different properties when expressed in Xenopus oocytes, they share one significant feature: The transport of glutamate is combined with a mechanism for increasing chloride permeability. It has been suggested that this anionic conductance may be important for preventing a reduction in the rate of transport due to the depolarization that would occur as a result of electrogenic glutamate uptake (Eliasof and Jahr, 1996).

4.2.3. GABA Transport

The electrophysiology of the Müller cell GABA transporter provides a number of parallels, as well as differences, when compared with results obtained for glutamate transport. In both skate and mammalian retina, radiolabeled GABA accumulates almost exclusively in Müller cells (Section 4.1.1). GABA uptake is known to be mediated by a sodium-dependent, elec-

trogenic mechanism (Keyman and Kanner, 1988; Malchow and Ripps, 1990; Qian et al., 1993; Faude et al., 1995), and studies in Xenopus oocytes expressing the GABA transporter GAT-1 indicate that each GABA molecule is co-transported with two sodium ions and one chloride ion (Mager et al., 1996).

The current generated by GABA transport in Müller cells appears often to be much smaller than that resulting from glutamate uptake. For example, application of 100 ^M GABA to Müller cells from guinea pig retina elicits an inward current of about 12 pA with the cells voltage-clamped at -80 mV (Biedermann et al., 1994); this is about 6-fold less than the current induced by only 20 ^M glutamate at a comparable holding potential in rabbit Müller cells (Sarantis and Attwell, 1990). It is not known whether this is due to fewer GABA transport molecules on Müller cell membranes, to the differential loss of protein as a result of the dissociation procedure, or simply to species differences.

In the skate, a more unusual situation is encountered—a good example ofthe care that must be exercised in identifying the cell types involved in transmitter uptake. We noted earlier that based on autoradiographic findings, 3H-GABA uptake in skate retina occurs almost exclusively in Müller cells, with little or no uptake into horizontal cells or other neurons (see Fig. 4.1). On the other hand, voltage-clamp recordings from skate horizontal and Müller cells give diametrically opposite results. The GABA-induced membrane currents of Müller cells appear to result solely from activation of GABAa receptors (Fig. 4.8C,D; Malchow et al., 1989), whereas the GABA currents of horizontal cells are mediated by a high affinity electrogenic uptake mechanism (Fig. 4.8A,B; Malchow and Ripps, 1990).

It is not easy to reconcile these findings. The apparent lack of GABA uptake into horizontal cells in the intact retina could possibly be explained by their limited access to exogenous GABA, i.e., most of the applied 3H-GABA may have been removed by Müller cells at the site of application. Indeed, if the autoradiographic findings are correct, GABA uptake must surely occur in skate Müller cells. Why then do we fail to record GABA-induced currents that are consistent with the presence of a transport mechanism? As it turned out, the large currents generated by activation of the GABAA receptors on Müller cells completely masked the small current elicited by the GABA transport process. Using a clever experimental approach that exploits the sodium dependence of the GABA transporter (cf. Akaike et al., 1987), it is possible to demonstrate electrophysiological^ the GABA transport activity of Müller cells (Qian et al., 1993).

The strategy is quite straightforward. Applying very low concentrations of GABA, and comparing responses obtained in the presence and in the absence of sodium, serves to expose the competition between the GABA receptor and the GABA transporter for the available GABA. As illustrated in

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