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Figure 4.5. Glutamate uptake currents recorded from isolated Müller cells of the tiger salamander retina. A. The amplitude of the inward current increases with membrane hyperpolar-ization. B. The I-V curve shows no reversal as the cell is voltage-clamped to depolarizing potentials. C. Sodium-dependence of the transport current measured at holding potentials from 0-100 mV (indicated to the right of the data points). Note that the greater the hyper-polarization and the lower the [Na]o, the smaller is the current response to 100 ^M glutamate; in the absence of extracellular Na+ (0 [Na]o on the scale of abscissae) the responses to glutamate are blocked at all holding potentials. D. Glutamate-induced uptake responses of rabbit Müller cells are mediated by a high affinity transporter with a Km of 5 ^M (Brew and Attwell, 1987; Sarantis and Attwell, 1990). (A, B, C copyright 1987 Macmillan Magazines Ltd., reprinted with permission. D copyright 1990 Elsevier Science, reprinted with permission.)

to axolotl Müller cells elicits an inward current that decreases in amplitude as the holding potential is moved from -120 mV to +47 mV (Brew and Attwell, 1987). However, even with the cell depolarized to +47 mV, there is no sign of reversal to an outward current (Fig. 4.5B), which would typically occur near 0 mV for glutamate-gated ion channels (cf. Wyllie et al., 1991). These findings, and the observation that the glutamate-induced responses are not accompanied by an increase in membrane current noise, argue for the presence of an electrogenic glutamate uptake carrier rather than the opening of ion channels through activation of glutamate receptors. Consistent with this interpretation are results indicating that removal of extracellular Na+ completely abolishes the glutamate-evoked current (Fig. 4.5C), and that glutamate analogs are far less effective than L-glutamate in inducing a membrane current (cf. Brew and Attwell, 1987; Schwartz and Tachibana, 1990; Barbour et al., 1991).

Na+-dependent glutamate uptake is mediated by a high affinity transporter with a Km ofabout 20 M for axolotl (Brew and Attwell, 1987) and tiger salamander Müller cells (Schwartz and Tachibana, 1990; Barbour et al., 1991), and by one of even higher affinity (Km = 5 ^M) in rabbit Müller cells (Fig. 4.5D) (Sarantis and Attwell, 1990). Because glutamate is a negatively charged amino acid, the fact that transporter activity is associated with an inward current indicates that Na+, or other cationic species, contributes a net positive charge that accompanies glutamate transport into the cell.

It appears now that in addition to its dependence upon sodium, the carrier requires the outward movement of intracellular K+. As Fig. 4.6A shows, removing intracellular K+ (or raising [K+]o) greatly reduces the glutamate-induced current (Sarantis and Attwell, 1990; Barbour et al., 1988, 1991; Szatkowski et al., 1991). Thus, there is an ordered process of ion exchange by the glutamate transporter: After sodium and glutamate are released into the cell, potassium binds to the carrier and is transported outward before a new cycle begins (Kanner and Bendahan, 1982).

However, the stoichiometry of the glutamate transporter in Müller cells appears to be even more complex. The previously cited data, together with the results obtained from measurements of intra- and extracellular pH (Bouvier et al., 1992), and a consideration of the carrier-associated anion conductance (Billups et al., 1996; see Section 4.2.2), strongly suggest that glutamate uptake induces an intracellular acidification by transporting H+ into the cell. Accordingly, for each cycle of the carrier, the inward current derives from cotransport into the cell of one glutamate anion together with two sodium ions and one proton, and the countertransport of one POtassium ion (Fig. 4.6B). As a result, there is a net transport of one positive charge per glutamate anion; i.e., the electrogenic current is proportional to the number of glutamate ions entering the cell.

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