RESULTADOS SATISFACCIÓN LABORAL

All five glutamate transporter types are expressed in the CNS (and some also in other parts of the body), but are distributed in different brain areas and in different cell types (Danbolt, 2001). Originally it was thought that GLAST and GLT-1 were exclusively expressed in glial cells, whereas EAAC-1, EAAT-4 and EAAT-5 were thought to be neuronal, but this categorization might be less rigid than previously thought. The following information is from several publications, which investigated

the regional and cellular distribution and the developmental expression profile of different glutamate transporters in the CNS (Rauen and Kanner, 1994; Rothstein et al., 1994; Chaudhry et al., 1995; Lehre et al., 1995; Rauen et al., 1996; Yamada et al., 1996; Furuta et al., 1997b; Furuta et al., 1997a; Ullensvang et al., 1997; Conti et al., 1998; Dehnes et al., 1998; Eliasof et al., 1998b; Eliasof et al., 1998a; Lehre and Danbolt, 1998; Mennerick et al., 1998; Plachez et al., 2000; Schmitt et al., 2002).

(1) GLAST/EAAT-1 appears to be a major glutamate transporter in the cerebellum and the retina, where it is located mainly in Bergmann glia and Müller cells respectively. However, it is also present in astrocytes throughout the brain and can be transiently expressed by some cultured neurones. (2) GLT-l/EAAT-2 is the most abundant glutamate transporter in the forebrain and is expressed in astrocytes throughout the CNS. In the retina GLT-1 is expressed in neurones (cone axon terminals, some cone bipolar cells and some amacrine cells) but not Müller cells. GLT-1 can also be expressed by neurones in hippocampal culture and the splice variant GLT-1 v is expressed neuronally throughout the brain. (3) EAAC-1/EAAT-3 is found in neurones throughout the CNS and also in astrocytes of the cerebral cortex. (4) EAAT-4 is found mainly in cerebellar Purkinje cells, but is also present (though at very low levels) in neuronal dendrites and some astrocytes in the forebrain. (5) EAAT-5 is found only in the retina, in photoreceptor axon terminals, in some bipolar cells and in Müller cells.

The different glutamate transporter types can be expressed by the same cell. For example, most astrocytes contain GLAST and GLT-1 in their plasma membrane, in a ratio of about 1:4; by contrast in cerebellar Bergmann glia this ratio is 6:1 (Lehre and Danbolt, 1998). Retinal Müller cells express GLAST, GLT-1 and EAAT-5 (Eliasof et al., 1998b; Eliasof et al., 1998a), and Purkinje cells in the cerebellum express EAAC-1 as well as EAAT-4 (Furuta et al., 1997b). The distribution of the transporters in the cell is not random but can depend on the type of neighbouring structure. For example GLT-1 and GLAST are more abundant in regions of the

astrocyte membrane which are adjacent to nerve terminals, axons and dendritic spines than in regions of the cell which are facing other astrocytes, blood vessels, pia or dendritic shafts (Chaudhry et al, 1995). The location of the individual transporters is likely to serve certain purposes. Transporters located in glial cells may mainly function to maintain a low extracellular resting glutamate concentration, setting up a concentration gradient for glutamate to diffuse out of the synaptic cleft. Carriers located in the vicinity of synaptic areas, such as EAAT-4 and EAAC-1 in Purkinje cell spines, might play a more direct role in shaping synaptic transmission (Takahashi et al., 1996a; Otis et al., 1997; Auger and Attwell, 2000; Brasnjo and Otis, 2001).

The surface expression of the transporters can be regulated by various extracellular soluble factors; for example GLAST expression in cultured cells was upregulated by glutamate (Duan et al., 1999), expression of glial transporters was increased when they were co-cultured with neurones (Swanson et al., 1997; Schlag et al., 1998) and pituitary adenylate cyclase activating polypeptide (PACAP), a neuronally derived peptide, increased surface expression of GLAST and GLT-1 in nearby astrocytes (Figiel and Engele, 2000).

1.3.3 Stoichiometry and properties of glutamate transport and its anion conductance

Glutamate uptake is driven by the co-transport of ions down their electrochemical gradients. It has been shown that glutamate uptake depends strongly on the co-transport of sodium ions (Erecinska et al., 1983). For both the mainly glial GLT-1 and the mainly neuronal EAAT-3 it has been shown that three sodium ions (Zerangue and Kavanaugh, 1996; Levy et al., 1998) and one proton (Bouvier et al., 1992; Zerangue and Kavanaugh, 1996; Levy et al., 1998) are co-transported, whereas one potassium ion (Barbour et al., 1988; Amato et al., 1994) is counter-transported for each molecule of glutamate transported across the membrane. Glutamate uptake

is thus electrogenic, transporting two net positive charges into the cell. This results in the transport of glutamate being inhibited at depolarized membrane potentials (Brew and Attwell, 1987). Since transporters operate solely by means of the co-transport of ions down their electrochemical gradients, if these gradients change they can also run backwards and release glutamate with the same stoichiometry as described above (Szatkowski et al., 1990; Attwell et al., 1993; Billups and Attwell, 1996). This non- vesicular release of glutamate is responsible for the rise of glutamate to neurotoxic levels in severe brain ischaemia (Szatkowski et al., 1990; Attwell et al., 1993; Szatkowski and Attwell, 1994; Rossi et al., 2000).

In addition to transporting glutamate, glutamate transporters can also conduct anions {in vivo mainly chloride). The first to report such a chloride flux through

glutamate transporters, in cones of the salamander retina, were Sarantis et al. (1988). This transporter has subsequently been cloned (EAAT-5; Arriza et al., 1997) and its high chloride permeability confirmed in expression systems. A significant chloride flux through glutamate transporters was also reported when EAAT-4 was cloned from human cerebellum and expressed in Xenopus oocytes (Fairman et al., 1995), and this chloride flux has been shown to occur when EAAT-4 is activated in Purkinje cells (Otis et al., 1997). From measurements of glutamate transporter currents (due to glutamate translocation and anion flux) in Müller cells of the salamander retina (Billups et al., 1996; Eliasof and Jahr, 1996), Purkinje cells of the cerebellum (Otis and Jahr, 1998) and EAAT-1, EAAT-2 and EAAT-3 expressed in Xenopus oocytes (Wadiche et al., 1995a) it has emerged that, although all glutamate transporters have an anion conductance within their structure, the different transporters show different degrees of anion permeability. The anion channel conductance is particularly high in EAAT-4 and EAAT-5 (Sarantis et al, 1988; Fairman et a l, 1995; Arriza et al, 1997), but is much smaller in the other glutamate transporters when physiological anions (i.e. chloride) are present (Wadiche et a l, 1995a). Although chloride is the most physiological anion, other ions such as nitrate or perchlorate permeate the

transporter much better and are thus often used to examine the transporters’ channel properties. The anion conductance is thermodynamically uncoupled from the transport activity; this means that the movement of anions does not drive glutamate transport. The transporter can conduct anions although it does not translocate glutamate. The anion conductance is activated by (intra- or extracellular) glutamate and critically dependent on sodium; potassium does not need to be present (Figure 1.2). The physiological role of the anion conductance is unclear. In principle a glutamate activated chloride conductance might help to keep the membrane potential at negative values and thus maintain the driving force for glutamate uptake, but for most cells the current generated by the anion conductance is too small to significantly alter the membrane potential. The only exception to this is in cones, where EAAT-5 mediated chloride currents generate significant voltage changes (Sarantis et al., 1988; Picaud et al., 1995).