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    • The dramatic increase in the development of EAAT inhibitors

    2022-05-13

    The dramatic increase in the development of EAAT inhibitors and substrates over the last several years can likely be attributed to a number of experimental advances. Firstly, the isolation of the individual EAAT clones allowed each subtype to be selectively examined in well-characterized expression systems, such as Xenopus oocytes or mammalian cell lines (e.g., HEK-293, Cos7, MDCK, and C17A cells). Secondly, the electrogenic nature of EAAT-mediated transport permitted uptake to be followed in real time using electrophysiological recording techniques. One very important advantage of this approach is that it allows substrates, which induce currents, to be easily differentiated from non-substrate inhibitors, which do not induce a current by themselves but will block a substrate-induced current when co-applied. Previously, a direct demonstration of substrate activity would have been dependent upon either the synthesis of a radiolabeled derivative or the development of an analytical protocol, such as HPLC, to quantify sequestered substrate. (It should be noted that substrate activity can be assessed indirectly in some situations by exploiting the process of heteroexchange, see Koch et al., 1999b.) Thirdly, a concerted effort was made to develop conformationally constrained EAA analogues, an approach that had a proven track record in the production of EAA receptor agonists and antagonists (Chamberlin et al., 1998). This approach is based on the hypothesis that if critical functional groups can be appropriately positioned (“locked”) using ring systems or by introducing substituents that produce a conformational bias, it can result in enhanced binding. A valuable side product of this result is that in many of these conformationally restricted analogues the relative position of the functional groups can be mapped in 3D space and used to construct binding site pharmacophores. Further, limiting the number of obtainable conformations also holds the potential to reduce cross-reactivity, which is a considerable issue given the number of receptors, transporters, and Riociguat australia in a typical EAA synapse likely to contain a glutamate-binding site. Needless to say, this strategy has yielded some very useful compounds that provide insight into specificity differences between EAAT subtypes, as well as between substrates and non-substrate inhibitors. Examples of these compounds are discussed below and have been organized on the basis of the chemical structure used to produce the conformational restriction.
    Roles in physiology and pathology A discussion of the functional significance of glutamate transport under both normal and pathological conditions might best be framed within the context of a single question: Does uptake influence the amount of transmitter that actually reaches and activates EAA receptors? In the instance of normal excitatory signaling, this question relates to whether or not transport effects such things as the shape of postsynaptic signals or the activation of extrasynaptic receptors. Then again, from the perspective of CNS injury and disease, the question concerns the handling of excessive levels of l-glutamate and the consequences of impaired transport relative to excitotoxic-mediated neuronal injury. Not surprisingly, several of the compounds described above, particularly those that potently inhibit uptake without activating or antagonizing EAA receptors, have proven invaluable in resolving these roles. If, as has been hypothesized for many years, the excitatory signal of l-glutamate is indeed terminated by its rapid clearance from the synaptic cleft by the EAATs, then transport inhibitors should have the potential to increase the half-life and/or the concentration of transmitter in the cleft and thereby produce a slowing of the corresponding EPSC and/or an increase in its amplitude. Transporters appear capable of producing such effects, even if the actual rates of translocation into cells are slow (≈70 ms/cycle) compared to the time course of the excitatory signal (≈2 ms), because the initial binding to the substrate sites on the transporters has been found to occur at rates comparable to that of receptors (Clements et al., 1992, Jonas et al., 1993, Tong & Jahr, 1994, Wadiche et al., 1995b, Diamond & Jahr, 1997, Wadiche & Kavanaugh, 1998). Thus, if high densities of transporters are positioned in proximity to synaptic receptors, then the transport proteins should be capable of binding the transmitter and effectively buffering its access to the receptors. It follows that the introduction of an uptake blocker should attenuate this process and increase the amount of l-glutamate that reaches the receptors. Interestingly, the results of such studies indicate that the contribution of transport to the shaping of the postsynaptic signals is not constant, but varies among individual excitatory circuits. For example, the excitatory signaling between hippocampal CA3 and CA1 pyramidal neurons appears to be insensitive to uptake blockers, while at others, such as the AMPA receptor-mediated excitatory input on cerebellar Purkinje neurons, transport inhibitors markedly slow postsynaptic currents (Isaacson & Nicoll, 1993, Sarantis et al., 1993, Barbour et al., 1994, Wadiche & Jahr, 2001).