Substrate Selectivity and Inhibitor Sensitivity of Cloned Transporters

Studies of the cloned human 5-HT transporter expressed in HeLa cells indicate that the Kis for inhibition of [3H]5-HT uptake by dopamine and noradrenaline are approximately 20-fold greater than the KM for 5-HT.21 However, one complication is that three mRNA transcripts (8.2, 5.0 and 3.3 kb) have been identified in human post-mortem brain tissue, all of which hybridize to the cDNA probe for the 5-HT transporter. Although these mRNAs are thought to be derived from a single gene, there is some tissue specificity in their distribution.22 This could mean that different mRNAs yield translation products which differ in their substrate selectivity and/or inhibitor sensitivity. This is rendered all the more plausible by evidence that even single point mutations of the 5-HT transporter markedly affect both the kinetics of 5-HT uptake and the affinity of uptake inhibitors.23,24 It has even been suggested that abnormal populations of 5-HT transporters could underlie some psychiatric and neurological disorders.22,25

In contrast, noradrenaline and dopamine transporters show poor selectivity for their respective substrates, a feature which is entirely consistent with their approximately 75% amino acid sequence homology. In fact, dopamine seems to be the preferred substrate for noradrenaline transporters in human placental brush border membranes26 and cloned human noradrenaline transporters expressed in HeLa cells.27 Studies of cloned human noradrenaline and rat dopamine transporters expressed in LLC-PK1, COS-7 or SKN-M-C cells confirm that the affinity of dopamine for both these transporters is more than two-fold greater than that of noradrenaline.28,29 There is some dispute over whether or not the Vmax for dopamine uptake by the noradrenaline transporter is also greater than that for noradrenaline: this factor could determine whether or not dopamine uptake by the noradrenaline transporter actually exceeds that of noradrenaline. Nevertheless, it seems that the noradrenaline transporter, at least, is not at all substrate selective.

In view of the lack of substrate selectivity of the catecholamine transporters, the specificity of monoamine uptake inhibitors might provide an alternative, and possibly more reliable, criterion for the classification of different transporters. In line with their potent uptake blocking activity, SSRIs have a high (nanomolar) affinity for the cloned 5-HT transporter which is certainly higher than their (micromolar) affinity for catecholamine transporters (Table 10.2). The Kis for displacement of [3H]ligands bound to different cloned transporters have been estimated for a wide range of monoamine reuptake inhibitors and their ranking shows excellent agreement with those derived from measurement of synaptosomal [3H]monoamine uptake. However, of 37 compounds tested for displacement of the selective dopamine transporter ligand, [3H]2-P-carbomethoxy-3- P-(4-fluorophenyl)-tropane (WIN 35428), the lowest Kd (25 nM) was obtained with the SSRI, sertraline.31 In fact, the affinity of this SSRI is two-fold higher than that of nomifensin, a compound regarded

Table 10.2. Binding of SSRIs to cloned human monoamine transporters













Noradrenaline transporter3







Dopamine transportera






12Ka/ 6Kb

Values show aKds or bKs (nM). From data cited in refs 27,30,31.

Values show aKds or bKs (nM). From data cited in refs 27,30,31.

as a potent dopamine reuptake inhibitor. Findings such as these undermine the possibility that it is the inhibition of 5-HT reuptake by the 5-HT transporter which accounts for the antidepressant actions of SSRIs.

Another point to emerge from these displacement studies is that, even within individual reports, the absolute values for the estimated ^¡s depend on the choice of radioligand. Almost without exception, displacement of [3H]labeled uptake inhibitors yield lower K^s than those obtained with a [3H]labeled monoamine (e.g., ref. 32). The most likely explanation for this disparity is that different types of ligand bind to different sites on the transporter protein.33-35 As a result of this, a reduction in binding of a given radioligand is not necessarily due to its competitive displacement. Furthermore, different uptake inhibitors seem to modify transporter function in different ways.23 For instance, some compounds, notably sertraline, reduce the rate at which the radioligand dissociates from the noradrenaline transporter suggesting that they 'stabilize' the binding of substrates to the transporter.36

Contrasting with these detailed studies, the effects of SSRIs on the uptake of [3H]monoamines by different types of cloned catecholamine transporters have not been investigated systematically. The Ks for inhibition of [ 3H]noradrenaline uptake by paroxetine (312 nM) and citalopram (1 ^M), but not that of other SSRIs, by cloned noradrenaline transporters have been reported (see ref. 18) and these are reasonably close to those for inhibition of [3H]monoamine uptake by synaptosomes. However, the Kis of SSRIs for inhibition of [3H]dopamine uptake by the cloned dopamine transporter do not seem to have been reported for any of the SSRIs.

A further complication is that there are two mRNAs for noradrenaline transporters, both of which hybridize to the human noradrenaline transporter cDNA probe.27 This could mean that there is more than one transcription product of the gene (or two distinct but homologous genes) for the noradrenaline transporter, a possibility which is supported by evidence that these two mRNAs have different distributions in the brain. The larger species (5.8 kb) is prominent in the brainstem and adrenal gland while the smaller (3.6 kb) is thought to represent a glial transporter. Pharmacologically distinct modes of uptake (neuronal uptake, 'uptake1' and extraneuronal uptake, 'uptake2') have been recognized in the periphery for over 30 years but there is now evidence for the existence of several functionally distinct noradrenaline uptake sites. These have been found in rat liver,37 cultured human glia cells38

and cultured brain astrocytes.39 The underlying explanation for these different uptake processes is as yet unresolved but these findings could point the way to transporters which differ in their sensitivity to different types of monoamine reuptake inhibitors. Even if there turn out to be no overt differences in composition of these transporters, functional differences could arise from post-translational glycosylation or phosphorylation of a single gene product.19 Further evidence for functionally distinct noradrenaline uptake sites is discussed below.

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