Is Na + required for the binding of dopamine, amphetamine, tyramine, and octopamine to the human dopamine transporter?

Abstract

The role of Na(+) in the recognition of blockers by the dopamine transporter is accomodated by a model with a cation site that overlaps with the blocker binding domain, and a distal Na(+) site that interacts with this cation site and perhaps with the blocker binding domain itself. The present study addresses the application of this model to the recognition of substrates by the dopamine transporter, focusing on conditions that should reveal a stimulatory effect, if present, of Na(+) on substrate binding. Recognition was studied via the inhibition of binding of [(3)H]WIN 35,428 (2beta-carbomethoxy-3beta-(4-fluorophenyl) [(3)H]tropane), a cocaine analog, to the human dopamine transporter in human embryonic kidney 293 cells. Little or no changes in binding were noted for dopamine, d-amphetamine, p-tyramine, or dl-octopamine by increasing [Na(+)] from 2 mM to 20 mM with co-varying Br(-), both at pH 7.4 and 7.0. In 74-mM Tris-HBr or -HCl, only dopamine and d-amphetamine showed binding increases upon raising Na(+), leveling off with NO(3)(-) or SO(4)(2-) but not Br(-) as anion at approximately 60 mM Na(+), consonant with a partly stimulatory action of Br(-). An Na(+) free, low 5-mM Tris-HEPES buffer was used for studying Na(+) curves truly starting at 0 mM, and, with SO(4)(2-) as the anion, no stimulation of binding by Na(+) was observed. This suggested that the stimulations observed in high (74 mM) Tris(+) buffer by Na(+) were not a direct effect of Na(+) but rather a disinhibitory effect of Na(+) in removing Tris(+) inhibition that depended upon substrate. Tris(+) IC(50) values in Na(+) free buffer were not lower for dopamine and d-amphetamine than p-tyramine and dl-octopamine. No evidence was found for a stronger inhibitory effect of Na(+) for dopamine and dl-octopamine potentially offsetting Tris(+) disinhibition. All results together support the existence of a substrate domain overlapping with a cation site that also binds Tris(+); a distal Na(+) site interacts with this cation site and with the substrate domain by negative allosterism and is additionally impacted by Cl(-). Importantly, interactions between sites vary with the type of substrate, and, in membrane preparations, Na(+) is not required for, or stimulatory to, the binding of any of the four substrates studied unlike the binding of the cocaine analog WIN 35,428.

Evidence for Distinct Sodium-, Dopamine-, and Cocaine-dependent Conformational Changes in Transmembrane Segments 7 and 8 of the Dopamine Transporter

“…9). Because dopamine and other substrates have been shown to bind to the hDAT and related transporters with unchanged affinity even in the absence of Na ϩ (23)(24)(25), this observation strongly supports the idea that the protection of M371C observed only in the presence of Na ϩ is the consequence of a transport-associated conformational change. Data from our previous studies also argue against position 371 being a direct part of the substrate binding crevice.…”

“…8), we were able to assess whether dopamine protection of this construct was Na ϩ -dependent. Transport is strictly Na ϩ -dependent, but increasing evidence suggests that dopamine and other substrates can bind to hDAT and related transporters with unchanged affinity even in the absence of Na ϩ (23)(24)(25). Accordingly, assessing the Na ϩ dependence of dopamine protection should enable us to distinguish between direct steric protection and a dopamineinduced conformational change that decreases the accessibility of M371C to MTSET.…”

GABA released from cultured cortical neurons influences the modulation of t-[35S]butylbicyclophosphorothionate binding at the GABAA receptor

“…As far as we know there are no reports of the interaction of Tris buffers with the GABA transport, but Li et al (2002) have reported that Tris allosterically interact with the substrate site of the dopamine transporter, which shares with the GABA transporters the fact that both belong to the SLC6 gene family (Gether et al, 2006). Furthermore, lead inhibited the uptake of GABA and potentiated the spontaneous release of GABA from motoneurons when Tris-HCl but not phosphate or carbonate buffers were used (Spence et al, 1985).…”

The Role of N-Glycosylation in Function and Surface Trafficking of the Human Dopamine Transporter

“…Protein content was determined by the DC protein assay (Bio-Rad). 3 H]DA uptake were obtained with the nonlinear computer fitting program KELL as described by us previously (17). IC 50 values for inhibitors were converted to K i values by the Cheng-Prusoff equation (18).…”