Allosteric Regulation by Sodium of the Binding of [3H]Cocaine and [3H]GBR 12935 to Rat and Bovine Striata

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Abstract
Sodium regulation of ligand binding to the dopamine transporter of rat and/or bovine striata was investigated using a filtration binding assay. In low Na+ phosphate or bicarbonate-buffered sucrose (300 mOsm), the tissue exhibited high affinity for [3H]cocaine which was reduced by the addition of Na+ in a dose-dependent manner. However, [3H]GBR 12935 binding was insensitive to Na+ in these physiological buffers. Although binding of [3H]GBR 12935 was displaced by cocaine in a manner consistent with competitive displacement, a non-linear affinity shift of the displacement of [3H]GBR 12935 by cocaine suggests that the two liganäs bind to distinct sites. Binding of both radioligands was suppressed when measured in sodium-free 50 mM Tris-sucrose and increased with the addition of Na+. Scatchard analysis indicated that Bmax for [3H]cocaine binding in Tris plus 120 mM NaCl reached the same level as in the physiological buffers. In Krebs-Ringer buffer with phosphate, bicarbonate or Tris, which contained 120 mM NaCl, both [3H]cocaine and [3H]WIN 35428 binding exhibited lower affinities than in Na+-deficient phosphate buffer. It is suggested that the cation form of Tris binds to the dopamine transporter and that the Tris-receptor complex does not bind [3H]cocaine or [3H]GBR 12935. Na+ displaces Tris, forming a Na+-receptor complex which binds these ligands. Thus, it is suggested that the Na+-dependent binding of cocaine to the dopamine transporter is observed only in Tris.