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

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 B_max 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.

Key Words:

• Cocaine binding
• dopamine transporter
• allosteric regulation