L-659,989: A useful probe in the detection of multiple conformational states of PAF receptors

Abstract

L-659,989 is a potent, specific and competitive plateletactivating factor (PAF) receptor antagonist. The 2,5-tritium labeled L-659,989, similar to [³H]PAF, specifically binds to rabbit platelet membranes with an equilibrium dissociation constant (K_D) of 1.60 (±0.20) nM in 10 mM MgCl₂. However, guanosine 5′-triphosphate (GTP) and several cations affect the specific binding of [³H]PAF and of [³H]L-659,989 to rabbit platelet membranes in different ways. K⁺, Mg²⁺, Ca²⁺ and Mn²⁺ potentiate the specific binding of both ligands. Na⁺ and Li⁺ inhibit the specific [³H]PAF binding, but enhance the binding of [³H]L-659,989; GTP reduces the [³H]PAF binding but has no effect on the binding of [³H]-L-659,989. Ni²⁺ inhibits the [³H]L-659-989 binding, but has no effect on the binding of [³H]PAF. In the presence of 150 mM NaCl, [³H]L-659,989 exhibits identical K_D and detectable binding sites (B_max) values as those in the presence of 10 mM MgCl₂, while K _d And B_max values of [³H]PAF are dramatically reduced in the presence of 150 mM NaCl compared to those in 10 mM MgCl₂. These results suggest the existence of multiple conformational states of the PAF specific receptor and that PAF and L-659,989 bind differently to those states. In the presence of 150 mM NaCl and 1 mM GTP, receptors appear to exist in a single conformational state with an equilibrium dissociation constant (K_B) of 0.93 μM for PAF as derived from the Schild plot. In isolated rabbit platelets pretreated with 10 μM ETH 227, a Na⁺-specific ionophore, the detectable [³H]PAF binding sites drop from 260 to 100 binding sites per platelet, but the binding sites for [³H]L-659,989 remain roughly the same. The Na⁺ binding sites which modulate the conformation of PAF receptors are therefore protected from extracellular Na⁺ until ionophore is added, and are probably located on the cytoplasmic side of the plasma membrane.