Molecular properties of the apamin binding protein associated with a calcium activated potassium channel

Abstract

AbstractApamin, a 2‐kdalton peptide from bee venom, is a potent convulsant. Its neurotoxic action has been attributed to specific blockade, at nanomolar concentrations, of the slow Co2+ activated K+ current (IAHP) that underlies the late afterhyperpolarisation in certain neurons. Mono[125I] iodoapamin binds with high affinity to intact cultured neurons (Kd = 30 PM, Bmax = 500‐1000 sites/cell) and synoptic membranes (Kd = 30 PM, Bmax = 30 fmol mg protein−1). Apamin contains two primary amines (alpha cysl and epsilon lys4) that can be modified to produce photoactivatable arylazide derivatives. Photoaffinity labeling with these derivatives has identified receptor polypeptides of 86 and 59 kdaltons accessible from the alpha cysl position and 33 kdaltons accessible from the epsilon lys4 position of the bound ligand. This suggests that apamin binds at the interface between 3 putative K+ channel subunits. Radiation inactivation studies indicated a target size of 84‐115 kdaltons. The simplest interpret at ion is that the 86 kdalton chain alone carries the neurotoxin binding site.The apamin binding protein can be extracted from rat brain membranes using sodium cholate. Scatchard analysis of equilibrium binding data revealed a single class of non‐interacting sites with Kd = 40 pm and Bmax = 17 fmol mg protein−1. As in membrane‐inserted receptors, apamin binding is stimulated by occupation of a K+ ion site that saturates at low millimolar concentrations. Other cations can be substituted for K+ with an affinity sequence: K+ = Tl+ = Rb+ > Cs+ > NH+4 > Li+ or Na+. The K+ channel Mockers quinidine and tetraethylammonium + displaced apamin from its solubilised receptor at concentrations similar to those required to block flux through apamin‐sensitive channels. Analysis by sucrose gradient centrifugation showed that the receptor sediments with S20=20, corresponding to a molecular weight of about 700 kdaltons. However, as much as 50%of this could be contributed by the detergent.Receptor sites for [125I]‐apamin have also been detected and identified by photoaffinity labeling in primary cultured astrocytes and heart cells as well as in membrane preparations from liver and smooth muscle. A 86–87 kdalton componet was was present in all these tissues whereas the 57–59 and 33kdalton polypeptides were revealed in some but not in others, These observations support the hypothesis that the larger chain contains the apamin binding site while the two smaller polypeptides are noncovalently associated perpheral subunits.