Detection and photoaffinity labeling of the Ca 2+-activated K + channel-associated apamin receptor in cultured astrocytes from rat brain

Abstract

Apamin, an 18-amino acid bee venom peptide, is a specific blocker of a class of Ca 2+ activated K + channels. Mono 125I-iodoapamin was used to detect the K + channel-associated receptor site in cultured astrocytes from rat brain. Specific high-affinity binding to intact glial cells with a K d of about 90 pM at 1 °C and pH 7.5 was demonstrated by equilibrium and kinetic methods. The average receptor capacity was 3 fmol/mg cell protein which is 2 to 3-fold lower than in primary cultured neurons. Bindings was stimulated by K + ions, but to a lesser extent than with neuronal receptors. Photoaffinity labeling of receptor/ion channel components using an arylazide derivative of 125I-monoiodoapamin revealed the presence of the 86- and 33-kDa polypeptides, previously detected in neurones. However a 59-kDa peptide which is present in synaptic membrane preparations from adult rat brain, but not in cultured neurons, was also clearly labeled in intact astrocytes. This indicates that the 59-kDa polypeptide is not a proteolytic fragment of the 86-kDa chain but an associated subunit which is only accessible to photolabeling in certain apamin receptor preparations. Apamin-sensitive Ca 2+-activated K + channels in astrocytes may be one of the pathways by which glial cells redistribute K + in the central nervous system (CNS).