Abstract
Large conductance Ca(2+)-activated BK channels are important regulators of action potential duration and firing frequency in many neurons. As the pore-forming subunits of BK channels are encoded by a single gene, channel diversity is mainly generated by alternative splicing and interaction with auxiliary beta-subunits (BKbeta1-4). In hypothalamic neurons several BK channel subtypes have been described electrophysiologically; however, the distribution of BKbeta subunits is unknown so far. Therefore, an antibody against the large extracellular loop of the BKbeta1 subunit was raised, freed from cross-reactivity against BKbeta2-4 and affinity-purified. The resulting polyclonal monospecific BKbeta1 antibody was characterized by Western blot analysis, ELISA techniques and immunocytochemical staining of BKbeta1-4-transfected CHO and COS-1 cells. Regional and cellular distribution in the rat hypothalamus was analysed by immunocytochemistry and in situ hybridization experiments. Immunocytochemical staining of rat hypothalamic neurons indicates strong BKbeta1 expression in the supraoptic nucleus and the magno- and parvocellular parts of the paraventricular nucleus. Lower expression was found in periventricular nucleus, the arcuate nucleus and in the median eminence. Immunostaining was predominantly localized to somata. In addition, pericytes and ependymal epithelial cells showed BKbeta1 labelling. In all cases immunocytochemical results were supported by in situ hybridization.