Abstract
Ca2+ channel opening has been proposed to be induced in the plasma membrane of mast cells and basophils upon crosslinking their Fc epsilon receptors. Here we report direct conductance measurements on planar lipid bilayers containing membrane components of rat basophils (RBL-2H3 line). These studies identify the Ca2+ channel-forming membrane component as the cromolyn binding protein [CBP, in which cromolyn is the anti-asthmatic drug 1,3-bis(2-carboxy-chromon-5-yloxy)-2-hydroxypropane]. Planar membranes were first formed from lipid vesicles containing unfractionated plasma membrane components prepared from RBL-2H3 cells. Conductance of these bilayers was induced by crosslinking IgE bound to the Fc epsilon receptors of this membrane by a specific polyvalent antigen. Channel conductance in the presence of only Ca2+ ions (2 mM) was 2 pS. When only sodium ions were present (150 mM), conductance was 10 pS. Upon addition of Ca2+ (2 mM) to the Na+ ion-containing solution, the conductance decreased from 10 pS to that of the Ca2+ ions--namely, 2pS. Open channel times were in the range of several hundred milliseconds. Conductance amplitudes and time characteristics were independent of the applied voltage. As our earlier studies revealed the essential role of the CBP in Ca2+ conductance of basophil membranes, we formed planar bilayers containing this isolated protein alone. Crosslinking of the CBP by a monoclonal antibody specific to it resulted in the appearance of channel conductances. All characteristics of these channels exhibited great similarity to those observed in planar membranes containing unfractionated RBL-2H3 membrane components. Moreover, in the latter membranes, the monoclonal anti-CBP antibody induced channel conductances that display an even closer similarity to those observed in membranes containing CBP alone. Conductances of both types of planar membranes, irrespective of the mode of activation used, were inhibited by cromolyn. Furthermore, the conductance induced in RBL membranes by polyvalent antigen was inhibited on dissociation of the crosslinked aggregates by a monovalent hapten. The detailed resemblance in channel behavior observed in experiments with the two types of planar bilayers provides compelling evidence that the CBP is the essential and sufficient component forming Ca2+ channels in basophil plasma membranes.