Calcium specificity of the antigen-induced channels in rat basophilic leukemia cells

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Abstract
Ion channels, activated upon IgE-Fc.epsilon. receptor aggregation by specific antigen, were studied in micropipet-supported lipid bilayers. These bilayers were reconstituted with purified IgE-Fc.epsilon. receptor complex and the intact 110-kDa channel-forming protein, both isolated from plasma membranes of rat basophilic leukemia cells (line RBL-2H3). In order to identify the current carrier through these ion channels and to determine their ion selectivity, we investigated the currents flowing through the IgE-Fc.epsilon. recptor gated channels in the presence of a gradient of Ca2+ ions. Thus, the solution in which the micropipet-supported bilayer was immersed contained 1.8 mM CaCl2, while the interior of the micropipet contained 0.1 .mu.M Ca2+ (buffered with EGTA). Both solutions also contained 150 mM of a monovalent cation chloride salt (either K+ or Na+). The currents induced upon specific aggregation of the IgE (by either antigen or anti-IgE antibodies) were examined over a range of potentials imposed on the bilayer. The type of conductance event most frequently observed under the employed experimental conditions was a channel that has a slope conductance of 3 pS and a reversal potential practically identical with the calculated value for the reversal potential of calcium (134 .+-. 11 mV in the presence of sodium, 125 .+-. 13 mV in the presence of potassium). These results indicate that this channel is highly selective for calcium against the monovalent cations sodium and potassium. This same channel has a conductance of 4-5 pS in the presence of symmetrical solutions containing only 100 mM CaCl2 and 8 pS in the presence of 0.5 M NaCl with no calcium. A second type of conductance event appears under all the above-described conditions at much lower frequency (< 10% of all the resolved events). These events have a higher conductance (10-20 pS in the presence of different concentrations of calcium ions, 25 pS in the presence of 0.5 M sodium chloride with no calcium) but are less selective for calcium against either sodium or potassium, as determined from their reversal potential. Comparison of the conductance changes observed in the above experiments with the reported values for the amounts of Ca2+ ions taken up by the intact RBL-2H3 cells upon antigen stimulation suggests that these antigen-induced calcium channels can account for the increase in intracellular calcium that occurs upon antigen-stimulation of these cells.