MITOGEN-ACTIVATED CA-LYMPHOCYTES(+ CHANNELS IN HUMAN B)

Two complementary experimental methods have been used to examine mitog en-induced transmembrane conductances in human B cells using the Daudi cell line as a model for human B cell activation. Spectrofluorometry was used to investigate mitogen-induced changes in [Ca2+]i and transme mbrane potential. Activation of human B cells with anti-mu antibodies resulted in a biphasic rise in [Ca2+]i, the second phase being mediate d by the influx of extracellular Ca++. Ca++ influx was inhibited by hi gh [K+]e, suggesting that this influx was transmembrane potential sens itive. Membrane currents of Daudi cells were investigated using voltag e clamp techniques. Before mitogenic stirnulation, the cells were elec trically quiet. Within several minutes of the addition of anti-mu anti bodies to the bath solution, inward currents were observed at negative voltages. Whole-cell currents changed instantly with voltage steps an d were transmembrane potential sensitive in that at potentials more po sitive than -40 mV no currents were detectable. A similar conductance was also activated by the introduction of IP3 into the intracellular s olution, suggesting that IP3 generation after surface IgM crosslinking is involved in the activation of this conductance. Both anti-mu and I P3 induced currents were blocked by 1 mM La+++, which is known to bloc k Ca++ channels. These results strongly support the presence of membra ne Ca++ channels in human B cells that function in the early stages of activation. Changes in transmembrane potential appear to be important in regulating Ca++ influx. These mechanisms work in concert to regula te the level of [Ca++]i during the early phases of human B cell activa tion.