Clonal expansion and differentiation of B lymphocytes are essential for the maintainance of the humoral arm of host defense. We propose a model for B cell growth whereby the role of ion channels in the regulation of expansion can be studied. In this system resting B cells become responsive to the growth promoting activity of anti-Ig if precultured in the presence of B cell stimulting factor-1 (BSF-1). This model predicts that B cells precultured with BSF-1 (phase-1) become """"""""competent"""""""" to proliferate in response to anti-Ig (phase 2). The role of ion channels in each phase of activation will be determined. To enhance our understanding of the molecular events that trigger B lymphocyte progression through the cell cycle, we propose to study mIg and BSF-1-mediated generation and modulation of transmembrane currents in single resting B lymphocytes. Using the patch clamp technique voltage sensitivity, ion selectivity and agonist activation of single ion channels will be determined. A profile of membrane channel properties will be obtained from determinations of channel distribution, mean channel lifetime and reversal potentials in response to membrane voltages clamped at specific levels and in response to BSF-1 +/-antiIg. To support and extend the electrophysiological studies, the function of these ion channels in the growth of B lymphocytes will be investigated by the use of specific in channel blockers. In addition to defining ion channel-receptor coupling, evidence for co-redistribution of membrane receptors and ion channels will be sought by patch clamping """"""""capped"""""""" regions of the B cell surface. The existence and nature of second messengers which regulate the activity of single channel events will be determined by a novel application of patch clamp technology. Coupling of mIgM and mIgD to specific ion channels will be determined using monoclonal anti-isotype specific antibodies. By comparing ion channel currents induced by anti-IgM in neonatal and mature B cell membranes, mechanisms for B cell tolerance vs activation may be revealed. Using well-defined mAb's to stage B cell ontogency, the ontogenic development of ion conductance systems in B cells will be defined. Through these approaches we combine the resources and expertise of eletrobiology and cellular immunology to pioneer new areas of exploration into the mechanisms of receptor-mediated cellular events.
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