Functional channel formation associated with cytotoxic T-cell granules

Lymphocyte granules from cytotoxic T-lymphocyte lines A2, A11, and R8 were enriched by subcellular fractionation using a Percoll gradient. Granule-enriched fractions showed potent hemolytic activity in the presence of Ca²⁺. Isolated granules induced rapid Ca²⁺-dependent membrane depolarization of J774 macrophage-like cells. When tested in planar bilayers, granules induced the formation of Ca²⁺-dependent functional ion channels of large conductance steps of 1-6 nS in 0.1 M NaCl. Granule-induced channels were resistant to closing by an increase in transmembrane potential, with few channels shifting to the closed state only at voltages of >70 mV, following a Poisson process. These channels showed poor ion selectivity and were permeable to all monovalent and divalent ions (K⁺, Na⁺, Li⁺, Cl^-, Ca²⁺, Mg²⁺, Zn²⁺, Ba²⁺). Ultrastructural examination of soluble granule proteins incubated for 48 hr at 37° C in the presence of Ca²⁺ revealed ring-like structures of 150-200 Å. Structural and functional channel formation may be involved in cytolysis induced by cytotoxic T lymphocytes.