Bruce L. Kagan - US grants

The broad aim of this study is to examine the two fundamental processes of gating and translocation in ionic channels formed by microbial toxins. The diphtheria toxin (DT) channel is the prototype for this class of channels. Channels will be studied using lipid bilayer, lipid vesicle, and patch clamp techniques. 1. Pharmacology of DT gating. The intracellular second messenger 1,4,5-inositol-triphosphate (IP3) bind to DT and stimulates channel formation from the trans side of the membrane. Dose-response curves for IP3, other inositol phosphates and other DT ligands including blockers of stimulation (ApUp, ATP) will be constructed. The mechanism of action of these ligands will be examined functionally at the macroscopic current and single channel level, and structurally through observing the effects of ligands on DT mutants, fragments and related toxins such as tetanus and Pseudomonas exotoxin A (PsA). Effects of inositol phosphates on the selectivity of the DT channel, especially with respect to calcium, will be examined. 2. Physiology of DT mutants-gating implications. Several DT mutants (called CRMs) for cross- reacting material and fragments are well characterized structurally and are known to form channels (CRM45, CRM50, CRM197, CB1 (cyanogen bromide fragment)). Only CRM45 has been well studied electrophysiologically and exhibits striking differences from the properties of the DT channel. The voltage and pH dependent properties of DT, its various fragments and mutants, and the related channel forming toxin (PsA) will be quantitatively characterized. Site specific mutants will be constructed in an attempt to alter the gating properties of DT via known structural changes. 3. Mechanism of translocation. These experiments will probe the molecular mechanism of protein translocation across membranes. The pore radius of the DT channel will be determined using non-electrolyte sieving in lipid vesicles in order to see if the size of DT is markedly different from CRM45 (d about 18A). Proteolytic digestion using endo- and exopeptidases of DT channels from cis and trans sides of the bilayer will be attempted to determine the location of the amino and carboxy terminals of DT with respect to the membrane. Differences in open and closed state topology will also be sought. Patch clamp experiments will attempt to record DT channels in Vero cells as they cross the cell membrane. Effects of inhibitors of DT entry such as SITs, and low Ca++ will be examined to elucidate the role of the DT receptor in DT translocation.

Behavioral, cognitive, and motor disorders are clinical manifestations of AIDs Dementia Complex (ADC). ADC is likely mediated, at least in part, by the cytokines tumor necrosis factor alpha (TNF-alpha) and lymphotoxin (LT). TNF and LT are pleiotropic cytokines secreted by macrophages and lymphocytes. TNF and LT possess a broad range of hormonal and cytotoxic actions, but the mechanism of action of TNF and LT is unknown. Our laboratory has made the recent discovery that TNF and LT can form ion-permeable channels in planar lipid membranes, and we have proposed that channel formation may explain some physiologic effects of TNF such as the killing of tumor cells and the depolarization of muscle cells. Several lines of evidence implicate TNF in the pathophysiology of AIDS including the facts that TNF levels are elevated in AIDS patients, TNF can cause cachexia, TNF can increase HIV replication, and TNF can kill HIV-infected lymphocytes. TNF can increase HIV replication, and TNF can kill HIV-infected lymphocytes. TNF has also been found in the CSF of AIDS patients and its ability to depolarize cells and demyelinate nerve fibers may contribute to the dementia complex of AIDS (ADC). The broad goal of this research is to understand the role of these channels in the pathophysiology of AIDS and to search for ways to modify the destructive actions of TNF and LT in AIDS. TNF and LT channels will be characterized using electrophysiologic techniques in lipid bilayer and whole cells (patch clamp).