Immoderate consumption of alcohol increases susceptibility to infection. Moreover, mortality and morbidity rates due to infectious disease are so high among alcoholic patients, that direct immunosuppression by the drug itself has long been suspected to occur. However, since alcoholic life- styles often predispose to other immunosuppressive sequelae, such as malnutrition and cirrhosis of the liver, it is difficult to study the immunomodulatory properties of alcohol in alcoholic patients. Therefore, we propose to reduce the problem to the molecular level and to investigate the effects of alcohol on cell-mediated immunity in vitro. T cell growth and proliferation is dependent upon the movement of specific ions across the plasma membrane. Ligand-gated calcium (Ca) channels open in response to stimulation of the T cell antigen receptor complex (TCR-CD3), and the influx of Ca which follows is necessary for cell cycle progression to proceed. Voltage-gated K channels set the membrane potential of T cells, and influence T cell proliferative responses by regulating Ca influx. Preliminary studies demonstrate that ethanol (35-50 mM) increases voltage- gated K channel activity and hyperpolarizes the membrane potential of human T cells. Thus, the intent of this proposal is to characterize ethanol-induced increases in T cell K conductance and to determine the biological significance of this enhancement. Electrophysiologic recordings will be performed using the gigaohm seal patch clamp technique. First, a complete characterization of the ion channel will be accomplished. Ion selectivity, single-channel slope conductance, and the large number of channels present in T cell membranes indicate that ethanol is increasing the open probability of n-type K channels. Additional studies on channel identification will include toxin sensitivity, divalent ion sensitivity, voltage-dependent inactivation, and stimulation by cyclic adenosine monophosphate (cAMP) elevating agents. Second, alcohol-induced alterations in K channel characteristics and on T cell membrane potential measurements will be studied for a variety of alcohols including: methanol, ethanol, butanol, and pentanol. In addition, the effects of ethanol on T cell K channels will be investigated in various subpopulations by separating the cells according to cluster of differentiation (CD) designations. Subpopulations will include CD4+ helper cells, CD8+ suppressor cells, CD45RA+ naive cells, and CD45RO+ memory cells. Third, the mechanism(s) of alcohol-induced increases in T cell K channel activity will be investigated. Alternatives include key biochemical events during T cell signal transduction, G protein activation, and channel phosphorylation. Fourth, alcohol-induced stimulation of T cell K channels will be studied in relation to T cell proliferative response, cytokine production, and signal transduction.
