We propose research to investigate the mechanisms of how glycine and related amino acids prevent plasma membrane damage in energy compromised cells. The effects of amino acids are not related to metabolism. Moreover, they occur in spite of severe ATP depletion and break down of ion homeostasis. Our preliminary studies show that the cellular defect in ATP depleted cells that is specifically corrected by glycine is a unique plasma membrane abnormality. Analysis of the dysfunction revealed that the membrane behaves as if it is perforated by pores of molecular dimensions with definable size exclusion limits, although it remains structurally intact in other respects. This type of pathology is either suppressed or aggravated by a number of other agents which have activities at inhibitory glycine receptors in the central nervous system (CNS). The alkaloid Strychnine and the anthelmintic Avermectin protect cells, and their effects are additive to those of glycine. Other agents, (RU5135, pitrazepin and thebaine) antagonize the protective effects of glycine in a specific manner. The pharmacology of these effects suggests strongly that the beneficial effects of glycine may be explained by its actions on a target protein in the plasma membrane that is analogous to the CNS glycine receptor chloride channel. According to the hypothesis, ATP depletion leads to an unknown abnormality of the glycine receptor like protein which is responsible for the porous membrane defect. Dose responses to some of the pharmacological agents (Avermectin, RU5135, pitrazepin) suggest high affinity interactions in the ATP depletion model and point towards strategies that may be used to identify and isolate the protein at which their effects are directed. Our objectives are threefold. Firstly, we will characterize the glycine specific membrane defect by functional as well as morphological techniques. Secondly, we will develop methodology to obtain ultra pure preparations of plasma membranes. Thirdly, we will use derivatives of Avermectin B1a to affinity label and affinity purify plasma membrane proteins to which they may bind. We believe that this approach will succeed in the identification and purification of the putative target protein for glycine. The studies will be done in a cultured kidney epithelial cell line, but will be generally relevant to other cell types.
