Gap junctions are assemblages of cell-cell channels occurring in regions of cell contact. These channels permit direct intercellular communication by passive diffusion of low molecular weight hydrophilic molecules, including calcium, cyclic nucleotides, inositol phosphates and other signaling molecules. Several gap junction proteins, termed connexins, serve prominent roles in the nervous system. Among these, Connexin43 (Cx43) is expressed at high levels in astrocytes where they provide a mechanism of spatial buffering of ions and exert a modulatory influence on neuronal activity. Mutations of Cx32, found in myelin, lead to a progressive peripheral neuropathy termed X-linked Charcot-Marie-Tooth disease. Mutations in Cx26 underlie the most common forms of non-syndromic deafness. Interestingly, connexins are the only family of plasma membrane proteins that do not appear to be glycosylated. Preliminary studies of Cxs 43, 32 and 26 indicate that they are significantly more negatively charged than anticipated based upon their sequence and known covalent modifications. That these pI variants are indistinguishable by SDS-PAGE indicates that the molecular basis for acidic pI variants is of low molecular weight. The specific aims of this proposal are to (1) identify this modification and the altered amino acid residues and (2) determine its role in the assembly and functioning of gap junction channels. Our approach to determining the chemical basis of connexin will be biochemical, relying especially on mass spectrometry, and genetic engineering. The role of this modification will be assessed using pharmacology and site-direct mutagenesis. Identification of a low Mr anion covalently attached to connexins might provide the first target for pharmacological intervention in gap junction function. Knowledge of where charged residues exist will profoundly influence modeling studies of connexins and, likely, other membrane proteins, in which alterations of electrostatic interactions play a role in channel selectivity and gating. Many of the experiments will push experimental procedures for use in the study of membrane proteins. Establishing the basis of connexin charge alteration will introduce a new player in chemical modification of proteins and their properties.