Connexin proteins form gap junction channels that mediate direct intercellular molecular communication crucial in development, physiology and response to trauma/inflammation. Defects in connexins cause human pathologies. Elucidation of the molecular mechanisms that regulate connexin channel function is essential for understanding their roles in human physiology and pathophysiology, and to identify targets for translational and basic science studies. The long-term goal of this project is to understand the cytosolic interdomain interactions that control the gating of connexin channels. This has been characterized for Cx43, in which interactions between the cytoplasmic loop (CL) and the C-terminal domain (CT) mediate channel gating by pH and other factors. The CL-CT mediated regulation of Cx43 is crucial for its physiological function, and is a therapeutic target for cardiovascular pathologies. However, the role of CL-CT interactions in modulation of other connexin channels, just as likely to be biomedically important, and their mechanism of action have not been explored. We have found that Cx26 channels are modulated by CL-CT interactions. Cx26 and Cx43 are representative members of the two largest families of connexins. Though structurally analogous, the effects of CL-CT interaction on Cx26 channel function seem to be fundamentally different from those in Cx43, suggesting that CL-CT interaction is a common modulatory mechanism in connexins, yet operate in connexin-specific ways. We propose to elucidate the molecular mechanisms of CL-CT control of channel function and properties using Cx26 and its closely related isoform Cx32. Cx26 is the only connexin channel for which there is a high- resolution structure, making it the basis for structure-based studies of all connexin channels. The proposed studies explore the basis and mechanisms of CL-CT interactions and channel properties they modulate, using strategies successfully applied to other channels, including use of competing peptides, engineered disulfide linkages, macroscopic and single channel recordings, and mutational analysis. The experiments utilize intact channels, complemented by peptide NMR. We propose to (a) determine the involvement of CL-CT interactions in channel gating, (b) identify the sites of CL-CT interactions, and (c) determine how CL-CT interaction and its effects are altered by mutations that cause human disease. Cx26 and Cx32 are widely distributed in the body. Mutations of Cx26 are responsible for over half the inherited sensorineural deafness worldwide, and also cause serious disfiguring skin disorders. Mutations of Cx32 cause a peripheral demyelination. In both connexins, many disease-causing mutations are positioned to affect CL-CT interaction. Both connexins are implicated tumor progression and a wide variety of pathological and physiological processes. The combination of a high resolution structure, a large number of disease causing mutations and the extensive experience of both PIs in studying gating, permeability and regulation of Cx26 and Cx32 channels provide a basis for productive, comprehensive investigation of CL-CT interactions.
This project will provide essential information regarding an heretofore unexplored mechanism by which intercellular molecular signaling throughout the body is modulated. Defects in this signaling cause a multitude of human pathologies, so elucidation of the mechanisms that determine its key functional properties is essential for identification of sites of intervention. This project is specifically targeted at the modulation of intercellular signaling in proteins that, when mutated, cause human diseases (e.g., deafness in one case, and a neurological disorder in the other).
|Lopez, William; Ramachandran, Jayalakshmi; Alsamarah, Abdelaziz et al. (2016) Mechanism of gating by calcium in connexin hemichannels. Proc Natl Acad Sci U S A 113:E7986-E7995|
|Tong, Xuhui; Lopez, William; Ramachandran, Jayalakshmi et al. (2015) Glutathione release through connexin hemichannels: Implications for chemical modification of pores permeable to large molecules. J Gen Physiol 146:245-54|
|Harris, Andrew L; Contreras, Jorge E (2014) Motifs in the permeation pathway of connexin channels mediate voltage and Ca (2+) sensing. Front Physiol 5:113|