Sunday, May 11, 2025
5/11/2025
Mechanistic role of the NT-domain in substrate selectivity and lipid-induced channel closure in the lens gap junctions - Project Summary Gap junction channels, composed of the isoforms connexin-46/50 (Cx46/50), are essential to proper development and maintenance of transparency in the mammalian eye lens. These intercellular channels play critical roles in establishing a micro-circulatory system of this avascular organ, required for the delivery of nutrients and antioxidants, and removal of metabolic waste products. A large pore (~1.4 nm diameter) provides passage to an array of chemical information; including ions, metabolites, hormones, and other small molecules (e.g., water, K+/Na+, cyclic-AMP (cAMP), glutathione (GSH) and glucose). When channel function is disrupted by inherited mutations or chemical modifications that accumulate throughout our lifetimes, cataracts occur. Despite the large pore size established by these intercellular channels, each connexin isoform (21 in human) displays an impressive level of selectivity to various types of cytosolic messages. It is thought that the permeation and selectivity (perm-selectivity) properties of each isoform is finely tuned to the physiological demands of its local environment. However, mechanistic explanations for this behavior have been limited. Our laboratory has resolved the high-resolution structures of native Cx46/50 intercellular channels isolated from the eye lens, using single particle CryoEM. Using these models, I conducted comparative all-atom molecular dynamics (MD) simulations and demonstrated that the N-terminal (NT)-domain of Cx46/50 adopts a more stable open-state conformation, as compared to the previously described connexin-26 (Cx26) crystal structure. I further showed that the NT-domain plays a critical role in establishing isoform-specific differences in perm-selectivity to electrically charged ions (Myers and Haddad et al. Nature 2018). Here, I propose state-of-the-art methods in computational modeling and MD simulation, coupled with emerging structural information obtained by single particle CryoEM, to extend my characterization of gap junction perm-selectivity to the universal cell-signaling molecule, cAMP and antioxidant GSH (Aim 1), and to understand how the local lipid environment is coupled to the stability of the NT-domain (Aim 2). This work will be conducted under the dual mentorship of Prof.'s Steve Reichow (PSU/OHSU) and Dan Zuckerman (OHSU), and through close collaboration with experts in gap junction physiology to directly correlate theory to function. These studies aim to provide atomic-level explanations for perm-selectivity in gap junctions and have the promise to unveil a completely new mechanism of modulating gap junction activity through the local lipid environment. Mechanistic models developed by these studies will fill critical gaps in knowledge for understanding how gap junctions mediate the cell-type specific transfer of chemical information and potentially open up new methods for computational design of gap junction-mediated (tissue- specific) delivery of small molecule drugs for the treatments of cancer, cataracts and other diseases.
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