Portland State University. Department of Chemistry
Term of Graduation
Date of Publication
Doctor of Philosophy (Ph.D.) in Chemistry
1 online resource (viii, 321 pages)
Gap junctions make up a class of intercellular channels that characteristically connect the cytoplasm of directly apposed cells through large assemblies, or plaques, constituted by a multitude of intercellular channels. Gap junction mediated intercellular communication is critical for a variety of physiological functions, from coordinating electrical impulses in the heart and brain to maintaining homeostasis in most tissues. There are 21 isoforms of connexins, the constituent subunit of the gap junction, expressed in a tissue dependent manner. Gap junctions formed from different isoforms exhibit distinct biophysical properties, such as gating kinetics and sensitivity, as well as unique permeability and selectivity to solutes suited to the needs of the cell. The molecular mechanism underlying the physiological roles of the gap junctions are poorly understood, due in large part to a lack of high-resolution structures.
The Reichow lab has recently elucidated the structure of two closely related gap junction channels, composed of isoforms expressed in the eye lens (Cx46 and Cx50) using single particle electron cryo-microscopy (CryoEM), providing a means for comparative structural analyses. However, these static structural models provide limited information on the dynamic behaviors that give rise to their complex functions. For my dissertation research, I have utilized molecular dynamics (MD) simulations -- a computational microscope that is unrestricted to the limits of electron microscopy -- to investigate the dynamic and thermodynamic mechanisms underlying gap junction permeability and selectivity. Using comparative equilibrium MD, I confirmed that Cx46 and Cx50 adopt a stable open state, distinct from the previously determined structure of Cx26; thus, underscoring the need for high-resolution structures of multiple isoforms. From these simulations, I identified the n-terminal helix (NTH) as the site of isoform-specific behaviors of substrate selectivity. Furthermore, I characterized multiple putative ion binding sites, which were hidden from view in the CryoEM reconstruction due to their transient nature.
A technological advance in membrane protein CryoEM studies, known as a lipid nanodiscs, allowed for my colleague to elucidate a sub-2 Å structure of Cx46 and Cx50 -- resolving ordered water molecules and concentric annular layers of lipids within the extracellular leaflet. Equilibrium MD simulations of Cx46 and Cx50 corroborated more than 80% of the solvent densities and recapitulated the extracellular leaflet stabilization -- indicating a novel protein-induced gel-phase lipid transition. Moreover, the simulations explained the absence of resolved lipid head-groups in the ensemble reconstructions.
In collaboration with gap junction electrophysiologists, I utilized in-silico mutagenesis to swap residues within the NTH between Cx46 and Cx50 to investigate the molecular determinants underlying isoform specific conductance and gating properties of these isoforms. These simulations predicted a swap in junctional conductance, matching closely to experiment, and explained a loss of function in the NTH-swapped Cx46. Additional analysis using computational electrophysiology characterized conductance through gap junctions composed of Cx46, Cx50, and Cx46/50; revealing the permeation mechanism and thermodynamics underlying channel rectification and cation selectivity. In this dissertation, I demonstrate the power of molecular dynamics simulation as a tool which corroborates and extends our knowledge and understanding of gap junction structure and function gained through experimental CryoEM and electrophysiology.
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Haddad, Bassam George, "Mechanisms of Connexin-46 and -50 Intercellular Channel Function and Stability by Molecular Dynamics Simulations" (2021). Dissertations and Theses. Paper 5757.
Available for download on Wednesday, August 02, 2023