Biology subjects

Myers, J. B.

Publications and source records attributed to Myers, J. B..

5 recordsLinked to original sources

bioRxiv2026

Distinct modes of N-terminal remodeling through a conserved inhibitor pocket in connexin-46/50

Selective pharmacological control of gap junction communication remains challenging because existing small-molecule connexin inhibitors act across multiple isoforms. The structural basis of this broad activity remains incompletely understood. Here, we used single-particle cryo-electron microscopy to examine native connexin-46/50 gap junction channels treated with two commonly used and chemically distinct inhibitors, 2-aminoethoxydiphenyl borate (2-APB) and carbenoxolone (CBX). Both treatments perturb the hydrophobic N-terminal (NT)-docking groove, but produce distinct conformational ensembles. 2-APB yields ordered destabilized-open and gated NT states resembling conformations observed during physiological pH- and Ca2+-dependent gating, with ligand- and lipid-associated densities accompanying the gated state. In contrast, CBX-compatible density occupies the same groove and is associated with loss of an ordered NT conformation, consistent with steric competition and altered physicochemical complementarity at the NT-docking site. Comparison with inhibitor-treated connexin-32 and connexin-36 structures identifies this region as a structurally conserved, pharmacologically sensitive pocket. These findings reveal how chemically diverse inhibitors perturb a conserved element of the native gating machinery while producing divergent NT-gating ensembles, providing a structural rationale for broad cross-isoform activity and identifying sequence-variable pocket margins that may support more selective modulation.

biophysics↗

bioRxiv2026

Lipids intercalate and mediate multi-channel assemblies of connexin-46/50 gap junctions

Gap junction channels enable direct electrical and metabolic exchange between adjacent cells and tissues, where they organize into dense plaques containing tens to thousands of channels. Although plaque formation is known to modulate junctional conductance, the structural basis for channel-channel organization within a membrane environment remains poorly defined. Here, we reconstitute native lens connexin-46/50 (Cx46/50) gap junction channels into MSP-based lipid nanodiscs that incorporate multiple channels to create miniature plaque-like complexes suitable for single-particle cryo-electron microscopy (cryo-EM). We determine high-resolution structures of dual-channel assemblies in two distinct configurations and find no ordered protein-protein contacts across the interface. Instead, the inter-channel space is occupied by ordered lipid density, indicating that channel packing in these assemblies is lipid mediated. These channel-channel interfaces stabilize discrete lipid populations, including an interstitial lipid intercalated between subunits and positioned near the N-terminal gating domain, suggesting a route by which channel organization could promote lipid occupancy near the pore. Additionally, we leverage this dataset to refine the Cx46/50 single-channel structure to 1.8 [A] resolution, revealing exceptional chemical detail of the pore-lining landscape in the stabilized open-state. Together, these results define principles of lipid-mediated multi-channel organization and suggest how plaque-like packing may tune gap junction function through specific lipid interactions.

biophysics↗

bioRxiv2026

Validation and testing of an in vitro model to study medical treatments for anterior urethral stricture disease: assessing the potential efficacy of phosphodiesterase-4 (PDE4) inhibition and testosterone

ObjectiveTo 1) determine the expression and distribution of all PDE4 isozymes (A-D) along the length of the anterior urethra, 2) culture fibroblasts and epithelial cells from healthy and strictured urethras, 3) investigate an in vitro model of anterior urethral stricture disease (aUSD), and 4) assess the therapeutic potential of phosphodiesterase-4 (PDE4) inhibitors and testosterone compared to paclitaxel. MethodsThe presence and relative abundance of PDE4 isozymes (A-D) was confirmed using immunohistochemistry on 5 male cadaveric urethras. Human urethral fibroblasts (FBs) were cultured from healthy control urethras of patients undergoing vaginoplasty (n=3) and from idiopathic bulbar urethral strictures (L2S1E2) of patients undergoing urethroplasty (n=3). Epithelial cells (ECs) were cultured from a healthy control urethra and two urethral strictures. To investigate a model of aUSD, Control FBs were stimulated with TGF{beta}1 and compared to Stricture FBs on assays of cell proliferation and expression of genes relevant to aUSD pathophysiology. To test therapeutics, Stricture FBs were treated with the PDE4 inhibitor, roflumilast, testosterone (T), or paclitaxel and compared to Control FBs on the previously mentioned assays and cell viability. ResultsPDE4- A, B, and D were detected along the length of the urethra. Expression levels did not differ between urethral regions. TGF{beta}1 altered proliferation and gene expression in a dose-dependent manner. Roflumilast and T preserved cell viability and proliferation and decreased expression of genes positively associated with auSD. ConclusionUrethral FBs and ECs can be cultured from healthy and strictured surgical specimens, enabling in vitro research. PDE4 inhibitors and T may be non-cytotoxic alternatives or additions to paclitaxel for aUSD. HighlightsO_LIPDE4 isozymes A, B, and D are expressed in adult anterior urethras C_LIO_LIPDE4 is expressed equally from proximal bulbar to meatal urethra C_LIO_LIEpithelial cells and fibroblasts can be cultured from healthy and stricture urethra C_LIO_LITGF{beta}1 may not be an optimal method to model aUSD in vitro C_LIO_LIUnlike paclitaxel, roflumilast and testosterone are not toxic to urethral cells C_LI

pharmacology and toxicology↗

bioRxiv2025

Reversible lipid mediated pH-gating of connexin-46/50 by cryo-EM

Gap junctions, formed by connexin proteins, establish direct electrical and metabolic coupling between cells, enabling coordinated tissue responses. These channels universally respond to intracellular pH changes, closing under acidic conditions to limit the spread of cytotoxic signals during cellular stress, such as ischemia. Using cryo-electron microscopy (cryo-EM), we uncover insights into the structural mechanism of pH-gating in native lens connexin-46/50 (Cx46/50) gap junctions. Mild acidification drives lipid infiltration into the channel pore, displacing the N-terminal (NT) domain and stabilizing pore closure. Lipid involvement is both essential and fully reversible, with structural transitions involving an ensemble of gated-states formed through non-cooperative NT domain movement as well as minor populations of a distinct destabilized open-state. These findings provide molecular insights into pH-gating dynamics, illustrating how structural changes may regulate gap junction function under cellular stress and linking Cx46/50 dysregulation to age-related cataract formation.

biophysics↗

bioRxiv2020

Connexin-46/50 in a dynamic lipid environment resolved by CryoEM at 1.9 A

Gap junctions establish direct pathways for connected cells and tissues to transfer metabolic and electrical messages1. The local lipid environment is known to affect the structure, stability and intercellular channel activity of gap junctions2-5; however, the molecular basis for these effects remains unknown. To gain insight toward how gap junctions interact with their local membrane environment, we used lipid nanodisc technology to incorporate native connexin-46/50 (Cx46/50) intercellular channels into a dual lipid membrane system, closely mimicking a native cell-to-cell junction. Structural characterization of Cx46/50 lipid-embedded channels by single particle CryoEM revealed a lipid-induced stabilization to the channel, resulting in a 3D reconstruction at 1.9 [A] resolution. Together with all-atom molecular dynamics (MD) simulations and 3D heterogeneity analysis of the ensemble CryoEM data, it is shown that Cx46/50 in turn imparts long-range stabilization to the dynamic local lipid environment that is specific to the extracellular lipid leaflet of the two opposed membranes. In addition, nearly 400 water molecules are resolved in the CryoEM map, localized throughout the intercellular permeation pathway and contributing to the channel architecture. These results illustrate how the aqueous-lipid environment is integrated with the architectural stability, structure and function of gap junction communication channels, and demonstrates the ability of CryoEM to effectively characterize dynamical protein-lipid interactions.

biophysics↗