Kraujaliene, L. | Search bioRxiv

bioRxiv2025

Model-based Evaluation of Connexin Hemichannel Permeability

Connexin (Cx) hemichannels represent a growing facet of Cx research. Hemichannels are hexamers of Cx subunits that are historically thought of as substrates of intercellular gap junction channels formed when two hemichannels from neighboring cells dock. Hemichannels, however, can function in the absence of docking and have been shown to play important roles in transmembrane signaling. There are 21 different Cx isoforms in humans and a host of disease-causing mutations have been identified. Although Cxs constitute large-pore channels, they can differ substantially in their conductance and permeability characteristics, thereby affecting the nature of signals that are transmitted. In this study, we present a methodology for quantifying and comparing the permeabilities of hemichannels formed by different Cx isoforms using a combination of fluorescence imaging, electrophysiological recordings and mathematical modeling. Fluorescence imaging, coupled with mathematical modeling based on Ficks law and/or the Goldman-Hodgkin-Katz current equation, enables estimation of tracer diffusion rates. These data are integrated with independently obtained electrophysiological measurements of hemichannel activity into a unified statistical model based on the likelihood ratio test. Simulation-based analyses demonstrate that our approach can reliably detect [~]2-fold differences in hemichannel permeability using datasets of moderate size. Crucially, this approach requires only a minimal amount of time-intensive electrophysiological recording, leveraging higher-throughout fluorescence measurements, which can be further streamlined using AI-based tools for automated cell detection and data extraction. We apply this method to compare the permeability of hemichannels formed by wild-type Cx26 and a pore-lining variant, Cx26*A49E. Our results show a significant increase in DAPI permeability in Cx26*A49E hemichannels, consistent with previous findings. This methodology can be extended to assess the permeabilities of other large-pore channels.

biophysics↗

bioRxiv2019

4-state model for simulating kinetic and steady-state voltage-dependent gating of gap junctions

Gap junction (GJ) channels, formed of connexin (Cx) proteins, provide a direct pathway for metabolic and electrical cell-to-cell communication. These specialized channels are not just passive conduits for the passage of ions and metabolites, but have been shown to gate robustly in response to transjunctional voltage, Vj, the voltage difference between two coupled cells and are regulated by various chemical factors. Voltage gating of GJs may play a physiological role, particularly in excitable cells which can exhibit large transients in membrane potential during the generation of an action potential. We present a mathematical/computational model of GJ channel voltage gating to assess properties of GJ channels that takes into account contingent gating of two series hemichannels and the distribution of Vj across each hemichannel. From electrophysiological recordings in cell cultures transfected with Cx43 and Cx45, isoforms that are expressed in cardiac tissue, data sets were fit simultaneously using global optimization. The results showed that the model is capable of describing both steady-state and kinetic properties of homotypic and heterotypic GJ channels composed of these connexins. Moreover, mathematical analyses showed that the model can be simplified to a reversible two-state system and solved analytically, using a rapid equilibrium assumption. Given that excitable cells are arranged in interconnected networks, the equilibrium assumption allows for a substantial reduction in computation time, which is useful in simulations of large clusters of coupled cells. Overall, this model can serve not just as a modeling tool, but also to provide a means of testing GJ channel gating behavior.\n\nSignificanceGap junction (GJ) channels gate in response to transjunctional voltage which provides the capacity for dynamic regulation of intercellular coupling. Kinetic properties of GJs in modeling studies have been infrequently addressed and we present a computational model of voltage gating that can account for both kinetic and steady-state changes in junctional conductance, gj. Although GJs possess two gating mechanisms, our analysis indicates that changes in gj for each voltage polarity can be adequately described by a kinetic scheme describing a single mechanism in each of the hemichannels, suggesting functional dominance of one mechanism over a substantial voltage range. This property allowed for model simplification that can be applied for efficient simulation of sizeable cell clusters and analyses of electrophysiological data.

biophysics↗