Abstract
Connexin (Cx) hemichannels form intercellular gap junction channels but can also function independently. These large-pore channels are permeable not only to ions but also to small signalling molecules and metabolites. This functional property is relevant to many physiological processes and can be altered by various biochemical factors or disease-causing mutations. 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 modelling. Fluorescence imaging, coupled with mathematical modelling based on Fick's law and/or the Goldman-Hodgkin-Katz current equation, enables assessment 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 this approach can reliably detect differences as low as two-fold in hemichannel permeability using datasets of moderate size (n < 100). Crucially, this approach requires only a minimal amount of time-intensive electrophysiological recording and leverages higher-throughput fluorescence measurements, which can be further streamlined using computational tools for automated cell detection and data extraction. We apply this methodology 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.