Evaluation of unitary conductance of gap junction channels based on stationary fluctuation analysis

Gap junction (GJ) channels, formed of connexin (Cx) protein, enable direct intercellular communication in most vertebrate tissues. One of the key biophysical characteristics of these channels is their unitary conductance, which can be affected by mutations in Cx genes and various biochemical factors, such as posttranslational modifications. Due to the unique intercellular configuration of GJ channels, recording single-channel currents is challenging, and precise data on unitary conductances of some Cx isoforms remain limited. In this study, we applied stationary noise analysis, a method successfully used for ion channels with very low unitary conductances, to GJ channels. We modified this technique to account for the residual conductance of GJ channels and present three strategies for estimating unitary conductance, including model-based evaluation of open-state probability and subtraction of residual conductance. To assess the validity, advantages, and limitations of these approaches, we performed mathematical analysis and simulation experiments. We also addressed practical issues such as the underestimation of sample variance in autocorrelated recordings and channel rundown, proposing solutions to these issues. Finally, we applied these strategies to electrophysiological data recorded from cells expressing Cx45. Our findings showed that noise-based estimates of Cx45 unitary conductance from macroscopic currents align well with those obtained from single-channel recordings.