From Flux to Function: Extracting Mechanistic Insights from Ion Channels via <i>I</i> – <i>V</i> and <i>I</i> –μ Analyses

The molecular origins of ion-channel current-voltage (<i>I</i>-<i>V</i>) relationships are often unclear, obscured by ensemble averaging in experimental analysis and persistent underestimation of single-channel ion currents in simulations. Here, we present a mathematical framework that relates ion channel properties to experimentally measured <i>I</i>-<i>V</i> and current-chemical potential (<i>I</i>-μ) relationships. By accounting for how rates change in response to electrochemical conditions in a multistate kinetic model of systems with sequential binding sites, this approach demonstrates how the spatial arrangement of sites and transition states, together with rate asymmetries in ion uptake, transfer, and release, manifest in distinct open-channel current and conductance profiles. Varying these properties in model systems reveals a molecular basis for understanding rectification and nonohmic open-channel flux. Application to models fit to experimental <i>I</i>-<i>V</i> curves demonstrates that these mechanistic trends hold in heterogeneous systems, suggesting a (potentially) transferable paradigm for open-channel flux in channels and transporters with two or more sequential binding sites. Together, these results establish a theoretical framework for open-channel current and foundation for mechanistically interpreting experimental <i>I</i>-<i>V</i> and <i>I</i>-μ assays.