Voltage versus concentration in ion channels: Physical signatures reveal rate-limiting steps

Within the field of electrophysiology, I-V curves are a primary experimental means of characterizing ion channel behavior. By directly measuring ionic currents across membranes, electrophysiological techniques quantify key properties, including conductance, ion selectivity, and voltage dependence. We demonstrate a method of linking experimentally observed ion flux with microscopic mechanistic properties of channels. Although voltage and chemical potential can provide the same net electrochemical driving force, they perturb ion transport differently: voltage modifies transition rates throughout the permeation pathway, whereas chemical potential primarily alters ion uptake probabilities. Consequently, uptake, transfer, and release respond differently to the two driving forces, producing distinct flux signatures and causing chemical potential-driven flux to plateau while voltage-driven flux continues to increase. These differences encode the rate-limiting step (RLS) in open-channel flux. We propose a protocol based on three standard I-V measurements obtained under symmetric and asymmetric ion concentrations for elucidating the RLS. Application of this analysis to experimental data from the KcsA potassium channel identifies release as the RLS for outward permeation, consistent with available structural and computational evidence. Together, these results reveal how the physics of the electrochemical driving force shapes ion transport and provide an experimentally accessible strategy for connecting macroscopic electrophysiology measurements to microscopic transport mechanisms.