Lumen charge governs gated ion transport in β-barrel nanopores.

β-Barrel nanopores are involved in crucial biological processes, from ATP export in mitochondria to bacterial resistance, and represent a promising platform for emerging sequencing technologies. However, in contrast to ion channels, the understanding of the fundamental principles governing ion transport through these nanopores remains largely unexplored. Here we integrate experimental, numerical and theoretical approaches to elucidate ion transport mechanisms in β-barrel nanopores. We identify and characterize two distinct nonlinear phenomena: open-pore rectification and gating. Through extensive mutation analysis of aerolysin nanopores, we demonstrate that open-pore rectification is caused by ionic accumulation driven by the distribution of lumen charges. In addition, we provide converging evidence suggesting that gating is controlled by electric fields dissociating counterions from lumen charges, promoting local structural deformations. Our findings establish a rigorous framework for characterizing and understanding ion transport processes in protein-based nanopores, enabling the design of adaptable nanofluidic biotechnologies. We illustrate this by optimizing an aerolysin mutant for computing applications.