Tuning Rectification and Gating in Biological Nanopores Through Lumen Charge Mutation

β-barrel nanopores are involved in crucial biological processes, from ATP export in mitochondria to antibiotic resistance in Gram-negative bacteria, 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 in its early stages. In this chapter, we describe the production and mutation of three major biological nanopores-aerolysin, MspA, and α-HL-as well as experimental approaches to elucidate ion transport mechanisms in these biological nanopores. Specifically, we provide tools to characterize two distinct nonlinear phenomena: open-pore rectification and gating, as well as guidelines to tune these phenomena to achieve a certain rectification or gating behavior.