Recent advances in structural techniques have provided unprecedented insights into protein structure and function. Yet, obtaining insights into the structure and function of transmembrane proteins, whether alone or in complex with modulators or other proteins, remains challenging. The aim of this chapter is to demonstrate how mutagenesis studies, in conjunction with sequence, structural, and computational information, can assist in identifying binding regions in ion channels and provide mechanistic insights into their modulation. The examples presented comprise various potassium channels and include the identification of binding sites for ions such as sodium and for lipids, including PI(4,5)P2 and cholesterol, as well as the characterization of channel-protein interactions involving the βγ subunits of G proteins. Further examples focus on elucidating modulatory mechanisms, such as the reversal of the effect of cholesterol on an ion channel from enhancement to suppression. Additionally, recent advances in double-mutant cycle analysis and deep learning are enabling more detailed mapping of interaction surfaces, quantification of interaction strengths, and more precise identification of key residues. Together, these studies exemplify the utility of mutagenesis as a powerful perturbation approach for probing ion channel function and modulation.