PROJECT SUMMARY KCa3.1, also known as SK4 or IK1, is a member of a family of small- to intermediate-conductance potassium channels in humans (KCa2.1–2.3, KCa3.1). KCa3.1 plays a crucial role in T-cell activation by effluxing potassium to maintain a negative membrane potential that allows for additional calcium influx. KCa3.1 is a potential therapeutic target for inflammatory bowel diseases such as Crohn's disease and ulcerative colitis and for organ transplant rejection. KCa3.1 (and KCa2.1–2.3) is homotetrameric and activated through calcium binding to calmodulin (CaM), which is constitutively associated with a cytoplasmic region of the channels (CaM binding domain, CaMBD). A novel feature of KCa3.1 activation is the additional requirement of phosphorylation of a specific histidine residue (His358), just downstream of the CaMBD, by NDPK-B (nucleoside diphosphate kinase-B). Although histidine phosphorylation is well characterized in two-component signaling systems in prokaryotes, the functional consequences of histidine phosphorylation in eukaryotes are just beginning to be understood. Recently, we showed that histidine phosphorylation of KCa3.1 is necessary to relieve copper inhibition of the channel. In this application, cryo-EM studies are proposed to elucidate the molecular mechanisms by which KCa3.1 is regulated by calcium and histidine phosphorylation.