CLC Cl- transport proteins are ubiquitous and perform essential and diverse physiological functions. Mutations in human CLC genes and genes encoding their accessory proteins underlie serious diseases including Dent's disease, Bartter syndrome, bone disease, neurodegeneration, deafness and myotonia. CLCs are regulated by phosphorylation, Ca2+, adenosine ligands and accessory proteins. However, little is known about the molecular mechanisms of regulation, which represents a major gap in the field and significant limitation in the development of pharmaceuticals that modulate CLC trafficking and function. Our laboratory, supported by DK51610, pioneered the use of the genetic model organism C. elegans to provide the first detailed molecular understanding of CLC regulatory mechanisms. CLH-3b is a C. elegans CLC-1/2/Ka/Kb anion channel subfamily member that is inactivated by GCK-3-mediated phosphorylation. GCK-3 is an ortholog/homolog of mammalian SPAK and OSR1 kinases, which play central roles in systemic fluid balance and are important targets for pharmaceutical development to treat blood pressure disorders. During the previous funding period, we began defining for the first time structure/function mechanisms of phosphorylation-dependent CLC regulation. The cytoplasmic C-terminus of CLCs includes two conserved CBS domains (CBS1 and CBS2) that dimerize to form a Bateman domain. An inter-CBS linker connects CBS1 and CBS2. The linker is an intrinsically disordered region (IDR) lacking rigid 3D structure. Much of the CLH-3b linker is dispensable for regulation. However, deletion of a 14 amino acid activation domain comprising two regulatory serine residues phosphorylated by GCK-3 inactivates CLH-3b to the same extent as phosphorylation. A newly identified CLC signal transduction domain comprising CBS2 and two membrane a-helices mediates phosphorylation-dependent intraprotein signaling that regulates channel activity. The overarching goal of this renewal application is to define for the first time the regulatory relationships between the CLC inter-CBS linker and the Bateman, signal transduction and membrane domains. Using NMR spectroscopy, patch clamp electrophysiology, biochemical and mutagenesis strategies; we will address three novel questions: Does the activation domain contain phosphorylation-sensitive secondary structure that modulates its regulatory interaction with the Bateman domain? Does activation domain phosphorylation induce regulatory conformational changes in the Bateman domain? Do CLC subunit interface conformational rearrangements mediate the effects of phosphorylation on channel activity? Proposed studies will yield new insights into regulatory structure/function relationships of CLC channels as well as those of CBS domains and IDRs, which are associated with numerous diseases and represent important targets for drug development. Results of our studies will provide an essential foundation for better understanding of CLC associated diseases and for development of pharmaceuticals that specifically target CLC intracellular regulatory domains.