ClC anion transport proteins are expressed in evolutionarily diverse organisms ranging from archaebacteria to mammals where they play essential roles in diverse processes such as systemic osmoregulation and regulation of cell and organelle Cl- and pH. Mutations in five of the nine human ClC genes give rise to or are associated with inherited muscle, bone, neurological and kidney disorders. Despite intensive study and their physiological importance, very little is known about how ClCs are regulated. We have exploited the genetic and molecular tractability of the nematode Caenorhabditis elegans to characterize the regulation and physiological roles of ClC channels that are assembled and operational in their native cellular environments. CLH-3b is a member of the mammalian ClC-1, 2, Ka and Kb anion channel subfamily, is expressed in the C. elegans oocyte and is activated by swelling and meiotic maturation via type 1 phosphatase mediated serine dephosphorylation. The Ste20 kinase GCK-3 binds to a 101 amino acid regulatory domain on the CLH-3b cytoplasmic C-terminus and functions to inhibit channel activity. Channel inhibition requires concomitant phosphorylation of two serine residues in the regulatory domain. Our studies of CLH-3b have provided the most detailed description of ClC channel regulation in the field. GCK-3 is a homolog of mammalian SPAK and OSR1. These kinases have emerged as critical regulators of diverse transport processes that play essential roles in cellular and systemic osmotic homeostasis. The SPAK/OSR1 signaling pathway is an important target for the development of new anti- hypertension drugs. We have shown that the role of GCK-3/SPAK/OSR1 signaling in osmosensing and systemic osmotic homeostasis is conserved from C. elegans to humans. The functional conservation of this signaling mechanism over hundreds of millions of years of evolution underscores its physiological significance. This renewal application builds on past successes of DK51610 and our unique understanding of CLH- 3b regulation to address two fundamental and unresolved questions: How do signaling events and conformational changes in the cytoplasmic C-terminus modulate and regulate ClC channel properties? How do cells and organisms detect osmotic perturbations and transduce those changes into specific responses? Our studies will use a variety of molecular, electrophysiological and biophysical approaches to characterize the signaling mechanisms by which GCK-3 and dephosphorylation control CLH-3b activity and to characterize conformational changes in the cytoplasmic C-terminus and outer pore that are induced by phosphorylation events. Detailed understanding of ClC biology is essential in order to fully define the role of these proteins in physiology and pathophysiology and their potential as therapeutic targets. Molecular understanding of cellular osmosensing represents a cornerstone for understanding and treating disturbances of salt and water balance that have a major impact on human health.

Public Health Relevance

ClC anion transport proteins carry out essential physiological functions and are associated with inherited muscle, bone, neurological and kidney disorders in humans. Studies described in this application will provide the first detailed description of how phosphorylation regulates ClC channel function and will provide new insights into mechanisms of cellular osmosensing. Detailed understanding of ClC regulation and cellular osmosensing is essential for understanding and treating disturbances of salt and water balance that have a major impact on human health.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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Special Emphasis Panel (ZRG1-DKUS-A (02))
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Ketchum, Christian J
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Mount Desert Island Biological Lab
Salsbury Cove
United States
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Yamada, Toshiki; Strange, Kevin (2018) Intracellular and extracellular loops of LRRC8 are essential for volume-regulated anion channel function. J Gen Physiol 150:1003-1015
Yamada, Toshiki; Wondergem, Robert; Morrison, Rebecca et al. (2016) Leucine-rich repeat containing protein LRRC8A is essential for swelling-activated Cl- currents and embryonic development in zebrafish. Physiol Rep 4:
Yamada, Toshiki; Strange, Kevin (2016) Regulatory Conformational Coupling between CLC Anion Channel Membrane and Cytoplasmic Domains. Biophys J 111:1887-1896
Strange, Kevin (2016) Drug Discovery in Fish, Flies, and Worms. ILAR J 57:133-143
Yamada, Toshiki; Krzeminski, Mickael; Bozoky, Zoltan et al. (2016) Role of CBS and Bateman Domains in Phosphorylation-Dependent Regulation of a CLC Anion Channel. Biophys J 111:1876-1886
Branicky, Robyn; Miyazaki, Hiroaki; Strange, Kevin et al. (2014) The voltage-gated anion channels encoded by clh-3 regulate egg laying in C. elegans by modulating motor neuron excitability. J Neurosci 34:764-75
Yamada, Toshiki; Bhate, Manasi P; Strange, Kevin (2013) Regulatory phosphorylation induces extracellular conformational changes in a CLC anion channel. Biophys J 104:1893-904
Miyazaki, Hiroaki; Strange, Kevin (2012) Differential regulation of a CLC anion channel by SPAK kinase ortholog-mediated multisite phosphorylation. Am J Physiol Cell Physiol 302:C1702-12
Miyazaki, Hiroaki; Yamada, Toshiki; Parton, Angela et al. (2012) CLC anion channel regulatory phosphorylation and conserved signal transduction domains. Biophys J 103:1706-18
Strange, Kevin; Yin, Viravuth P (2012) Rhythmic Ca²? signaling: keeping time with microRNAs. Curr Biol 22:R1000-1

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