Chloride channels and transporters of the CLC family play crucial roles in a myriad of physiological processes including regulation of electrical excitability of nerve and muscle cells, modulation of salt and water movement across epithelia and acidification of intracellular compartments. The human genome encodes for 9 CLCs, 5 of which are transporters and 4 are channels. Mutations in 5 of these genes lead to the synthesis of proteins with altered functionalities that cause genetically inherited disorders such as myotonia congenita, Bartter's syndrome, Dent's disease, osteopetrosis and epilepsy. The involvement of these proteins in such a wide array of physiological and pathological processes marks them as ideal targets for the development of therapeutic treatments and drug design. This progress is, however, stunted by our lack of knowledge of the basic structural and mechanistic underpinnings underlying CLC function. This proposal aims to provide a molecular description of how CLC proteins regulate transmembrane Cl- fluxes and how these are coupled to H+ movement. This goal will be pursued through the combined use of X-ray crystallography, electrophysiological recordings, flux measurements and, for the first time for CLC proteins, direct substrate binding measurements. CLC proteins are homodimers where each monomer forms an independent permeation pathway. All family members catalyze movement of Cl- ions across cellular membranes, but can do so via either of two thermodynamically opposing mechanisms: the CLC channels dissipate the Cl- electrochemical gradient, whereas the CLC transporters catalyze uphill Cl- movement at the expense of the H+ electrochemical gradient, or vice versa. Our first major aim is to identify the molecular steps that allow CLC transporters to catalyze the stoichiometric exchange of Cl- and H+ across cellular membranes. Transporters undergo a complex series of conformational changes that allow them to transform the energy stored in electrochemical gradients into uphill substrate movement. We will identify, isolate and characterize the crucial structural players in this process. Our second major aim is to identify the molecular basis of anionic selectivity in CLC proteins. Substrate specificity is of paramount importance to proper function of both channels and transporters. We have now identified several residues potentially crucial for this process. We will test this hypothesis by manipulating through mutagenesis of these residues the selectivity of binding and permeability in order to alter the substrate specificity of the CLC channels and transporters. It has been proposed that CLC transporters and channels share a common architecture. The third goal of this proposal is to test this hypothesis by transforming the former into the latter. We will accomplish this by pursuing two complementary approaches: first, we will identify and eliminate the physical barriers blocking Cl- movement through the transporters and, second, we will identify the key residues differentiating the channels and the transporters and mutate the ones into the others.

Public Health Relevance

The CLC channels and transporters mediate anion transport across cellular membranes to modulate the electrical excitability of muscle and nerve, to allow salt and water movement across epithelia, participate in acidification of vesicles along the endosomal- lysosomal pathway and of neurotransmitter release vesicles. Mutations in 5 of the 9 human CLC genes lead to genetic diseases. Because of the crucial role of these channels and transporters in all of these physiological processes, understanding the mechanism of function of these proteins will be therapeutically useful. By relating the structure of these proteins to their function, it may ultimately be possible to develop or identify pharmaceutical agents that could either enhance or inhibit Cl- transport, depending on the target and the ultimate goal. ??

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Chin, Jean
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Weill Medical College of Cornell University
Schools of Medicine
New York
United States
Zip Code
Basilio, Daniel; Noack, Kristin; Picollo, Alessandra et al. (2014) Conformational changes required for H(+)/Cl(-) exchange mediated by a CLC transporter. Nat Struct Mol Biol 21:456-63
Williams, P D; Dobson, A P; Dhondt, K V et al. (2014) Evidence of trade-offs shaping virulence evolution in an emerging wildlife pathogen. J Evol Biol 27:1271-8
Sakuma, Chisako; Kawauchi, Takeshi; Haraguchi, Shuka et al. (2014) Drosophila Strip serves as a platform for early endosome organization during axon elongation. Nat Commun 5:5180
Miller, Jean; Davis, Cheryl D (2013) Increasing frequency of feline cytauxzoonosis cases diagnosed in western Kentucky from 2001 to 2011. Vet Parasitol 198:205-8
Malvezzi, Mattia; Chalat, Madhavan; Janjusevic, Radmila et al. (2013) Ca2+-dependent phospholipid scrambling by a reconstituted TMEM16 ion channel. Nat Commun 4:2367
Hickok, Jason R; Vasudevan, Divya; Antholine, William E et al. (2013) Nitric oxide modifies global histone methylation by inhibiting Jumonji C domain-containing demethylases. J Biol Chem 288:16004-15
Chen, Yunqiu; Unger, Michelle; Ntai, Ioanna et al. (2013) Gobichelin A and B: Mixed-Ligand Siderophores Discovered Using Proteomics. Medchemcomm 4:233-238
Picollo, Alessandra; Xu, Yanyan; Johner, Niklaus et al. (2012) Synergistic substrate binding determines the stoichiometry of transport of a prokaryotic H(+)/Cl(-) exchanger. Nat Struct Mol Biol 19:525-31, S1
Hara, Yuji; Balci-Hayta, Burcu; Yoshida-Moriguchi, Takako et al. (2011) A dystroglycan mutation associated with limb-girdle muscular dystrophy. N Engl J Med 364:939-46
Ying, Shui-Wang; Tibbs, Gareth R; Picollo, Alessandra et al. (2011) PIP2-mediated HCN3 channel gating is crucial for rhythmic burst firing in thalamic intergeniculate leaflet neurons. J Neurosci 31:10412-23

Showing the most recent 10 out of 13 publications