This research is aimed at understanding in chemical detail the fundamental mechanisms by which proteins belonging to the """"""""CLC family of Cl- channels"""""""" work. CLC channels are widespread throughout the biological world and are essential to the proper functioning of mammalian cell membranes in many different physiological contexts. Mutations or disruptions of any of the nine CLC isoforms inhabiting the human genome lead to numerous diseases, from skeletal muscle myotonias to compromised renal control of blood pressure to improper reabsorption of bone. As a result of recent work we now know that the CLC family includes isoforms that operate by fundamentally different Cl- transport mechanisms; while the long-studied muscle type CLCs are passive channels, a bacterial homolog was surprisingly found to be a secondary active transporter in which uphill Cl- transport is coupled to downhill H+ transport. I propose to investigate the ramifications of this perplexing discovery by carrying out a detailed structure-function study of the bacterial CLC homolog. We will investigate the mechanism of proton coupling to Cl- transport, will attempt to transform, by directed mutagenesis, the active transporter into a true channel, and will work to clone, express, and reconstitute a set of prokaryotic CLCs carrying a long c-terminal domain.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Biophysics of Synapses, Channels, and Transporters Study Section (BSCT)
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Shapiro, Bert I
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Brandeis University
Schools of Arts and Sciences
United States
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Jayaram, Hariharan; Robertson, Janice L; Wu, Fang et al. (2011) Structure of a slow CLC Cl?/H+ antiporter from a cyanobacterium. Biochemistry 50:788-94
Robertson, Janice L; Kolmakova-Partensky, Ludmila; Miller, Christopher (2010) Design, function and structure of a monomeric ClC transporter. Nature 468:844-7
Miller, Christopher; Nguitragool, Wang (2009) A provisional transport mechanism for a chloride channel-type Cl-/H+ exchanger. Philos Trans R Soc Lond B Biol Sci 364:175-80
Fang, Yiling; Jayaram, Hariharan; Shane, Tania et al. (2009) Structure of a prokaryotic virtual proton pump at 3.2 A resolution. Nature 460:1040-3
Nguitragool, Wang; Miller, Christopher (2007) Inaugural Article: CLC Cl /H+ transporters constrained by covalent cross-linking. Proc Natl Acad Sci U S A 104:20659-65
Nguitragool, Wang; Miller, Christopher (2006) Uncoupling of a CLC Cl-/H+ exchange transporter by polyatomic anions. J Mol Biol 362:682-90
Accardi, Alessio; Lobet, Severine; Williams, Carole et al. (2006) Synergism between halide binding and proton transport in a CLC-type exchanger. J Mol Biol 362:691-9
Accardi, Alessio; Walden, Michael; Nguitragool, Wang et al. (2005) Separate ion pathways in a Cl-/H+ exchanger. J Gen Physiol 126:563-70
Nimigean, Crina M; Shane, Tania; Miller, Christopher (2004) A cyclic nucleotide modulated prokaryotic K+ channel. J Gen Physiol 124:203-10
Kienker, Paul K; Jakes, Karen S; Blaustein, Robert O et al. (2003) Sizing the protein translocation pathway of colicin Ia channels. J Gen Physiol 122:161-76

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