Structure of the CLC-1 chloride channel from Homo sapiens.

CLC channels mediate passive Cl- conduction, while CLC transporters mediate active Cl- transport coupled to H+ transport in the opposite direction. The distinction between CLC-0/1/2 channels and CLC transporters seems undetectable by amino acid sequence. To understand why they are different functionally we determined the structure of the human CLC-1 channel. Its 'glutamate gate' residue, known to mediate proton transfer in CLC transporters, adopts a location in the structure that appears to preclude it from its transport function. Furthermore, smaller side chains produce a wider pore near the intracellular surface, potentially reducing a kinetic barrier for Cl- conduction. When the corresponding residues are mutated in a transporter, it is converted to a channel. Finally, Cl- at key sites in the pore appear to interact with reduced affinity compared to transporters. Thus, subtle differences in glutamate gate conformation, internal pore diameter and Cl- affinity distinguish CLC channels and transporters

Mouse Bestrophin-2 Is a Bona fide Cl− Channel: Identification of a Residue Important in Anion Binding and Conduction

Bestrophins have recently been proposed to comprise a new family of Cl− channels. Our goal was to test whether mouse bestrophin-2 (mBest2) is a bona fide Cl− channel. We expressed mBest2 in three different mammalian cell lines. mBest2 was trafficked to the plasma membrane as shown by biotinylation and immunoprecipitation, and induced a Ca2+-activated Cl− current in all three cell lines (EC50 for Ca2+ = 230 nM). The permeability sequence was SCN−: I−: Br−: Cl−: F− (8.2: 1.9: 1.4: 1: 0.5). Although SCN− was highly permeant, its conductance was ∼10% that of Cl− and SCN− blocked Cl− conductance (IC50 = 12 mM). Therefore, SCN− entered the pore more easily than Cl−, but bound more tightly than Cl−. Mutations in S79 altered the relative permeability and conductance for SCN− as expected if S79 contributed to an anion binding site in the channel. PSCN/PCl = 8.2 ± 1.3 for wild-type and 3.9 ± 0.4 for S79C. GSCN/GCl = 0.14 ± 0.03 for wild-type and 0.94 ± 0.04 for S79C. In the S79 mutants, SCN− did not block Cl− conductance. This suggested that the S79C mutation altered the affinity of an anion binding site for SCN−. Additional evidence that S79 was located in the conduction pathway was provided by the finding that modification of the sulfhydryl group in S79C with MTSET+ or MTSES− increased conductance significantly. Because the effect of positively and negatively charged MTS reagents was similar, electrostatic interactions between the permeant anion and the channel at this residue were probably not critical in anion selectivity. These data provide strong evidence that mBest2 forms part of the novel Cl− conduction pathway in mBest2-transfected cells and that S79 plays an important role in anion binding in the pore of the channel

Molecular insights into the mechanisms of transport and energy coupling in membrane transport proteins

Membrane transport proteins are the main gatekeepers controlling the traffic of molecules in and out the cell. The mechanism by which they mediate selective and regulated transport across the membrane is of broad physiological and biophysical relevance. In this dissertation, several critical aspects of the transport process have been studied through molecular dynamics (MD) simulations, including ion binding and its coupling to chemical processes such as H+ transport, translocation of the transported substrate and cotransported ions, dynamics of the catalytic site, coordinated motions of the remote regions, as well as other molecular events facilitating the transport of the cargo. The first part of the dissertation covers topics on a Cl-/H+ transporter from the CLC superfamily, which catalyzes stoichiometrically coupled exchange of Cl- and H+ across biological membranes. CLC transporters exchange H+ for halides and certain polyatomic anions, but exclude cations, F-, and larger physiological anions, such as PO4^3- and SO4^2-. Despite comparable transport rates of different anions, the H+ coupling in CLC transporters varies significantly depending on the chemical nature of the transported anion. Although the molecular mechanism of exchange remains unknown, studies on bacterial ClC-ec1 transporter have revealed that Cl- binding to the central anion-binding site is crucial for the anion-coupled H+ transport. This study shows that Cl-, F-, NO3-, and SCN- display distinct binding coordinations at the central site and are hydrated in different manners. Consistent with the observation of differential bindings, ClC-ec1 exhibits markedly variable ability to support the formation of the transient water wires, which are necessary to support the connection of the two H+ transfer sites (Gluin and Gluex), in the presence of different anions. These findings provide structural details of anion binding in ClC-ec1 and reveal a putative atomic-level mechanism for the decoupling of H+ transport to the transport of anions other than Cl-. Another important question concerning the functional mechanism of CLC transporters is that no large conformational change have been detected crystallographically, even though transporters usually undergo global conformational change to alternately expose substrate-binding sites to opposite sides of the membrane. The collaborative work here demonstrates the formation of a previously uncharacterized `outward-facing open' state enrich by high H+ concentration, which involves global structural changes ~20 A away from the outer gate. This long distance conformational change highlights the coupled motions as well as the relevance of global structural changes in CLC transport cycle. The second part of the dissertation focus on a phospholipid scramblase which mediates rapid transbilayer redistribution (scrambling) of phospholipids at plasma membrane. This process dissipates lipid asymmetry in response to signals for critical cellular events like apoptosis that elevate cytoplasmic Ca^2+ concentration. The work here shows that the hydrophilic aqueduct on the surface of the fungal scramblase nhTMEM16 serves as the path for lipid translocation, and that Ca$^{2+}$ binding plays a key role in determining an open conformation of the path for lipid diffusion. The fully occupied lipid track connects the inner and outer leaflets and forms a “proteolipidic” pore, which allows ion conduction through the aqueous pathway formed between the protein and lipid headgroups under transmembrane electric potentials. Supporting this mechanism, site-specific mutagenesis experiments show that nhTMEM16 ionic currents are synergistically linked to phospholipid scrambling. To further validate the idea that ions permeate through TMEM16s via the same structural pathway taken by phospholipids, two specific residues in the pore region were pinpointed, which are able to convert TMEM16A Ca^2+-activated Cl- channel (CaCC) into robust scramblase upon point mutations. This novel view of flexible pore structure explains a number of unusual features of the TMEM16 ionic currents, especially the highly variable ionic selectivity and the ability to permeate large ions, which also provides crucial information on the functional dichotomy in TMEM16s.Ope

CLC channel function and dysfunction in health and disease

CLC channels and transporters are expressed in most tissues and fulfill diverse functions. There are four human CLC channels, ClC-1, ClC-2, ClC-Ka and ClC-Kb, and five CLC transporters, ClC-3 through -7. Some of the CLC channels additionally associate with accessory subunits. Whereas barttin is mandatory for the functional expression of CLC-K, GlialCam is a facultative subunit of ClC-2 which modifies gating and thus increases the functional variability within the CLC family. Isoform-specific ion conduction and gating properties optimize distinct CLC channels for their cellular tasks. ClC-1 preferentially conducts at negative voltages, and the resulting inward rectification provides a large resting chloride conductance without interference with the muscle action potential. Exclusive opening at voltages negative to the chloride reversal potential allows for ClC-2 to regulate intracellular chloride concentrations. ClC-Ka and ClC-Kb are equally suited for inward and outward currents to support transcellular chloride fluxes. Every human CLC channel gene has been linked to a genetic disease, and studying these mutations has provided much information about the physiological roles and the molecular basis of CLC channel function. Mutations in the gene encoding ClC-1 cause myotonia congenita, a disease characterized by sarcolemmal hyperexcitability and muscle stiffness. Loss-of-function of ClC-Kb/barttin channels in patients suffering from Bartter syndrome identified the determinants of chloride conductances in the limb of Henle. Mutations in CLCN2 were found in patients with CNS disorders but the functional role of this isoform is still not understood. Recent links between ClC-1 and epilepsy and ClC-Ka and heart failure suggested novel cellular functions of these proteins. This review aims to survey the knowledge about physiological and pathophysiological functions of human CLC channels in the light of recent discoveries from biophysical, physiological and genetic studies

Irreversible Zinc Block of the Swelling-activated Chloride Current in DI TNC1 Astrocytes

The swelling-activated chloride current, commonly referred to as ICl,swell, is an outwardly-rectifying anion current that plays an important role in cell volume regulation, among other capacities. Despite several decades of research, the molecular identity of the channel responsible for this chloride current remains controversial. Recent indications that key endogenous sulfhydryl groups are capable of modifying the current led us to assess the effects of several divalent cations, including zinc, on ICl,swell. Zinc is known to tightly associate with sulfhydryl groups such as in zinc finger proteins. We found that extracellular zinc irreversibly inhibited ICl,swell at a site downstream in the signaling cascade. Moreover, zinc blocking kinetics were voltage dependent, suggesting interaction with a site within the electric field, across the pore of the channel responsible for ICl,swell. The importance of sulfhydryl groups was confirmed by demonstrating irreversible block by N-ethylmaleimide, a sulfhydryl alkylating reagent. In contrast, nickel failed to block ICl,swell, and as noted in previous studies, cadmium preferentially blocked the time-dependent component of ICl,swell. These data confirm the importance of sulfhydryl groups in the function of ICl,swell. Moreover, by demonstrating the voltage-dependence of block, the data strongly suggest the critical sulfhydryl group is within the channel pore. These biophysical characteristics of native ICl,swell are markers that should be recapitulated in expressed proteins claimed to be responsible for ICl,swell

Three-Dimensional Model for the Human Cl−/HCO3− Exchanger, AE1, by Homology to the E. coli ClC Protein

AbstractAE1 mediates electroneutral 1:1 exchange of bicarbonate for chloride across the plasma membrane of erythrocytes and type A cells of the renal collecting duct. No high-resolution structure is available for the AE1 membrane domain, which alone is required for its transport activity. A recent electron microscopy structure of the AE1 membrane domain was proposed to have a similar protein fold to ClC chloride channels. We developed a three-dimensional homology model of the AE1 membrane domain, using the Escherichia coli ClC channel structure as a template. This model agrees well with a long list of biochemically established spatial constraints for AE1. To investigate the AE1 transport mechanism, we created point mutations in regions corresponding to E. coli ClC transport mechanism residues. When expressed in HEK293 cells, several mutants had Cl−/HCO3− exchange rates significantly different from that of wild-type AE1. When further assessed in Xenopus laevis oocytes, there were significant changes in the transport activity of several AE1 point mutants as assessed by changes in pH. None of the mutants, however, added an electrogenic component to AE1 transport activity. This indicates that the AE1 point mutants altered the transport activity of AE1, without changing its electrogenicity and stoichiometry. The homology model successfully identified residues in AE1 that are critical to AE1 transport activity. Thus, we conclude that AE1 has a similar protein fold to ClC chloride channels

STRUCTURAL AND FUNCTIONAL CHARACTERIZATION OF MULITDRUG RESISTANCE TRANSPORTER AND REGULATOR

Drug resistant bacteria pathogen poses a severe threat to human health. Bacterial drug efflux pumps are transporter proteins involved in the export of antibiotics out of cells. Efflux by transporters is one of the major drug resistant mechanisms. Multidrug efflux pumps can transport multiple classes of antibiotics and are associated with bacteria multiple drug resistance (MDR). Overproduction of these pumps reduces susceptibility of bacteria to a variety of antibiotics. MDR regulators are cytoplasmic proteins that control the expression level of MDR transporters in response to the cellular concentration of antibiotics. This thesis research focuses on three main directions in the area of bacteria drug resistance: the structural and functional study of a MDR transporter, the characterization of a novel MDR regulator protein, and the development of a sensing method for the detection of glycopeptide antibiotics. Acriflavine resistance protein B (AcrB) in Escherichia coli belongs to resistance nodulation division (RND) superfamily of efflux transporters. It plays an important role in confering multidrug resistance in Gram-negative bacteria. The functional unit of AcrB is a trimer in vivo. However, the relationship between AcrB trimer stability and functionality remains elusive. In chapter 2, a residue that is critical for AcrB trimerization, Pro 223, was identified. The replacement of Pro 223 by other residues destabilized AcrB trimer, and thus decreased its activity. The loss of transport activity could be partially recovered when the AcrB trimer was stabilized by the introduction of a pair of inter-subunit disulfide bond. In chapter 3, a systematically alanine-scanning study of the producing loop (amino acid residues 211-240) was conducted. Five residues in the loop were found to be important for AcrB activity. These residues form a collar or belt in the loop close to the tip. These mutation studies revealed new insight into the conformation of the loop during AcrB trimerization. In chapter 4, residue Arg 780 was identified to be crucial for the pump function of AcrB. The study results indicated that Pro 223 serves as a “wedge” and Arg 780 as a “lock” via hydrogen bonding between the backbone carbonyl oxygen of Pro 223 and side chain of Arg780. Similar as Pro 223, replacement of Arg 780 by other residues drastically decreased the activity of AcrB. Dissociation of the AcrB trimer also contributed to the decrease of activity. However, the introduction of inter-subunit disulfide bond could not restore the function of the mutant, indicating that Arg 780 plays multiples roles in the operation of AcrB. In chapter 5, a MDR regulator ST1710 from the archaeon Sulfolobus tokodaii, homologous to the multiple-antibiotic resistance repressor (MarR) family bacterial regulators, was characterized in vitro. The binding affinities of ligands and double strand (ds) DNA for ST1710 were measured. The presence of substrates suppressed the interaction between ST1710 and dsDNA, which indicated that ST1710 functioned as a repressor in vivo. Finally, in chapter 6, a direct fluorescence polarization based method for the detection of glycopeptide antibiotics is developed. Briefly, the acetylated tripeptide L-Lys-D-Ala-D-Ala was labeled with a fluorophore (fluorescein isothiocyanate or AlexaFluor 680) to create a peptide probe. The fluorescence polarization signal of the peptide probe increased upon binding with glycopeptide antibiotics in a concentration dependent manner. The detection is highly selective toward glycopeptide antibiotics. The designed method is expected it to have broad applications in both research and clinical settings