Chair, Provost's Advisory Committee on Postdoctoral Affairs, Stanford University (2012 - Present)
Honors & Awards
Faculty Scholar, Esther Ehrman Lazard (2003-2005)
Scientist Development Award, American Heart Association (2004-2007)
Cranefield Award, Society of General Physiologists (2008)
Spark Scholar, Stanford University (2010)
Boards, Advisory Committees, Professional Organizations
Associate Editor, Journal of General Physiology (2014 - Present)
Ph.D., UCSD, Chemistry & Biochemistry (1995)
B.S., Wheaton College, Chemistry (1989)
Current Research and Scholarly Interests
Ion transport across the hydrophobic barrier of the cell membrane is a primary challenge faced by all cells. Such transport sets up and exploits ion gradients, thus providing the basic energy and signaling events that are the foundation of life. My laboratory studies the molecular mechanisms of ion channels and transporters, the proteins that catalyze this transport. Our major research focus is on the chloride-selective CLC family, which contains both types ion-transport protein. CLC proteins are expressed ubiquitously and perform diverse physiological functions in cardiovascular, neuronal, muscular, and epithelial function. We use a combination of biophysical methods to investigate membrane-protein structure and dynamics together with electrophysiological analyses to directly measure function.
A major direction includes developing NMR approaches for studying CLC structure and dynamics and combining these studies with molecular dynamics simulations (in collaboration with Emad Tajkhorshid, University of Illinois). As part of this endeavor, we are part of the Membrane Protein Structural Dynamics Consortium, a multidisciplinary team focused on elucidating the relationship between structure, dynamics and function in a variety of membrane proteins - http://memprotein.org/.
We are also developing novel small-molecule inhibitors as tools for studying the CLC proteins, in collaboration with Prof Justin Du Bois (Chemistry). These molecules will be used as biophysical probes to advance our understanding of CLC mechanisms, as cellular probes to study CLC-mediated physiological processes, and as lead compounds for treating several types of CLC-related diseases. The latter effort is in collaboration with Prof Alan Pao (Medicine) and funded by the Stanford SPARK program.
In a new multidisciplinary team effort, we are working in the emerging field of non-invasive ultrasonic neural modulation, with the goal of developing ultrasound technology for non-invasive brain stimulation in experimental and clinical applications. Research is in collaboration with Profs Baccus (Neurobiology), Butts-Pauly (Radiology) and Khuri-Yakub (Electrical Engineering). Our lab is addressing the problem and the molecular level, studying the effects of ultrasound on ion channels in reduced systems using electrophysiological recording techniques.
- How Cells Work: Energetics, Compartments, and Coupling in Cell Biology
MCP 156, MCP 256 (Spr)
- Neuroscience Journal Club and Professional Development Series
MCP 300, NEPR 280 (Aut, Win)
Independent Studies (9)
- Directed Reading in Biophysics
BIOPHYS 399 (Aut, Win, Spr, Sum)
- Directed Reading in Molecular and Cellular Physiology
MCP 299 (Aut, Win, Spr, Sum)
- Directed Reading in Neurosciences
NEPR 299 (Aut, Win, Spr, Sum)
- Graduate Research
BIOPHYS 300 (Aut, Win, Spr, Sum)
- Graduate Research
MCP 399 (Aut, Win, Spr, Sum)
- Graduate Research
NEPR 399 (Aut, Win, Spr, Sum)
- Medical Scholars Research
MCP 370 (Aut, Win, Spr, Sum)
- Out-of-Department Graduate Research
BIO 300X (Aut)
- Undergraduate Research
MCP 199 (Aut, Spr, Sum)
- Directed Reading in Biophysics
Prior Year Courses
- Neuroscience Journal Club and Professional Development Series
MCP 300 (Aut, Win, Spr)
- How Cells Work: Energetics, Compartments, and Coupling in Cell Biology
MCP 156, MCP 256 (Win)
- Imaging: Biological Light Microscopy
BIO 152, MCP 222 (Win)
- Professional Development and Integrity in Neuroscience
NBIO 300 (Aut, Win, Spr)
- Neuroscience Journal Club and Professional Development Series
C-13 NMR detects conformational change in the 100-kD membrane transporter ClC-ec1
JOURNAL OF BIOMOLECULAR NMR
2015; 61 (3-4): 209-226
CLC transporters catalyze the exchange of Cl(-) for H(+) across cellular membranes. To do so, they must couple Cl(-) and H(+) binding and unbinding to protein conformational change. However, the sole conformational changes distinguished crystallographically are small movements of a glutamate side chain that locally gates the ion-transport pathways. Therefore, our understanding of whether and how global protein dynamics contribute to the exchange mechanism has been severely limited. To overcome the limitations of crystallography, we used solution-state (13)C-methyl NMR with labels on methionine, lysine, and engineered cysteine residues to investigate substrate (H(+)) dependent conformational change outside the restraints of crystallization. We show that methyl labels in several regions report H(+)-dependent spectral changes. We identify one of these regions as Helix R, a helix that extends from the center of the protein, where it forms the part of the inner gate to the Cl(-)-permeation pathway, to the extracellular solution. The H(+)-dependent spectral change does not occur when a label is positioned just beyond Helix R, on the unstructured C-terminus of the protein. Together, the results suggest that H(+) binding is mechanistically coupled to closing of the intracellular access-pathway for Cl(-).
View details for DOI 10.1007/s10858-015-9898-7
View details for Web of Science ID 000352711900004
View details for PubMedID 25631353
Water access points and hydration pathways in CLC H+/Cl- transporters
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2014; 111 (5): 1819-1824
CLC transporters catalyze transmembrane exchange of chloride for protons. Although a putative pathway for Cl(-) has been established, the pathway of H(+) translocation remains obscure. Through a highly concerted computational and experimental approach, we characterize microscopic details essential to understanding H(+)-translocation. An extended (0.4 µs) equilibrium molecular dynamics simulation of membrane-embedded, dimeric ClC-ec1, a CLC from Escherichia coli, reveals transient but frequent hydration of the central hydrophobic region by water molecules from the intracellular bulk phase via the interface between the two subunits. We characterize a portal region lined by E202, E203, and A404 as the main gateway for hydration. Supporting this mechanism, site-specific mutagenesis experiments show that ClC-ec1 ion transport rates decrease as the size of the portal residue at position 404 is increased. Beyond the portal, water wires form spontaneously and repeatedly to span the 15-Å hydrophobic region between the two known H(+) transport sites [E148 (Gluex) and E203 (Gluin)]. Our finding that the formation of these water wires requires the presence of Cl(-) explains the previously mystifying fact that Cl(-) occupancy correlates with the ability to transport protons. To further validate the idea that these water wires are central to the H(+) transport mechanism, we identified I109 as the residue that exhibits the greatest conformational coupling to water wire formation and experimentally tested the effects of mutating this residue. The results, by providing a detailed microscopic view of the dynamics of water wire formation and confirming the involvement of specific protein residues, offer a mechanism for the coupled transport of H(+) and Cl(-) ions in CLC transporters.
View details for DOI 10.1073/pnas.1317890111
View details for Web of Science ID 000330587600048
View details for PubMedID 24379362
- Dynamic Response of Model Lipid Membranes to Ultrasonic Radiation Force PLOS ONE 2013; 8 (10)
Novel diuretic targets
AMERICAN JOURNAL OF PHYSIOLOGY-RENAL PHYSIOLOGY
2013; 305 (7): F931-F942
As the molecular revolution continues to inform a deeper understanding of disease mechanisms and pathways, there exist unprecedented opportunities for translating discoveries at the bench into novel therapies for improving human health. Despite the availability of several different classes of antihypertensive medications, only about half of the 67 million Americans with hypertension manage their blood pressure appropriately. A broader selection of structurally diverse antihypertensive drugs acting through different mechanisms would provide clinicians with greater flexibility in developing effective treatment regimens for an increasingly diverse and aging patient population. An emerging body of physiological, genetic, and pharmacological evidence has implicated several renal ion-transport proteins, or regulators thereof, as novel, yet clinically unexploited, diuretic targets. These include the renal outer medullary potassium channel, ROMK (Kir1.1), Kir4.1/5.1 potassium channels, ClC-Ka/b chloride channels, UTA/B urea transporters, the chloride/bicarbonate exchanger pendrin, and the STE20/SPS1-related proline/alanine-rich kinase (SPAK). The molecular pharmacology of these putative targets is poorly developed or lacking altogether; however, recent efforts by a few academic and pharmaceutical laboratories have begun to lessen this critical barrier. Here, we review the evidence in support of the aforementioned proteins as novel diuretic targets and highlight examples where progress toward developing small-molecule pharmacology has been made.
View details for DOI 10.1152/ajprenal.00230.2013
View details for Web of Science ID 000325353900001
View details for PubMedID 23863472
Dynamic response of model lipid membranes to ultrasonic radiation force.
2013; 8 (10)
Low-intensity ultrasound can modulate action potential firing in neurons in vitro and in vivo. It has been suggested that this effect is mediated by mechanical interactions of ultrasound with neural cell membranes. We investigated whether these proposed interactions could be reproduced for further study in a synthetic lipid bilayer system. We measured the response of protein-free model membranes to low-intensity ultrasound using electrophysiology and laser Doppler vibrometry. We find that ultrasonic radiation force causes oscillation and displacement of lipid membranes, resulting in small (<1%) changes in membrane area and capacitance. Under voltage-clamp, the changes in capacitance manifest as capacitive currents with an exponentially decaying sinusoidal time course. The membrane oscillation can be modeled as a fluid dynamic response to a step change in pressure caused by ultrasonic radiation force, which disrupts the balance of forces between bilayer tension and hydrostatic pressure. We also investigated the origin of the radiation force acting on the bilayer. Part of the radiation force results from the reflection of the ultrasound from the solution/air interface above the bilayer (an effect that is specific to our experimental configuration) but part appears to reflect a direct interaction of ultrasound with the bilayer, related to either acoustic streaming or scattering of sound by the bilayer. Based on these results, we conclude that synthetic lipid bilayers can be used to study the effects of ultrasound on cell membranes and membrane proteins.
View details for DOI 10.1371/journal.pone.0077115
View details for PubMedID 24194863
A Designed Inhibitor of a CLC Antiporter Blocks Function through a Unique Binding Mode
CHEMISTRY & BIOLOGY
2012; 19 (11): 1460-1470
The lack of small-molecule inhibitors for anion-selective transporters and channels has impeded our understanding of the complex mechanisms that underlie ion passage. The ubiquitous CLC "Chloride Channel" family represents a unique target for biophysical and biochemical studies because its distinctive protein fold supports both passive chloride channels and secondary-active chloride-proton transporters. Here, we describe the synthesis and characterization of a specific small-molecule inhibitor directed against a CLC antiporter (ClC-ec1). This compound, 4,4'-octanamidostilbene-2,2'-disulfonate (OADS), inhibits ClC-ec1 with low micromolar affinity and has no specific effect on a CLC channel (ClC-1). Inhibition of ClC-ec1 occurs by binding to two distinct intracellular sites. The location of these sites and the lipid dependence of inhibition suggest potential mechanisms of action. This compound will empower research to elucidate differences between antiporter and channel mechanisms and to develop treatments for CLC-mediated disorders.
View details for DOI 10.1016/j.chembiol.2012.09.017
View details for Web of Science ID 000312047800013
View details for PubMedID 23177200
Biochemistry to the Rescue: A CIC-2 Auxiliary Subunit Provides a Tangible Link to Leukodystrophy
2012; 73 (5): 855-857
ClC-2 is a broadly distributed chloride channel with an enigmatic neurophysiological function. In this issue of Neuron, Jeworutzki et al. (2012) use a biochemical approach to identify GlialCAM, a protein with a defined link to leukodystrophy, as a ClC-2 auxiliary subunit.
View details for DOI 10.1016/j.neuron.2012.02.012
View details for Web of Science ID 000301558600001
View details for PubMedID 22405196
Substrate-driven conformational changes in ClC-ec1 observed by fluorine NMR
2009; 28 (20): 3090-3102
The CLC 'Cl(-) channel' family consists of both Cl(-)/H(+) antiporters and Cl(-) channels. Although CLC channels can undergo large, conformational changes involving cooperativity between the two protein subunits, it has been hypothesized that conformational changes in the antiporters may be limited to small movements localized near the Cl(-) permeation pathway. However, to date few studies have directly addressed this issue, and therefore little is known about the molecular movements that underlie CLC-mediated antiport. The crystal structure of the Escherichia coli antiporter ClC-ec1 provides an invaluable molecular framework, but this static picture alone cannot depict the protein movements that must occur during ion transport. In this study we use fluorine nuclear magnetic resonance (NMR) to monitor substrate-induced conformational changes in ClC-ec1. Using mutational analysis, we show that substrate-dependent (19)F spectral changes reflect functionally relevant protein movement occurring at the ClC-ec1 dimer interface. Our results show that conformational change in CLC antiporters is not restricted to the Cl(-) permeation pathway and show the usefulness of (19)F NMR for studying conformational changes in membrane proteins of known structure.
View details for DOI 10.1038/emboj.2009.259
View details for Web of Science ID 000271008200004
View details for PubMedID 19745816
Proton-coupled gating in chloride channels
PHILOSOPHICAL TRANSACTIONS OF THE ROYAL SOCIETY B-BIOLOGICAL SCIENCES
2009; 364 (1514): 181-187
The physiologically indispensable chloride channel (CLC) family is split into two classes of membrane proteins: chloride channels and chloride/proton antiporters. In this article we focus on the relationship between these two groups and specifically review the role of protons in chloride-channel gating. Moreover, we discuss the evidence for proton transport through the chloride channels and explore the possible pathways that the protons could take through the chloride channels. We present results of a mutagenesis study, suggesting the feasibility of one of the pathways, which is closely related to the proton pathway proposed previously for the chloride/proton antiporters. We conclude that the two groups of CLC proteins, although in principle very different, employ similar mechanisms and pathways for ion transport.
View details for DOI 10.1098/rstb.2008.0123
View details for Web of Science ID 000261697300006
View details for PubMedID 18957380
Thinking outside the crystal Complementary approaches for examining transporter conformational change
2008; 2 (5): 373-379
As the number of high-resolution structures of membrane proteins continues to rise, so has the necessity for techniques to link this structural information to protein function. In the case of transporters, function is achieved via coupling of conformational changes to substrate binding and release. Static structural data alone cannot convey information on these protein movements, but it can provide a high-resolution foundation on which to interpret lower resolution data obtained by complementary approaches. Here, we review selected biochemical and spectroscopic methods for assessing transporter conformational change. In addition to more traditional techniques, we present ¹?F-NMR as an attractive method for characterizing conformational change in transporters of known structure. Using biosynthetic labeling, multiple, non-perturbing fluorine-labeled amino acids can be incorporated throughout a protein to serve as reporters of conformational change. Such flexibility in labeling allows characterization of movement in protein regions that may not be accessible via other methods.
View details for Web of Science ID 000262078700012
View details for PubMedID 18989097
The ClC-0 chloride channel is a 'broken' Cl-/H+ antiporter
NATURE STRUCTURAL & MOLECULAR BIOLOGY
2008; 15 (8): 805-810
Ion channels have historically been viewed as distinct from secondary active transporters. However, the recent discovery that the CLC 'chloride channel' family is made up of both channels and active transporters has led to the hypothesis that the ion-transport mechanisms of these two types of membrane proteins may be similar. Here we use single-channel analysis to demonstrate that ClC-0 channel gating (opening and closing) involves the transmembrane movement of protons. This result indicates that ClC-0 is a 'broken' Cl(-)/H(+) antiporter in which one of the conformational states has become leaky for chloride ions. This finding clarifies the evolutionary relationship between the channels and transporters and conveys that similar mechanisms and analogous protein movements are used by both.
View details for DOI 10.1038/nsmb.1466
View details for Web of Science ID 000258191100014
View details for PubMedID 18641661
A Cytoplasmic Domain Mutation in ClC-Kb Affects Long-Distance Communication Across the Membrane
2008; 3 (7)
ClC-Kb and ClC-Ka are homologous chloride channels that facilitate chloride homeostasis in the kidney and inner ear. Disruption of ClC-Kb leads to Bartter's Syndrome, a kidney disease. A point mutation in ClC-Kb, R538P, linked to Bartter's Syndrome and located in the C-terminal cytoplasmic domain was hypothesized to alter electrophysiological properties due to its proximity to an important membrane-embedded helix.Two-electrode voltage clamp experiments were used to examine the electrophysiological properties of the mutation R538P in both ClC-Kb and ClC-Ka. R538P selectively abolishes extracellular calcium activation of ClC-Kb but not ClC-Ka. In attempting to determine the reason for this specificity, we hypothesized that the ClC-Kb C-terminal domain had either a different oligomeric status or dimerization interface than that of ClC-Ka, for which a crystal structure has been published. We purified a recombinant protein corresponding to the ClC-Kb C-terminal domain and used multi-angle light scattering together with a cysteine-crosslinking approach to show that the dimerization interface is conserved between the ClC-Kb and ClC-Ka C-terminal domains, despite the fact that there are several differences in the amino acids that occur at this interface.The R538P mutation in ClC-Kb, which leads to Bartter's Syndrome, abolishes calcium activation of the channel. This suggests that a significant conformational change--ranging from the cytoplasmic side of the protein to the extracellular side of the protein--is involved in the Ca(2+)-activation process for ClC-Kb, and shows that the cytoplasmic domain is important for the channel's electrophysiological properties. In the highly similar ClC-Ka (90% identical), the R538P mutation does not affect activation by extracellular Ca(2+). This selective outcome indicates that ClC-Ka and ClC-Kb differ in how conformational changes are translated to the extracellular domain, despite the fact that the cytoplasmic domains share the same quaternary structure.
View details for DOI 10.1371/journal.pone.0002746
View details for Web of Science ID 000264302900017
View details for PubMedID 18648499
Discovery of potent CLC chloride channel inhibitors
ACS CHEMICAL BIOLOGY
2008; 3 (7): 419-428
Anion-transport proteins are central to all of physiology, for processes ranging from regulating bone-density, muscle excitability, and blood pressure, to facilitating extreme-acid survival of pathogenic bacteria. 4,4-Diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) has been used as an anion-transport inhibitor for decades. In this study, we demonstrate that polythiourea products derived from DIDS hydrolysis inhibit three different CLC chloride-transport proteins, ClC-ec1, ClC-0, and ClC-Ka, more effectively than DIDS itself. The structures of the five major products were determined by NMR spectroscopy, mass spectrometry, and chemical synthesis. These compounds bind directly to the CLC proteins, as evidenced by the fact that inhibition of ClC-0 occurs only from the intracellular side and inhibition of ClC-Ka is prevented by the point mutation N68D. These polythioureas are the highest affinity inhibitors known for the CLCs and provide a new class of chemical probes for dissecting the molecular mechanisms of chloride transport.
View details for DOI 10.1021/cb800083a
View details for Web of Science ID 000257793500004
View details for PubMedID 18642799
The role of a conserved lysine in chloride- and voltage-dependent ClC-0 fast gating
JOURNAL OF GENERAL PHYSIOLOGY
2007; 130 (4): 351-363
ClC-0 is a chloride channel whose gating is sensitive to voltage, chloride, and pH. In a previous publication, we showed that the K149C mutation causes a +70-mV shift in the voltage dependence of ClC-0 fast gating. In this paper we analyze the effects of a series of mutations at K149 on the voltage and chloride dependence of gating. By fitting our data to the previously proposed four-state model for ClC-0 fast gating, we show which steps in fast-gate opening are likely to be affected by these mutations. Computational analysis of mutant ClC-0 homology models show electrostatic contributions to chloride binding that may partially account for the effects of K149 on gating. The analysis of gating kinetics in combination with the available structural information suggests some of the structural changes likely to underpin fast-gate opening.
View details for DOI 10.1085/jgp.200709760
View details for Web of Science ID 000249950900002
View details for PubMedID 17846165
The mechanism of fast-gate opening in ClC-0
JOURNAL OF GENERAL PHYSIOLOGY
2007; 130 (4): 335-349
ClC-0 is a chloride channel whose gating is sensitive to both voltage and chloride. Based on analysis of gating kinetics using single-channel recordings, a five-state model was proposed to describe the dependence of ClC-0 fast-gate opening on voltage and external chloride (Chen, T.-Y., and C. Miller. 1996. J. Gen. Physiol. 108:237-250). We aimed to use this five-state model as a starting point for understanding the structural changes that occur during gating. Using macroscopic patch recordings, we were able to reproduce the effects of voltage and chloride that were reported by Chen and Miller and to fit our opening rate constant data to the five-state model. Upon further analysis of both our data and those of Chen and Miller, we learned that in contrast to their conclusions, (a) the features in the data are not adequate to rule out a simpler four-state model, and (b) the chloride-binding step is voltage dependent. In order to be able to evaluate the effects of mutants on gating (described in the companion paper, see Engh et al. on p. 351 of this issue), we developed a method for determining the error on gating model parameters, and evaluated the sources of this error. To begin to mesh the kinetic model(s) with the known CLC structures, a model of ClC-0 was generated computationally based on the X-ray crystal structure of the prokaryotic homolog ClC-ec1. Analysis of pore electrostatics in this homology model suggests that at least two of the conclusions derived from the gating kinetics analysis are consistent with the known CLC structures: (1) chloride binding is necessary for channel opening, and (2) chloride binding to any of the three known chloride-binding sites must be voltage dependent.
View details for DOI 10.1085/jgp.200709759
View details for Web of Science ID 000249950900001
View details for PubMedID 17846164
The CLC 'chloride channel' family: revelations from prokaryotes
MOLECULAR MEMBRANE BIOLOGY
2007; 24 (5-6): 342-350
Members of the CLC 'chloride channel' family play vital roles in a wide variety of physiological settings. Research on prokaryotic CLC homologues provided long-anticipated high-resolution structures as well as the unexpected discovery that some CLCs are not chloride channels, but rather are proton-chloride antiporters. Hence, CLCs encompass two functional classes of transport proteins once thought to be fundamentally different from one another. In this review, we discuss the structural features and molecular mechanisms of CLC channels and antiporters. We focus on ClC-0, the most thoroughly studied CLC channel, and ClC-ec1, the prokaryotic antiporter of known structure. We highlight some striking similarities between these CLCs and discuss compelling questions that remain to be addressed. Prokaryotic CLCs will undoubtedly continue to shed light upon this understudied family of proteins.
View details for DOI 10.1080/09687680701413874
View details for Web of Science ID 000248853200004
View details for PubMedID 17710638
Side-dependent inhibition of a prokaryotic CIC by DIDS
2005; 89 (3): 1721-1730
The x-ray structure of the Escherichia coli chloride/proton antiporter ClC-ec1 provides a structural paradigm for the widespread and diverse ClC family of chloride channels and transporters. To maximize the usefulness of this paradigm, it is important to directly relate structure to function via studies of ClC-ec1 itself; however, few functional studies of this protein have been performed. In an endeavor to develop new tools for functional analysis of ClC-ec1, we have discovered that this transporter is inhibited by the stilbenedisulfonate 4,4-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). In planar lipid bilayers, DIDS inhibits ClC-ec1 activity reversibly, with an apparent affinity in the micromolar range. Since ClC-ec1 is randomly oriented in the bilayers, ascertaining whether DIDS inhibits from the intracellular or extracellular side required an indirect approach. Using the ClC-ec1 structure as a guide, we designed a strategy in which modification of Y445C was monitored in conjunction with inhibition by DIDS. We found that DIDS inhibits transporters specifically from the intracellular side. Transporters with their extracellular side exposed to DIDS function normally, maintaining stoichiometric proton/chloride antiport over a wide range of proton and chloride concentrations. The side-dependent nature of DIDS inhibition will be useful for generating "functionally oriented" preparations of ClC-ec1, in which DIDS is used to silence transporters in one orientation but not the other.
View details for DOI 10.1529/biophysj.105.066522
View details for Web of Science ID 000231502800029
View details for PubMedID 15994902
Cysteine accessibility in ClC-0 supports conservation of the ClC intracellular vestibule
JOURNAL OF GENERAL PHYSIOLOGY
2005; 125 (6): 601-617
ClC chloride channels, which are ubiquitously expressed in mammals, have a unique double-barreled structure, in which each monomer forms its own pore. Identification of pore-lining elements is important for understanding the conduction properties and unusual gating mechanisms of these channels. Structures of prokaryotic ClC transporters do not show an open pore, and so may not accurately represent the open state of the eukaryotic ClC channels. In this study we used cysteine-scanning mutagenesis and modification (SCAM) to screen >50 residues in the intracellular vestibule of ClC-0. We identified 14 positions sensitive to the negatively charged thiol-modifying reagents sodium (2-sulfonatoethyl)methanethiosulfonate (MTSES) or sodium 4-acetamido-4'-maleimidylstilbene-2'2-disulfonic acid (AMS) and show that 11 of these alter pore properties when modified. In addition, two MTSES-sensitive residues, on different helices and in close proximity in the prokaryotic structures, can form a disulfide bond in ClC-0. When mapped onto prokaryotic structures, MTSES/AMS-sensitive residues cluster around bound chloride ions, and the correlation is even stronger in the ClC-0 homology model developed by Corry et al. (2004). These results support the hypothesis that both secondary and tertiary structures in the intracellular vestibule are conserved among ClC family members, even in regions of very low sequence similarity.
View details for DOI 10.1085/jgp.200509258
View details for Web of Science ID 000230003100008
View details for PubMedID 15897295
The poststructural festivities begin
2003; 38 (1): 1-3
ClC chloride channels orchestrate the movement of chloride necessary for proper neuronal, muscular, cardiovascular, and epithelial function. In this issue of Neuron, Jentsch, Pusch, and colleagues use the structure of a bacterial ClC homolog to guide a mutagenic analysis of inhibitor binding to ClC-0, ClC-1, and ClC-2.
View details for Web of Science ID 000182202300001
View details for PubMedID 12691656
Projection structure of a CIC-type chloride channel at 6.5 angstrom resolution
2001; 409 (6817): 219-223
Virtually all cells in all eukaryotic organisms express ion channels of the ClC type, the only known molecular family of chloride-ion-selective channels. The diversity of ClC channels highlights the multitude and range of functions served by gated chloride-ion conduction in biological membranes, such as controlling electrical excitability in skeletal muscle, maintaining systemic blood pressure, acidifying endosomal compartments, and regulating electrical responses of GABA (gamma-aminobutyric acid)-containing interneurons in the central nervous system. Previously, we expressed and purified a prokaryotic ClC channel homologue. Here we report the formation of two-dimensional crystals of this ClC channel protein reconstituted into phospholipid bilayer membranes. Cryo-electron microscopic analysis of these crystals yields a projection structure at 6.5 A resolution, which shows off-axis water-filled pores within the dimeric channel complex.
View details for Web of Science ID 000166316200051
View details for PubMedID 11196649
CIC chloride channels
2001; 2 (2)
Chloride-conducting ion channels of the ClC family are emerging as critical contributors to a host of biological processes. These polytopic membrane proteins form aqueous pathways through which anions are selectively allowed to pass down their concentration gradients. The ClCs are found in nearly all organisms, with members in every mammalian tissue, yet relatively little is known about their mechanism or regulation. It is clear, however, that they are fundamentally different in molecular construction and mechanism from the well-known potassium-, sodium-, and calcium-selective channels. The medical importance of ClC channels - four inherited diseases have been blamed on familial ClC dysfunction to date - highlights their diverse physiological functions and provides strong motivation for further study.
View details for Web of Science ID 000207583600004
View details for PubMedID 11182894
A decade of CLC chloride channels: Structure, mechanism, and many unsettled questions
ANNUAL REVIEW OF BIOPHYSICS AND BIOMOLECULAR STRUCTURE
2000; 29: 411-438
ClC-type chloride channels are ubiquitous throughout the biological world. Expressed in nearly every cell type, these proteins have a host of biological functions. With nine distinct homologues known in eukaryotes, the ClCs represent the only molecularly defined family of chloride channels. ClC channels exhibit features of molecular architecture and gating mechanisms unprecedented in other types of ion channels. They form two-pore homodimers, and their voltage-dependence arises not from charged residues in the protein, but rather via coupling of gating to the movement of chloride ions within the pore. Because the functional characteristics of only a few ClC channels have been studied in detail, we are still learning which properties are general to the whole family. New approaches, including structural analyses, will be crucial to an understanding of ClC architecture and function.
View details for Web of Science ID 000088492300014
View details for PubMedID 10940254
High-level expression, functional reconstitution, and quaternary structure of a prokaryotic ClC-type chloride channel
JOURNAL OF GENERAL PHYSIOLOGY
1999; 114 (5): 713-722
ClC-type anion-selective channels are widespread throughout eukaryotic organisms. BLAST homology searches reveal that many microbial genomes also contain members of the ClC family. An Escherichia coli-derived ClC Cl(-) channel homologue, "EriC," the product of the yadQ gene, was overexpressed in E. coli and purified in milligram quantities in a single-step procedure. Reconstitution of purified EriC into liposomes confers on these membranes permeability to anions with selectivity similar to that observed electrophysiologically in mammalian ClC channels. Cross-linking studies argue that EriC is a homodimer in both detergent micelles and reconstituted liposomes, a conclusion corroborated by gel filtration and analytical sedimentation experiments.
View details for Web of Science ID 000083531500007
View details for PubMedID 10539975
Formation of CLC-0 chloride channels from separated transmembrane and cytoplasmic domains
1998; 37 (5): 1315-1321
CLC-0, a member of the CLC family of Cl(-)-conducting ion channels, consists of an N-terminal hydrophobic core and a C-terminal region that is thought to be cytoplasmic. This study provides evidence that the C-terminal region is a mechanistically relevant cytoplasmic domain of the CLC-0 ion channel. Both a point mutation and a 37-residue deletion in this region cause drastic alterations in voltage-dependent gating of CLC-0 current expressed in Xenopus oocytes. CLC-0 current is not observed when the entire C-terminal region is deleted, but functional channels are efficiently reconstituted by co-injection of separate cRNA constructs encoding the N-terminal transmembrane and the C-terminal cytoplasmic domains. Moreover, reconstitution of CLC-0 can be achieved by co-injection of cRNA encoding the transmembrane domain along with Escherichia coli-expressed C-terminal domain polypeptide.
View details for Web of Science ID 000072048200020
View details for PubMedID 9477958
IMPORT OF A MITOCHONDRIAL PRESEQUENCE INTO P-DENITRIFICANS - INSIGHT INTO THE EVOLUTION OF PROTEIN-TRANSPORT
1994; 337 (1): 9-13
According to the endosymbiont hypothesis, mitochondria are descended from ancient aerobic bacteria that were engulfed by protoeukaryotic cells. Experiments described here show that a synthetic peptide corresponding to a yeast mitochondrial targeting sequence can be imported into Paracoccus denitrificans, a soil bacterium thought to be closely related to the protomitochondrion. The import is very similar to that observed with isolated yeast mitochondria. The results suggest that the protomitochondrion may have been inherently able to translocate mitochondrial presequences. This ability may partly explain the development of the protein import process during the evolution of the mitochondrion.
View details for Web of Science ID A1994NA14700002
View details for PubMedID 8276120
IMPORT OF A MITOCHONDRIAL PRESEQUENCE INTO PROTEIN-FREE PHOSPHOLIPID-VESICLES
1993; 260 (5106): 364-367
A synthetic mitochondrial presequence has been shown to translocate across pure phospholipid bilayers. The presequence was fluorescently labeled so that its association with membranes could be monitored spectroscopically. In the presence of large unilamellar vesicles, the presequence showed time- and potential-dependent protection from reaction with added trypsin and dithionite. The protection was rapidly reversed by treatment of the vesicles with detergent. If the vesicles contained trypsin, the added presequence became sensitive to digestion by the protease. The results show that a mitochondrial presequence can translocate across phospholipid bilayers that lack a hydrophilic translocation pore.
View details for Web of Science ID A1993KX80000042
View details for PubMedID 8385804