Administrative Appointments

  • Associate Director, Molecular and Cellular Physiology Graduate Program (2021 - Present)
  • Co-director, Neurosciences Interdisciplinary Graduate Program (2021 - Present)
  • President, Society of General Physiologists (2018 - 2019)
  • Executive Committee, Biophysics Interdisciplinary Program (2012 - 2021)
  • Chair, Provost's Advisory Committee on Postdoctoral Affairs, Stanford University (2012 - 2016)

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

  • Founding Curator, Biophysics Colab (2021 - Present)
  • Editor, Biophysical Journal (2020 - Present)
  • Reviewing Editor, eLife (2019 - Present)
  • Associate Editor, Journal of General Physiology (2014 - 2019)
  • Editorial Board, Journal of General Physiology (2010 - Present)

Professional Education

  • Ph.D., UCSD, Chemistry & Biochemistry (1995)
  • B.S., Wheaton College, Chemistry, summa cum laude (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. A 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. We additionally apply our expertise on ion-transport mechanisms to interdisciplinary collaborations to create novel chemical tools, to develop CLC-targeted therapies, and to understand the mechanism by which ultrasound modulates ion-transport to effect neuromodulation.


  • Mechanisms of CLC Channels and Transporters, Stanford University


    Stanford, CA

  • Small-molecule probes for study of CLC-2 chloride-channel function in the central nervous system, Stanford University


    Stanford, CA


    • Justin Du Bois, Stanford University
    • John Huguenard, Professor of Neurology, of Neurosurgery and, by courtesy, of Molecular and Cellular Physiology, Stanford University
  • BRAIN Initiative - Neurostimulation by Ultrasound: Physical, Biophysical, and Neural Mechanisms, Stanford University


    Stanford, CA


  • Structure-based strategy for developing inhibitors of the kidney chloride channel CLC-Ka


    279 Campus Drive West


    • Alan Pao, Associate Professor, Stanford University
    • Wah Chiu, Wallenberg-Bienenstock Professor and Professor of Bioengineering and of Microbiology and Immunology, Stanford University
    • Mark Smith, Stanford University
    • Ron Dror, Associate Professor of Computer Science and, by courtesy, of Molecular and Cellular Physiology and of Structural Biology, Stanford University
  • Inhibiting the CLC-Ka Cl- channel, a novel therapeutic target for hyponatremia, Stanford University


    279 Campus Drive West


2023-24 Courses

Stanford Advisees

All Publications

  • Towards an ion-channel-centric approach to ultrasound neuromodulation. Current opinion in behavioral sciences Prieto, M. L., Maduke, M. 2024; 56


    Ultrasound neuromodulation is a promising technology that could revolutionize study and treatment of brain conditions ranging from mood disorders to Alzheimer's disease and stroke. An understanding of how ultrasound directly modulates specific ion channels could provide a roadmap for targeting specific neurological circuits and achieving desired neurophysiological outcomes. Although experimental challenges make it difficult to unambiguously identify which ion channels are sensitive to ultrasound in vivo, recent progress indicates that there are likely several different ion channels involved, including members of the K2P, Piezo, and TRP channel families. A recent result linking TRPM2 channels in the hypothalamus to induction of torpor by ultrasound in rodents demonstrates the feasibility of targeting a specific ion channel in a specific population of neurons.

    View details for DOI 10.1016/j.cobeha.2024.101355

    View details for PubMedID 38505510

    View details for PubMedCentralID PMC10947167

  • CryoEM structures of the human CLC-2 voltage-gated chloride channel reveal a ball-and-chain gating mechanism. eLife Xu, M., Neelands, T., Powers, A. S., Liu, Y., Miller, S. D., Pintilie, G. D., Bois, J. D., Dror, R. O., Chiu, W., Maduke, M. 2024; 12


    CLC-2 is a voltage-gated chloride channel that contributes to electrical excitability and ion homeostasis in many different tissues. Among the nine mammalian CLC homologs, CLC-2 is uniquely activated by hyperpolarization, rather than depolarization, of the plasma membrane. The molecular basis for the divergence in polarity of voltage gating among closely related homologs has been a long-standing mystery, in part because few CLC channel structures are available. Here, we report cryoEM structures of human CLC-2 at 2.46 - 2.76 Å, in the presence and absence of the selective inhibitor AK-42. AK-42 binds within the extracellular entryway of the Cl--permeation pathway, occupying a pocket previously proposed through computational docking studies. In the apo structure, we observed two distinct conformations involving rotation of one of the cytoplasmic C-terminal domains (CTDs). In the absence of CTD rotation, an intracellular N-terminal 15-residue hairpin peptide nestles against the TM domain to physically occlude the Cl--permeation pathway. This peptide is highly conserved among species variants of CLC-2 but is not present in other CLC homologs. Previous studies suggested that the N-terminal domain of CLC-2 influences channel properties via a "ball-and-chain" gating mechanism, but conflicting data cast doubt on such a mechanism, and thus the structure of the N-terminal domain and its interaction with the channel has been uncertain. Through electrophysiological studies of an N-terminal deletion mutant lacking the 15-residue hairpin peptide, we support a model in which the N-terminal hairpin of CLC-2 stabilizes a closed state of the channel by blocking the cytoplasmic Cl--permeation pathway.

    View details for DOI 10.7554/eLife.90648

    View details for PubMedID 38345841

  • Novel CLC-Kb pore mutation associated with defective glycosylation and renal tubulopathy Sharma, Y., Dong, W., Liao, X., Venkataraman, A., Qiao, Y., Francisco, C. V., Maduke, M., Charu, V., Kambham, N., Pochynyuk, O., Govaerts, C., Bhalla, V. OXFORD UNIV PRESS INC. 2022: 1026-1027
  • Development and validation of a potent and specific inhibitor for the CLC-2 chloride channel. Proceedings of the National Academy of Sciences of the United States of America Koster, A. K., Reese, A. L., Kuryshev, Y., Wen, X., McKiernan, K. A., Gray, E. E., Wu, C., Huguenard, J. R., Maduke, M., Du Bois, J. 2020


    CLC-2 is a voltage-gated chloride channel that is widely expressed in mammalian tissues. In the central nervous system, CLC-2 appears in neurons and glia. Studies to define how this channel contributes to normal and pathophysiological function in the central nervous system raise questions that remain unresolved, in part due to the absence of precise pharmacological tools for modulating CLC-2 activity. Herein, we describe the development and optimization of AK-42, a specific small-molecule inhibitor of CLC-2 with nanomolar potency (IC50 = 17 ± 1 nM). AK-42 displays unprecedented selectivity (>1,000-fold) over CLC-1, the closest CLC-2 homolog, and exhibits no off-target engagement against a panel of 61 common channels, receptors, and transporters expressed in brain tissue. Computational docking, validated by mutagenesis and kinetic studies, indicates that AK-42 binds to an extracellular vestibule above the channel pore. In electrophysiological recordings of mouse CA1 hippocampal pyramidal neurons, AK-42 acutely and reversibly inhibits CLC-2 currents; no effect on current is observed on brain slices taken from CLC-2 knockout mice. These results establish AK-42 as a powerful tool for investigating CLC-2 neurophysiology.

    View details for DOI 10.1073/pnas.2009977117

    View details for PubMedID 33277431

  • LRRC8A:C/E Heteromeric Channels Are Ubiquitous Transporters of cGAMP. Molecular cell Lahey, L. J., Mardjuki, R. E., Wen, X., Hess, G. T., Ritchie, C., Carozza, J. A., Bohnert, V., Maduke, M., Bassik, M. C., Li, L. 2020


    Extracellular 2'3'-cyclic-GMP-AMP (cGAMP) is an immunotransmitter exported by diseased cells and imported into host cells to activate the innate immune STING pathway. We previously identified SLC19A1 as a cGAMP importer, but its use across human cell lines is limited. Here, we identify LRRC8A heteromeric channels, better known as volume-regulated anion channels (VRAC), as widely expressed cGAMP transporters. LRRC8A forms complexes with LRRC8C and/or LRRC8E, depending on their expression levels, to transport cGAMP and other 2'3'-cyclic dinucleotides. In contrast, LRRC8D inhibits cGAMP transport. We demonstrate that cGAMP is effluxed or influxed via LRRC8 channels, as dictated by the cGAMP electrochemical gradient. Activation of LRRC8A channels, which can occur under diverse stresses, strongly potentiates cGAMP transport. We identify activator sphingosine 1-phosphate and inhibitor DCPIB as chemical tools to manipulate channel-mediated cGAMP transport. Finally, LRRC8A channels are key cGAMP transporters in resting primary human vasculature cells and universal human cGAMP transporters when activated.

    View details for DOI 10.1016/j.molcel.2020.10.021

    View details for PubMedID 33171122

  • Spike frequency-dependent inhibition and excitation of neural activity by high-frequency ultrasound. The Journal of general physiology Prieto, M. L., Firouzi, K., Khuri-Yakub, B. T., Madison, D. V., Maduke, M. 2020; 152 (11)


    Ultrasound can modulate action potential firing in vivo and in vitro, but the mechanistic basis of this phenomenon is not well understood. To address this problem, we used patch-clamp recording to quantify the effects of focused, high-frequency (43 MHz) ultrasound on evoked action potential firing in CA1 pyramidal neurons in acute rodent hippocampal brain slices. We find that ultrasound can either inhibit or potentiate firing in a spike frequency-dependent manner: at low (near-threshold) input currents and low firing frequencies, ultrasound inhibits firing, while at higher input currents and higher firing frequencies, ultrasound potentiates firing. The net result of these two competing effects is that ultrasound increases the threshold current for action potential firing, the slope of frequency-input curves, and the maximum firing frequency. In addition, ultrasound slightly hyperpolarizes the resting membrane potential, decreases action potential width, and increases the depth of the after-hyperpolarization. All of these results can be explained by the hypothesis that ultrasound activates a sustained potassium conductance. According to this hypothesis, increased outward potassium currents hyperpolarize the resting membrane potential and inhibit firing at near-threshold input currents but potentiate firing in response to higher-input currents by limiting inactivation of voltage-dependent sodium channels during the action potential. This latter effect is a consequence of faster action potential repolarization, which limits inactivation of voltage-dependent sodium channels, and deeper (more negative) after-hyperpolarization, which increases the rate of recovery from inactivation. Based on these results, we propose that ultrasound activates thermosensitive and mechanosensitive two-pore-domain potassium (K2P) channels through heating or mechanical effects of acoustic radiation force. Finite-element modeling of the effects of ultrasound on brain tissue suggests that the effects of ultrasound on firing frequency are caused by a small (<2°C) increase in temperature, with possible additional contributions from mechanical effects.

    View details for DOI 10.1085/jgp.202012672

    View details for PubMedID 33074301

  • Expanding the membrane-protein NMR toolkit. Nature chemical biology Cheng, R. C., Maduke, M. 2020

    View details for DOI 10.1038/s41589-020-0597-7

    View details for PubMedID 32665639

  • An international gathering of physiologists in Valparaiso, Chile. The Journal of general physiology Maduke, M. C., Rothberg, B. S. 2020; 152 (6)

    View details for DOI 10.1085/jgp.202012626

    View details for PubMedID 32347297

  • Special Issue: Molecular Biophysics of Membranes and Membrane Proteins Preface BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES Thompson, L. K., Maduke, M. 2020; 1862 (1): 183116

    View details for DOI 10.1016/j.bbamem.2019.183116

    View details for Web of Science ID 000503314800001

    View details for PubMedID 31810503

  • A CLC-ec1 mutant reveals global conformational change and suggests a unifying mechanism for the Cl-/H+ transport cycle. eLife Chavan, T. S., Cheng, R. C., Jiang, T. n., Mathews, I. I., Stein, R. A., Koehl, A. n., Mchaourab, H. S., Tajkhorshid, E. n., Maduke, M. n. 2020; 9


    Among coupled exchangers, CLCs uniquely catalyze the exchange of oppositely charged ions (Cl- for H+). Transport-cycle models to describe and explain this unusual mechanism have been proposed based on known CLC structures. While the proposed models harmonize with many experimental findings, gaps and inconsistencies in our understanding have remained. One limitation has been that global conformational change - which occurs in all conventional transporter mechanisms - has not been observed in any high-resolution structure. Here, we describe the 2.6 Å structure of a CLC mutant designed to mimic the fully H+-loaded transporter. This structure reveals a global conformational change to improve accessibility for the Cl- substrate from the extracellular side and new conformations for two key glutamate residues. Together with DEER measurements, MD simulations, and functional studies, this new structure `provides evidence for a unified model of H+ /Cl- transport that reconciles existing data on all CLC-type proteins.

    View details for DOI 10.7554/eLife.53479

    View details for PubMedID 32310757

  • Dynamical model of the CLC-2 ion channel reveals conformational changes associated with selectivity-filter gating. PLoS computational biology McKiernan, K. A., Koster, A. K., Maduke, M. n., Pande, V. S. 2020; 16 (3): e1007530


    This work reports a dynamical Markov state model of CLC-2 "fast" (pore) gating, based on 600 microseconds of molecular dynamics (MD) simulation. In the starting conformation of our CLC-2 model, both outer and inner channel gates are closed. The first conformational change in our dataset involves rotation of the inner-gate backbone along residues S168-G169-I170. This change is strikingly similar to that observed in the cryo-EM structure of the bovine CLC-K channel, though the volume of the intracellular (inner) region of the ion conduction pathway is further expanded in our model. From this state (inner gate open and outer gate closed), two additional states are observed, each involving a unique rotameric flip of the outer-gate residue GLUex. Both additional states involve conformational changes that orient GLUex away from the extracellular (outer) region of the ion conduction pathway. In the first additional state, the rotameric flip of GLUex results in an open, or near-open, channel pore. The equilibrium population of this state is low (∼1%), consistent with the low open probability of CLC-2 observed experimentally in the absence of a membrane potential stimulus (0 mV). In the second additional state, GLUex rotates to occlude the channel pore. This state, which has a low equilibrium population (∼1%), is only accessible when GLUex is protonated. Together, these pathways model the opening of both an inner and outer gate within the CLC-2 selectivity filter, as a function of GLUex protonation. Collectively, our findings are consistent with published experimental analyses of CLC-2 gating and provide a high-resolution structural model to guide future investigations.

    View details for DOI 10.1371/journal.pcbi.1007530

    View details for PubMedID 32226009

  • Spike-frequency dependent inhibition and potentiation of neural activity by ultrasound BioRxiv Prieto, M. L., Firouzi, K., Khuri-Yakub, B. R., Madison, D. V., Maduke, M. 2020
  • Development and validation of a potent and specific inhibitor for the CLC-2 chloride channel BioRxiv Koster, A. K., Reese, A. L., Kuryshev, Y., Wen, X., McKiernan, K. A., Gray, E. E., Wu, C., Huguenard, J. R., Maduke, M., Du Bois, J. 2020
  • A selective class of inhibitors for the CLC-Ka chloride ion channel PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Koster, A. K., Wood, C. P., Thomas-Tran, R., Chavan, T. S., Almqvist, J., Choi, K., Du Bois, J., Maduke, M. 2018; 115 (21): E4900–E4909


    CLC proteins are a ubiquitously expressed family of chloride-selective ion channels and transporters. A dearth of pharmacological tools for modulating CLC gating and ion conduction limits investigations aimed at understanding CLC structure/function and physiology. Herein, we describe the design, synthesis, and evaluation of a collection of N-arylated benzimidazole derivatives (BIMs), one of which (BIM1) shows unparalleled (>20-fold) selectivity for CLC-Ka over CLC-Kb, the two most closely related human CLC homologs. Computational docking to a CLC-Ka homology model has identified a BIM1 binding site on the extracellular face of the protein near the chloride permeation pathway in a region previously identified as a binding site for other less selective inhibitors. Results from site-directed mutagenesis experiments are consistent with predictions of this docking model. The residue at position 68 is 1 of only ∼20 extracellular residues that differ between CLC-Ka and CLC-Kb. Mutation of this residue in CLC-Ka and CLC-Kb (N68D and D68N, respectively) reverses the preference of BIM1 for CLC-Ka over CLC-Kb, thus showing the critical role of residue 68 in establishing BIM1 selectivity. Molecular docking studies together with results from structure-activity relationship studies with 19 BIM derivatives give insight into the increased selectivity of BIM1 compared with other inhibitors and identify strategies for further developing this class of compounds.

    View details for PubMedID 29669921

  • Activation of Piezo1 but Not NaV1.2 Channels by Ultrasound at 43 MHz. Ultrasound in medicine & biology Prieto, M. L., Firouzi, K. n., Khuri-Yakub, B. T., Maduke, M. n. 2018


    Ultrasound (US) can modulate the electrical activity of the excitable tissues, but the mechanisms underlying this effect are not understood at the molecular level or in terms of the physical modality through which US exerts its effects. Here, we report an experimental system that allows for stable patch-clamp recording in the presence of US at 43 MHz, a frequency known to stimulate neural activity. We describe the effects of US on two ion channels proposed to be involved in the response of excitable cells to US: the mechanosensitive Piezo1 channel and the voltage-gated sodium channel NaV1.2. Our patch-clamp recordings, together with finite-element simulations of acoustic field parameters indicate that Piezo1 channels are activated by continuous wave US at 43 MHz and 50 or 90 W/cm2through cell membrane stress caused by acoustic streaming. NaV1.2 channels were not affected through this mechanism at these intensities, but their kinetics could be accelerated by US-induced heating.

    View details for PubMedID 29525457

  • Protein ligands for studying ion channel proteins. journal of general physiology Chavan, T., Maduke, M., Swartz, K. 2017

    View details for DOI 10.1085/jgp.201711776

    View details for PubMedID 28270405

    View details for PubMedCentralID PMC5379924

  • CLC Chloride/Proton Transporters Maduke, M. CELL PRESS. 2017: 308A
  • Molecular Basis for Differential Anion Binding and Proton Coupling in the Cl(-)/H(+) Exchanger ClC-ec1. Journal of the American Chemical Society Jiang, T., Han, W., Maduke, M., Tajkhorshid, E. 2016; 138 (9): 3066-3075

    View details for DOI 10.1021/jacs.5b12062

    View details for PubMedID 26880377

  • An Outward-Facing Open Conformational State in a CLC Transporter Abraham, S. J., Chavan, T., Cheng, R. C., Fenollar-Ferrer, C., Han, W., Islam, S. M., Jiang, T., Khantwal, C. M., Mathews, I. I., Stein, R. A., Roux, B., Forrest, L. R., Mchaourab, H. S., Tajkhorshid, E., Maduke, M. CELL PRESS. 2016: 178A
  • Revealing an outward-facing open conformational state in a CLC Cl-/H+ exchange transporter ELIFE Khantwal, C. M., Abraham, S. J., Han, W., Jiang, T., Chavan, T. S., Cheng, R. C., Elvington, S. M., Liu, C. W., Mathews, I. I., Steins, R. A., Mchaourab, H. S., Tajkhorshid, E., Maduke, M. 2016; 5


    CLC secondary active transporters exchange Cl(-) for H(+). Crystal structures have suggested that the conformational change from occluded to outward-facing states is unusually simple, involving only the rotation of a conserved glutamate (Gluex) upon its protonation. Using (19)F NMR, we show that as [H(+)] is increased to protonate Gluex and enrich the outward-facing state, a residue ~20 Å away from Gluex, near the subunit interface, moves from buried to solvent-exposed. Consistent with functional relevance of this motion, constriction via inter-subunit cross-linking reduces transport. Molecular dynamics simulations indicate that the cross-link dampens extracellular gate-opening motions. In support of this model, mutations that decrease steric contact between Helix N (part of the extracellular gate) and Helix P (at the subunit interface) remove the inhibitory effect of the cross-link. Together, these results demonstrate the formation of a previously uncharacterized 'outward-facing open' state, and highlight the relevance of global structural changes in CLC function.

    View details for DOI 10.7554/eLife.11189

    View details for Web of Science ID 000373794800001

    View details for PubMedID 26799336

    View details for PubMedCentralID PMC4769167

  • C-13 NMR detects conformational change in the 100-kD membrane transporter ClC-ec1 JOURNAL OF BIOMOLECULAR NMR Abraham, S. J., Cheng, R. C., Chew, T. A., Khantwal, C. M., Liu, C. W., Gong, S., Nakamoto, R. K., Maduke, M. 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

    View details for PubMedCentralID PMC4398623

  • Water access points and hydration pathways in CLC H+/Cl- transporters PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Han, W., Cheng, R. C., Maduke, M. C., Tajkhorshid, E. 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

    View details for PubMedCentralID PMC3918786

  • Monitoring Substrate-Driven Structural Changes in a CLC Chloride-Proton Antiporter with Double Electron-Electron Resonance Spectroscopy Cheng, R. C., Chang, P., Fenollar-Ferrer, C., Stein, R. A., Trone, K., Forrest, L. R., Mchaourab, H. S., Maduke, M. C. CELL PRESS. 2014: 145A
  • Dynamic Response of Model Lipid Membranes to Ultrasonic Radiation Force PLOS ONE Prieto, M. L., Oralkan, O., Khuri-Yakub, B. T., Maduke, M. C. 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 Web of Science ID 000326037000042

    View details for PubMedID 24194863

    View details for PubMedCentralID PMC3806737

  • Novel diuretic targets AMERICAN JOURNAL OF PHYSIOLOGY-RENAL PHYSIOLOGY Denton, J. S., Pao, A. C., Maduke, M. 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

    View details for PubMedCentralID PMC3798746

  • Monitoring Substrate-driven Structural Changes in a CLC Chloride-Proton Antiporter with Double Electron-Electron Resonance Spectroscopy Cheng, R. C., Chang, P., Stein, R. A., Trone, K., Mchaourab, H. S., Maduke, M. C. ROCKEFELLER UNIV PRESS. 2013: 5A
  • Dynamic response of model lipid membranes to ultrasonic radiation force. PloS one Prieto, M. L., Ömer, O., Khuri-Yakub, B. T., Maduke, M. C. 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 Howery, A. E., Elvington, S., Abraham, S. J., Choi, K., Dworschak-Simpson, S., Phillips, S., Ryan, C. M., Sanford, R. L., Almqvist, J., Tran, K., Chew, T. A., Zachariae, U., Andersen, O. S., Whitelegge, J., Matulef, K., Du Bois, J., Maduke, M. C. 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

    View details for PubMedCentralID PMC3508466

  • Structural investigations of ClC-ec1, a large integral membrane protein, using solution-state NMR and nanodisc technology Experimental Biology Meeting 2012 Chew, T., Abraham, S., Elvington, S., Maduke, M. FEDERATION AMER SOC EXP BIOL. 2012
  • Biochemistry to the Rescue: A CIC-2 Auxiliary Subunit Provides a Tangible Link to Leukodystrophy NEURON Maduke, M. C., Reimer, R. J. 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

  • Characterization of a Novel CLC-ec1 Inhibitor Matulef, K. I., Howery, A., Sanford, R., Phillips, S., Simpson, S., Abraham, S., Whitelegge, J., Du Bois, J., Andersen, O. S., Maduke, M. CELL PRESS. 2012: 520A
  • Ultrasound-Induced Currents in Planar Lipid Blayers: Origins and Potential Physiological Significance Prieto, M. L., Oralkan, O., Khuri-Yakub, B. T., Maduke, M. C. CELL PRESS. 2012: 34A
  • CLC Transporters: The Search for Conformational Change Maduke, M. CELL PRESS. 2012: 407A
  • Monitoring Substrate-Driven Conformational Changes of ClC-ec1 by [Methyl-13C] Methionine NMR Cheng, R. K., Elvington, S. M., Maduke, M. C. CELL PRESS. 2012: 520A–521A
  • Substrate-driven conformational changes in ClC-ec1 observed by fluorine NMR EMBO JOURNAL Elvington, S. M., Liu, C. W., Maduke, M. C. 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

    View details for PubMedCentralID PMC2771095

  • Proton-coupled gating in chloride channels PHILOSOPHICAL TRANSACTIONS OF THE ROYAL SOCIETY B-BIOLOGICAL SCIENCES Lisal, J., Maduke, M. 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

    View details for PubMedCentralID PMC2674089

  • Ion Channel Targets - Select Biosciences' Fourth Annual Conference IDRUGS Holmqvist, M., Maduke, M. 2008; 11 (11): 795–98


    Select Biosciences' Fourth Annual Ion Channel Targets conference brought together scientists from industry and academia who are interested in the discovery of therapeutics targeted to various ion channels implicated in human disease. Topics addressed included methodological aspects of screening for ion channel drugs, the discovery of novel inhibitors and activators of ion channels that are drug candidates, and suggestions of potential new ion channel targets.

    View details for Web of Science ID 000260643900007

    View details for PubMedID 18988121

  • Thinking outside the crystal Complementary approaches for examining transporter conformational change CHANNELS Elvington, S. M., Maduke, M. 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 ¹⁹F-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 Lisal, J., Maduke, M. 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

    View details for PubMedCentralID PMC2559860

  • A Cytoplasmic Domain Mutation in ClC-Kb Affects Long-Distance Communication Across the Membrane PLOS ONE Martinez, G. Q., Maduke, M. 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

    View details for PubMedCentralID PMC2447174

  • Discovery of potent CLC chloride channel inhibitors ACS CHEMICAL BIOLOGY Matulef, K., Howery, A. E., Tan, L., Kobertz, W. R., Du Bois, J., Maduke, M. 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

    View details for PubMedCentralID PMC2556891

  • The role of a conserved lysine in chloride- and voltage-dependent ClC-0 fast gating JOURNAL OF GENERAL PHYSIOLOGY Engh, A. M., Faraldo-Gomez, J. D., Maduke, M. 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

    View details for PubMedCentralID PMC2151651

  • The mechanism of fast-gate opening in ClC-0 JOURNAL OF GENERAL PHYSIOLOGY Engh, A. M., Faraldo-Gomez, J. D., Maduke, M. 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

    View details for PubMedCentralID PMC2151655

  • The CLC 'chloride channel' family: revelations from prokaryotes MOLECULAR MEMBRANE BIOLOGY Matulef, K., Maduke, M. 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

  • Inhibition of CLC-ec1 by DIDS hydrolysis products 51st Annual Meeting of the Biophysical-Society Matulef, K., Howery, A. E., Ganesan, R., Martinez, G., Du Bois, J., Maduke, M. CELL PRESS. 2007: 347A–347A
  • Side-dependent inhibition of a prokaryotic CIC by DIDS BIOPHYSICAL JOURNAL Matulef, K., Maduke, M. 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

    View details for PubMedCentralID PMC1366676

  • Cysteine accessibility in ClC-0 supports conservation of the ClC intracellular vestibule JOURNAL OF GENERAL PHYSIOLOGY Engh, A. M., Maduke, M. 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

    View details for PubMedCentralID PMC2234078

  • The poststructural festivities begin NEURON Maduke, M., Mindell, J. A. 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 NATURE Mindell, J. A., Maduke, M., Miller, C., Grigorieff, N. 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 GENOME BIOLOGY Mindell, J. A., Maduke, M. 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 Maduke, M., Miller, C., Mindell, J. A. 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 Maduke, M., Pheasant, D. J., Miller, C. 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 BIOCHEMISTRY Maduke, M., Williams, C., Miller, C. 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



    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



    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