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    • In Vivo Dual-Channel Widefield GCaMP Imaging using Transparent Ultrasound Transducer

      Kim, Y., Prieto, M., Lin, C., Zhang, Y., Firouzi, K., Maduke, M., Pauly, K., Khuri-Yakub, P., IEEE

      ABSTRACT
      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.
      https://www.ncbi.nlm.nih.gov/pubmed/33171122 https://doi.org/10.1109/UFFC-JS60046.2024.10793885
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    • CryoEM structures of the human CLC-2 voltage-gated chloride channel reveal a ball-and-chain gating mechanism.

      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.

      eLife

      ABSTRACT
      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.
      https://www.ncbi.nlm.nih.gov/pubmed/38345841 https://doi.org/10.7554/eLife.90648
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    • Towards an ion-channel-centric approach to ultrasound neuromodulation.

      Prieto, M. L., Maduke, M.

      Current opinion in behavioral sciences

      ABSTRACT
      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.
      https://www.ncbi.nlm.nih.gov/pubmed/38505510 https://doi.org/10.1016/j.cobeha.2024.101355
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    • 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.

      ABSTRACT
      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.
      https://www.ncbi.nlm.nih.gov/pubmed/33277431 https://doi.org/10.1073/pnas.2009977117
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    • Development and validation of a potent and specific inhibitor for the CLC-2 chloride channel

      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.

      BioRxiv

      ABSTRACT
      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.
      https://www.ncbi.nlm.nih.gov/pubmed/32310757 http://dx.doi.org/10.1101/2020.01.07.897785
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    • Expanding the membrane-protein NMR toolkit.

      Cheng, R. C., Maduke, M.

      Nature chemical biology

      ABSTRACT
      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.
      https://www.ncbi.nlm.nih.gov/pubmed/32665639 https://doi.org/10.1038/s41589-020-0597-7
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    • Dynamical model of the CLC-2 ion channel reveals conformational changes associated with selectivity-filter gating.

      McKiernan, K. A., Koster, A. K., Maduke, M. n., Pande, V. S.

      PLoS computational biology

      ABSTRACT
      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.
      https://www.ncbi.nlm.nih.gov/pubmed/32226009 https://doi.org/10.1371/journal.pcbi.1007530
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    • LRRC8A:C/E Heteromeric Channels Are Ubiquitous Transporters of cGAMP.

      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.

      Molecular cell

      ABSTRACT
      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.
      https://www.ncbi.nlm.nih.gov/pubmed/33171122 https://doi.org/10.1016/j.molcel.2020.10.021
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    • Spike frequency-dependent inhibition and excitation of neural activity by high-frequency ultrasound.

      Prieto, M. L., Firouzi, K., Khuri-Yakub, B. T., Madison, D. V., Maduke, M.

      The Journal of general physiology

      ABSTRACT
      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.
      https://www.ncbi.nlm.nih.gov/pubmed/33074301 https://doi.org/10.1085/jgp.202012672
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    • A CLC-ec1 mutant reveals global conformational change and suggests a unifying mechanism for the Cl-/H+ transport cycle.

      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.

      eLife

      ABSTRACT
      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.
      https://www.ncbi.nlm.nih.gov/pubmed/32310757 https://doi.org/10.7554/eLife.53479
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    • Development and validation of a potent and specific inhibitor for the CLC-2 chloride channel.

      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.

      Proceedings of the National Academy of Sciences of the United States of America

      ABSTRACT
      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.
      https://www.ncbi.nlm.nih.gov/pubmed/33277431 https://doi.org/10.1073/pnas.2009977117
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    • An international gathering of physiologists in Valparaiso, Chile.

      Maduke, M. C., Rothberg, B. S.

      The Journal of general physiology

      ABSTRACT
      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.
      https://www.ncbi.nlm.nih.gov/pubmed/32347297 https://doi.org/10.1085/jgp.202012626
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    • Spike-frequency dependent inhibition and potentiation of neural activity by ultrasound

      Prieto, M. L., Firouzi, K., Khuri-Yakub, B. R., Madison, D. V., Maduke, M.

      BioRxiv

      ABSTRACT
      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.
      https://www.ncbi.nlm.nih.gov/pubmed/31810503 http://dx.doi.org/10.1101/2020.06.01.128710
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    • Special Issue: Molecular Biophysics of Membranes and Membrane Proteins Preface

      Thompson, L. K., Maduke, M.

      BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES

      ABSTRACT
      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.
      https://www.ncbi.nlm.nih.gov/pubmed/31810503 https://doi.org/10.1016/j.bbamem.2019.183116
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    • Activation of Piezo1 but Not NaV1.2 Channels by Ultrasound at 43 MHz.

      Prieto, M. L., Firouzi, K. n., Khuri-Yakub, B. T., Maduke, M. n.

      Ultrasound in medicine & biology

      ABSTRACT
      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.
      https://www.ncbi.nlm.nih.gov/pubmed/29525457 https://dx.doi.org/10.1085/jgp.201711776
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    • A selective class of inhibitors for the CLC-Ka chloride ion channel

      Koster, A. K., Wood, C. P., Thomas-Tran, R., Chavan, T. S., Almqvist, J., Choi, K., Du Bois, J., Maduke, M.

      PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA

      ABSTRACT
      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.
      https://www.ncbi.nlm.nih.gov/pubmed/29669921 https://doi.org/10.1016/j.bpj.2016.11.1666
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    • Protein ligands for studying ion channel proteins.

      Chavan, T., Maduke, M., Swartz, K.

      journal of general physiology

      ABSTRACT
      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(-).
      https://www.ncbi.nlm.nih.gov/pubmed/28270405 https://dx.doi.org/10.1085/jgp.201711776
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    • CLC Chloride/Proton Transporters

      Maduke, M.

      ABSTRACT
      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.
      https://www.ncbi.nlm.nih.gov/pubmed/26880377 https://doi.org/10.1016/j.bpj.2016.11.1666
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    • Revealing an outward-facing open conformational state in a CLC Cl-/H+ exchange transporter

      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.

      ELIFE

      ABSTRACT
      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.
      https://www.ncbi.nlm.nih.gov/pubmed/26799336 https://dx.doi.org/10.7554/eLife.11189
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    • 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.

      ABSTRACT
      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.
      https://www.ncbi.nlm.nih.gov/pubmed/26799336 https://doi.org/10.1016/j.bpj.2015.11.994
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