DOCSLIB.ORG

Molecular Architecture of Full-Length Kcsa Role of Cytoplasmic Domains in Ion Permeation and Activation Gating

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Molecular Architecture of Full-Length Kcsa Role of Cytoplasmic Domains in Ion Permeation and Activation Gating Molecular Architecture of Full-Length KcsA Role of Cytoplasmic Domains in Ion Permeation and Activation Gating D. Marien Cortes, Luis G. Cuello, and Eduardo Perozo From the Department of Molecular Physiology and Biological Physics and Center for Structural Biology, University of Virginia Health Sciences Center, Charlottesville, Virginia 22906 abstract The molecular architecture of the NH2 and COOH termini of the prokaryotic potassium channel KcsA has been determined using site-directed spin-labeling methods and paramagnetic resonance EPR spectros- copy. Cysteine mutants were generated (residues 5–24 and 121–160) and spin labeled, and the X-band CW EPR spectra were obtained from liposome-reconstituted channels at room temperature. Data on probe mobility Ϫ1 (⌬Ho ), accessibility parameters (⌸O2 and ⌸NiEdda), and inter-subunit spin-spin interaction (⍀) were used as structural constraints to build a three-dimensional folding model of these cytoplasmic domains from a set of simu- lated annealing and restrained molecular dynamics runs. 32 backbone structures were generated and averaged us- ing fourfold symmetry, and a final mean structure was obtained from the eight lowest energy runs. Based on the Downloaded from present data, together with information from the KcsA crystal structure, a model for the three-dimensional fold of full-length KcsA was constructed. In this model, the NH2 terminus of KcsA forms an ␣-helix anchored at the mem- brane–water interface, while the COOH terminus forms a right-handed four-helix bundle that extend some 40–50 Å towards the cytoplasm. Functional analysis of COOH-terminal deletion constructs suggest that, while the COOH terminus does not play a substantial role in determining ion permeation properties, it exerts a modulatory role in the pH-dependent gating mechanism. key words: KcsA • cytoplasmic domains • EPR spectroscopy • three-dimensional fold jgp.rupress.org INTRODUCTION together with spectroscopic approaches it has also of- fered a glimpse into the molecular events underlying ac- Our knowledge of ion channel function has advanced tivation gating (Perozo et al., 1999). Ironically, func- on August 24, 2017 dramatically since the discovery of KcsA, a small tional understanding of this channel has lagged our cur- prokaryotic Kϩ channel first identified in the gram-pos- rent structural knowledge, since reliable functional itive bacterium Streptomyces lividans (Schrempf et al., studies in KcsA were made possible only after experi- 1995). With only 160 residues, KcsA shares consider- ments with reconstituted KcsA showed that channel ac- able similarity with the core or “pore domain” of volt- tivity can be modulated by pH levels (Cuello et al., age-dependent (Kv)1 channels. Its ability to express at 1998). Under these conditions, KcsA displays all of the high levels in Escherichia coli (Schrempf et al., 1995; hallmarks of other well-characterized eukaryotic Kϩ- Cortes and Perozo, 1997; Heginbotham et al., 1997), as selective channels, including high selectivity against Naϩ well as its remarkable oligomeric stability in detergents (Cuello et al., 1998; Heginbotham et al., 1999) and (Cortes and Perozo, 1997; Heginbotham et al., 1997), block by Ba2ϩ and quaternary ammonium ions (Cuello paved the way to the breakthrough determination of its et al., 1998; Heginbotham et al., 1999; Meuser et al., three-dimensional structure, recently solved by x-ray 1999). Reconstituted KcsA displays a predominant large crystallographic methods (Doyle et al., 1998). -pS in 250 mM Kϩ) with rectify 140ف) conductance level The KcsA crystal structure has led to an understand- ing properties at large negative potentials (Cuello et al., ing of the physical basis of ion permeation and selectiv- 1998; Heginbotham et al., 1999; Meuser et al., 1999). ity (Doyle et al., 1998; Jiang and MacKinnon, 2000), and Gating mechanisms of eukaryotic channels are often Address corrrespondence to Eduardo Perozo, Department of Molec- subject to strict regulatory control by means of phos- ular Physiology and Biological Physics and Center for Structural Biol- phorylation cascades, ligand binding, or direct interac- ogy, University of Virginia Health Sciences Center, Box 449, Jordan tion with other cytoplasmic proteins such as heterotri- Hall, Charlottesville, VA 22906-0011. Fax: (804) 982-1616; E-mail: ep- meric G proteins (Wickman and Clapham, 1995; Jonas [email protected] 1Abbreviations used in this paper: EPR, paramagnetic resonance; Kv and Kaczmarek, 1996; Hilgemann, 1997; Gray et al., channel, voltage-dependent channel; ␣PI, periodicity index; SDSL, 1998). In channels belonging to the voltage-dependent site-directed spin labeling; TM2, second transmembrane segment. channel super-family, such regulatory mechanisms typi- 165 J. Gen. Physiol. © The Rockefeller University Press • 0022-1295/2001/02/165/16 $5.00 Volume 117 February 2001 165–180 http://www.jgp.org/cgi/content/full/117/2/165 cally involve extensive cytoplasmic regions found at the (Talon resin; CLONTECH Laboratories, Inc.). Unless noted oth- erwise, the purified mutant protein was spin labeled overnight NH2 and COOH termini end of the molecule. These cytoplasmic regions participate as modulators of chan- with methanethiosulfonate spin label (Toronto Research) at a 10:1 label/channel molar ratio and reconstituted at a 500:1 nel function, determine homo- or heterosubunit tet- lipid/channel molar ratio by dilution in PBS (Cuello et al., ramerization during folding and assembly, or help es- 1998). tablish the specific targeting of channels to particular cellular regions (Sheng and Kim, 1996). In prokary- Rb Influx and Stability Assays ϩ otes, it is likely that many of the K channels identified The functional state of individual spin-labeled mutants was as- from whole-genome sequencing projects will also be sessed by measuring the extent of 86Rbϩ influx into proteolipo- subject to regulatory control from the multiple signal somes containing KcsA, as previously described (Cuello et al., transduction cascades found in bacteria and archaea 1998). Each 86Rbϩ uptake experiment was reported relative to (Bourret et al., 1991; Alex and Simon, 1994; Goudreau that of unlabeled, wild-type KcsA in the presence and absence of 5 mM Ba2ϩ, a known Kϩ channel blocker. and Stock, 1998), although the true function of their The effects of spin labeling on the oligomeric stability of the cytoplasmic domains remains to be established. mutant channels were evaluated from the changes in the ener- In the current KcsA structure, features of the trans- getics of the tetramer-to-monomer transition during thermal de- membrane and extracellular regions of the channel are naturation according to Perozo et al. (1998). Destabilization clearly resolved (Doyle et al., 1998). However, due to a ⌬⌬Gs were derived from the temperature dependence of dena- turation in the presence of SDS using simple gel-shift assays. The lack of defined electron density, and to the require- mid-point of the denaturation curve was obtained after numeri- ments imposed by the crystallization conditions that cally fitting a Boltzmann function to the data and corresponded Downloaded from produced high quality crystals, no structural informa- to the melting temperature of the tetramer. tion exists on the cytoplasmic domains of this channel. These highly charged domains are likely to play a role Liposome Patch-Clamp Recordings in controlling or modulating KcsA gating, and could Liposome-reconstituted KcsA was patch clamped following the potentially influence the permeation properties of the method of Delcour et al. (1989), with some modifications. KcsA was reconstituted as above at protein-to-lipid rations varying from open channel. Using site-directed spin labeling (SDSL) jgp.rupress.org and electron paramagnetic resonance (EPR) spectros- 1:1,000 to 1:10,000. The proteoliposome suspension was centri- fuged for 1 h at 100,000 g, and the pellet, corresponding to 10 copy, we have systematically probed regions in the NH2 mg of lipids, was resuspended in 60 ␮l of rehydration buffer. Typ- and COOH termini of KcsA predicted to be soluble and ically, three drops of the suspension were dried overnight in a h, at which time 20 24ف exposed to the cytoplasm. EPR analysis of spin-labeled desiccation chamber under vacuum for ␮l of rehydration buffer were applied to each dried drop. Rehy- mutants yields three types of structural information: on August 24, 2017 spin-label dynamics, solvent accessibility of the attached dration was allowed proceed for 5 h, yielding liposomes suitable for patch clamp. All patch-clamp measurements were done in spin label, and inter-spin proximities. This multidimen- symmetrical conditions: 200 mM KCl and MOPS buffer, pH 4.0, sional data set can be used to derive both global folding at room temperature. Single-channel currents were recorded patterns and conformational changes in proteins (for with a Dagan 3900 patch clamp amplifier, and currents were sam- reviews see Hubbell et al., 1998; Mchaourab and Per- pled at 40 kHz with analogue filter set to 5 kHz (Ϫ3 dB). Pipette ⍀ ozo, 2000). Using this data set, together with the known resistances were 5–10 M . coordinates from the crystal structure, we have devel- EPR Spectroscopy and Data Analysis oped a backbone three-dimensional model of full- length KcsA to provide a structural basis for under- X-band CW EPR spectra were obtained in a Bruker EMX spec- standing activation gating in this channel. trometer equipped with a loop-gap resonator under the follow- ing conditions: 2 mW incident power, 100 kHz modulation fre- quency, and 1 G modulation amplitude. Power saturation curves MATERIALS AND METHODS were obtained for each spin-labeled mutant after equilibration in Mutagenesis, Expression, and Spin Labeling of KcsA N2, air (21% O2), and N2 in the presence of 10 mM Ni-Edda. Data were analyzed and converted to the accessibility parameter Cysteine mutants were generated for residues 5–24 and 121–160 ⌸ according to Farahbakhsh et al. (1992). To determine what ef- in KcsA, covering most of the NH2- and COOH-terminal ends of fects, if any, would the presence of the 6ϫ His tag have on the the channel.
Load more

Recommended publications
  • Inactivation in the Potassium Channel Kcsa T ⁎ Yunyao Xu, Ann E
    Journal of Structural Biology: X 3 (2019) 100009 Contents lists available at ScienceDirect Journal of Structural Biology: X journal homepage: www.journals.elsevier.com/journal-of-structural-biology-x This article is part of the Special Issue on Solid-State NMR of Biomolecular Assemblies ☆ Inactivation in the potassium channel KcsA T ⁎ Yunyao Xu, Ann E. McDermott Department of Chemistry, Columbia University, New York, NY 10027, United States ARTICLE INFO ABSTRACT Keywords: Inactivation, the slow cessation of transmission after activation, is a general feature of potassium channels. It is Potassium channel essential for their function, and malfunctions in inactivation leads to numerous pathologies. The detailed me- C-type inactivation chanism for the C-type inactivation, distinct from the N-type inactivation, remains an active area of investiga- Solid state NMR tion. Crystallography, computational simulations, and NMR have greatly enriched our understanding of the Allosteric coupling process. Here we review the major hypotheses regarding C-type inactivation, particularly focusing on the key KcsA role played by NMR studies of the prokaryotic potassium channel KcsA, which serves as a good model for voltage gated mammalian channels. 1. Introduction treatment in a wide range of disorders including vasodilation and blood pressure regulation, arrhythmias, neurodegenerative diseases and psy- Potassium channels form the second largest family of membrane chiatric treatments (Parekh et al., 2018). Deletion of one of the back- proteins, and control numerous metabolic processes. Their primary ground potassium channels TREK-1 results in a depression-resistant function is to facilitate passive diffusion of potassium ions through low phenotype (Heurteaux et al., 2006). Potassium channel mutations and dielectric membrane, with great selectivity with respect to other ions malfunction (“channelopathies”) result in heart timing defects such as (McCoy and Nimigean, 2012; Yellen, 2002).
    [Show full text]
  • Structural Characterization of Bacterial Collagenases Ryan Bauer University of Arkansas, Fayetteville
    University of Arkansas, Fayetteville ScholarWorks@UARK Theses and Dissertations 8-2017 Structural Characterization of Bacterial Collagenases Ryan Bauer University of Arkansas, Fayetteville Follow this and additional works at: http://scholarworks.uark.edu/etd Part of the Biochemistry Commons Recommended Citation Bauer, Ryan, "Structural Characterization of Bacterial Collagenases" (2017). Theses and Dissertations. 2431. http://scholarworks.uark.edu/etd/2431 This Dissertation is brought to you for free and open access by ScholarWorks@UARK. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of ScholarWorks@UARK. For more information, please contact [email protected], [email protected]. Structural Characterization of Bacterial Collagenases A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Chemistry by Ryan Bauer University of Arkansas Bachelor of Science in Chemistry, 2006 August 2017 University of Arkansas This dissertation is approved for recommendation to the Graduate Council ________________________________ Dr. Joshua Sakon Dissertation Director _______________________________ _______________________________ Dr. Colin Heyes Dr. Wesley Stites Committee Member Committee Member _______________________________ Dr. Suresh Thallapuranam Committee Member Abstract Clostridium histolyticum secretes collagenases ColG and ColH to cause extensive tissue destruction during myonecrosis. The collagenases are multi-domain enzymes consisting of a N- terminal collagenase module, s1, polycystic kidney disease (PKD)-like domains (s2 in ColG; s2a and s2b in ColH) and collagen-binding domains (CBD) (s3a and s3b in ColG; s3 in ColH). The individual CBD and PKD-like domains chelate calcium to modulate stability and domain rearrangement. Though used by bacteria to break down collagen in the extracellular matrix during infection, therapeutic use of the enzyme to break down excess connective tissue has been approved by the FDA.
    [Show full text]
  • Twisting Conformational Changes of Single Kcsa Potassium Channel Upon Gating Using Diffracted X-Ray Tracking
    Life science : Medical Biology Twisting Conformational Changes of Single KcsA Potassium Channel upon Gating using Diffracted X-ray Tracking Ion channels are signal transduction molecules were exploited in this study to contrast the changes in that selectively permeate ions and generate electrical motion in a different gating status. signals through turning on and off of ion permeation When channels were in the closed state at neutral pathways. Channels respond to various stimuli, such pH, diffraction spots moved radially. This represents as membrane voltage and chemical substances. With bending motions of channels in the range of a few sensor domains, channel molecules transduce various degrees. When channels were actively gated at acidic stimuli into conformational changes of ion permeation pH, diffraction spots moved circumferentially (Fig. pathways, leading to ion-conductive (open) and non- 3(a,b)). These motions of the spots correspond to the conductive (closed) states. Single-channel current rotation of the channel around the longitudinal axis. recordings have elucidated the random nature of Random clockwise and counter-clockwise rotations opening and closing transitions (gating; Fig. 1(a)). were recorded. We found that the range of the Genes of potassium channels exist from bacteria to rotations was several tens of degrees, which are humans and constitute one of the largest gene family. unexpectedly large values. The direction of the Ten years ago, the crystal structure of a potassium rotational motions was sometimes reversed during channel from Streptomyces lividans (KcsA channel) recordings. We call this type of motion as twisting. was resolved (Fig. 1(b)). The three-dimensional This is the first observation of a large twisting in the structure provided the architecture of the ion conformational change of channel molecules upon permeation pathway and insights on how ions are gating.
    [Show full text]
  • Studies of the Structure of Potassium Channel Kcsa in the Open Conformation and the Effect of Anionic Lipids on Channel Inactivation
    Studies of the structure of potassium channel KcsA in the open conformation and the effect of anionic lipids on channel inactivation Dongyu (Allison) Zhang Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Graduate School of Arts and Sciences COLUMBIA UNIVERSITY 2019 © 2019 Dongyu Zhang All rights reserved Abstract Studies of the structure of potassium channel KcsA in the open conformation and the effect of anionic lipids on channel inactivation Dongyu Zhang Membrane proteins play a vital role in cellular processes. In this thesis, we use KcsA, a prokaryotic potassium channel, as a model to investigate the gating mechanism of ion channels and the effect of anionic lipids on the channel activity using solid-state NMR spectroscopy. KcsA activity is known to be highly dependent on the presence of negatively charged lipids. Multiple crystal structures combined with biochemistry assays suggest that KcsA is co-purified with anionic lipids with phosphatidylglycerol headgroup. Here, we identified this specifically bound, isotopically labeled lipid in the protein 13C-13C correlation spectra. Our results reveal that the lipid cross peaks show stronger intensity when the channel is in the inactivated state compared to the activated state, which indicates a stronger protein-lipid interaction when KcsA is inactivated. In addition, our data shows that including anionic lipids into proteoliposomes leads to a weaker potassium ion affinity at the selectivity filter. Considering ion loss as a model of inactivation, our results suggest anionic lipids promote channel inactivation. However, the surface charge is not the only physical parameter that regulates channel gating or conformational preference.
    [Show full text]
  • Selectivity Filter Modalities and Rapid Inactivation of the Herg1 Channel
    Selectivity filter modalities and rapid inactivation of the hERG1 channel Williams E. Mirandaa, Kevin R. DeMarcob, Jiqing Guoc, Henry J. Duffc, Igor Vorobyovb,d, Colleen E. Clancyb,d,1, and Sergei Yu. Noskova,1 aCentre for Molecular Simulation, Department of Biological Sciences, University of Calgary, Calgary, AB T2N 1N4, Canada; bDepartment of Physiology and Membrane Biology, University of California, Davis, CA 95616; cLibin Cardiovascular Institute of Alberta, Cumming School of Medicine, University of Calgary, Calgary, AB T2N 4N1, Canada; and dDepartment of Pharmacology, University of California, Davis, CA 95616 Edited by Michael L. Klein, Temple University, Philadelphia, PA, and approved December 19, 2019 (received for review May 28, 2019) The human ether-á-go-go–related gene (hERG1) channel conducts slow-inactivating current observed from its bacterial counterparts small outward K+ currents that are critical for cardiomyocyte mem- (15–17) and voltage-gated channels from the Shaker family brane repolarization. The gain-of-function mutation N629D at the (18–20). Moreover, the electrophysiological properties of the outer mouth of the selectivity filter (SF) disrupts inactivation and hERG1 channel are highly sensitive to mutations altering local K+-selective transport in hERG1, leading to arrhythmogenic pheno- electrical fields or changing the interaction network topology in + types associated with long-QT syndrome. Here, we combined com- the vicinity of canonical K binding sites in the SF region. For putational electrophysiology with Markov state model analysis to example, the mutation N629D adjacent to the SF signature se- investigate how SF-level gating modalities control selective cation quence 626GFG628 of the hERG1 channel was the first reported to transport in wild-type (WT) and mutant (N629D) hERG1 variants.
    [Show full text]
  • Modulation of Function, Structure and Clustering of K Channels by Lipids: Lessons Learnt from Kcsa
    International Journal of Molecular Sciences Review Modulation of Function, Structure and Clustering of K+ Channels by Lipids: Lessons Learnt from KcsA María Lourdes Renart 1 , Ana Marcela Giudici 1, Clara Díaz-García 2 , María Luisa Molina 1 , Andrés Morales 3, José M. González-Ros 1,* and José Antonio Poveda 1,* 1 Instituto de Investigación, Desarrollo e Innovación en Biotecnología Sanitaria de Elche (IDiBE), and Instituto de Biología Molecular y Celular (IBMC), Universidad Miguel Hernández, Elche, E-03202 Alicante, Spain; [email protected] (M.L.R.); [email protected] (A.M.G.); [email protected] (M.L.M.) 2 iBB-Institute for Bioengineering and Bioscience, Instituto Superior Técnico, Universidade de Lisboa, 1049-001 Lisboa, Portugal; [email protected] 3 Departamento de Fisiología, Genética y Microbiología, Universidad de Alicante, E-03080 Alicante, Spain; [email protected] * Correspondence: [email protected] (J.M.G.-R.); [email protected] (J.A.P.) Received: 5 March 2020; Accepted: 5 April 2020; Published: 7 April 2020 Abstract: KcsA, a prokaryote tetrameric potassium channel, was the first ion channel ever to be structurally solved at high resolution. This, along with the ease of its expression and purification, made KcsA an experimental system of choice to study structure–function relationships in ion channels. In fact, much of our current understanding on how the different channel families operate arises from earlier KcsA information. Being an integral membrane protein, KcsA is also an excellent model to study how lipid–protein and protein–protein interactions within membranes, modulate its activity and structure. In regard to the later, a variety of equilibrium and non-equilibrium methods have been used in a truly multidisciplinary effort to study the effects of lipids on the KcsA channel.
    [Show full text]
  • Exploring Blocker Binding to a Homology Model of the Open Herg K+ Channel Using Docking and Molecular Dynamics Methods
    FEBS 29575 FEBS Letters 579 (2005) 2939–2944 Exploring blocker binding to a homology model of the open hERG K+ channel using docking and molecular dynamics methods Fredrik O¨ sterberg, Johan A˚ qvist* Department of Cell and Molecular Biology, Uppsala University, Biomedical Center, Box 596, SE-751 24 Uppsala, Sweden Received 1 March 2005; revised 7 April 2005; accepted 15 April 2005 Available online 29 April 2005 Edited by Gianni Cesareni to rely on homology modelling based on the bacterial K+ Abstract Binding of blockers to the human voltage-gated hERG potassium channel is studied using a combination of channel structures that have been solved [6]. homology modelling, automated docking calculations and molec- The voltage-dependent hERG potassium (Kv) channel has a ular dynamics simulations, where binding affinities are evaluated 50% sequence identity to the Drosophila melanogaster ether a´ using the linear interaction energy method. A homology model go-go (Eag) gene [7] and has also six membrane spanning seg- was constructed based on the available crystal structure of the ments per each of its four subunits. Segments S1–S4 form the bacterial KvAP channel and the affinities of a series of sertindole voltage sensor part where the S4 helix is thought to be the most analogues predicted using this model. The calculations reproduce important of the four with its several charged arginines. The the relative binding affinities of these compounds very well and sequences spanning through S5–P–S6 form the central channel indicate that both polar interactions near the intracellular open- cavity (Fig. 1) where blockers bind. Between the two helices P ing of the selectivity filter as well as hydrophobic complementar- and S6 one finds the highly conserved selectivity filter (SF) ity in the region around F656 are important for blocker binding.
    [Show full text]
  • Unraveling of a Strongly Correlated Dynamical Network of Residues Controlling the Permeation of Potassium in Kcsa Ion Channel
    entropy Article Unraveling of a Strongly Correlated Dynamical Network of Residues Controlling the Permeation of Potassium in KcsA Ion Channel Salvatore M. Cosseddu †, Eunju Julia Choe and Igor A. Khovanov * School of Engineering, University of Warwick, Coventry CV4 7AL, UK; [email protected] (S.M.C.); [email protected] (E.J.C.) * Correspondence: [email protected] † Current address: Viseca Payment Services SA, Hagenholzstrasse 56, 8050 Zürich, Switzeland. Abstract: The complicated patterns of the single-channel currents in potassium ion channel KcsA are governed by the structural variability of the selectivity filter. A comparative analysis of the dynamics of the wild type KcsA channel and several of its mutants showing different conducting patterns was performed. A strongly correlated dynamical network of interacting residues is found to play a key role in regulating the state of the wild type channel. The network is centered on the aspartate D80 which plays the role of a hub by strong interacting via hydrogen bonds with residues E71, R64, R89, and W67. Residue D80 also affects the selectivity filter via its backbones. This network further compromises ions and water molecules located inside the channel that results in the mutual influence: the permeation depends on the configuration of residues in the network, and the dynamics of network’s residues depends on locations of ions and water molecules inside the selectivity filter. Some features of the network provide a further understanding of experimental results describing the KcsA activity. In particular, the necessity of anionic lipids to be present for functioning the channel is explained by the interaction between the lipids and the arginine residues R64 and R89 that prevents destabilizing the structure of the selectivity filter.
    [Show full text]
  • Conformational Equilibrium Shift Underlies Altered K+ Channel Gating As Revealed by NMR
    ARTICLE https://doi.org/10.1038/s41467-020-19005-3 OPEN Conformational equilibrium shift underlies altered K+ channel gating as revealed by NMR Yuta Iwahashi1, Yuki Toyama1, Shunsuke Imai 1, Hiroaki Itoh 1, Masanori Osawa 1,3, Masayuki Inoue 1 & ✉ Ichio Shimada 1,2 The potassium ion (K+) channel plays a fundamental role in controlling K+ permeation across the cell membrane and regulating cellular excitabilities. Mutations in the transmem- + 1234567890():,; brane pore reportedly affect the gating transitions of K channels, and are associated with the onset of neural disorders. However, due to the lack of structural and dynamic insights into the functions of K+ channels, the structural mechanism by which these mutations cause K+ channel dysfunctions remains elusive. Here, we used nuclear magnetic resonance spectro- scopy to investigate the structural mechanism underlying the decreased K+-permeation caused by disease-related mutations, using the prokaryotic K+ channel KcsA. We demon- strated that the conformational equilibrium in the transmembrane region is shifted toward the non-conductive state with the closed intracellular K+-gate in the disease-related mutant. We also demonstrated that this equilibrium shift is attributable to the additional steric contacts in the open-conductive structure, which are evoked by the increased side-chain bulkiness of the residues lining the transmembrane helix. Our results suggest that the alteration in the conformational equilibrium of the intracellular K+-gate is one of the fun- damental mechanisms underlying the dysfunctions of K+ channels caused by disease-related mutations. 1 Graduate School of Pharmaceutical Sciences, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
    [Show full text]
  • Synchrotron Radiation Circular Dichroism Spectroscopy- Defined Structure of the C-Terminal Domain of Nachbac and Its Role in Channel Assembly
    Synchrotron radiation circular dichroism spectroscopy- defined structure of the C-terminal domain of NaChBac and its role in channel assembly Andrew M. Powl, Andrias O. O’Reilly, Andrew J. Miles, and B. A. Wallace1 Department of Crystallography, Institute of Structural and Molecular Biology, Birkbeck College, University of London, London WC1E 7HX, United Kingdom Edited by Robert Baldwin, Stanford University, Stanford, CA, and approved June 24, 2010 (received for review February 16, 2010) Extramembranous domains play important roles in the structure channels (7). In the KcsA potassium channel the CTD has been and function of membrane proteins, contributing to protein stabi- shown to be critical for expression, tetramerization, and stability lity, forming association domains, and binding ancillary subunits of the closed channel (8, 9) as well as having a role in pH sensing and ligands. However, these domains are generally flexible, (10, 11). The structure of the CTD of KcsA has recently been making them difficult or unsuitable targets for obtaining high- defined crystallographically (12). It forms a right-handed four- resolution X-ray and NMR structural information. In this study helix coiled-coil bundle of approximately 40 residues in length we show that the highly sensitive method of synchrotron radiation that projects 70 Å beyond the membrane interface, not interact- circular dichroism (SRCD) spectroscopy can be used as a powerful ing significantly with the TM regions of the protein, and is stabi- tool to investigate the structure of the extramembranous C-term- lized by a hydrophobic core in addition to a network of hydrogen inal domain (CTD) of the prokaryotic voltage-gated sodium channel bonds and salt-bridges between adjacent helices.
    [Show full text]
  • Structural Correlates of Selectivity and Inactivation in Potassium Channels☆
    Biochimica et Biophysica Acta 1818 (2012) 272–285 Contents lists available at SciVerse ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbamem Review Structural correlates of selectivity and inactivation in potassium channels☆ Jason G. McCoy a, Crina M. Nimigean a,b,c,⁎ a Department of Anesthesiology, Weill Cornell Medical College, 1300 York Avenue, New York, NY 10021, USA b Department of Physiology and Biophysics, Weill Cornell Medical College, 1300 York Avenue, New York, NY 10021, USA c Department of Biochemistry, Weill Cornell Medical College, 1300 York Avenue, New York, NY 10021, USA article info abstract Article history: Potassium channels are involved in a tremendously diverse range of physiological applications requiring dis- Received 17 July 2011 tinctly different functional properties. Not surprisingly, the amino acid sequences for these proteins are di- Received in revised form 7 September 2011 verse as well, except for the region that has been ordained the “selectivity filter”. The goal of this review is Accepted 9 September 2011 to examine our current understanding of the role of the selectivity filter and regions adjacent to it in speci- Available online 16 September 2011 fying selectivity as well as its role in gating/inactivation and possible mechanisms by which these processes are coupled. Our working hypothesis is that an amino acid network behind the filter modulates selectivity in Keywords: Selectivity channels with the same signature sequence while at the same time affecting channel inactivation properties. Inactivation This article is part of a Special Issue entitled: Membrane protein structure and function. Potassium channel © 2011 Elsevier B.V.
    [Show full text]
  • Ion Permeation Efficiency Through Potassium Channels
    Tuesday, February 18, 2014 539a closely related potassium channels display differences of up to two orders of ions in the selectivity filter was found during permeation events, indicating magnitude in their single channel conductance. The substitution of Proline that multiple permeation mechanisms concurrently underlie efficient ion 475 by Aspartate increases Shaker Kþ transport rate by 7-8 fold. Previous permeation. work of our lab suggests that such a dramatic increase in Kþ transport rate could arise from increased pore occupancy (Moscoso et al 2012). We decided 2730-Pos Board B422 to test the occupancy hypothesis by introducing charged residues along the pore Strategies to Achieve Selective Conductance in K- and Na- Selective Ion of Shaker in order to fill the permeation pathway and compare their maximal Channels transport rate to that of BK channels (600pS). The occupancy was tested Yibo Wang, Chunfeng Zhao, Sergei Yu. Noskov. with Molecular Dynamic simulations while the channel conductance was tested Center for Molecular Simulations, Department of Biological Sciences, by single channel recordings at several Kþ concentrations. Fully occupied University of Calgary, Calgary, AB, Canada. Shaker variants were still far below of BK single channel conductance values. Ion channels are proteins spanning the membrane that conduct the ions across A possible explanation for the latter is that inner entrance dimensions could the cell membranes. They play a vital role in generating and regulating the elec- limit the maximal ion transport rate to 1/3 of BK channel. To test the latter trical singling in living organisms such as pace-making, neuronal signaling and we determined the radius capture of our Shaker channels by measuring the smooth muscle function.
    [Show full text]