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1.
Front Pharmacol ; 15: 1401599, 2024.
Article in English | MEDLINE | ID: mdl-39050757

ABSTRACT

With over 450 genes, solute carriers (SLCs) constitute the largest transporter superfamily responsible for the uptake and efflux of nutrients, metabolites, and xenobiotics in human cells. SLCs are associated with a wide variety of human diseases, including cancer, diabetes, and metabolic and neurological disorders. They represent an important therapeutic target class that remains only partly exploited as therapeutics that target SLCs are scarce. Additionally, many small molecules reported in the literature to target SLCs are poorly characterized. Both features may be due to the difficulty of developing SLC transport assays that fulfill the quality criteria for high-throughput screening. Here, we report one of the main limitations hampering assay development within the RESOLUTE consortium: the lack of a resource providing high-quality information on SLC tool compounds. To address this, we provide a systematic annotation of tool compounds targeting SLCs. We first provide an overview on RESOLUTE assays. Next, we present a list of SLC-targeting compounds collected from the literature and public databases; we found that most data sources lacked specificity data. Finally, we report on experimental tests of 19 selected compounds against a panel of 13 SLCs from seven different families. Except for a few inhibitors, which were active on unrelated SLCs, the tested inhibitors demonstrated high selectivity for their reported targets. To make this knowledge easily accessible to the scientific community, we created an interactive dashboard displaying the collected data in the RESOLUTE web portal (https://re-solute.eu). We anticipate that our open-access resources on assays and compounds will support the development of future drug discovery campaigns for SLCs.

2.
Nat Commun ; 15(1): 5503, 2024 Jun 29.
Article in English | MEDLINE | ID: mdl-38951531

ABSTRACT

Proline is widely known as the only proteogenic amino acid with a secondary amine. In addition to its crucial role in protein structure, the secondary amino acid modulates neurotransmission and regulates the kinetics of signaling proteins. To understand the structural basis of proline import, we solved the structure of the proline transporter SIT1 in complex with the COVID-19 viral receptor ACE2 by cryo-electron microscopy. The structure of pipecolate-bound SIT1 reveals the specific sequence requirements for proline transport in the SLC6 family and how this protein excludes amino acids with extended side chains. By comparing apo and substrate-bound SIT1 states, we also identify the structural changes that link substrate release and opening of the cytoplasmic gate and provide an explanation for how a missense mutation in the transporter causes iminoglycinuria.


Subject(s)
Angiotensin-Converting Enzyme 2 , Cryoelectron Microscopy , Proline , SARS-CoV-2 , Angiotensin-Converting Enzyme 2/metabolism , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/genetics , Proline/metabolism , Humans , SARS-CoV-2/metabolism , SARS-CoV-2/genetics , COVID-19/virology , COVID-19/metabolism , Amino Acid Transport Systems, Neutral/metabolism , Amino Acid Transport Systems, Neutral/genetics , Amino Acid Transport Systems, Neutral/chemistry , Models, Molecular
3.
Structure ; 32(7): 849-850, 2024 Jul 11.
Article in English | MEDLINE | ID: mdl-38996509

ABSTRACT

In this issue of Structure, Schneider et al.1 report multiple structures of the low-affinity inorganic-phosphate transporter Pho90 from Saccharomyces cerevisiae. With remarkable resolution of the Divalent Anion Sodium Symporter family member, their cryo-EM studies of this fungal protein reveal new modes of sodium, substrate, and lipid binding.


Subject(s)
Phosphates , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Phosphates/metabolism , Phosphates/chemistry , Cryoelectron Microscopy , Sodium/metabolism
4.
J Mol Biol ; 436(16): 168665, 2024 Aug 15.
Article in English | MEDLINE | ID: mdl-38878854

ABSTRACT

Transporters of the solute carrier superfamily (SLCs) are responsible for the transmembrane traffic of the majority of chemical substances in cells and tissues and are therefore of fundamental biological importance. As is often the case with membrane proteins that can be heavily glycosylated, a lack of reliable high-affinity binders hinders their functional analysis. Purifying and reconstituting transmembrane proteins in their lipidic environments remains challenging and standard approaches to generate binders for multi-transmembrane proteins, such as SLCs, channels or G protein-coupled receptors (GPCRs) are lacking. While generating protein binders to 27 SLCs, we produced full length protein or cell lines as input material for binder generation by selected binder generation platforms. As a result, we obtained 525 binders for 22 SLCs. We validated the binders with a cell-based validation workflow using immunofluorescent and immunoprecipitation methods to process all obtained binders. Finally, we demonstrated the potential applications of the binders that passed our validation pipeline in structural, biochemical, and biological applications using the exemplary protein SLC12A6, an ion transporter relevant in human disease. With this work, we were able to generate easily renewable and highly specific binders against SLCs, which will greatly facilitate the study of this neglected protein family. We hope that the process will serve as blueprint for the generation of binders against the entire superfamily of SLC transporters.


Subject(s)
Protein Binding , Solute Carrier Proteins , Humans , Solute Carrier Proteins/metabolism , Membrane Transport Proteins/metabolism , Membrane Transport Proteins/genetics , Membrane Transport Proteins/chemistry , HEK293 Cells
5.
ACS Chem Neurosci ; 15(7): 1424-1431, 2024 04 03.
Article in English | MEDLINE | ID: mdl-38478848

ABSTRACT

Excitatory amino acid transporters (EAATs) are important regulators of amino acid transport and in particular glutamate. Recently, more interest has arisen in these transporters in the context of neurodegenerative diseases. This calls for ways to modulate these targets to drive glutamate transport, EAAT2 and EAAT3 in particular. Several inhibitors (competitive and noncompetitive) exist to block glutamate transport; however, activators remain scarce. Recently, GT949 was proposed as a selective activator of EAAT2, as tested in a radioligand uptake assay. In the presented research, we aimed to validate the use of GT949 to activate EAAT2-driven glutamate transport by applying an innovative, impedance-based, whole-cell assay (xCELLigence). A broad range of GT949 concentrations in a variety of cellular environments were tested in this assay. As expected, no activation of EAAT3 could be detected. Yet, surprisingly, no biological activation of GT949 on EAAT2 could be observed in this assay either. To validate whether the impedance-based assay was not suited to pick up increased glutamate uptake or if the compound might not induce activation in this setup, we performed radioligand uptake assays. Two setups were utilized; a novel method compared to previously published research, and in a reproducible fashion copying the methods used in the existing literature. Nonetheless, activation of neither EAAT2 nor EAAT3 could be observed in these assays. Furthermore, no evidence of GT949 binding or stabilization of purified EAAT2 could be observed in a thermal shift assay. To conclude, based on experimental evidence in the present study GT949 requires specific assay conditions, which are difficult to reproduce, and the compound cannot simply be classified as an activator of EAAT2 based on the presented evidence. Hence, further research is required to develop the tools needed to identify new EAAT modulators and use their potential as a therapeutic target.


Subject(s)
Excitatory Amino Acid Transporter 2 , Glutamic Acid , Excitatory Amino Acid Transporter 2/metabolism , Electric Impedance , Glutamic Acid/metabolism , Biological Transport , Excitatory Amino Acid Transporter 3/metabolism
6.
Nat Commun ; 15(1): 2533, 2024 Mar 21.
Article in English | MEDLINE | ID: mdl-38514618

ABSTRACT

Small-molecule modulators of diverse voltage-gated K+ (Kv) channels may help treat a wide range of neurological disorders. However, developing effective modulators requires understanding of their mechanism of action. We apply an orthogonal approach to elucidate the mechanism of action of an imidazolidinedione derivative (AUT5), a highly selective positive allosteric modulator of Kv3.1 and Kv3.2 channels. AUT5 modulation involves positive cooperativity and preferential stabilization of the open state. The cryo-EM structure of the Kv3.1/AUT5 complex at a resolution of 2.5 Å reveals four equivalent AUT5 binding sites at the extracellular inter-subunit interface between the voltage-sensing and pore domains of the channel's tetrameric assembly. Furthermore, we show that the unique extracellular turret regions of Kv3.1 and Kv3.2 essentially govern the selective positive modulation by AUT5. High-resolution apo and bound structures of Kv3.1 demonstrate how AUT5 binding promotes turret rearrangements and interactions with the voltage-sensing domain to favor the open conformation.


Subject(s)
Shaw Potassium Channels , Binding Sites , Shaw Potassium Channels/metabolism
7.
J Vis Exp ; (199)2023 09 29.
Article in English | MEDLINE | ID: mdl-37843272

ABSTRACT

Solute carriers (SLCs) are membrane transporters that import and export a range of endogenous and exogenous substrates, including ions, nutrients, metabolites, neurotransmitters, and pharmaceuticals. Despite having emerged as attractive therapeutic targets and markers of disease, this group of proteins is still relatively underdrugged by current pharmaceuticals. Drug discovery projects for these transporters are impeded by limited structural, functional, and physiological knowledge, ultimately due to the difficulties in the expression and purification of this class of membrane-embedded proteins. Here, we demonstrate methods to obtain high-purity, milligram quantities of human SLC transporter proteins using codon-optimized gene sequences. In conjunction with a systematic exploration of construct design and high-throughput expression, these protocols ensure the preservation of the structural integrity and biochemical activity of the target proteins. We also highlight critical steps in the eukaryotic cell expression, affinity purification, and size-exclusion chromatography of these proteins. Ultimately, this workflow yields pure, functionally active, and stable protein preparations suitable for high-resolution structure determination, transport studies, small-molecule engagement assays, and high-throughput in vitro screening.


Subject(s)
Membrane Transport Proteins , Solute Carrier Proteins , Humans , Membrane Transport Proteins/genetics , Membrane Transport Proteins/metabolism , Solute Carrier Proteins/chemistry , Solute Carrier Proteins/metabolism , Drug Discovery/methods , High-Throughput Screening Assays , Membrane Proteins/metabolism , Pharmaceutical Preparations
8.
Mol Cell ; 83(15): 2739-2752.e5, 2023 08 03.
Article in English | MEDLINE | ID: mdl-37499662

ABSTRACT

Solute carrier spinster homolog 2 (SPNS2), one of only four known major facilitator superfamily (MFS) lysolipid transporters in humans, exports sphingosine-1-phosphate (S1P) across cell membranes. Here, we explore the synergistic effects of lipid binding and conformational dynamics on SPNS2's transport mechanism. Using mass spectrometry, we discovered that SPNS2 interacts preferentially with PI(4,5)P2. Together with functional studies and molecular dynamics (MD) simulations, we identified potential PI(4,5)P2 binding sites. Mutagenesis of proposed lipid binding sites and inhibition of PI(4,5)P2 synthesis reduce S1P transport, whereas the absence of the N terminus renders the transporter essentially inactive. Probing the conformational dynamics of SPNS2, we show how synergistic binding of PI(4,5)P2 and S1P facilitates transport, increases dynamics of the extracellular gate, and stabilizes the intracellular gate. Given that SPNS2 transports a key signaling lipid, our results have implications for therapeutic targeting and also illustrate a regulatory mechanism for MFS transporters.


Subject(s)
Lysophospholipids , Sphingosine , Humans , Anion Transport Proteins/genetics , Anion Transport Proteins/metabolism
9.
Nat Commun ; 13(1): 2644, 2022 05 12.
Article in English | MEDLINE | ID: mdl-35551191

ABSTRACT

The Na+-dependent dicarboxylate transporter from Vibrio cholerae (VcINDY) is a prototype for the divalent anion sodium symporter (DASS) family. While the utilization of an electrochemical Na+ gradient to power substrate transport is well established for VcINDY, the structural basis of this coupling between sodium and substrate binding is not currently understood. Here, using a combination of cryo-EM structure determination, succinate binding and site-directed cysteine alkylation assays, we demonstrate that the VcINDY protein couples sodium- and substrate-binding via a previously unseen cooperative mechanism by conformational selection. In the absence of sodium, substrate binding is abolished, with the succinate binding regions exhibiting increased flexibility, including HPinb, TM10b and the substrate clamshell motifs. Upon sodium binding, these regions become structurally ordered and create a proper binding site for the substrate. Taken together, these results provide strong evidence that VcINDY's conformational selection mechanism is a result of the sodium-dependent formation of the substrate binding site.


Subject(s)
Dicarboxylic Acid Transporters , Vibrio cholerae , Binding Sites , Dicarboxylic Acid Transporters/chemistry , Dicarboxylic Acid Transporters/genetics , Dicarboxylic Acid Transporters/metabolism , Sodium/metabolism , Succinic Acid/metabolism , Vibrio cholerae/metabolism
10.
Nat Chem Biol ; 18(7): 706-712, 2022 07.
Article in English | MEDLINE | ID: mdl-35361990

ABSTRACT

Membrane protein efflux pumps confer antibiotic resistance by extruding structurally distinct compounds and lowering their intracellular concentration. Yet, there are no clinically approved drugs to inhibit efflux pumps, which would potentiate the efficacy of existing antibiotics rendered ineffective by drug efflux. Here we identified synthetic antigen-binding fragments (Fabs) that inhibit the quinolone transporter NorA from methicillin-resistant Staphylococcus aureus (MRSA). Structures of two NorA-Fab complexes determined using cryo-electron microscopy reveal a Fab loop deeply inserted in the substrate-binding pocket of NorA. An arginine residue on this loop interacts with two neighboring aspartate and glutamate residues essential for NorA-mediated antibiotic resistance in MRSA. Peptide mimics of the Fab loop inhibit NorA with submicromolar potency and ablate MRSA growth in combination with the antibiotic norfloxacin. These findings establish a class of peptide inhibitors that block antibiotic efflux in MRSA by targeting indispensable residues in NorA without the need for membrane permeability.


Subject(s)
Methicillin-Resistant Staphylococcus aureus , Staphylococcal Infections , Anti-Bacterial Agents/chemistry , Bacterial Proteins/metabolism , Cryoelectron Microscopy , Humans , Microbial Sensitivity Tests , Multidrug Resistance-Associated Proteins/chemistry , Multidrug Resistance-Associated Proteins/metabolism , Multidrug Resistance-Associated Proteins/pharmacology , Staphylococcus aureus/metabolism
11.
FEBS J ; 289(6): 1515-1523, 2022 03.
Article in English | MEDLINE | ID: mdl-34403567

ABSTRACT

The divalent anion sodium symporter (DASS) family contains both sodium-driven anion cotransporters and anion/anion exchangers. The family belongs to a broader ion transporter superfamily (ITS), which comprises 24 families of transporters, including those of AbgT antibiotic efflux transporters. The human proteins in the DASS family play major physiological roles and are drug targets. We recently determined multiple structures of the human sodium-dependent citrate transporter (NaCT) and the succinate/dicarboxylate transporter from Lactobacillus acidophilus (LaINDY). Structures of both proteins show high degrees of structural similarity to the previously determined VcINDY fold. Conservation between these DASS protein structures and those from the AbgT family indicates that the VcINDY fold represents the overall protein structure for the entire ITS. The new structures of NaCT and LaINDY are captured in the inward- or outward-facing conformations, respectively. The domain arrangements in these structures agree with a rigid body elevator-type transport mechanism for substrate translocation across the membrane. Two separate NaCT structures in complex with a substrate or an inhibitor allowed us to explain the inhibition mechanism and propose a detailed classification scheme for grouping disease-causing mutations in the human protein. Structural understanding of multiple kinetic states of DASS proteins is a first step toward the detailed characterization of their entire transport cycle.


Subject(s)
Membrane Transport Proteins , Symporters , Anions/metabolism , Dicarboxylic Acid Transporters/genetics , Dicarboxylic Acid Transporters/metabolism , Humans , Membrane Transport Proteins/genetics , Sodium/metabolism , Symporters/metabolism
12.
Nature ; 591(7848): 157-161, 2021 03.
Article in English | MEDLINE | ID: mdl-33597751

ABSTRACT

Citrate is best known as an intermediate in the tricarboxylic acid cycle of the cell. In addition to this essential role in energy metabolism, the tricarboxylate anion also acts as both a precursor and a regulator of fatty acid synthesis1-3. Thus, the rate of fatty acid synthesis correlates directly with the cytosolic concentration of citrate4,5. Liver cells import citrate through the sodium-dependent citrate transporter NaCT (encoded by SLC13A5) and, as a consequence, this protein is a potential target for anti-obesity drugs. Here, to understand the structural basis of its inhibition mechanism, we determined cryo-electron microscopy structures of human NaCT in complexes with citrate or a small-molecule inhibitor. These structures reveal how the inhibitor-which binds to the same site as citrate-arrests the transport cycle of NaCT. The NaCT-inhibitor structure also explains why the compound selectively inhibits NaCT over two homologous human dicarboxylate transporters, and suggests ways to further improve the affinity and selectivity. Finally, the NaCT structures provide a framework for understanding how various mutations abolish the transport activity of NaCT in the brain and thereby cause epilepsy associated with mutations in SLC13A5 in newborns (which is known as SLC13A5-epilepsy)6-8.


Subject(s)
Carrier Proteins/antagonists & inhibitors , Carrier Proteins/chemistry , Citric Acid/metabolism , Cryoelectron Microscopy , Malates/pharmacology , Phenylbutyrates/pharmacology , Symporters/antagonists & inhibitors , Symporters/chemistry , Binding Sites , Brain/metabolism , Carrier Proteins/genetics , Carrier Proteins/ultrastructure , Citric Acid/chemistry , Dicarboxylic Acid Transporters/chemistry , Dicarboxylic Acid Transporters/metabolism , Epilepsy/genetics , Epilepsy/metabolism , Humans , Malates/chemistry , Models, Molecular , Mutation , Phenylbutyrates/chemistry , Protein Multimerization , Sodium/metabolism , Substrate Specificity/drug effects , Substrate Specificity/genetics , Symporters/genetics , Symporters/ultrastructure
13.
Elife ; 92020 09 01.
Article in English | MEDLINE | ID: mdl-32869741

ABSTRACT

Citrate, α-ketoglutarate and succinate are TCA cycle intermediates that also play essential roles in metabolic signaling and cellular regulation. These di- and tricarboxylates are imported into the cell by the divalent anion sodium symporter (DASS) family of plasma membrane transporters, which contains both cotransporters and exchangers. While DASS proteins transport substrates via an elevator mechanism, to date structures are only available for a single DASS cotransporter protein in a substrate-bound, inward-facing state. We report multiple cryo-EM and X-ray structures in four different states, including three hitherto unseen states, along with molecular dynamics simulations, of both a cotransporter and an exchanger. Comparison of these outward- and inward-facing structures reveal how the transport domain translates and rotates within the framework of the scaffold domain through the transport cycle. Additionally, we propose that DASS transporters ensure substrate coupling by a charge-compensation mechanism, and by structural changes upon substrate release.


Subject(s)
Dicarboxylic Acid Transporters/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli/metabolism , Cryoelectron Microscopy , Crystallography, X-Ray , Lactobacillus acidophilus/metabolism , Molecular Dynamics Simulation
14.
Bioinformatics ; 35(18): 3224-3231, 2019 09 15.
Article in English | MEDLINE | ID: mdl-30689741

ABSTRACT

MOTIVATION: Optimal growth temperature is a fundamental characteristic of all living organisms. Knowledge of this temperature is central to the study of a prokaryote, the thermal stability and temperature dependent activity of its genes, and the bioprospecting of its genome for thermally adapted proteins. While high throughput sequencing methods have dramatically increased the availability of genomic information, the growth temperatures of the source organisms are often unknown. This limits the study and technological application of these species and their genomes. Here, we present a novel method for the prediction of growth temperatures of prokaryotes using only genomic sequences. RESULTS: By applying the reverse ecology principle that an organism's genome includes identifiable adaptations to its native environment, we can predict a species' optimal growth temperature with an accuracy of 5.17°C root-mean-square error and a coefficient of determination of 0.835. The accuracy can be further improved for specific taxonomic clades or by excluding psychrophiles. This method provides a valuable tool for the rapid calculation of organism growth temperature when only the genome sequence is known. AVAILABILITY AND IMPLEMENTATION: Source code, genomes analyzed and features calculated are available at: https://github.com/DavidBSauer/OGT_prediction. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.


Subject(s)
Temperature , Genomics , High-Throughput Nucleotide Sequencing , Sequence Analysis, DNA , Software
15.
Biophys J ; 109(7): 1420-8, 2015 Oct 06.
Article in English | MEDLINE | ID: mdl-26445442

ABSTRACT

Ex vivo stability is a valuable protein characteristic but is laborious to improve experimentally. In addition to biopharmaceutical and industrial applications, stable protein is important for biochemical and structural studies. Taking advantage of the large number of available genomic sequences and growth temperature data, we present two bioinformatic methods to identify a limited set of amino acids or positions that likely underlie thermostability. Because these methods allow thousands of homologs to be examined in silico, they have the advantage of providing both speed and statistical power. Using these methods, we introduced, via mutation, amino acids from thermoadapted homologs into an exemplar mesophilic membrane protein, and demonstrated significantly increased thermostability while preserving protein activity.


Subject(s)
Computational Biology/methods , Mutation , Protein Stability , Temperature , Amino Acids/chemistry , Amino Acids/genetics , Anti-Bacterial Agents/pharmacology , Antiporters/chemistry , Antiporters/genetics , Bacillus subtilis , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Escherichia coli , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Tetracycline/pharmacology , Transfection
16.
Nat Commun ; 4: 2721, 2013.
Article in English | MEDLINE | ID: mdl-24217363

ABSTRACT

Potassium channels selectively conduct K(+), primarily to the exclusion of Na(+), despite the fact that both ions can bind within the selectivity filter. Here we perform crystallographic titration and single-channel electrophysiology to examine the competition of Na(+) and K(+) binding within the filter of two NaK channel mutants; one is the potassium-selective NaK2K mutant and the other is the non-selective NaK2CNG, a CNG channel pore mimic. With high-resolution structures of these engineered NaK channel constructs, we explicitly describe the changes in K(+) occupancy within the filter upon Na(+) competition by anomalous diffraction. Our results demonstrate that the non-selective NaK2CNG still retains a K(+)-selective site at equilibrium, whereas the NaK2K channel filter maintains two high-affinity K(+) sites. A double-barrier mechanism is proposed to explain K(+) channel selectivity at low K(+) concentrations.


Subject(s)
Ion Channel Gating , Potassium Channels/metabolism , Potassium/metabolism , Sodium/metabolism , Bacillus cereus/metabolism , Bacillus subtilis/metabolism , Bacterial Proteins/metabolism , Binding Sites , Binding, Competitive , Crystallography, X-Ray , Electrophysiology , Escherichia coli/metabolism , Liposomes/metabolism , Mutation , Protein Conformation
17.
Science ; 335(6069): 686-90, 2012 Feb 10.
Article in English | MEDLINE | ID: mdl-22323814

ABSTRACT

Sodium/calcium (Na(+)/Ca(2+)) exchangers (NCX) are membrane transporters that play an essential role in maintaining the homeostasis of cytosolic Ca(2+) for cell signaling. We demonstrated the Na(+)/Ca(2+)-exchange function of an NCX from Methanococcus jannaschii (NCX_Mj) and report its 1.9 angstrom crystal structure in an outward-facing conformation. Containing 10 transmembrane helices, the two halves of NCX_Mj share a similar structure with opposite orientation. Four ion-binding sites cluster at the center of the protein: one specific for Ca(2+) and three that likely bind Na(+). Two passageways allow for Na(+) and Ca(2+) access to the central ion-binding sites from the extracellular side. Based on the symmetry of NCX_Mj and its ability to catalyze bidirectional ion-exchange reactions, we propose a structure model for the inward-facing NCX_Mj.


Subject(s)
Archaeal Proteins/chemistry , Calcium/metabolism , Methanococcales/chemistry , Methanococcales/metabolism , Sodium-Calcium Exchanger/chemistry , Sodium-Calcium Exchanger/metabolism , Sodium/metabolism , Amino Acid Sequence , Archaeal Proteins/metabolism , Binding Sites , Crystallization , Crystallography, X-Ray , Ion Transport , Ligands , Models, Molecular , Molecular Sequence Data , Protein Conformation , Protein Structure, Secondary
18.
Proc Natl Acad Sci U S A ; 108(40): 16634-9, 2011 Oct 04.
Article in English | MEDLINE | ID: mdl-21933962

ABSTRACT

The structural and functional conversion of the nonselective NaK channel to a K(+) selective channel (NaK2K) allows us to identify two key residues, Tyr and Asp in the filter sequence of TVGYGD, that participate in interactions central to stabilizing the K(+) channel selectivity filter. By using protein crystallography and channel electrophysiology, we demonstrate that the K(+) channel filter exists as an energetically strained structure and requires these key protein interactions working in concert to hold the filter in the precisely defined four-sited configuration that is essential for selective K(+) permeation. Disruption of either interaction, as tested on both the NaK2K and eukaryotic K(v)1.6 channels, can reduce or completely abolish K(+) selectivity and in some cases may also lead to channel inactivation due to conformational changes at the filter. Additionally, on the scaffold of NaK we recapitulate the protein interactions found in the filter of the Kir channel family, which uses a distinct interaction network to achieve similar stabilization of the filter.


Subject(s)
Models, Molecular , Potassium/chemistry , Protein Conformation , Sodium-Potassium-Exchanging ATPase/metabolism , Amino Acid Motifs , Crystallography , Electrophysiology , Sodium-Potassium-Exchanging ATPase/genetics
19.
Proc Natl Acad Sci U S A ; 108(2): 598-602, 2011 Jan 11.
Article in English | MEDLINE | ID: mdl-21187421

ABSTRACT

Selective ion conduction across ion channel pores is central to cellular physiology. To understand the underlying principles of ion selectivity in tetrameric cation channels, we engineered a set of cation channel pores based on the nonselective NaK channel and determined their structures to high resolution. These structures showcase an ensemble of selectivity filters with a various number of contiguous ion binding sites ranging from 2 to 4, with each individual site maintaining a geometry and ligand environment virtually identical to that of equivalent sites in K(+) channel selectivity filters. Combined with single channel electrophysiology, we show that only the channel with four ion binding sites is K(+) selective, whereas those with two or three are nonselective and permeate Na(+) and K(+) equally well. These observations strongly suggest that the number of contiguous ion binding sites in a single file is the key determinant of the channel's selectivity properties and the presence of four sites in K(+) channels is essential for highly selective and efficient permeation of K(+) ions.


Subject(s)
Ions/chemistry , Bacillus subtilis/metabolism , Binding Sites , Carbon/chemistry , Crystallography, X-Ray/methods , Electrophysiology/methods , Ligands , Mutation , Oxygen/chemistry , Potassium/chemistry , Potassium Channels/chemistry , Protein Binding , Protein Conformation , Sodium/chemistry
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