The Journal of General Physiology最新文献

{"title":"The connexin26 human mutation N14K disrupts cytosolic intersubunit interactions and promotes channel opening.","authors":"Juan M Valdez Capuccino,&nbsp;Payal Chatterjee,&nbsp;Isaac E García,&nbsp;Wesley M Botello-Smith,&nbsp;Han Zhang,&nbsp;Andrew L Harris,&nbsp;Yun Luo,&nbsp;Jorge E Contreras","doi":"10.1085/jgp.201812219","DOIUrl":"https://doi.org/10.1085/jgp.201812219","url":null,"abstract":"<p><p>A group of human mutations within the N-terminal (NT) domain of connexin 26 (Cx26) hemichannels produce aberrant channel activity, which gives rise to deafness and skin disorders, including keratitis-ichthyosis-deafness (KID) syndrome. Structural and functional studies indicate that the NT of connexin hemichannels is folded into the pore, where it plays important roles in permeability and gating. In this study, we explore the molecular basis by which N14K, an NT KID mutant, promotes gain of function. In macroscopic and single-channel recordings, we find that the N14K mutant favors the open conformation of hemichannels, shifts calcium and voltage sensitivity, and slows deactivation kinetics. Multiple copies of MD simulations of WT and N14K hemichannels, followed by the Kolmogorov-Smirnov significance test (KS test) of the distributions of interaction energies, reveal that the N14K mutation significantly disrupts pairwise interactions that occur in WT hemichannels between residue K15 of one subunit and residue E101 of the adjacent subunit (E101 being located at the transition between transmembrane segment 2 [TM2] and the cytoplasmic loop [CL]). Double mutant cycle analysis supports coupling between the NT and the TM2/CL transition in WT hemichannels, which is disrupted in N14K mutant hemichannels. KS tests of the α carbon correlation coefficients calculated over MD trajectories suggest that the effects of the N14K mutation are not confined to the K15-E101 pairs but extend to essentially all pairwise residue correlations between the NT and TM2/CL interface. Together, our data indicate that the N14K mutation increases hemichannel open probability by disrupting interactions between the NT and the TM2/CL region of the adjacent connexin subunit. This suggests that NT-TM2/CL interactions facilitate Cx26 hemichannel closure.</p>","PeriodicalId":173753,"journal":{"name":"The Journal of General Physiology","volume":" ","pages":"328-341"},"PeriodicalIF":3.8,"publicationDate":"2019-03-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1085/jgp.201812219","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36754521","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Interpretation of spectroscopic data using molecular simulations for the secondary active transporter BetP.","authors":"Vanessa Leone,&nbsp;Izabela Waclawska,&nbsp;Katharina Kossmann,&nbsp;Caroline Koshy,&nbsp;Monika Sharma,&nbsp;Thomas F Prisner,&nbsp;Christine Ziegler,&nbsp;Burkhard Endeward,&nbsp;Lucy R Forrest","doi":"10.1085/jgp.201812111","DOIUrl":"https://doi.org/10.1085/jgp.201812111","url":null,"abstract":"<p><p>Mechanistic understanding of dynamic membrane proteins such as transporters, receptors, and channels requires accurate depictions of conformational ensembles, and the manner in which they interchange as a function of environmental factors including substrates, lipids, and inhibitors. Spectroscopic techniques such as electron spin resonance (ESR) pulsed electron-electron double resonance (PELDOR), also known as double electron-electron resonance (DEER), provide a complement to atomistic structures obtained from x-ray crystallography or cryo-EM, since spectroscopic data reflect an ensemble and can be measured in more native solvents, unperturbed by a crystal lattice. However, attempts to interpret DEER data are frequently stymied by discrepancies with the structural data, which may arise due to differences in conditions, the dynamics of the protein, or the flexibility of the attached paramagnetic spin labels. Recently, molecular simulation techniques such as EBMetaD have been developed that create a conformational ensemble matching an experimental distance distribution while applying the minimal possible bias. Moreover, it has been proposed that the work required during an EBMetaD simulation to match an experimentally determined distribution could be used as a metric with which to assign conformational states to a given measurement. Here, we demonstrate the application of this concept for a sodium-coupled transport protein, BetP. Because the probe, protein, and lipid bilayer are all represented in atomic detail, the different contributions to the work, such as the extent of protein backbone movements, can be separated. This work therefore illustrates how ranking simulations based on EBMetaD can help to bridge the gap between structural and biophysical data and thereby enhance our understanding of membrane protein conformational mechanisms.</p>","PeriodicalId":173753,"journal":{"name":"The Journal of General Physiology","volume":" ","pages":"381-394"},"PeriodicalIF":3.8,"publicationDate":"2019-03-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1085/jgp.201812111","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36926259","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Membrane physiologists of all kinds meet at Woods Hole.","authors":"Janice L Robertson,&nbsp;José D Faraldo-Gómez","doi":"10.1085/jgp.201912340","DOIUrl":"https://doi.org/10.1085/jgp.201912340","url":null,"abstract":"A special issue of JGP celebrates the 2018 Symposium of the Society of General Physiologists, where diversity and world-class science went hand in hand.","PeriodicalId":173753,"journal":{"name":"The Journal of General Physiology","volume":" ","pages":"273"},"PeriodicalIF":3.8,"publicationDate":"2019-03-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1085/jgp.201912340","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36991617","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Novel alanine serine cysteine transporter 2 (ASCT2) inhibitors based on sulfonamide and sulfonic acid ester scaffolds.","authors":"Elias Ndaru,&nbsp;Rachel-Ann A Garibsingh,&nbsp;YueYue Shi,&nbsp;Evan Wallace,&nbsp;Paul Zakrepine,&nbsp;Jiali Wang,&nbsp;Avner Schlessinger,&nbsp;Christof Grewer","doi":"10.1085/jgp.201812276","DOIUrl":"https://doi.org/10.1085/jgp.201812276","url":null,"abstract":"<p><p>The neutral amino acid transporter alanine serine cysteine transporter 2 (ASCT2) belongs to the solute carrier 1 (SLC1) family of transport proteins and transports neutral amino acids, such as alanine and glutamine, into the cell in exchange with intracellular amino acids. This amino acid transport is sodium dependent, but not driven by the transmembrane Na⁺ concentration gradient. Glutamine transport by ASCT2 is proposed to be important for glutamine homoeostasis in rapidly growing cancer cells to fulfill the energy and nitrogen demands of these cells. Thus, ASCT2 is thought to be a potential anticancer drug target. However, the pharmacology of the amino acid binding site is not well established. Here, we report on the synthesis and characterization of a novel class of ASCT2 inhibitors based on an amino acid scaffold with a sulfonamide/sulfonic acid ester linker to a hydrophobic group. The compounds were designed based on an improved ASCT2 homology model using the human glutamate transporter hEAAT1 crystal structure as a modeling template. The compounds were shown to inhibit with a competitive mechanism and a potency that scales with the hydrophobicity of the side chain. The most potent compound binds with an apparent affinity, <i>K</i> _i, of 8 ± 4 µM and can block the alanine response with a <i>K</i> _i of 40 ± 23 µM at 200 µM alanine concentration. Computational analysis predicts inhibitor interactions with the binding site through molecular docking. In conclusion, the sulfonamide/sulfonic acid ester scaffold provides facile synthetic access to ASCT2 inhibitors with a potentially large variability in chemical space of the hydrophobic side chain. These inhibitors will be useful chemical tools to further characterize the role of ASCT2 in disease as well as improve our understanding of inhibition mechanisms of this transporter.</p>","PeriodicalId":173753,"journal":{"name":"The Journal of General Physiology","volume":" ","pages":"357-368"},"PeriodicalIF":3.8,"publicationDate":"2019-03-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1085/jgp.201812276","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36976930","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Unlocking the gating mechanism of Kv2.1 using guangxitoxin.","authors":"Marco A Navarro,&nbsp;Lorin S Milescu,&nbsp;Mirela Milescu","doi":"10.1085/jgp.201812254","DOIUrl":"https://doi.org/10.1085/jgp.201812254","url":null,"abstract":"Navarro et al discuss new work using the gating-modifier toxin GxTx to investigate the molecular mechanism of Kv2.1 channel gating.","PeriodicalId":173753,"journal":{"name":"The Journal of General Physiology","volume":" ","pages":"275-278"},"PeriodicalIF":3.8,"publicationDate":"2019-03-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1085/jgp.201812254","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36795848","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The rhomboid protease GlpG has weak interaction energies in its active site hydrogen bond network.","authors":"Kristen A Gaffney,&nbsp;Heedeok Hong","doi":"10.1085/jgp.201812047","DOIUrl":"https://doi.org/10.1085/jgp.201812047","url":null,"abstract":"<p><p>Intramembrane rhomboid proteases are of particular interest because of their function to hydrolyze a peptide bond of a substrate buried in the membrane. Crystal structures of the bacterial rhomboid protease GlpG have revealed a catalytic dyad (Ser201-His254) and oxyanion hole (His150/Asn154/the backbone amide of Ser201) surrounded by the protein matrix and contacting a narrow water channel. Although multiple crystal structures have been solved, the catalytic mechanism of GlpG is not completely understood. Because it is a serine protease, hydrogen bonding interactions between the active site residues are thought to play a critical role in the catalytic cycle. Here, we dissect the interaction energies among the active site residues His254, Ser201, and Asn154 of <i>Escherichia coli</i> GlpG, which form a hydrogen bonding network. We combine double mutant cycle analysis with stability measurements using steric trapping. In mild detergent, the active site residues are weakly coupled with interaction energies (ΔΔ<i>G</i> _Inter) of ‒1.4 kcal/mol between His254 and Ser201 and ‒0.2 kcal/mol between Ser201 and Asn154. Further, by analyzing the propagation of single mutations of the active site residues, we find that these residues are important not only for function but also for the folding cooperativity of GlpG. The weak interaction between Ser and His in the catalytic dyad may partly explain the unusually slow proteolysis by GlpG compared with other canonical serine proteases. Our result suggests that the weak hydrogen bonds in the active site are sufficient to carry out the proteolytic function of rhomboid proteases.</p>","PeriodicalId":173753,"journal":{"name":"The Journal of General Physiology","volume":" ","pages":"282-291"},"PeriodicalIF":3.8,"publicationDate":"2019-03-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1085/jgp.201812047","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36658410","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Models to determine the kinetic mechanisms of ion-coupled transporters.","authors":"Juke S Lolkema, Dirk J Slotboom","doi":"10.1085/jgp.201812055","DOIUrl":"10.1085/jgp.201812055","url":null,"abstract":"<p><p>With high-resolution structures available for many ion-coupled (secondary active) transporters, a major challenge for the field is to determine how coupling is accomplished. Knowledge of the kinetic mechanism of the transport reaction, which defines the binding order of substrate and co-ions, together with the sequence with which all relevant states are visited by the transporter, will help to reveal this coupling mechanism. Here, we derived general mathematical models that can be used to analyze data from steady-state transport measurements and show how kinetic mechanisms can be derived. The models describe how the apparent maximal rate of substrate transport depends on the co-ion concentration, and vice versa, in different mechanisms. Similarly, they describe how the apparent affinity for the transported substrate is affected by the co-ion concentration and vice versa. Analyses of maximal rates and affinities permit deduction of the number of co-ions that bind before, together with, and after the substrate. Hill analysis is less informative, but in some mechanisms, it can reveal the total number of co-ions transported with the substrate. However, prior knowledge of the number of co-ions from other experimental approaches is preferred when deriving kinetic mechanisms, because the models are generally overparameterized. The models we present have wide applicability for the study of ion-coupled transporters.</p>","PeriodicalId":173753,"journal":{"name":"The Journal of General Physiology","volume":" ","pages":"369-380"},"PeriodicalIF":0.0,"publicationDate":"2019-03-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/f9/d8/JGP_201812055.PMC6400521.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36852798","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Interrogating the conformational dynamics of BetP transport.","authors":"Janice L Robertson","doi":"10.1085/jgp.201812315","DOIUrl":"https://doi.org/10.1085/jgp.201812315","url":null,"abstract":"Robertson reflects on a new study showing how ensemble-biased metadynamics can be used to interpret BetP protein dynamics data.","PeriodicalId":173753,"journal":{"name":"The Journal of General Physiology","volume":" ","pages":"279-281"},"PeriodicalIF":3.8,"publicationDate":"2019-03-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1085/jgp.201812315","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36963056","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The tarantula toxin GxTx detains K⁺ channel gating charges in their resting conformation.","authors":"Drew C Tilley,&nbsp;Juan M Angueyra,&nbsp;Kenneth S Eum,&nbsp;Heesoo Kim,&nbsp;Luke H Chao,&nbsp;Anthony W Peng,&nbsp;Jon T Sack","doi":"10.1085/jgp.201812213","DOIUrl":"https://doi.org/10.1085/jgp.201812213","url":null,"abstract":"<p><p>Allosteric ligands modulate protein activity by altering the energy landscape of conformational space in ligand-protein complexes. Here we investigate how ligand binding to a K⁺ channel's voltage sensor allosterically modulates opening of its K⁺-conductive pore. The tarantula venom peptide guangxitoxin-1E (GxTx) binds to the voltage sensors of the rat voltage-gated K⁺ (Kv) channel Kv2.1 and acts as a partial inverse agonist. When bound to GxTx, Kv2.1 activates more slowly, deactivates more rapidly, and requires more positive voltage to reach the same K⁺-conductance as the unbound channel. Further, activation kinetics are more sigmoidal, indicating that multiple conformational changes coupled to opening are modulated. Single-channel current amplitudes reveal that each channel opens to full conductance when GxTx is bound. Inhibition of Kv2.1 channels by GxTx results from decreased open probability due to increased occurrence of long-lived closed states; the time constant of the final pore opening step itself is not impacted by GxTx. When intracellular potential is less than 0 mV, GxTx traps the gating charges on Kv2.1's voltage sensors in their most intracellular position. Gating charges translocate at positive voltages, however, indicating that GxTx stabilizes the most intracellular conformation of the voltage sensors (their resting conformation). Kinetic modeling suggests a modulatory mechanism: GxTx reduces the probability of voltage sensors activating, giving the pore opening step less frequent opportunities to occur. This mechanism results in K⁺-conductance activation kinetics that are voltage-dependent, even if pore opening (the rate-limiting step) has no inherent voltage dependence. We conclude that GxTx stabilizes voltage sensors in a resting conformation, and inhibits K⁺ currents by limiting opportunities for the channel pore to open, but has little, if any, direct effect on the microscopic kinetics of pore opening. The impact of GxTx on channel gating suggests that Kv2.1's pore opening step does not involve movement of its voltage sensors.</p>","PeriodicalId":173753,"journal":{"name":"The Journal of General Physiology","volume":" ","pages":"292-315"},"PeriodicalIF":3.8,"publicationDate":"2019-03-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1085/jgp.201812213","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36638356","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A forward genetic screen identifies chaperone CNX-1 as a conserved biogenesis regulator of <i>ERG</i> K⁺ channels.","authors":"Xue Bai,&nbsp;Kai Li,&nbsp;Li Yao,&nbsp;Xin-Lei Kang,&nbsp;Shi-Qing Cai","doi":"10.1085/jgp.201812025","DOIUrl":"https://doi.org/10.1085/jgp.201812025","url":null,"abstract":"<p><p>The human ether-a-go-go-related gene (hERG) encodes a voltage-gated potassium channel that controls repolarization of cardiac action potentials. Accumulating evidence suggests that most disease-related hERG mutations reduce the function of the channel by disrupting protein biogenesis of the channel in the endoplasmic reticulum (ER). However, the molecular mechanism underlying the biogenesis of ERG K⁺ channels is largely unknown. By forward genetic screening, we identified an ER-located chaperone CNX-1, the worm homologue of mammalian chaperone Calnexin, as a critical regulator for the protein biogenesis of UNC-103, the ERG-type K⁺ channel in <i>Caenorhabditis elegans</i> Loss-of-function mutations of <i>cnx-1</i> decreased the protein level and current density of the UNC-103 K⁺ channel and suppressed the behavioral defects caused by a gain-of-function mutation in <i>unc-103</i> Moreover, CNX-1 facilitated tetrameric assembly of UNC-103 channel subunits in a liposome-assisted cell-free translation system. Further studies showed that CNX-1 act in parallel to DNJ-1, another ER-located chaperone known to regulate maturation of UNC-103 channels, on controlling the protein biogenesis of UNC-103. Importantly, Calnexin interacted with hERG proteins in the ER in HEK293T cells. Deletion of <i>calnexin</i> reduced the expression and current densities of endogenous hERG K⁺ channels in SH-SY5Y cells. Collectively, we reveal an evolutionarily conserved chaperone CNX-1/Calnexin controlling the biogenesis of ERG-type K⁺ channels.</p>","PeriodicalId":173753,"journal":{"name":"The Journal of General Physiology","volume":" ","pages":"1189-1201"},"PeriodicalIF":3.8,"publicationDate":"2018-08-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1085/jgp.201812025","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36256722","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}