The pH sensor of the plant K+-uptake channel KAT1 is built from a sensory cloud rather than from single key amino acids

2012 ◽  
Vol 442 (1) ◽  
pp. 57-63 ◽  
Author(s):  
Wendy González ◽  
Janin Riedelsberger ◽  
Samuel E. Morales-Navarro ◽  
Julio Caballero ◽  
Jans H. Alzate-Morales ◽  
...  
Keyword(s):  
Ph Sensor ◽  
Structural Models ◽  
Voltage Sensor ◽  
Activation Mechanism ◽  
Potassium Ions ◽  
Acid Activation ◽  
Extracellular Loop ◽  
K Uptake ◽  
Transmembrane Segments ◽  
Ph Dependency

The uptake of potassium ions (K+) accompanied by an acidification of the apoplasm is a prerequisite for stomatal opening. The acidification (approximately 2–2.5 pH units) is perceived by voltage-gated inward potassium channels (Kin) that then can open their pores with lower energy cost. The sensory units for extracellular pH in stomatal Kin channels are proposed to be histidines exposed to the apoplasm. However, in the Arabidopsis thaliana stomatal Kin channel KAT1, mutations in the unique histidine exposed to the solvent (His267) do not affect the pH dependency. We demonstrate in the present study that His267 of the KAT1 channel cannot sense pH changes since the neighbouring residue Phe266 shifts its pKa to undetectable values through a cation–π interaction. Instead, we show that Glu240 placed in the extracellular loop between transmembrane segments S5 and S6 is involved in the extracellular acid activation mechanism. Based on structural models we propose that this region may serve as a molecular link between the pH- and the voltage-sensor. Like Glu240, several other titratable residues could contribute to the pH-sensor of KAT1, interact with each other and even connect such residues far away from the voltage-sensor with the gating machinery of the channel.

scholarly journals The extracellular loop of the membrane permease VraG interacts with GraS to sense cationic antimicrobial peptides in Staphylococcus aureus

2021 ◽  
Vol 17 (3) ◽  
pp. e1009338
Author(s):  
Junho Cho ◽  
Stephen K. Costa ◽  
Rachel M. Wierzbicki ◽  
William F. C. Rigby ◽  
Ambrose L. Cheung
Keyword(s):  
Positive Charge ◽  
Efflux Pump ◽  
Survival Strategies ◽  
Extracellular Loop ◽  
Major Question ◽  
Defense Proteins ◽  
Bacterial Membranes ◽  
Transmembrane Segments ◽  
Charge Interaction ◽  
Sense And Respond

Host defense proteins (HDPs), aka defensins, are a key part of the innate immune system that functions by inserting into the bacterial membranes to form pores to kill invading and colonizing microorganisms. To ensure survival, microorganism such as S. aureus has developed survival strategies to sense and respond to HDPs. One key strategy in S. aureus is a two-component system (TCS) called GraRS coupled to an efflux pump that consists of a membrane permease VraG and an ATPase VraF, analogous to the BceRS-BceAB system of Bacillus subtilis but with distinct differences. While the 9 negatively charged amino acid extracellular loop of the membrane sensor GraS has been shown to be involved in sensing, the major question is how such a small loop can sense diverse HDPs. Mutation analysis in this study divulged that the vraG mutant phenocopied the graS mutant with respect to reduced activation of downstream effector mprF, reduction in surface positive charge and enhanced 2 hr. killing with LL-37 as compared with the parental MRSA strain JE2. In silico analysis revealed VraG contains a single 200-residue extracellular loop (EL) situated between the 7th and 8th transmembrane segments (out of 10). Remarkably, deletion of EL in VraG enhanced mprF expression, augmented surface positive charge and improved survival in LL-37 vs. parent JE2. As the EL of VraG is rich in lysine residues (16%), in contrast to a preponderance of negatively charged aspartic acid residues (3 out of 9) in the EL of GraS, we divulged the role of charge interaction by showing that K380 in the EL of VraG is an important residue that likely interacts with GraS to interfere with GraS-mediated signaling. Bacterial two-hybrid analysis also supported the interaction of EL of VraG with the EL of GraS. Collectively, we demonstrated an interesting facet of efflux pumps whereby the membrane permease disrupts HDP signaling by inhibiting GraS sensing that involves charged residues in the EL of VraG.

scholarly journals Molecular basis of plant-specific acid activation of K+ uptake channels

1997 ◽  
Vol 94 (9) ◽  
pp. 4806-4810 ◽  
Author(s):  
S. Hoth ◽  
I. Dreyer ◽  
P. Dietrich ◽  
D. Becker ◽  
B. Muller-Rober ◽  
...  
Keyword(s):  
Molecular Basis ◽  
Acid Activation ◽  
K Uptake

scholarly journals Functionality of the voltage-gated proton channel truncated in S4

2009 ◽  
Vol 107 (5) ◽  
pp. 2313-2318 ◽  
Author(s):  
Souhei Sakata ◽  
Tatsuki Kurokawa ◽  
Morten H. H. Nørholm ◽  
Masahiro Takagi ◽  
Yoshifumi Okochi ◽  
...  
Keyword(s):  
Voltage Sensor ◽  
Proton Channel ◽  
Voltage Sensing ◽  
Proton Channels ◽  
Voltage Gated ◽  
Transmembrane Segments ◽  
Cysteine Scanning ◽  
Dog Pancreas ◽  
Voltage Gated Ion Channels ◽  
Channel Properties

The voltage sensor domain (VSD) is the key module for voltage sensing in voltage-gated ion channels and voltage-sensing phosphatases. Structurally, both the VSD and the recently discovered voltage-gated proton channels (Hv channels) voltage sensor only protein (VSOP) and Hv1 contain four transmembrane segments. The fourth transmembrane segment (S4) of Hv channels contains three periodically aligned arginines (R1, R2, R3). It remains unknown where protons permeate or how voltage sensing is coupled to ion permeation in Hv channels. Here we report that Hv channels truncated just downstream of R2 in the S4 segment retain most channel properties. Two assays, site-directed cysteine-scanning using accessibility of maleimide-reagent as detected by Western blotting and insertion into dog pancreas microsomes, both showed that S4 inserts into the membrane, even if it is truncated between the R2 and R3 positions. These findings provide important clues to the molecular mechanism underlying voltage sensing and proton permeation in Hv channels.

Abstract 3499: Extracellular Loop (Domain I, S5-S6) of α1D L-type Ca Channel is an Antigenic Site in Autoimmune Associated Congenital Heart Block

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Eddy Karnabi ◽  
Yongxia Qu ◽  
Raj Wadgaonkar ◽  
Salvatore Mancarella ◽  
Yunkun Yue ◽  
...  
Keyword(s):  
Heart Block ◽  
Congenital Heart ◽  
Antigenic Site ◽  
Congenital Heart Block ◽  
Ca Channel ◽  
Extracellular Loop ◽  
Channel Protein ◽  
Transmembrane Segments ◽  
Electrophysiological Characterization ◽  
Domain I

Congenital heart block (CHB) is an autoimmune disease associated with autoantibodies against intracellular ribonucleoproteins SSB/La and SSA/Ro. The hallmark of CHB is complete atrioventricular block. We have established that anti-SSA/Ro -SSB/La autoantibodies inhibit L-type α 1D Ca current, I Ca-L and cross-react with the α 1D Ca channel protein. This study aims at identifying the possible binding sites on α 1D protein for autoantibodies from sera of mothers with CHB children. GST fusion proteins of the extracellular regions between the transmembrane segments (S5-S6) of each of the four α 1D Ca channel protein domains I–IV were prepared and tested for reactivity with sera from mothers with CHB children and controls using ELISA. Sera from 118 mothers with CHB children and 28 control healthy sera were used in this study. Seventeen of 118 (14.4%) maternal CHB sera reacted with the extracellular loop of Domain I S5-S6 region (E1). In contrast, 2 of 28 (7%) healthy control sera reacted with the E1 loop. Preincubation of E1 loop with the positive sera decreased the O.D. reading of the positive sera establishing the specificity of the response. Electrophysiological characterization of the ELISA positive sera demonstrated inhibition (44.1%) of the α 1D I Ca-L expressed in tsA201 cells. The inhibition was abolished when the sera were pre-incubated with E1 extracellular loop fusion protein. The results identified the extracellular loop of domain I S5-S6 of L-type Ca channel α 1D subunit as a target for autoantibodies from mothers with CHB children. This novel finding provides insights into the development of therapeutic peptides that could bind to the pathogenic antibodies.

scholarly journals Mg2+ Modulates Voltage-Dependent Activation in Ether-à-Go-Go Potassium Channels by Binding between Transmembrane Segments S2 and S3

2000 ◽  
Vol 116 (5) ◽  
pp. 663-678 ◽  
Author(s):  
William R. Silverman ◽  
Chih-Yung Tang ◽  
Allan F. Mock ◽  
Kyung-Bong Huh ◽  
Diane M. Papazian
Keyword(s):  
Potassium Channels ◽  
Binding Site ◽  
Voltage Sensor ◽  
Wild Type ◽  
Activation Kinetics ◽  
Fast Kinetics ◽  
K Channels ◽  
Transmembrane Segments ◽  
Acidic Residues ◽  
Voltage Dependent

Extracellular Mg2+ directly modulates voltage-dependent activation in ether-à-go-go (eag) potassium channels, slowing the kinetics of ionic and gating currents (Tang, C.-Y., F. Bezanilla, and D.M. Papazian. 2000. J. Gen. Physiol. 115:319-337). To exert its effect, Mg2+ presumably binds to a site in or near the eag voltage sensor. We have tested the hypothesis that acidic residues unique to eag family members, located in transmembrane segments S2 and S3, contribute to the Mg2+-binding site. Two eag-specific acidic residues and three acidic residues found in the S2 and S3 segments of all voltage-dependent K+ channels were individually mutated in Drosophila eag, mutant channels were expressed in Xenopus oocytes, and the effect of Mg2+ on ionic current kinetics was measured using a two electrode voltage clamp. Neutralization of eag-specific residues D278 in S2 and D327 in S3 eliminated Mg2+-sensitivity and mimicked the slowing of activation kinetics caused by Mg2+ binding to the wild-type channel. These results suggest that Mg2+ modulates activation kinetics in wild-type eag by screening the negatively charged side chains of D278 and D327. Therefore, these residues are likely to coordinate the bound ion. In contrast, neutralization of the widely conserved residues D284 in S2 and D319 in S3 preserved the fast kinetics seen in wild-type eag in the absence of Mg2+, indicating that D284 and D319 do not mediate the slowing of activation caused by Mg2+ binding. Mutations at D284 affected the eag gating pathway, shifting the voltage dependence of Mg2+-sensitive, rate limiting transitions in the hyperpolarized direction. Another widely conserved residue, D274 in S2, is not required for Mg2+ sensitivity but is in the vicinity of the binding site. We conclude that Mg2+ binds in a water-filled pocket between S2 and S3 and thereby modulates voltage-dependent gating. The identification of this site constrains the packing of transmembrane segments in the voltage sensor of K+ channels, and suggests a molecular mechanism by which extracellular cations modulate eag activation kinetics.

scholarly journals Topology of transmembrane segments 1–4 in the human chloride/bicarbonate anion exchanger 1 (AE1) by scanning N-glycosylation mutagenesis

2005 ◽  
Vol 390 (1) ◽  
pp. 137-144 ◽  
Author(s):  
Joanne C. Cheung ◽  
Jing Li ◽  
Reinhart A. F. Reithmeier
Keyword(s):  
Plasma Membrane ◽  
Anion Exchanger ◽  
Physiological Role ◽  
Mutant Form ◽  
Extracellular Loop ◽  
Anion Exchanger 1 ◽  
Hek 293 ◽  
Human Embryonic Kidney Cells ◽  
Transmembrane Segments

Human AE1 (anion exchanger 1), or Band 3, is an abundant membrane glycoprotein found in the plasma membrane of erythrocytes. The physiological role of the protein is to carry out chloride/bicarbonate exchange across the plasma membrane, a process that increases the carbon-dioxide-carrying capacity of blood. To study the topology of TMs (transmembrane segments) 1–4, a series of scanning N-glycosylation mutants were created spanning the region from EC (extracellular loop) 1 to EC2 in full-length AE1. These constructs were expressed in HEK-293 (human embryonic kidney) cells, and their N-glycosylation efficiencies were determined. Unexpectedly, positions within putative TMs 2 and 3 could be efficiently glycosylated. In contrast, the same positions were very poorly glycosylated when present in mutant AE1 with the SAO (Southeast Asian ovalocytosis) deletion (ΔA400–A408) in TM1. These results suggest that the TM2–3 region of AE1 may become transiently exposed to the endoplasmic reticulum lumen during biosynthesis, and that there is a competition between proper folding of the region into the membrane and N-glycosylation at introduced sites. The SAO deletion disrupts the proper integration of TMs 1–2, probably leaving the region exposed to the cytosol. As a result, engineered N-glycosylation acceptor sites in TM2–3 could not be utilized by the oligosaccharyltransferase in this mutant form of AE1. The properties of TM2–3 suggest that these segments form a re-entrant loop in human AE1.

scholarly journals A simplified methodology: pH sensing using an in situ fabricated Ir electrode under neutral conditions

2021 ◽  
Author(s):  
Yuqi Chen ◽  
Xiuting Li ◽  
Danlei Li ◽  
Christopher Batchelor-McAuley ◽  
Richard G. Compton
Keyword(s):  
Ph Sensor ◽  
Iridium Oxide ◽  
Ph Sensitive ◽  
Ph Sensing ◽  
Ph Dependency ◽  
Acidic Conditions ◽  
Ph Range ◽  
Neutral Conditions ◽  
Electrochemical Fabrication

AbstractHerein, a simplified fabrication method for the producing of a pH-sensitive iridium electrode is developed. The in situ electrochemical fabrication of an iridium oxide film is optimized and shown to be achievable under neutral conditions rather than the acidic conditions hitherto employed. The formation of a pH sensitive Ir(III/IV) hydrous film is confirmed via XPS. The amperometric pH-sensing properties of this electrochemically generated material were investigated using square wave voltammetry. In the pH range 2–13, the iridium oxide redox signal has a pH dependency of 86.1 ± 1.1 mV per pH unit for midpoint potentials with uncertainties being ± 0.01–0.05 pH. Finally, the newly developed pH sensor was used to measure the pH of a natural water sample with excellent results as compared to a conventional glass pH probe.

scholarly journals Acid activation mechanism of the influenza A M2 proton channel

2016 ◽  
Vol 113 (45) ◽  
pp. E6955-E6964 ◽  
Author(s):  
Ruibin Liang ◽  
Jessica M. J. Swanson ◽  
Jesper J. Madsen ◽  
Mei Hong ◽  
William F. DeGrado ◽  
...  
Keyword(s):  
Free Energy ◽  
Proton Transport ◽  
Influenza A ◽  
Conduction Mechanism ◽  
Transmembrane Domain ◽  
Proton Conduction ◽  
Proton Flux ◽  
Activation Mechanism ◽  
Proton Channel ◽  
Acid Activation

The homotetrameric influenza A M2 channel (AM2) is an acid-activated proton channel responsible for the acidification of the influenza virus interior, an important step in the viral lifecycle. Four histidine residues (His37) in the center of the channel act as a pH sensor and proton selectivity filter. Despite intense study, the pH-dependent activation mechanism of the AM2 channel has to date not been completely understood at a molecular level. Herein we have used multiscale computer simulations to characterize (with explicit proton transport free energy profiles and their associated calculated conductances) the activation mechanism of AM2. All proton transfer steps involved in proton diffusion through the channel, including the protonation/deprotonation of His37, are explicitly considered using classical, quantum, and reactive molecular dynamics methods. The asymmetry of the proton transport free energy profile under high-pH conditions qualitatively explains the rectification behavior of AM2 (i.e., why the inward proton flux is allowed when the pH is low in viral exterior and high in viral interior, but outward proton flux is prohibited when the pH gradient is reversed). Also, in agreement with electrophysiological results, our simulations indicate that the C-terminal amphipathic helix does not significantly change the proton conduction mechanism in the AM2 transmembrane domain; the four transmembrane helices flanking the channel lumen alone seem to determine the proton conduction mechanism.

scholarly journals Access of Extracellular Cations to their Binding Sites in Na,K-ATPase: Role of the Second Extracellular Loop of the α Subunit

2006 ◽  
Vol 127 (3) ◽  
pp. 341-352 ◽  
Author(s):  
Oihana Capendeguy ◽  
Pierre Chodanowski ◽  
Olivier Michielin ◽  
Jean-Daniel Horisberger
Keyword(s):  
Binding Sites ◽  
Structural Model ◽  
Maximum Activity ◽  
Extracellular Loop ◽  
Animal Cells ◽  
Α Subunit ◽  
Transmembrane Segments ◽  
Gating Mechanism ◽  
Extracellular Side ◽  
Active Transport System

Na,K-ATPase, the main active transport system for monovalent cations in animal cells, is responsible for maintaining Na+ and K+ gradients across the plasma membrane. During its transport cycle it binds three cytoplasmic Na+ ions and releases them on the extracellular side of the membrane, and then binds two extracellular K+ ions and releases them into the cytoplasm. The fourth, fifth, and sixth transmembrane helices of the α subunit of Na,K-ATPase are known to be involved in Na+ and K+ binding sites, but the gating mechanisms that control the access of these ions to their binding sites are not yet fully understood. We have focused on the second extracellular loop linking transmembrane segments 3 and 4 and attempted to determine its role in gating. We replaced 13 residues of this loop in the rat α1 subunit, from E314 to G326, by cysteine, and then studied the function of these mutants using electrophysiological techniques. We analyzed the results using a structural model obtained by homology with SERCA, and ab initio calculations for the second extracellular loop. Four mutants were markedly modified by the sulfhydryl reagent MTSET, and we investigated them in detail. The substituted cysteines were more readily accessible to MTSET in the E1 conformation for the Y315C, W317C, and I322C mutants. Mutations or derivatization of the substituted cysteines in the second extracellular loop resulted in major increases in the apparent affinity for extracellular K+, and this was associated with a reduction in the maximum activity. The changes produced by the E314C mutation were reversed by MTSET treatment. In the W317C and I322C mutants, MTSET also induced a moderate shift of the E1/E2 equilibrium towards the E1(Na) conformation under Na/Na exchange conditions. These findings indicate that the second extracellular loop must be functionally linked to the gating mechanism that controls the access of K+ to its binding site.

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