Variants with increased negative electrostatic potential in the Cx50 gap junction pore increased unitary channel conductance and magnesium modulation

2018 ◽  
Vol 475 (21) ◽  
pp. 3315-3330 ◽  
Author(s):  
Mary Grace Tejada ◽  
Swathy Sudhakar ◽  
Nicholas K. Kim ◽  
Hiroshi Aoyama ◽  
Brian H. Shilton ◽  
...  
Keyword(s):  
Gap Junction ◽  
Structural Models ◽  
Channel Conductance ◽  
Intracellular Magnesium ◽  
Amino Terminal ◽  
Molecular Determinants ◽  
Charged Residues ◽  
Voltage Dependent ◽  
Pore Lining ◽  
Positively Charged

Gap junction (GJ) channels are oligomers of connexins forming channels linking neighboring cells. GJs formed by different connexins show distinct unitary channel conductance (γj), transjunctional voltage-dependent gating (Vj-gating) properties, and modulation by intracellular magnesium ([Mg2+]i). The underlying molecular determinants are not fully clear. Previous experimental evidence indicates that residues in the amino terminal (NT) and initial segment of the first extracellular (E1) domain influence the γj, Vj-gating, and/or [Mg2+]i modulation in several GJs. Increasing negatively charged residues in Cx50 (connexin50) E1 (G46D or G46E) increased γj, while increasing positively charged residue (G46K) reduced the γj. Sequence alignment of Cx50 and Cx37 in the NT and E1 domains revealed that in Cx50 G8 and V53, positions are negatively charged residues in Cx37 (E8 and E53, respectively). To evaluate these residues together, we generated a triple variant in Cx50, G8E, G46E, and V53E simultaneously to study its γj, Vj-gating properties, and modulation by [Mg2+]i. Our data indicate that the triple variant and individual variants G8E, G46E, and V53E significantly increased Cx50 GJ γj without a significant change in the Vj gating. In addition, elevated [Mg2+]i reduced γj in Cx50 and all the variant GJs. These results and our homology structural models suggest that these NT/E1 residues are likely to be pore-lining and the variants increased the negative electrostatic potentials along the GJ pore to facilitate the γj of this cation-preferring GJ channel. Our results indicate that electrostatic properties of the Cx50 GJ pore are important for the γj and the [Mg2+]i modulation.

scholarly journals Charge-Based Inhibitors of Amylin Fibrillization and Toxicity

2015 ◽  
Vol 2015 ◽  
pp. 1-13 ◽  
Author(s):  
Sharadrao M. Patil ◽  
Andrei T. Alexandrescu
Keyword(s):  
Type 2 Diabetes ◽  
Ionic Strength ◽  
Mouse Model ◽  
Electrostatic Interactions ◽  
Amyloid Fibril ◽  
Structural Models ◽  
Electrostatic Repulsion ◽  
Charged Residues ◽  
Positively Charged

To test the hypothesis that electrostatic repulsion is an important force opposing amyloid fibril assembly, we designed peptides that substitute strings of positively or negatively charged residues into the sequence of the amyloidogenic hormone amylin, which contributes to type 2 diabetes pathology. Arg-1 and Arg-2 substitute four positively charged arginines for segments that in structural models of amylin fibrils form the end of strandβ1 and the beginning of strandβ2, respectively. Mem-T substitutes negatively charged aspartates for the peptide segment with the largest avidity for membranes. All three charge-loaded peptides fibrillize poorly on their own and inhibit fibril elongation of WT-amylin at physiological ionic strength. The inhibition of WT-amylin fibril elongation rates is salt-dependent indicating that the analogs act through electrostatic interactions. Arg-1 protects against WT-amylin cytotoxicity towards a MIN6 mouse model of pancreaticβ-cells, and Arg-2 protects at higher concentrations, whereas Mem-T has no effect. The most effective variant, Arg-1, inhibits WT-amylin fibril elongation rates with an IC50of ~1 µM and cytotoxicity with an IC50of ~50 µM, comparable to other types of fibrillization inhibitors reported in the literature. Taken together, these results suggest that electrostatic interactions can be exploited to develop new types of inhibitors of amyloid fibrillization and toxicity.

scholarly journals Role of the Negative Charges in the Cytosolic Domain of TOM22 in the Import of Precursor Proteins into Mitochondria

1998 ◽  
Vol 18 (6) ◽  
pp. 3173-3181 ◽  
Author(s):  
Frank E. Nargang ◽  
Doron Rapaport ◽  
R. Gary Ritzel ◽  
Walter Neupert ◽  
Roland Lill
Keyword(s):  
Outer Membrane ◽  
Mitochondrial Outer Membrane ◽  
Wild Type ◽  
Binding Studies ◽  
Amino Terminal ◽  
Precursor Proteins ◽  
Mutant Strains ◽  
Cytosolic Domain ◽  
Charged Residues ◽  
Positively Charged

ABSTRACT TOM22 is an essential mitochondrial outer membrane protein required for the import of precursor proteins into the organelles. The amino-terminal 84 amino acids of TOM22 extend into the cytosol and include 19 negatively and 6 positively charged residues. This region of the protein is thought to interact with positively charged presequences on mitochondrial preproteins, presumably via electrostatic interactions. We constructed a series of mutant derivatives of TOM22 in which 2 to 15 of the negatively charged residues in the cytosolic domain were changed to their corresponding amido forms. The mutant constructs were transformed into a sheltered Neurospora crassa heterokaryon bearing atom22::hygromycin R disruption in one nucleus. All constructs restored viability to the disruption-carrying nucleus and gave rise to homokaryotic strains containing mutanttom22 alleles. Isolated mitochondria from three representative mutant strains, including the mutant carrying 15 neutralized residues (strain 861), imported precursor proteins at efficiencies comparable to those for wild-type organelles. Precursor binding studies with mitochondrial outer membrane vesicles from several of the mutant strains, including strain 861, revealed only slight differences from binding to wild-type vesicles. Deletion mutants lacking portions of the negatively charged region of TOM22 can also restore viability to the disruption-containing nucleus, but mutants lacking the entire region cannot. Taken together, these data suggest that an abundance of negative charges in the cytosolic domain of TOM22 is not essential for the binding or import of mitochondrial precursor proteins; however, other features in the domain are required.

scholarly journals Electrostatics in the Cytoplasmic Pore Produce Intrinsic Inward Rectification in Kir2.1 Channels

2005 ◽  
Vol 126 (6) ◽  
pp. 551-562 ◽  
Author(s):  
Shih-Hao Yeh ◽  
Hsueh-Kai Chang ◽  
Ru-Chi Shieh
Keyword(s):  
Cell Types ◽  
Inward Rectifier ◽  
Inward Rectification ◽  
Membrane Excitability ◽  
Channel Inhibition ◽  
Charged Residues ◽  
Voltage Dependent ◽  
Inwardly Rectifying ◽  
Kir2.1 Channel ◽  
Positively Charged

Inward rectifier K+ channels are important in regulating membrane excitability in many cell types. The physiological functions of these channels are related to their unique inward rectification, which has been attributed to voltage-dependent block. Here, we show that inward rectification can also be induced by neutral and positively charged residues at site 224 in the internal vestibule of tetrameric Kir2.1 channels. The order of extent of inward rectification is E224K mutant > E224G mutant > wild type in the absence of internal blockers. Mutating the glycines at the equivalent sites to lysines also rendered weak inward rectifier Kir1.1 channels more inwardly rectifying. Also, conjugating positively charged methanethiosulfonate to the cysteines at site 224 induced strong inward rectification, whereas negatively charged methanethiosulfonate alleviated inward rectification in the E224C mutant. These results suggest that charges at site 224 may control inward rectification in the Kir2.1 channel. In a D172N mutant, spermine interacting with E224 and E299 induced channel inhibition during depolarization but did not occlude the pore, further suggesting that a mechanism other than channel block is involved in the inward rectification of the Kir2.1 channel. In this and our previous studies we showed that the M2 bundle crossing and selectivity filter were not involved in the inward rectification induced by spermine interacting with E224 and E299. We propose that neutral and positively charged residues at site 224 increase a local energy barrier, which reduces K+ efflux more than K+ influx, thereby producing inward rectification.

scholarly journals Clusters of Charged Residues at the C Terminus of MotA and N Terminus of MotB Are Important for Function of the Escherichia coli Flagellar Motor

2008 ◽  
Vol 190 (15) ◽  
pp. 5517-5521 ◽  
Author(s):  
Edan R. Hosking ◽  
Michael D. Manson
Keyword(s):  
Escherichia Coli ◽  
Flagellar Motor ◽  
Protein Levels ◽  
Partner Protein ◽  
C Terminus ◽  
N Terminus ◽  
Charged Residues ◽  
Positively Charged ◽  
Terminal Cluster

ABSTRACT MotA contains a conserved C-terminal cluster of negatively charged residues, and MotB contains a conserved N-terminal cluster of positively charged residues. Charge-altering mutations affecting these residues impair motility but do not diminish Mot protein levels. The motility defects are reversed by second-site mutations targeting the same or partner protein.

scholarly journals Membrane Topogenesis of a Type I Signal-Anchor Protein, Mouse Synaptotagmin Ii, on the Endoplasmic Reticulum

2000 ◽  
Vol 150 (4) ◽  
pp. 719-730 ◽  
Author(s):  
Yuichiro Kida ◽  
Masao Sakaguchi ◽  
Mitsunori Fukuda ◽  
Katsuhiko Mikoshiba ◽  
Katsuyoshi Mihara
Keyword(s):  
Endoplasmic Reticulum ◽  
Endoplasmic Reticulum Membrane ◽  
Type I ◽  
Hydrophobic Region ◽  
Anchor Protein ◽  
Nascent Polypeptide ◽  
Charged Residues ◽  
Er Targeting ◽  
Signal Anchor ◽  
Positively Charged

Synaptotagmin II is a type I signal-anchor protein, in which the NH2-terminal domain of 60 residues (N-domain) is located within the lumenal space of the membrane and the following hydrophobic region (H-region) shows transmembrane topology. We explored the early steps of cotranslational integration of this molecule on the endoplasmic reticulum membrane and demonstrated the following: (a) The translocation of the N-domain occurs immediately after the H-region and the successive positively charged residues emerge from the ribosome. (b) Positively charged residues that follow the H-region are essential for maintaining the correct topology. (c) It is possible to dissect the lengths of the nascent polypeptide chains which are required for ER targeting of the ribosome and for translocation of the N-domain, thereby demonstrating that different nascent polypeptide chain lengths are required for membrane targeting and N-domain translocation. (d) The H-region is sufficiently long for membrane integration. (e) Proline residues preceding H-region are critical for N-domain translocation, but not for ER targeting. The proline can be replaced with amino acid with low helical propensity.

scholarly journals Voltage-dependent block of endothelial volume-regulated anion channels by calix[4]arenes

1998 ◽  
Vol 275 (3) ◽  
pp. C646-C652 ◽  
Author(s):  
Guy Droogmans ◽  
Jean Prenen ◽  
Jan Eggermont ◽  
Thomas Voets ◽  
Bernd Nilius
Keyword(s):  
Open Channel ◽  
Single Channel ◽  
Anion Channel ◽  
Anion Channels ◽  
Channel Conductance ◽  
Open Channel Block ◽  
Maximum Inhibition ◽  
Voltage Dependent ◽  
A Site ◽  
Maximal Block

We have studied the effects of calix[4]arenes on the volume-regulated anion channel (VRAC) currents in cultured calf pulmonary artery endothelial cells. TS- and TS-TM-calix[4]arenes induced a fast inhibition at positive potentials but were ineffective at negative potentials. Maximal block occurred at potentials between 30 and 50 mV. Lowering extracellular pH enhanced the block and shifted the maximum inhibition to more negative potentials. Current inhibition was also accompanied by an increased current noise. From the analysis of the calix[4]arene-induced noise, we obtained a single-channel conductance of 9.3 ± 2.1 pS ( n = 9) at +30 mV. The voltage- and time-dependent block were described using a model in which calix[4]arenes bind to a site at an electrical distance of 0.25 inside the channel with an affinity of 220 μM at 0 mV. Binding occludes VRAC at moderately positive potentials, but calix[4]arenes permeate the channel at more positive potentials. In conclusion, our data suggest an open-channel block of VRAC by calix[4]arenes that also depends on the protonation of the binding site within the pore.

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