Nonpolar residues in the presumptive pore-lining helix of mechanosensitive channel MSL10 influence channel behavior and establish a nonconducting function.
Ontology highlight
ABSTRACT:
SUBMITTER: Maksaev G
PROVIDER: S-EPMC6261518 | biostudies-literature | 2018 Jun
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Mechanosensitive (MS) ion channels are widespread mechanisms for cellular mechanosensation that can be directly activated by increasing membrane tension. The well-studied MscS family of MS ion channels is found in bacteria, archaea, and plants. MscS-Like (MSL)1 is localized to the inner mitochondrial membrane of Arabidopsis thaliana, where it is required for normal mitochondrial responses to oxidative stress. Like Escherichia coli MscS, MSL1 has a pore-lining helix that is kinked. However, in MSL1 this kink is comprised of two charged pore-lining residues, R326 and D327. Using single-channel patch-clamp electrophysiology in E. coli, we show that altering the size and charge of R326 and D327 leads to dramatic changes in channel kinetics. Modest changes in gating pressure were also observed while no effects on channel rectification or conductance were detected. MSL1 channel variants had differing physiological function in E. coli hypoosmotic shock assays, without clear correlation between function and particular channel characteristics. Taken together, these results demonstrate that altering pore-lining residue charge and size disrupts normal channel state stability and gating transitions, and led us to propose the "sweet spot" model. In this model, the transition to the closed state is facilitated by attraction between R326 and D327 and repulsion between R326 residues of neighboring monomers. In the open state, expansion of the channel reduces inter-monomeric repulsion, rendering open state stability influenced mainly by attractive forces. This work provides insight into how unique charge-charge interactions can be combined with an otherwise conserved structural feature to help modulate MS channel function.
| S-EPMC7757850 | biostudies-literature
Project description:Type 1 ryanodine receptors (RyR1s) release Ca(2+) from the sarcoplasmic reticulum to initiate skeletal muscle contraction. The role of RyR1-G4934 and -G4941 in the pore-lining helix in channel gating and ion permeation was probed by replacing them with amino acid residues of increasing side chain volume. RyR1-G4934A, -G4941A, and -G4941V mutant channels exhibited a caffeine-induced Ca(2+) release response in HEK293 cells and bound the RyR-specific ligand [(3)H]ryanodine. In single channel recordings, significant differences in the number of channel events and mean open and close times were observed between WT and RyR1-G4934A and -G4941A. RyR1-G4934A had reduced K(+) conductance and ion selectivity compared with WT. Mutations further increasing the side chain volume at these positions (G4934V and G4941I) resulted in reduced caffeine-induced Ca(2+) release in HEK293 cells, low [(3)H]ryanodine binding levels, and channels that were not regulated by Ca(2+) and did not conduct Ca(2+) in single channel measurements. Computational predictions of the thermodynamic impact of mutations on protein stability indicated that although the G4934A mutation was tolerated, the G4934V mutation decreased protein stability by introducing clashes with neighboring amino acid residues. In similar fashion, the G4941A mutation did not introduce clashes, whereas the G4941I mutation resulted in intersubunit clashes among the mutated isoleucines. Co-expression of RyR1-WT with RyR1-G4934V or -G4941I partially restored the WT phenotype, which suggested lessening of amino acid clashes in heterotetrameric channel complexes. The results indicate that both glycines are important for RyR1 channel function by providing flexibility and minimizing amino acid clashes.
| S-EPMC4498087 | biostudies-literature
Project description:The influenza B virus BM2 proton-selective ion channel is essential for virus uncoating, a process that occurs in the acidic environment of the endosome. The BM2 channel causes acidification of the interior of the virus particle, which results in dissociation of the viral membrane protein from the ribonucleo-protein core. The BM2 protein is similar to the A/M2 protein ion channel of influenza A virus (A/M2) in that it contains an HXXXW motif. Unlike the A/M2 protein, the BM2 protein is not inhibited by the antiviral drug amantadine. We used mutagenesis to ascertain the pore-lining residues of the BM2 ion channel. The specific activity (relative to wild type), reversal voltage, and susceptibility to modification by (2-aminoethyl)-methane thiosulfonate and N-ethylmaleimide of cysteine mutant proteins were measured in oocytes. It was found that mutation of transmembrane domain residues Ser(9), Ser(12), Phe(13), Ser(16), His(19), and Trp(23) to cysteine were most disruptive for ion channel function. These cysteine mutants were also most susceptible to (2-aminoethyl)-methane thiosulfonate and N-ethylmaleimide modification. Furthermore, considerable amounts of dimer were formed in the absence of oxidative reagents when cysteine was introduced at positions Ser(9), Ser(12), Ser(16), or Trp(23). Based on these experimental data, a BM2 transmembrane domain model is proposed. The presence of polar residues in the pore is a probable explanation for the amantadine insensitivity of the BM2 protein and suggests that related but more polar compounds might serve as useful inhibitors of the protein.
| S-EPMC2414288 | biostudies-literature
Project description:The water permeability of aquaporins (AQPs) varies by more than an order of magnitude even though the pore structure, geometry, as well as the channel lining residues are highly conserved. However, channel gating by pH, divalent ions or phosphorylation was only shown for a minority of AQPs. Structural and in silico indications of water flux modulation by flexible side chains of channel lining residues have not been experimentally confirmed yet. Hence, the aquaporin "open state" is still considered to be a continuously open pore with water molecules permeating in a single-file fashion. Using protein mutations outside the selectivity filter in the aqua(glycerol)facilitator GlpF of Escherichia coli we, to the best of our knowledge, for the first time, modulate the position of the highly conserved Arg in the selectivity filter. This in turn enhances or reduces the unitary water permeability of GlpF as shown in silico by molecular dynamics (MD) simulations and in vitro with purified and reconstituted GlpF. This finding suggests that AQP water permeability can indeed be regulated by lipid bilayer asymmetry and the transmembrane potential. Strikingly, our long-term MD simulations reveal that not only the conserved Arg in the selectivity filter, but the position and dynamics of multiple other pore lining residues modulate water passage through GlpF. This finding is expected to trigger a wealth of future investigations on permeability and regulation of AQPs among others with the aim to tune water permeability for biotechnological applications.
| S-EPMC9490802 | biostudies-literature
Project description:The p7 protein from hepatitis C virus is critical for the assembly and secretion of infectious virus, making it an attractive drug target. It is thought to be a viroporin with a demonstrated ion channel activity when reconstituted into planar lipid bilayers. Electron microscopy experiments suggest that p7 oligomers coexist as hexamers and heptamers. Proposed models of p7 oligomers assume the N-terminal helix to be the pore lining helix. Here, we demonstrate, via electrophysiology, that Cu(2+) has an inhibitory effect on the p7 ion channel and that the amino acid responsible for this inhibition is one histidine in each monomer. This information coupled with the p7 sequence data suggests that the N-terminal helix of p7 does indeed form the transmembrane pore and that this histidine is pore-lining. The information will aid in the construction of oligomeric pore-models and the interpretation of electron microscopy data.
| S-EPMC2716475 | biostudies-literature
Project description:The Caenorhabditis elegans MEC-4/MEC-10 channel mediates the worm's response to gentle body touch and is activated by laminar shear stress (LSS) when expressed in Xenopus oocytes. Substitutions at multiple sites within the second transmembrane domain (TM2) of MEC-4 or MEC-10 abolish the gentle touch response in worms, but the roles of these residues in mechanosensing are unclear. The present study therefore examined the role of specific MEC-4 and MEC-10 TM2 residues in the channel's response to LSS. We found that introducing mutations within the TM2 of MEC-4 or MEC-10 not only altered channel activity, but also affected the channel's response to LSS. This response was enhanced by Cys substitutions at selected MEC-4 sites (Phe715, Gly716, Gln718, and Leu719) between the degenerin and the putative amiloride-binding sites in this subunit. In contrast, the LSS response was largely blunted in MEC-10 variants bearing single Cys substitutions in the regions preceding and following the amiloride-binding site (Gly677-Leu681), as well as with four MEC-10 touch-deficient mutations that introduced charged residues into the TM2 domain. An enhanced response to LSS was observed with a MEC-10 mutation in the putative selectivity filter. Overall, MEC-4 or MEC-10 mutants that altered the channel's LSS response are primarily clustered between the degenerin site and the selectivity filter, a region that probably forms the narrowest portion of the channel pore. Our results suggest that pore-lining residues of MEC-4 and MEC-10 have important yet different roles in tuning the channel's response to mechanical forces.
| S-EPMC6036219 | biostudies-literature
Project description:The Ca(2+)-activated potassium channel KCa3.1 is emerging as a therapeutic target for a large variety of health disorders. One distinguishing feature of KCa3.1 is that the channel open probability at saturating Ca(2+) concentrations (Pomax) is low, typically 0.1-0.2 for KCa3.1 wild type. This observation argues for the binding of Ca(2+) to the calmodulin (CaM)-KCa3.1 complex, promoting the formation of a preopen closed-state configuration leading to channel opening. We have previously shown that the KCa3.1 active gate is most likely located at the level of the selectivity filter. As Ca(2+)-dependent gating of KCa3.1 originates from the binding of Ca(2+) to CaM in the C terminus, the hypothesis of a gate located at the level of the selectivity filter requires that the conformational change initiated in the C terminus be transmitted to the S5 and S6 transmembrane helices, with a resulting effect on the channel pore helix directly connected to the selectivity filter. A study was thus undertaken to determine to what extent the interactions between the channel pore helix with the S5 and S6 transmembrane segments contribute to KCa3.1 gating. Molecular dynamics simulations first revealed that the largest contact area between the pore helix and the S5 plus S6 transmembrane helices involves residue F248 at the C-terminal end of the pore helix. Unitary current recordings next confirmed that modulating aromatic-aromatic interactions between F248 and W216 of the S5 transmembrane helical segment and/or perturbing the interactions between F248 and residues in S6 surrounding the glycine hinge G274 cause important changes in Pomax. This work thus provides the first evidence for a key contribution of the pore helix in setting Pomax by stabilizing the channel closed configuration through aromatic-aromatic interactions involving F248 of the pore helix. We propose that the interface pore helix/S5 constitutes a promising site for designing KCa3.1 potentiators.
| S-EPMC4001770 | biostudies-literature
Project description:The human innate immunity factor apolipoprotein L-I (APOL1) protects against infection by several protozoan parasites, including Trypanosoma brucei brucei Endocytosis and acidification of high-density lipoprotein-associated APOL1 in trypanosome endosomes leads to eventual lysis of the parasite due to increased plasma membrane cation permeability, followed by colloid-osmotic swelling. It was previously shown that recombinant APOL1 inserts into planar lipid bilayers at acidic pH to form pH-gated nonselective cation channels that are opened upon pH neutralization. This corresponds to the pH changes encountered during endocytic recycling, suggesting APOL1 forms a cytotoxic cation channel in the parasite plasma membrane. Currently, the mechanism and domains required for channel formation have yet to be elucidated, although a predicted helix-loop-helix (H-L-H) was suggested to form pores by virtue of its similarity to bacterial pore-forming colicins. Here, we compare recombinant human and baboon APOL1 orthologs, along with interspecies chimeras and individual amino acid substitutions, to identify regions required for channel formation and pH gating in planar lipid bilayers. We found that whereas neutralization of glutamates within the H-L-H may be important for pH-dependent channel formation, there was no evidence of H-L-H involvement in either pH gating or ion selectivity. In contrast, we found two residues in the C-terminal domain, tyrosine 351 and glutamate 355, that influence pH gating properties, as well as a single residue, aspartate 348, that determines both cation selectivity and pH gating. These data point to the predicted transmembrane region closest to the APOL1 C terminus as the pore-lining segment of this novel channel-forming protein.
| S-EPMC7504933 | biostudies-literature
Project description:The mechanosensitive channel MscS functions as an osmolyte emergency release-valve in the event of a sudden decrease in external environmental osmolarity. MscS has served as a paradigm for studying how channel proteins detect and respond to mechanical stimuli. However, the inter-domain interactions and structural rearrangements that occur in the MscS gating process remain largely unknown. Here, we determined the interactions between the transmembrane domain and cytoplasmic domain of MscS. Using in vivo cellular viability, single-channel electrophysiological recording, and cysteine disulfide trapping, we demonstrated that N117 of the TM3b helix and N167 of the Cyto-helix are critical residues that function at the TM3b-Cyto helix interface. In vivo downshock assays showed that double cysteine substitution at N117 and N167 failed to rescue the osmotic-lysis phenotype of cells in acute osmotic downshock. Single-channel recordings demonstrated that cysteine cross-linking of N117C and N167C led to a non-conductive channel. Consistently, coordination of the histidines of N117H and N167H caused a decrease in channel gating. Moreover, cross-linked N117 and N167 altered the gating of the severe gain-of-function mutant L109S. Our results demonstrate that N117-N167 interactions stabilize the inactivation state by an association of TM3b segments with β-domains of the cytoplasmic region, providing further insights into the gating mechanism of the MscS channel.
| S-EPMC6174206 | biostudies-literature
Project description:Compared to other aquaporins (AQPs), lens-specific AQP0 is a poor water channel, and its permeability was reported to be pH-dependent. To date, most water conduction studies on AQP0 were performed on protein expressed in Xenopus oocytes, and the results may therefore also reflect effects introduced by the oocytes themselves. Experiments with purified AQP0 reconstituted into liposomes are challenging because the water permeability of AQP0 is only slightly higher than that of pure lipid bilayers. By reconstituting high amounts of AQP0 and using high concentrations of cholesterol to reduce the permeability of the lipid bilayer, we improved the signal-to-noise ratio of water permeability measurements on AQP0 proteoliposomes. Our measurements show that mutation of two pore-lining tyrosine residues, Tyr-23 and Tyr-149 in sheep AQP0, to the corresponding residues in the high-permeability water channel AQP1 have additive effects and together increase the water permeability of AQP0 40-fold to a level comparable to that of AQP1. Molecular dynamics simulations qualitatively support these experimental findings and suggest that mutation of Tyr-23 changes the pore profile at the gate formed by residue Arg-187.
| S-EPMC5355496 | biostudies-literature