The mobility of single-file water molecules is governed by the number of H-bonds they may form with channel-lining residues.
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ABSTRACT:
SUBMITTER: Horner A
PROVIDER: S-EPMC4496530 | biostudies-literature |
REPOSITORIES: biostudies-literature
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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:Water molecules lose two of their four bulk neighbours when entering single-file channels. This process may be sensitive to the presence of positive and negative charges at the channel mouth, since the costs for dehydrating cations and anions differ by a large margin. However, it is not known whether entrance charges affect the single channel water permeability (pf). So far, pf is only known to be governed by H-bond formation between permeating water molecules and wall-lining residues. Here we compare the pf values of five different aquaporin species (AQP1, AQPZ, AQP4 wild type, and two phosphorylation mimicking AQP4 mutants) that offer the same number of hydrogen bond donating and receiving residues in their single-file region but display different entrance charges. The pf measurements were performed with reconstituted lipid vesicles. We assessed (i) the osmotically induced vesicle deflation from the light scattering intensity in a stopped-flow device and (ii) the aquaporin abundance by fluorescence correlation spectroscopy. Substitution of serine at positions 111 and 180 in AQP4 for aspartic acid showed only a marginal effect on pf, suggesting that negative entrance charges are of minor importance. In contrast, the total number of positively charged amino acid side chains at entrances and exits correlates with pf: a total of three, four and seven charges of AQP4, AQPZ, and AQP1 translate into pf values of 1.1, 1.8, and 3.2 × 10-13 cm3 s-1, respectively. Thus, positive interfacial charges boost the pf value of AQP1 to three times the value of AQP4. Nevertheless, the number of hydrogen bond donating and receiving residues in the single-file region remains the major determinant of pf. Their effect on pf may be a hundredfold larger than that of interfacial charges.
| S-EPMC6161257 | biostudies-literature
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:Colicin Ia is a bactericidal protein of 626 amino acid residues that kills its target cell by forming a channel in the inner membrane; it can also form voltage-dependent channels in planar lipid bilayer membranes. The channel-forming activity resides in the carboxy-terminal domain of approximately 177 residues. In the crystal structure of the water-soluble conformation, this domain consists of a bundle of 10 alpha-helices, with eight mostly amphipathic helices surrounding a hydrophobic helical hairpin (helices H8-H9). We wish to know how this structure changes to form a channel in a lipid bilayer. Although there is evidence that the open channel has four transmembrane segments (H8, H9, and parts of H1 and H6-H7), their arrangement relative to the pore is largely unknown. Given the lack of a detailed structural model, it is imperative to better characterize the channel-lining protein segments. Here, we focus on a segment of 44 residues (573-616), which in the crystal structure comprises the H8-H9 hairpin and flanking regions. We mutated each of these residues to a unique cysteine, added the mutant colicins to the cis side of planar bilayers to form channels, and determined whether sulfhydryl-specific methanethiosulfonate reagents could alter the conduction of ions through the open channel. We found a pattern of reactivity consistent with parts of H8 and H9 lining the channel as alpha-helices, albeit rather short ones for spanning a lipid bilayer (12 residues). The effects of the reactions on channel conductance and selectivity tend to be greater for residues near the amino terminus of H8 and the carboxy terminus of H9, with particularly large effects for G577C, T581C, and G609C, suggesting that these residues may occupy a relatively constricted region near the cis end of the channel.
| S-EPMC2585860 | biostudies-literature
Project description:Mechanosensitive (MS) ion channels provide a universal mechanism for sensing and responding to increased membrane tension. MscS-like (MSL) 10 is a relatively well-studied MS ion channel from Arabidopsis thaliana that is implicated in cell death signaling. The relationship between the amino acid sequence of MSL10 and its conductance, gating tension, and opening and closing kinetics remains unstudied. Here, we identify several nonpolar residues in the presumptive pore-lining transmembrane helix of MSL10 (TM6) that contribute to these basic channel properties. F553 and I554 are essential for wild type channel conductance and the stability of the open state. G556, a glycine residue located at a predicted kink in TM6, is essential for channel conductance. The increased tension sensitivity of MSL10 compared to close homolog MSL8 may be attributed to F563, but other channel characteristics appear to be dictated by more global differences in structure. Finally, MSL10 F553V and MSL10 G556V provided the necessary tools to establish that MSL10's ability to trigger cell death is independent of its ion channel function.
| S-EPMC6261518 | 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:Regulated ion diffusion across biological membranes is vital for cell function. In a nanoscale ion channel, the active role of discrete water molecules in modulating hydrodynamic behaviors of individual ions is poorly understood because of the technical challenge of tracking water molecules through the channel. Here we report the results of a hydroxyl radical footprinting analysis of the zinc-selective channel ZIPB from the Gram-negative bacterium, Bordetella bronchiseptica Irradiating ZIPB by microsecond X-ray pulses activated water molecules to form covalent hydroxyl radical adducts at nearby residues, which were identified by bottom-up proteomics to detect residues that interact either with zinc or water in response to zinc binding. We found a series of residues exhibiting reciprocal changes in water accessibility attributed to alternating zinc and water binding. Mapping these residues to the previously reported crystal structure of ZIPB, we identified a water-reactive pathway that superimposed on a zinc translocation pathway consisting of two binuclear metal centers and an interim zinc-binding site. The cotranslocation of zinc and water suggested that pore-lining residues undergo a mode switch between zinc coordination and water binding to confer zinc mobility. The unprecedented details of water-mediated zinc transport identified here highlight an essential role of solvated waters in driving zinc coordination dynamics and transmembrane crossing.
| S-EPMC6737223 | biostudies-literature
Project description:The mechanisms underlying the selective permeation of ions through channel molecules are a fundamental issue related to understanding how neurons exert their functions. The "knock-on" mechanism, in which multiple ions in the selectivity filter are hit by an incoming ion, is one of the leading concepts. This mechanism has been supported by crystallographic studies that demonstrated ion distribution in the structure of the Streptomyces lividans (KcsA) potassium channel. These still pictures under equilibrium conditions, however, do not provide a snapshot of the actual, ongoing permeation processes. To understand the dynamics of permeation, we determined the ratio of the ion and water flow [the water-ion coupling ratio (CR(w-i))] through the KcsA channel by measuring the streaming potential (V(stream)) electrophysiologically. The V(stream) value was converted to the CR(w-i) value, which reveals how individual ion and water molecules are queued in the narrow and short filter during permeation. At high K(+) concentrations, the CR(w-i) value was 1.0, indicating that turnover between the alternating ion and water arrays occurs in a single-file manner. At low K(+), the CR(w-i) value was increased to a point over 2.2, suggesting that the filter contained mostly one ion at a time. These average behaviors of permeation were kinetically analyzed for a more detailed understanding of the permeation process. Here, we envisioned the permeation as queues of ion and water molecules and sequential transitions between different patterns of arrays. Under physiological conditions, we predicted that the knock-on mechanism may not be predominant.
| S-EPMC6623228 | 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