Cis Side Research Articles

Functional Reconstitution of a GIRK1-Chimera and its Regulation by the βγ Subunits of G Proteins

The molecular details of ion channel regulation by G proteins remain unknown. A first step in this direction is to define the characteristics of interacting proteins of known structure in isolation and in complex form. The three-dimensional structure of a GIRK1-chimera determined by single particle electron microscopy at 25A is consistent with the crystal structure (Nishida et al., 2007, EMBO J 26:4005-15). We have functionally reconstituted this GIRK1-chimera into a 1:1 ratio of phosphatidylethanolamine to phosphatidylserine planar lipid bilayers. The GIRK1-chimera produces a conductance of approximately 23 pS that shows Mg2+-dependent inwardly rectifying K+ currents and an absolute requirement on the presence of phosphatidylinositol-4,5-bisphosphate for activation. These currents are blocked by PIP2 antibody and poly-lysine applied from the cis but not the trans side. Moreover, the channel shows a high affinity for diC8-PIP2 (EC50 ∼ 7.5 μM). GIRK1-chimera channel currents are blocked by Ba2+ and the GIRK peptide blocker tertiapin when applied from the trans but not the cis side. Interestingly, Gβγ applied from the cis side inhibits GIRK1-chimera currents and shifts phosphoinositide sensitivity by decreasing the apparent affinity to PIP2. This is in contrast to the Gβγ effects on full-length GIRK1∗ channels assayed in Xenopus oocytes or planar lipid bilayers.

Free energy landscape for the translocation of polymer through an interacting pore

Free energy landscapes for polymer chain translocating through an interacting pore are calculated by using exact enumeration method. A potential barrier exists at weak attractive or repulsive polymer-pore interaction and it changes to a potential well with the increase in the attraction. The result reveals that there is a free translocation point where polymer is free of energy barrier. Using the free energy landscape, the translocation time tau for polymer worming through the pore and the migration time tau(m) for polymer migrating from cis side to trans side are calculated with the Fokker-Plank equation. It shows that a moderate attractive polymer-pore interaction accelerates the migration of polymer from cis side to trans side.

Calreticulin-dependent recycling in the early secretory pathway mediates optimal peptide loading of MHC class I molecules

Calreticulin is a lectin chaperone of the endoplasmic reticulum (ER). In calreticulin-deficient cells, major histocompatibility complex (MHC) class I molecules travel to the cell surface in association with a sub-optimal peptide load. Here, we show that calreticulin exits the ER to accumulate in the ER-Golgi intermediate compartment (ERGIC) and the cis-Golgi, together with sub-optimally loaded class I molecules. Calreticulin that lacks its C-terminal KDEL retrieval sequence assembles with the peptide-loading complex but neither retrieves sub-optimally loaded class I molecules from the cis-Golgi to the ER, nor supports optimal peptide loading. Our study, to the best of our knowledge, demonstrates for the first time a functional role of intracellular transport in the optimal loading of MHC class I molecules with antigenic peptide.

The Membrane Topography of the Diphtheria Toxin T Domain Linked to the A Chain Reveals a Transient Transmembrane Hairpin and Potential Translocation Mechanisms

The diphtheria toxin T domain helps translocate the A chain of the toxin across membranes. To gain insight into translocation, the membrane topography of key residues in T domain attached to the A chain (AT protein) was compared to that in the isolated T domain using fluorescence techniques. This study demonstrates that residues in T domain hydrophobic helices (TH5-TH9) tended to be less exposed to aqueous solution in the AT protein than in the isolated T domain. Under conditions in which the loop connecting TH5 to TH6/7 is located stably on the cis (insertion) side of the membrane in the isolated T domain, it moves between the cis and trans sides of the membrane in the AT protein. This is indicative of the formation of a dynamic, transient transmembrane hairpin topography by TH5-TH7 in the AT protein. Since TH8 and TH9 also form a transmembrane hairpin, this means that TH5-TH9 may form a cluster of transmembrane helices. These helices have a nonpolar surface likely to face the lipid bilayer in a helix cluster and a surface rich in uncharged hydrophilic residues which in a helix cluster would likely be facing inward (and perhaps be pore-lining). This uncharged hydrophilic surface could play a crucial role in translocation, interacting transiently with the translocating A chain. A similar motif can be found in, and may be important for, other protein translocation systems.

H2S and HS– donor NaHS inhibits intracellular chloride channels

We have characterized the effect of H(2)S on single channel properties of the chloride channels derived from the rat heart lysosomal vesicles incorporated into a bilayer lipid membrane. The single chloride channel currents were measured in 250 : 50 mmol/l KCl cis/trans solutions. H(2)S inhibited the chloride channels by decreasing the channel open probability in a concentration-dependent manner. The inhibitory effect of H(2)S was side-dependent with the IC(50) values of 42 and 75 micromol/l for the trans and the cis sides, respectively. The mixture of H2S with the NO donor S-nitroso-N-acetyl-DL-penicillamine had smaller effect (IC(50) = 180 micromol/l) than H(2)S alone. We assume that the inhibitory effect of H(2)S on chloride channels may be responsible for some of its numerous biological effects.

Translocation dynamics with attractive nanopore-polymer interactions

Using Langevin dynamics simulations, we investigate the influence of polymer-pore interactions on the dynamics of biopolymer translocation through nanopores. We find that an attractive interaction can significantly change the translocation dynamics. This can be understood by examining the three components of the total translocation time tau approximately tau1+tau2+tau3 corresponding to the initial filling of the pore, transfer of polymer from the cis side to the trans side, and emptying of the pore, respectively. We find that the dynamics for the last process of emptying of the pore changes from nonactivated to activated in nature as the strength of the attractive interaction increases, and tau3 becomes the dominant contribution to the total translocation time for strong attraction. This leads to nonuniversal dependence of tau as a function of driving force and chain length. Our results are in good agreement with recent experimental findings, and provide a plausible explanation for the different scaling behavior observed in solid state nanopores vs that for the natural alpha-hemolysin channel.

Orientation of the Monomeric Porin OmpG in Planar Lipid Bilayers

Outer membrane protein G (OmpG) is a non-selective porin from Escherichia coli. OmpG is a monomer, which makes it unusual among porins, and suggests that it may be useful in biotechnology. In planar lipid bilayers, individual OmpG pores reconstituted by insertion from detergent exhibit pronounced asymmetry in current-voltage relationships and voltage-dependent gating. Here, this asymmetry is used to deduce the orientation of OmpG in the bilayers. We introduced two cysteines into the extracellular loops of OmpG. Cleavage of the disulfide bond formed by these residues significantly increases spontaneous gating of the pore. By adding DTT to one side of the bilayer or the other, we demonstrated that pores showing a quiet trace at negative potentials have a "trans" conformation (extracellular loops on the trans side of the bilayer), while pores showing a quiet trace at positive potentials have a "cis" conformation (extracellular loops on the cis side). With this knowledge, we examined the binding of a cyclodextrin to OmpG. When the cyclodextrin was presented to the extracellular face of the pore, transient multisite interactions were observed. In contrast, when the cyclodextrin was presented to the periplasmic face, a more stable single-site interaction occurred. Because the cyclodextrin can act as a molecular adapter by binding analytes, this information serves to advance the use of OmpG as a biosensor.

Identification of Channel-lining Amino Acid Residues in the Hydrophobic Segment of Colicin Ia

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 ∼177 residues. In the crystal structure of the water-soluble conformation, this domain consists of a bundle of 10 α-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 α-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.

Hard Sphere Diffusion Behaviour of Polymer Translocating through Interacting Pores

The translocation of polymer chain through a small pore from a high concentration side (cis side) to a low concentration side (trans side) is simulated by using Monte Carlo technique. The effect of the polymer-pore interaction on the translocation is studied. We find a special interaction at which the decay of the number of polymer chain, N, at the cis side obeys Fick's law, i.e. N decreases exponentially with time. The behaviour is analogous to the diffusion of hard sphere.

Carbon Dioxide Transport through Membranes

Several membrane channels, like aquaporin-1 (AQP1) and the RhAG protein of the rhesus complex, were hypothesized to be of physiological relevance for CO(2) transport. However, the underlying assumption that the lipid matrix imposes a significant barrier to CO(2) diffusion was never confirmed experimentally. Here we have monitored transmembrane CO(2) flux (J(CO2)) by imposing a CO(2) concentration gradient across planar lipid bilayers and detecting the resulting small pH shift in the immediate membrane vicinity. An analytical model, which accounts for the presence of both carbonic anhydrase and buffer molecules, was fitted to the experimental pH profiles using inverse problems techniques. At pH 7.4, the model revealed that J(CO2) was entirely rate-limited by near-membrane unstirred layers (USL), which act as diffusional barriers in series with the membrane. Membrane tightening by sphingomyelin and cholesterol did not alter J(CO2) confirming that membrane resistance was comparatively small. In contrast, a pH-induced shift of the CO(2) hydration-dehydration equilibrium resulted in a relative membrane contribution of about 15% to the total resistance (pH 9.6). Under these conditions, a membrane CO(2) permeability (3.2 +/- 1.6 cm/s) was estimated. It indicates that cellular CO(2) uptake (pH 7.4) is always USL-limited, because the USL size always exceeds 1 mum. Consequently, facilitation of CO(2) transport by AQP1, RhAG, or any other protein is highly unlikely. The conclusion was confirmed by the observation that CO(2) permeability of epithelial cell monolayers was always the same whether AQP1 was overexpressed in both the apical and basolateral membranes or not.

Heterotypic tubular connections at the endoplasmic reticulum–Golgi complex interface

Electron microscopy and cryoimmunocytochemistry were used to characterize tubular connections in the secretory pathway using rat spermatids as model. Our results support the existence of a complex tubular network enriched in the Golgi matrix protein GM130 that transiently joins the cis-Golgi side and the endoplasmic reticulum. These tubules occasionally contain the endoplasmic reticulum resident protein PDI but not COPII complexes or KDEL receptor. At the lateral edges of the stacks tubules were seen to connect cisternae belonging to the same or adjacent stacks. These connections were observed in all cisternae but preferentially on the cis side. Giantin, Gos28 and Rab6 were detected in the tubules; importantly, we reported the presence of cis-trans heterotypic connections between cisternae. On the trans-Golgi side, we occasionally observed tubules highly immunoreactive for Rab6 connecting the stack with the forming acrosome. Together, our results support the existence of transient continuities throughout the secretory pathways.

Factors Affecting Polymer Translocation Through a Nanopore in a Membrane

Monte Carlo simulations were used to study the translocation of a flexible polymer through a pore in a membrane, assuming an attractive interaction between the monomers and the membrane on the trans side of the membrane and no interaction on the cis side. For the case TTc (the temperature corresponding to the minimum in the translocation time ), the value of decreases with increasing temperature, whereas for T>Tc, increases with increasing temperature. The translocation time depends on the absorbed energy u0 in a nontrivial way. The value of increases initially upon increasing u0 before it begins to decrease. The variation of the translocation time with respect to the solvent quality was also studied. It showed that there is a transition, as the solvent quality improves from "poor" to "good": when ABc (the interaction energy corresponding to the minimum in ), decreases with increasing the value of AB; when AB>c, increases with increasing AB. When the chain length was changed, it was found that when the absorbed energy u0 was greater than uc, was proportional to N1.602; for u0uc, N2.248. As the solvent quality improved from "poor" to "good," the translocation probability increased initially before becoming stable.

Molecular structure of trans-cinnamaldehyde as determined by gas electron diffraction aided by DFT calculations

The molecular structure of trans-cinnamaldehyde (( E)-3-phenyl-2-propenal) was determined by means of gas electron diffraction. The nozzle temperature was 165 °C. The results of B3LYP calculations with the 6-31G ∗∗ basis set were used as Supporting information. It was found that this molecule has two stable conformers, s- cis and s- trans, which differ in the orientation of the CH O group. Their abundances at 165 °C were determined to be 25 ± 19% and 75%, for the s- cis and s- trans, respectively. This conformational composition is consistent with the prediction by the theoretical calculations. The determined structural parameters ( r g and ∠ α) of the more abundant conformer, s- trans, of trans-cinnamaldehyde are as follows: < r(C C) ring> = 1.398(1) Å; r(C C) = 1.348 (←) Å; r(C 1 C) = 1.470(8) Å; r(C C( O)) = 1.473(←) Å; r(C O) = 1.225(6) Å; < r(C H)> = 1.116(6) Å; ∠C 6 C 1 C 2 = 118.6(3)°; ∠C 1 C 2 C = 121.0(←)°; ∠C C 6 C 1 = 121.4(←)°; ∠C 2 C 1 C( C) = 122.0(26)°; ∠C 1 C C = 128.3(26)°; ∠C C CO = 115.3(27)°; ∠C C O = 126.6(19)°. The C 1, C 2 and C 6 atoms are on the ring with the C 1 attached to the CH CH CHO group, and the C 2 and C 6 are on the cis and trans sides to the C C bond, respectively. Angle brackets denote average values; parenthesized values are the estimated limits of error (3 σ) referring to the last significant digit; left arrows in the parentheses mean that the differences to the preceding parameters are fixed.

Binding of N-terminal fragments of anthrax edema factor (EF N) and lethal factor (LF N) to the protective antigen pore

Anthrax toxin consists of three different molecules: the binding component protective antigen (PA, 83 kDa), and the enzymatic components lethal factor (LF, 90 kDa) and edema factor (EF, 89 kDa). The 63 kDa C-terminal part of PA, PA 63, forms heptameric channels that insert in endosomal membranes at low pH, necessary to translocate EF and LF into the cytosol of target cells. In many studies, about 30 kDa N-terminal fragments of the enzymatic components EF (254 amino acids) and LF (268 amino acids) were used to study their interaction with PA 63-channels. Here, in experiments with artificial lipid bilayer membranes, EF N and LF N show block of PA 63-channels in a dose, voltage and ionic strength dependent way with high affinity. However, when compared to their full-length counterparts EF and LF, they exhibit considerably lower binding affinity. Decreasing ionic strength and, in the case of EF N, increasing transmembrane voltage at the cis side of the membranes, resulted in a strong decrease of half saturation constants. Our results demonstrate similarities but also remarkable differences between the binding kinetics of both truncated and full-length effectors to the PA 63-channel.

How Many Ways Through the Golgi Maze?

The secretory route in eukaryotic cells has been regarded as one common pathway from the endoplasmic reticulum (ER) through the Golgi cisternae to the trans Golgi network where recognition, sorting and exit of cargo molecules are thought to occur. Morphologically, the ribosome-coated ER is observed throughout the cytoplasm, while the Golgi apparatus usually is confined to a perinuclear position in mammalian cells. However, Golgi outposts have been observed in neuronal dendrites and dispersed Golgi elements in skeletal muscle myofibers. In insects, like in Drosophila melanogaster imaginal disc cells and epidermal cells of Tobacco and Arabidopsis leafs, individual Golgi stacks are distributed throughout the cytoplasm. Golgi stacks do not only differ in their intracellular localization but also in the number of stacks from one to several hundreds. Each stack consists of closely aligned, flattened, membrane-limited cisternae. The number of cisternae in a Golgi stack is also variable, 2-3 in some ciliates, 10 in many plant cell types and up to 30 in certain euglenoids. The yeast Saccharomyces cerevisiae has a Golgi structure of minimal complexity with scattered solitary cisternae. It is assumed that the number of Golgi cisternae reflects the overall complexity of the enzymatic reactions that occur in their lumen, while the number of stacks reflects the load of macromolecules arriving at the cis side. In this review, we will focus on how the available morphological and biochemical data fit with the current view of protein sorting in the secretory pathway, particularly in polarized cells like neuronal and epithelial cells.

Translocation of polymer chains through interacting nanopores

The effect of the interaction between nanopore and chain segment on the translocation of polymer chains through an interacting nanopore from a confined environment (cis side, high concentration of chain) to a spacious environment (trans side, zero concentration) was studied by using dynamic Monte Carlo simulations. Results showed that a moderate attractive pore–polymer interaction accelerates the translocation of chain. The optimal interaction at which translocation is the fastest increases with the concentration of chain on the cis side. The dependence of microscopic behaviors of chain translocation on the interaction was investigated.

Pore formation by the Bordetella adenylate cyclase toxin in lipid bilayer membranes: Role of voltage and pH

The bifunctional adenylate cyclase toxin (ACT or CyaA) of Bordetella pertussis invades target cells via transport through the cytoplasmic membrane. The membrane potential represents thereby an important factor for the uptake in vivo. Previous studies demonstrated that adenylate cyclase (AC) delivery into cells requires a negative membrane potential inside the cells. The results of lipid bilayer experiments with ACT presented here indicated that two different types of pore-like structures are formed by ACT dependent on the orientation of the electrical potential across the membranes. Pore formation at a positive potential at the cis side of the membranes, the side of the addition of the toxin, was fast and its conductance had a defined size, whereas at negative potential the pores were not defined, had a reduced pore-forming activity and a very short lifetime. Fluctuations inserted at positive potentials showed asymmetric current–voltage relationships for positive and negative voltages. Positive potentials at the cis side resulted in an increasing current, whereas at negative potentials the current decreased or remained at a constant level. Calcium ions enhanced the voltage dependence of the ACT pores when they were added to the cis side. The single-pore conductance was strongly affected by the variation of the pH value and increased in 1M KCl with increasing pH from about 4 pS at pH 5 to about 60 pS at pH 9. The ion selectivity remained unaffected by pH. Experiments with ACT mutants revealed, that the adenylate cyclase (AC) and repeat (RT) domains were not involved in voltage and pH sensing.

Inhibition of anion channels derived from mitochondrial membranes of the rat heart by stilbene disulfonate—DIDS

The objective of this work was to characterize in more detail the inhibition effect of diisothiocyanatostilbene-2',2-disulfonic acid (DIDS) on anion channels isolated from the rat heart mitochondria. The channels reconstituted into a planar lipid membrane displayed limited powers of discrimination between anions and cations and the ion conductance measured under asymmetric (250/50 mM KCl, cis/trans) and symmetric (150 mM KCl) conditions was approximately 100 pS. DIDS caused a dramatic decrease in the channel activity (IC(50) = 11.7 +/- 3.1 microM) only when it was added to the cis side of a planar lipid membrane. The inhibition was accompanied by the significant prolongation of closings and the shortening of openings within the burst as well as gaps between bursts were prolonged and durations of bursts were reduced. The blockade was complete and irreversible when concentration of DIDS was increased up to 200 microM. Our data indicate that DIDS is an allosteric blocker of mitochondrial anion channels and this specific effect could be used as a tool for reliable identification of anion channels on the functional level.

Nitroxyl: A positive inotrope enhancing SR CA2+ cycling in isolated myocytes

Objectives. Nitroxyl (HNO) is a positive inotrope in vivo. Here we aim to elucidate the mechanisms for HNO-induced inotropy. We demonstrate that HNO enhances sarcoplasmic reticular (SR) Ca2+ cycling, in a thiol-sensitive manner. Methods and results. In murine isolated myocytes, the HNO donor Angeli's Salt (AS) increased sarcomere shortening in a dose-dependent manner (for instance, the increase with 1 mM AS was 117±25%; p<.01 vs. base). This effect was not mimicked by the NO donor DEA/NO. Preincubation with reduced glutathione (GSH, 4 mM, 3 h) incremented intracellular thiol content by 6% (p<.05), and AS/HNO response was markedly blunted in these cells: 58±19% (p=.05 vs. AS 1 mM alone). AS/HNO activated cardiac ryanodine receptor (RyR2). In reconstituted RyR2, HNO increased RyR2 open probability (Po) without altering the unitary conductance: Po increased from 0.16±0.03 to 0.25±0.05, 0.46±0.07 and 0.69±0.11 after 0.1, 0.3, and 1 mM AS, respectively. These changes were fully reverted by addition of the reducing agent DTT (2 mM) to the cis side (0.11±0.04). In addition, HNO dose-dependently increased Ca2+ sparks frequency (CSF): with 0.5 mM AS/HNO CSF was 26±3 vs. 4±1 sparks/100 μm/s (p<.01). Again, pretreatment with cell-permeable GSH abrogated HNO-induced increase in CSF. Finally, to assess whether AS also affects SERCA2a function, AS (250 μM) was added to isolated cardiac mouse SR vesicles. HNO markedly increased the rate of SR Ca2+ uptake. Conclusions. Thus, HNO increases and sustain inotropy by enhancing SR Ca2+ cycling, without altering diastolic Ca2+ levels and/or SR Ca2+ load.

The Thylakoid Proton Gradient Promotes an Advanced Stage of Signal Peptide Binding Deep within the Tat Pathway Receptor Complex

Tat (twin arginine translocation) systems transport folded proteins across the thylakoid membrane of chloroplasts and the plasma membrane of most bacteria. Tat precursors are targeted by hydrophobic cleavable signal peptides with twin arginine (RR) motifs. Bacterial precursors possess an extended consensus, (S/T)RRXFLK, of which the two arginines and the phenylalanine are essential for efficient transport. Thylakoid Tat precursors possess twin arginines but lack the consensus phenylalanine. Here, we have characterized two stages of precursor binding to the thylakoid Tat signal peptide receptor, the 700-kDa cpTatC-Hcf106 complex. The OE17 precursor tOE17 binds to the receptor by RR-dependant electrostatic interactions and partially dissociates during blue native gel electrophoresis. In addition, the signal peptide of thylakoid-bound tOE17 is highly exposed to the membrane surface, as judged by accessibility to factor Xa of cleavage sites engineered into signal peptide flanking regions. By contrast, tOE17 containing a consensus phenylalanine in place of Val(-20) (V - 20F) binds the receptor more strongly and is completely stable during blue native gel electrophoresis. Thylakoid bound V - 20F is also completely protected from factor Xa at the identical sites. This suggests that the signal peptide is buried deeply in the cpTatC-Hcf106 binding site. We further provide evidence that the proton gradient, which is required for translocation, induces a tighter interaction between tOE17 and the cpTat machinery, similar to that exhibited by V - 20F. This implies that translocation involves a very intimate association of the signal peptide with the receptor complex binding site.