Comparing the Temperature-Dependent Conductance of the Two Structurally Similar E. coli Porins OmpC and OmpF

The temperature-dependent ion conductance of OmpC, a major outer membrane channel of Escherichia coli, is predicted using all-atom molecular dynamics simulations and experimentally verified. To generalize previous results, OmpC is compared to its structural homolog OmpF at different KCl concentrations, pH values, and a broad temperature range. At low salt concentrations and up to room temperature, the molecular modeling predicts the experimental conductance accurately. At high salt concentrations above 1 M KCl and above room temperature, the simulations underestimate the conductance. Moreover, the temperature dependence of the channel conductance is different from that of the bulk, both in experiment and simulation, indicating a strong contribution of surface effects to the ion conductance. With respect to OmpC, subconductance levels can be observed in experiments only. Subconductance and gating levels can be clearly distinguished by their differences in conductance values and temperature-dependent behavior. With increasing temperature, the probability of a subconductance state to occur, increases, while the dwell time is decreased. The open probability, frequency, and dwell time of such states is largely pH- and KCl concentration-independent, while their amplitudes show a lower increase with increasing salt concentration than gating amplitudes. Voltage dependence of subconductance has been found to be negligible within the uncertainty of the measurements.     

OmpC-like porin from outer membrane of Yersinia enterocolitica: Molecular structure and functional activity

OmpC-like porin was isolated from the outer membrane (OM) of Yersinia enterocolitica cultured at 37°C (the “warm” variant) and its physicochemical and functional properties were studied. The amino acid sequence of OmpC porin was established, and the primary structure and transmembrane topology of this protein were analyzed in comparison with the OmpF porin isolated from Y. enterocolitica cultured at 6°C (the “cold” variant). Both porins of Y. enterocolitica had a high homology degree (65%) between themselves and with OmpC and OmpF porins from OM of Escherichia coli (58 and 76% homology, respectively). The secondary structure of OmpC and OmpF porins from OM of Y. enterocolitica consists of 16 β-strands connected by short “periplasmic” and longer “extracellular” loops with disordered structure, according to the topological model developed for porins of E. coli. The molecular structures of OmpC and OmpF porins of Y. enterocolitica have significant differences in the structure of the “extracellular” loops and in the position of one of three tryptophan residues. Using the bilayer lipid membrane (BLM) technique, pores formed by OmpC porin of Y. enterocolitica were shown to differ in electrophysiological characteristics from channels of OmpF protein of this microorganism. The isolated OmpC porin reconstructed into BLM displayed functional plasticity similarly to OmpF protein and nonspecific porins of other enterobacteria. The conductivity level of the channels formed by this protein in the BLM was regulated by value of the applied potential.     

Conductance and selectivity properties of a substate of the rabbit sarcoplasmic reticulum channel.

In this paper I describe the occurrence and properties of a subconductance state of the cation-selective channel of the sarcoplasmic reticulum. The substate conductance is 60% of the major state conductance in every salt solution examined. When single channel conductance is plotted vs. ion concentration (for potassium or thallium salt solutions) the Michaelis-Menten constant is nearly the same for both conductance states, while the maximum conductance is reduced for the subconductance state. Both conductance states show anomalous conductance behavior in mixed potassium-thallium solutions that may be modeled in the same way. These results indicate that the ionic selectivity of the channel is the same for both open states.     

Mutant OmpF porins of Yersinia pseudotuberculosis with deletions of external loops: Structure–functional and immunochemical properties

Recombinant mutant OmpF porins from Yersinia pseudotuberculosis outer membrane were obtained using site-directed mutagenesis. Here we used four OmpF mutants where single extracellular loops L1, L4, L6, and L8 were deleted one at a time. The proteins were expressed in Escherichia coli at levels comparable to full-sized recombinant OmpF porin and isolated from the inclusion bodies. Purified trimers of the mutant porins were obtained after dialysis and consequent ion-exchange chromatography. Changes in molecular and spatial structure of the mutants obtained were studied using SDS–PAGE and optical spectroscopy (circular dichroism and intrinsic protein fluorescence). Secondary and tertiary structure of the mutant proteins was found to have some features in comparison with that of the full-sized recombinant OmpF. As shown by bilayer lipid membrane technique, the pore-forming activity of purified mutant porins was identical to OmpF porin isolated from the bacterial outer membrane. Lacking of the external loops mentioned above influenced significantly upon the antigenic structure of the porin as demonstrated using ELISA.     

Understanding Ion Conductance on a Molecular Level: An All-Atom Modeling of the Bacterial Porin OmpF

All-atom molecular dynamics simulations of the ion current through OmpF, the major porin in the outer membrane of Escherichia coli, were performed. Starting from the crystal structure, the all-atom modeling allows us to calculate a parameter-free ion conductance in semiquantitative agreement with experiment. Discrepancies between modeling and experiment occur, e.g., at salt concentrations above 1 M KCl or at high temperatures. At lower salt concentrations, the ions have separate pathways along the channel surface. The constriction zone in the channel contains, on one side, a series of positively charges (R42, R82, R132), and on the opposite side, two negatively charged residues (D113, E117). Mutations generated in the constriction zone by removing cationic residues enhance the otherwise small cation selectivity, whereas removing the anionic residues reverses the selectivity. Reduction of the negatively charged residues decreases the conductance by half, whereas cationic residues enhance the conductance. Experiments on mutants confirm the results of the molecular-level simulations.     

Conductance and selectivity fluctuations in D127 mutants of the bacterial porin OmpF

A recent molecular dynamics study questioned the protonation state and physiological role of aspartate 127 (D127) of E. coli porin OmpF. To address that question we isolated two OmpF mutants with D127 either neutralized (D127N) or replaced by a positively charged lysine (D127K). The charge state of the residue at position 127 has clear effects on both conductance and selectivity. The D127K but not the D127N mutant expresses resilient conductance and selectivity fluctuations. These fluctuations reflect, we think, either changes in the ionization state of K127 and/or transitions between unstable subconformations as induced by the electrostatic repulsion between two positively charged residues, K127 and the nearby R167. Our results slightly favor the view that in WT OmpF residue D127 is deprotonated. As for the role of D127 in OmpF functionality, the gating of both mutants shows very similar sensitivity toward voltage as WT OmpF. Moreover, the current fluctuations of the D127K mutant were observed also in the absence of an applied electric field. We therefore dismiss D127 as a key residue in the control mechanism of the voltage-dependent gating of OmpF.     

Ablation of skeletal muscle triadin impairs FKBP12/RyR1 channel interactions essential for maintaining resting cytoplasmic Ca2+

Previously, we have shown that lack of expression of triadins in skeletal muscle cells results in significant increase of myoplasmic resting free Ca(2+) ([Ca(2+)](rest)), suggesting a role for triadins in modulating global intracellular Ca(2+) homeostasis. To understand this mechanism, we study here how triadin alters [Ca(2+)](rest), Ca(2+) release, and Ca(2+) entry pathways using a combination of Ca(2+) microelectrodes, channels reconstituted in bilayer lipid membranes (BLM), Ca(2+), and Mn(2+) imaging analyses of myotubes and RyR1 channels obtained from triadin-null mice. Unlike WT cells, triadin-null myotubes had chronically elevated [Ca(2+)](rest) that was sensitive to inhibition with ryanodine, suggesting that triadin-null cells have increased basal RyR1 activity. Consistently, BLM studies indicate that, unlike WT-RyR1, triadin-null channels more frequently display atypical gating behavior with multiple and stable subconductance states. Accordingly, pulldown analysis and fluorescent FKBP12 binding studies in triadin-null muscles revealed a significant impairment of the FKBP12/RyR1 interaction. Mn(2+) quench rates under resting conditions indicate that triadin-null cells also have higher Ca(2+) entry rates and lower sarcoplasmic reticulum Ca(2+) load than WT cells. Overexpression of FKBP12.6 reverted the null phenotype, reducing resting Ca(2+) entry, recovering sarcoplasmic reticulum Ca(2+) content levels, and restoring near normal [Ca(2+)](rest). Exogenous FKBP12.6 also reduced the RyR1 channel P(o) but did not rescue subconductance behavior. In contrast, FKBP12 neither reduced P(o) nor recovered multiple subconductance gating. These data suggest that elevated [Ca(2+)](rest) in triadin-null myotubes is primarily driven by dysregulated RyR1 channel activity that results in part from impaired FKBP12/RyR1 functional interactions and a secondary increased Ca(2+) entry at rest.     

Cytoplasmic acidosis induces multiple conductance states in ATP-sensitive potassium channels of cardiac myocytes

We studied the effect of cytoplasmic acidosis on the ionic conducting states of ATP-sensitive potassium channels in heart ventricular cells of guinea pigs and rabbits by using a patch-clamp technique with inside-out patch configuration. Under normal conditions (pH 7.4), the channel alternated between a closed state and a main open state in the absence of nucleotides on the cytoplasmic side. As internal pH was reduced below 6.5, the single channel current manifested distinct subconductance levels. The probability of the appearance of these subconductance levels was pH dependent with a greater probability of subconductance states at lower pH. A variance-mean amplitude analysis technique revealed two subconductance levels approximately equally spaced between the main open level and the closed level (63 and 33%). A current-voltage plot of the two subconductance levels and the main level showed that they had similar reversal potentials and rectification properties. An intrinsic flickering gating property characteristic of these ATP-sensitive channels was found unchanged in the 63% subconductance state, suggesting that this subconductance state and the main conductance state share similar ion pore properties (including ion selection and block) and similar gating mechanisms. The appearance of the subconductance states decreased as ionic strength was increased, and the subconductance states were also slightly voltage dependent, suggesting an electrostatic interaction between the protons and the negative surface charge in the vicinity of the binding sites, which may be close to the inner entrance of the ion pore. Proteolytic modification of the channel on the cytoplasmic side with trypsin did not abolish the subconductance levels. External acidosis did not induce subconductance levels. These results suggest that protons bound to the negatively charged group at the inner entrance of the channel ion pore may induce conformational changes, leading to partially reduced conductance states.     

High Salt Concentrations Increase Permeability through OmpC Channels of Escherichia coli

OmpF and OmpC porin channels are responsible for the passage of small hydrophilic solutes across the outer membrane of Escherichia coli. Although these channels are two of the most extensively studied porin channels, what had yet remained elusive was the reason why OmpC shows markedly lower permeability than OmpF, despite having little difference in its channel size. The OmpC channel, however, is known to contain a larger number of ionizable residues than the OmpF channel. In this study, we examined the channel property of OmpF and OmpC using the intact cell of E. coli, and we found that the permeability of several β-lactams and lactose through OmpC became increased to the level comparable with OmpF with up to 0.3 m salt that may increase the Debye-Hückel shielding or with 2% ethanol or 0.3 m urea that may perturb the short range ordering of water molecules. Replacing 10 pore-lining residues that show different ionization behavior between OmpC and OmpF led to substantial conversion of channel property with respect to their permeability and response to external salt concentration. We thus propose that the overall configuration of ionizable residues in the channel that may orient water molecules and the electrostatic profile of the channel play a decisive role in defining the channel property of the OmpC porin rather than its channel size.     

Gating Kinetics of E. coli Poly-3-Hydroxybutyrate/Polyphosphate Channels in Planar Bilayer Membranes

Nonproteinaceous calcium channel complexes from Escherichia coli, composed of poly-(R)-3-hydroxybutyrate (PHB) and inorganic polyphosphate (polyP), exhibit two distinct gating modes (modes 1 and 2) in planar lipid bilayers. Here we report the kinetic characterization of the channel in mode 2, a mode characterized by two well-defined conductance levels, a fully open state (87 ± 3 pS), and a major subconductance state (56 ± 2 pS). Other subconductance states and full closures are rare (<0.5% of total time). Several kinetic properties of the channel showed asymmetric voltage-dependence indicating an asymmetry in the channel structure. Accordingly, single channels responded to potential change in one of two mirror-image patterns, postulated to arise from opposite orientations of the asymmetrical channel complex in the bilayer. The fraction of time spent in each conductance level was strongly voltage-sensitive. For channels reported in this study, presumably all oriented in the same direction, residence time in the fully open state increased as clamping potentials became more positive whereas residence time in the major subconductance state increased at more negative potentials. Analysis of open time distributions revealed existence of two kinetically distinct states for each level. The shorter time constants for both conductance states exhibited weak voltage-sensitivity; however, the longer time constants were strongly voltage-sensitive. A kinetic scheme, consistent with the complex voltage dependence of the channel, is proposed. Received: 1 February 1999/Revised: 2 April 1999     

Subconductance Gating and Voltage Sensitivity of Sarcoplasmic Reticulum K+ Channels: A Modeling Approach

Sarcoplasmic reticulum (SR) K⁺ channels are voltage-regulated channels that are thought to be actively gating when the membrane potential across the SR is close to zero as is expected physiologically. A characteristic of SR K⁺ channels is that they gate to subconductance open states but the relevance of the subconductance events and their contribution to the overall current flowing through the channels at physiological membrane potentials is not known. We have investigated the relationship between subconductance and full conductance openings and developed kinetic models to describe the voltage sensitivity of channel gating. Because there may be two subtypes of SR K⁺ channels (TRIC-A and TRIC-B) present in most tissues, to conduct our study on a homogeneous population of SR K⁺ channels, we incorporated SR vesicles derived from Tric-a knockout mice into artificial membranes to examine the remaining SR K⁺ channel (TRIC-B) function. The channels displayed very low open probability (Po) at negative potentials (≤0 mV) and opened predominantly to subconductance open states. Positive holding potentials primarily increased the frequency of subconductance state openings and thereby increased the number of subsequent transitions into the full open state, although a slowing of transitions back to the sublevels was also important. We investigated whether the subconductance gating could arise as an artifact of incomplete resolution of rapid transitions between full open and closed states; however, we were not able to produce a model that could fit the data as well as one that included multiple distinct current amplitudes. Our results suggest that the apparent subconductance openings will provide most of the K⁺ flux when the SR membrane potential is close to zero. The relative contribution played by openings to the full open state would increase if negative charge developed within the SR thus increasing the capacity of the channel to compensate for ionic imbalances.     

Effects of pore mutations and permeant ion concentration on the spontaneous gating activity of OmpC porin

Porins are trimers of beta-barrels that form channels for ions and other hydrophilic solutes in the outer membrane of Gram-negative bacteria. The X-ray structures of OmpF and PhoE show that each monomeric pore is constricted by an extracellular loop that folds into the channel vestibule, a motif that is highly conserved among bacterial porins. Electrostatic calculations have suggested that the distribution of ionizable groups at the constriction zone (or eyelet) may establish an intrinsic transverse electrostatic field across the pore, that is perpendicular to the pore axis. In order to study the role that electrostatic interactions between pore residues may have in porin function, we used spontaneous mutants and engineered site-directed mutants that have an altered charge distribution at the eyelet and compared their electrophysiological behavior with that of wild-type OmpC. We found that some mutations lead to changes in the spontaneous gating activity of OmpC porin channels. Changes in the concentration of permeant ions also altered this activity. These results suggest that the ionic interactions that exist between charged residues at the constriction zone of porin may play a role in the transitions between the channel's closed and open states.     

Rectification properties and pH-dependent selectivity of meningococcal class 1 porin

We studied the current rectification properties and selectivity of class 1 porin (PorA) from Neisseria meningitidis (strain H44/76 Δ3Δ4) reconstituted in planar lipid membranes varying salt concentrations and pH. PorA channel shows voltage gating with a characteristic time remarkably longer than other porins. Its current-voltage asymmetry, evaluated as the current rectification ratio, changes nonmonotonically with salt concentration. Interestingly, it reaches its maximum value at physiological concentration. Porin selectivity, quantified by reversal potential measurements, is also significantly asymmetric. Depending on the direction of the salt gradient, the channel becomes more or less selective (10:1 vs. 5:1 Na⁺/Cl⁻). Besides, the reversal potential measurements suggest that porin inserts directionally following the concentration gradient. Measurements over a wide range of pH show that although PorA is strongly cation selective at pH >6, its selectivity gradually changes to anionic in an acidic medium (pH < 4). We show that a continuum electrodiffusion model quantitatively accounts for conductance and reversal potential measurements at positive and negative applied voltages.     

Subconductance States of a Mutant NMDA Receptor Channel Kinetics,Calcium, and Voltage Dependence

The kinetic properties of main and subconductance states of a mutant mouse N-methyl-d-aspartate (NMDA) receptor channel were examined. Recombinant receptors made of ζ-ε₂ (NR1-NR2B) subunits having asparagine-to-glutamine mutations in the M2 segment (ζN598Q /ε₂N589Q) were expressed in Xenopus oocytes. Single channel currents recorded from outside-out patches were analyzed using hidden Markov model techniques. In Ca²⁺-free solutions, an open receptor channel occupies a main conductance (93 pS) and a subconductance (62 pS) with about equal probability. There are both brief and long-lived subconductance states, but only a single main level state. At −80 mV, the lifetime of the main and the longer-lived sub level are both ∼3.3 ms. The gating of the pore and the transition between conductance levels are essentially independent processes. Surprisingly, hyperpolarization speeds both the sub-to-main and main-to-sub transition rate constants (∼120 mV/e-fold change), but does not alter the equilibrium occupancies. Extracellular Ca²⁺ does not influence the transition rate constants. We conclude that the subconductance levels arise from fluctuations in the energetics of ion permeation through a single pore, and that the voltage dependence of these fluctuations reflects the modulation by the membrane potential of the barrier between the main and subconductance conformations of the pore.     

Examination of subconductance levels arising from a single ion channel.

Single-channel records often show frequent currents at a main conductance level and occasional currents at subconductance levels. In some instances, the conductances occur at regular levels that are multiples of a minimum conductance. It is well-appreciated that multiple conductance levels may arise either from the co-operative gating of more than one pore or from changes that occur in a single pore. In this paper, we used theoretical models of ion permeation to examine subconductances arising in a single-pore channel. In particular, the work focuses on the following question: how can an ion channel that provides only one aqueous pore through the membrane produce regular subconductances and a main conductance that all have the same selectivity and the same ion binding affinity? The three types of ion permeation models used in this study showed that a single-pore channel can have subconductances because of long-lived conformational states, because of alterations in rapid fluctuations between conformational states, or because of slight alterations in the electrostatic properties in the channel's entrance vestibules. Regular subconductances with the same selectivity and binding affinity can arise in a single pore even if the energy profile changes do not meet the constant peak offset condition. The results show that the appearance of regular subconductance levels in a single-channel recording is not sufficient evidence to conclude that identical pores have co-operative gating, as would arise in a channel that is a multi-pore complex.     

Distinct sensitivities of OmpF and PhoE porins to charged modulators

The inhibition of the anion-selective PhoE porin by ATP and of the cation-selective OmpF porin by polyamines has been previously documented. In the present study, we have extended the comparison of the inhibitor-porin pairs by investigating the effect of anions (ATP and aspartate) and positively charged polyamines (spermine and cadaverine) on both OmpF and PhoE with the patch-clamp technique, and by comparing directly the gating kinetics of the channels modulated by their respective substrates. The novel findings reported here are (1) that the activity of PhoE is completely unaffected by polyamines, and (2) that the kinetic changes induced by ATP on PhoE or polyamines on OmpF suggest different mechanisms of inhibition. ATP induces a high degree of flickering in the PhoE-mediated current and appears to behave as a blocker of ion flow during its presumed transport through PhoE. Polyamines modulate the kinetics of openings and closings of OmpF, in addition to promoting a blocker-like flickering activity. The strong correlation between sensitivity to inhibitors and ion selectivity suggests that some common molecular determinants are involved in these two properties and is in agreement with the hypothesis that polyamines bind inside the pore of cationic porins.     

E.coli PhoE porin has an opposite voltage-dependence to the homologous OmpF.

We used patch clamp analysis to compare the electrophysiological behavior of two related porins from Escherichia coli, the anion-specific PhoE and the cation-selective OmpF. Outer membrane fractions were obtained from strains expressing just one of these porin types, and the channels were reconstituted into liposomes without prior purification. We show that the orientation of the reconstituted channels is not random and is the same for both PhoE and OmpF. Like cation-selective porins, PhoE shows fast and slow gating to closed levels of various amplitudes, testifying that the channels visit multiple functional states and behave as cooperative entities. The voltage-dependence of PhoE closure is asymmetric, but strikingly, occurs at voltages of inverse polarity from those promoting closures of OmpC and OmpF. Both slow kinetics and inverse voltage-dependence are removed when 70 amino acids from the N-terminal of OmpF are introduced into the homologous region of PhoE. This novel observation regarding the voltage-dependence of the two channel types, along with published results on PhoE and OmpF mutants, allows us to propose a molecular mechanism for voltage sensing and sensor charge movements in bacterial porins. It also offers new cues on the possible physiological relevance in bacteria of this common form of channel modulation.     

Imaging the electrostatic potential of transmembrane channels: atomic probe microscopy of OmpF porin

The atomic force microscope (AFM) was used to image native OmpF porin and to detect the electrostatic potential generated by the protein. To this end the OmpF porin trimers from Escherichia coli was reproducibly imaged at a lateral resolution of approximately 0.5 nm and a vertical resolution of approximately 0.1 nm at variable electrolyte concentrations of the buffer solution. At low electrolyte concentrations the charged AFM probe not only contoured structural details of the membrane protein surface but also interacted with local electrostatic potentials. Differences measured between topographs recorded at variable ionic strength allowed mapping of the electrostatic potential of OmpF porin. The potential map acquired by AFM showed qualitative agreement with continuum electrostatic calculations based on the atomic OmpF porin embedded in a lipid bilayer at the same electrolyte concentrations. Numerical simulations of the experimental conditions showed the measurements to be reproduced quantitatively when the AFM probe was included in the calculations. This method opens a novel avenue to determine the electrostatic potential of native protein surfaces at a lateral resolution better than 1 nm and a vertical resolution of approximately 0.1 nm.     

Selectivity Changes during Activation of Mutant Shaker Potassium Channels

Mutations of the pore-region residue T442 in Shaker channels result in large effects on channel kinetics. We studied mutations at this position in the backgrounds of NH₂-terminal–truncated Shaker H4 and a Shaker -NGK2 chimeric channel having high conductance (Lopez, G.A., Y.N. Jan, and L.Y. Jan. 1994. Nature (Lond.). 367: 179–182). While mutations of T442 to C, D, H, V, or Y resulted in undetectable expression in Xenopus oocytes, S and G mutants yielded functional channels having deactivation time constants and channel open times two to three orders of magnitude longer than those of the parental channel. Activation time courses at depolarized potentials were unaffected by the mutations, as were first-latency distributions in the T442S chimeric channel. The mutant channels show two subconductance levels, 37 and 70% of full conductance. From single-channel analysis, we concluded that channels always pass through the larger subconductance state on the way to and from the open state. The smaller subconductance state is traversed in ∼40% of activation time courses. These states apparently represent kinetic intermediates in channel gating having voltage-dependent transitions with apparent charge movements of ∼1.6 e₀. The fully open T442S chimeric channel has the conductance sequence Rb⁺ > NH₄ ⁺ > K⁺. The opposite conductance sequence, K⁺ > NH₄ ⁺ > Rb⁺, is observed in each of the subconductance states, with the smaller subconductance state discriminating most strongly against Rb⁺.     

Characterisation of channels induced in planar bilayer membranes by detergent solubilised Escherichia coli porins

Purified OmpF, OmpC, NmpC, PhoE and Lc (Protein 2) porins from the Escherichia coli outer membrane were incorporated into planar phospholipid bilayer membranes and the permeability properties of the pores studied. Triton X-100 solubilised porin samples showed large and reproducible increases in membrane conductivity composed of discreet single-channel events. The magnitude of the cation selectivity found for the porins was in the order OmpC greater than OmpF greater than NmpC = Lc; PhoE was anion selective. For the cation selective porins the cation/anion permeability ratios in a variety of solutes ranged from 6 to 35. Further information on the internal structure of the porins was obtained by examination of the single-channel conductance and this was used to interpret macroscopic observations and to estimate single-channel diameters. The same porins solubilised in SDS exhibited slight conductance increase with no observable single-channel activity. Use of on-line microcomputer techniques confirmed the ohmic current vs. voltage behaviour for all the single porin channels examined.