Channel Interior Research Articles

Ion-water and ion-polypeptide correlations in a gramicidin-like channel. A molecular dynamics study

This work describes a molecular dynamics study of ion-water and ion-polypeptide correlation in a model gramicidin-like channel (the polyglycine analogue) based upon interaction between polarizable, multipolar groups. The model suggests that the vicinity of the dimer junction and of the ethanolamine tail are regions of unusual flexibility. Cs+ binds weakly in the mouth of the channel: there it coordinates five water molecules and the #11CO group with which it interacts strongly and is ideally aligned. In the channel interior it is generally pentacoordinate; at the dimer junction, because of increased channel flexibility, it again becomes essentially hexacoordinate. The ion is also strongly coupled to the #13 CO but not to either #9 or #15, consistent with 13C NMR data. Water in the channel interior is strikingly different from bulk water; it has a much lower mean dipole moment. This correlates with our observation (which differs from that of previous studies) that water-water angular correlations do not persist within the channel, a result independent of ion occupancy or ionic polarity. In agreement with streaming potential measurements, there are seven single file water molecules associated with Cs+ permeation; one of these is always in direct contact with bulk water. At the mouth of an ion-free channel, there is a pattern of dipole moment alteration among the polar groups. Due to differential interaction with water, exo-carbonyls have unusually large dipole moments whereas those of the endo-carbonyls are low. The computed potential of mean force for CS+ translocation is qualitatively reasonable. However, it only exhibits a weakly articulated binding site and it does not quantitatively account for channel energetics. Correction for membrane polarization reduces, but does not eliminate, these problems.

Characterization of the nucleoside‐binding site inside the Tsx channel ofEscherichia coliouter membrane Reconstitution experiments with lipid bilayer membranes

Reconstitution of purified Tsx protein from Escherichia coli into lipid bilayer membranes showed that Tsx formed small ion-permeable channels with a single-channel conductance of 10 pS in 1 M KCl. The dependence of conductance versus salt concentration was linear, suggesting that Tsx has no binding site for ions. Conductance was inhibited by the addition of 20 mM adenosine. Titration of the Tsx-mediated membrane conductance with different solutes including free bases, nucleosides, and deoxynucleosides suggested that the channel contains a binding site for nucleosides but not for sugars or amino acids, and binding increased in the following order: free base, nucleoside, and deoxynucleoside. Among the five nucleosides the stability constant for the binding increased in the order of cytidine, guanosine, uridine, adenosine, and thymidine. Control experiments revealed that the binding of the nucleosides is independent of ion concentration in the aqueous phase, i.e. there was no competition between nucleosides and ions for the binding site inside the channel. The binding of the solutes to the channel interior can be explained by a one-site two-barrier model for the Tsx channel. The advantage of a binding site inside a specific porin for the permeation of solutes is discussed with respect to the properties of a general diffusion pore.

Mechanism of sugar transport through the sugar-specific LamB channel of Escherichia coli outer membrane.

Lipid bilayer experiments were performed with the sugar-specific LamB (maltoporin) channel of Escherichia coli outer membrane. Single-channel analysis of the conductance steps caused by LamB showed that there was a linear relationship between the salt concentration in the aqueous phase and the channel conductance, indicating only small or no binding between the ions and the channel interior. The total or the partial blockage of the ion movement through the LamB channel was not dependent on the ion concentration in the aqueous phase. Both results allowed the investigation of the sugar binding in more detail, and the stability constants of the binding of a large variety of sugars to the binding site inside the channel were calculated from titration experiments of the membrane conductance with the sugars. The channel was highly cation selective, both in the presence and absence of sugars, which may be explained by the existence of carbonyl groups inside the channel. These carbonyl groups may also be involved in the sugar binding via hydrogen bonds. The kinetics of the sugar transport through the LamB channel were estimated relative to maltose by assuming a simple one-site, two-barrier model from the relative rates of permeation taken from M. Luckey and H. Nikaido (Proc. Natl. Acad. Sci. USA 77:165-171 (1980a)) and the stability constants for the sugar binding given in this study.

External monovalent cations that impede the closing of K channels.

We have examined the effects of a variety of monovalent cations on K channel gating in squid giant axons. The addition of the permeant cations K, Rb, or Cs to the external medium decreases the channel closing rate and causes a negative shift of the conductance-voltage relationship. Both of these effects are larger in Rb than in K. The opening kinetics of the K channel are, on the other hand, unaffected by these monovalent cations. Other permeant species, like NH4 and Tl, slightly increase the closing rate, whereas the relatively impermeant cations Na, Li, and Tris have little or no effect on K channel gating. The permeant cations have different effects on the reversal potential and the shape of the instantaneous current-voltage relationship. These effects give information about entry and binding of the cations in K channels. Rb, for example, enters the pore readily (large shift of the reversal potential), but binds tightly to the channel interior, inhibiting current flow. We find a correlation between the occupancy of the channel by a monovalent cation and the closing rate, and conclude that the presence of a monovalent cation in the pore inhibits channel closing, and thereby causes a leftward shift in the activation-voltage curve. In causing these effects, the cations appear to bind near the inner surface of the membrane.

Non-equilibrium voltage noise generated by ion transport through pores.

In this paper, we describe a systematic approach to the theoretical analysis of non-equilibrium voltage noise that arises from ions moving through pores in membranes. We assume that an ion must cross one or two barriers in the pore in order to move from one side of the membrane to the other. In our analysis, we consider the following factors: a) surface charge as a variable in the kinetic equations, b) linearization of the kinetic equations, c) master equation approach to fluctuations. To analyze the voltage noise arising from ion movement through a two barrier (i.e., one binding site) pore, we included the effects of ions in the channel's interior on the voltage noise. The current clamp is considered as a white noise generating additional noise in the system. In contrast to what is found for current noise, at low frequencies the voltage noise intensity is reduced by increasing voltage across the membrane. With this approach, we demonstrate explicitly for the examples treated that, apart from additional noise generated by the current clamp, the non-equilibrium voltage fluctuations can be related to the current fluctuations by the complex admittance.

Do protons block Na+ channels by binding to a site outside the pore?

In a detailed structural model of the Na+ channel which has evolved over the past decade, the selectivity of the channel for cations1,2, the voltage-dependence of channel block by protons3 and other small cations2, and block by tetrodotoxin (TTX)4,5 all depend on the presence of a negatively charged site inside the channel that is part of a constriction called the ‘selectivity filter’. Much of the detail inherent in the model arises from Woodhull's quantitative desciption of the voltage dependence of Na+ channel block by protons3, in which the apparent relief of block observed at positive potentials occurs because the blocking site is located inside the sodium channel ∼25% of the way through the membrane voltage drop. I report here that proton block of skeletal muscle Na+ channels, determined from tail currents, is voltage-independent, and that the apparent voltage-dependent block of peak Na+ currents is instead an effect of low pH on channel kinetics. These results suggest that the proton binding site lies effectively outside the membrane field and thus at or outside the channel mouth. As a consequence, present models of the channel interior will need to be re-examined and substantially modified.

An Analysis of Critical Heat Flux in Flow Reversal Transients

A large inlet break Loss of Coolant Accident in a Pressurized Water Reactor (PWR) would cause the flow through the core to reverse within milliseconds. Currently approved methods of analysis conservatively assume that vapor blanketing of core heat transfer surfaces occurs upon this first reduction to zero flow. A coordinated experimental and analytical study has been conducted to determine when and where the vapor blanketing or Critical Heat Flux (CHF) conditions actually do develop in constant pressure rapid flow reversals. The results indicate that first occurrence of CHF is due not to low coolant velocities, but to flow stagnation in the channel interior with associated rapid channel voiding. Calculations indicate that good cooling should persist over large regions of the core for about 1 s longer than is currently assumed.

Operational analysis of the continuous dynode channel electron multiplier

A theory of operation is developed for the continuous dynode channel electron multiplier and its validity checked by comparison with experimentally measured data. Secondary emission, the multiplication mechanism inside the channel, is best described by a Maxwell distribution in energy and a Lambert distribution in direction of released electrons. Electron motion in the uniform axial field of the channel interior leads to three probability density functions describing at a fixed time interval after emission the electron's expected location. A graphical method is employed to relate the probability of being able to locate the electro with respect to the cylindrical channel geometry.