Enhanced ionic interaction in narrow pores and ion pair formation

The same physical phenomenon that gives rise to the increase in the electrostatic self-energy of an ion within a narrow water-filled pore is shown to result in interionic electrical interactions within the pore that are much stronger and of longer range than those between the same ions in the same solution in bulk. Because of the much enhanced attraction between ions of opposite charge within the pore the formation of ion pairs becomes likely, even for strong electrolytes that are fully dissociated in the same solution when not spatially confined. Some predicted consequences of ion pair formation in narrow pores that may be experimentally detected are discussed. It is shown that, in a simple passive pore, due to ion pair formation, an Ussing unidirectional flux ratio exponent of less than 1 is predicted. This is usually thought to characterize a carrier rather than a pore.     

Ion leakage through transient water pores in protein-free lipid membranes driven by transmembrane ionic charge imbalance

We have employed atomic-scale molecular dynamics simulations to address ion leakage through transient water pores in protein-free phospholipid membranes. Our results for phospholipid membranes in aqueous solution with NaCl and KCl salts show that the formation of transient water pores and the consequent ion leakage can be induced and be driven by a transmembrane ionic charge imbalance, an inherent feature in living cells. These processes take place if the gradient is large enough to develop a sufficiently significant potential difference across the membrane. The transport of cations and anions through the water pores is then seen; it discharges the transmembrane potential, considerably reduces the size of a water pore, and makes the water pore metastable, leading eventually to its sealing. The ion transport is found to be sensitive to the type of ions. It turns out that Na(+) and Cl(-) ions leak through a membrane at approximately the same ratio despite the fact that Na(+) ions are expected to experience a lower potential barrier for the permeation through the pore. This is because of strong interactions of sodium ions with the carbonyl region of a phospholipid membrane as well as with lipid headgroups forming pore "walls," considerably slowing down the permeation of sodium ions. In contrast, we observed a pronounced selectivity of a phospholipid membrane to the permeation of potassium ions as compared to chloride ions: Potassium ions, being larger than sodium ions, interact only weakly with phospholipid headgroups, so that these interactions are not able to compensate for a large difference in free-energy barriers for permeation of K(+) and Cl(-) ions. These findings are found to be robust to a choice of force-field parameters for ions (tested by Gromacs and Charmm force-fields for ions). What is more, a potassium ion is found to be able to permeate a membrane along an alternate, "water-defect-mediated" pathway without actual formation of a pore. The "water-defect-mediated" leakage involves formation of a single water defect only and is found to be at least one order of magnitude faster than the pore-mediated ion leakage.     

CHAP: A Versatile Tool for the Structural and Functional Annotation of Ion Channel Pores

The control of ion channel permeation requires the modulation of energetic barriers or “gates” within their pores. However, such barriers are often simply identified from the physical dimensions of the pore. Such approaches have worked well in the past, but there is now evidence that the unusual behavior of water within narrow hydrophobic pores can produce an energetic barrier to permeation without requiring steric occlusion of the pathway. Many different ion channels have now been shown to exploit “hydrophobic gating” to regulate ion flow, and it is clear that new tools are required for more accurate functional annotation of the increasing number of ion channel structures becoming available. We have previously shown how molecular dynamics simulations of water can be used as a proxy to predict hydrophobic gates, and we now present a new and highly versatile computational tool, the Channel Annotation Package (CHAP) that implements this methodology.     

Correlated ion flux through parallel pores: Application to channel subconductance states

Summary Many ion channels that normally gate fully open or shut have recently been observed occasionally to display well-defined subconductance states with conductances much less than those of the fully open channel. One model of this behavior is a channel consisting of several parallel pores with a strong correlation between the flux in each pore such that, normally, they all conduct together but, under special circumstances, the pores may transfer to a state in which only some of them conduct. This paper introduces a general technique for modeling correlated pores, and explores in detail by computer simulation a particular model based upon electric interaction between the pores. Correlation is obtained when the transient electric field of ions passing through the pores acts upon a common set of ionizable residues of the channel protein, causing transient changes in their effective pK and hence in their charged state. The computed properties of such a correlated parallel pore channel with single occupation of each pore are derived and compared to those predicted for a single pore that can contain more than one ion at a time and also to those predicted for a model pore with fluctuating barriers. Experiments that could distinguish between the present and previous models are listed.R.M.B. is grateful to the S.E.R.C. for the award of a graduate studentship.     

GCEMC Simulations of “Swiss Cheese” Ion Exchange Membranes in Dilute Solutions of Primitive Model 1:1 Electrolytes with Different Sizes of Ions or 2:1 Electrolytes with Different Bjerrum Parameters

Abstract

Monte Carlo simulations using a Markov process corresponding to a (generalized) Grand Canonical Ensemble have been performed for a number of spherical micropores in equilibrium with dilute external bulk solutions of primitive model electrolytes. Dilute solutions of 1:1 electrolytes with a Bjerrum parameter B = 1.546 with cations three times larger than the anions have been simulated. Also, dilute solutions of 2:1 electrolytes with ions of equal size and reduced Bjerrum parameters B_r = 1.546 and 3 have been simulated. The pores are primitive pores with hard walls and the same dielectric permittivity in the wall and in the pore solution. They range from a pore radius = 5 times the mean ionic diameter to 35 times this diameter, and they carry a fixed charge equal to + 5,0 and ?5 elementary charges. The fixed charge is modelled as smoothly distributed on the pore-wall interface. In addition to the electric potential of the interfacial charge and the electric potential of the spherical double layer, a potential Δ between the pore solution and the bulk solution may be deliberately added. For single pores we may take Δ = 0, but then the pore is generally not electroneutral. In a “Swiss cheese” membrane with a lot of (equally sized) pores, the membrane phase has to approach electroneutrality for growing size of the phase. This is approximated by means of a membrane generated potential Δ in each pore (from the electrostatic interactions with the other pores). The potential A so chosen to obtain electroneutrality is the GCEMC Donnan potential. These non-ideal Donnan potentials are compared to the ideal values (with activity coefficients equal to zero). From the mean occupation numbers of cations and anions in the pores, the average pore values of the mean ionic and the single ionic activity coefficients of the ions are calculated. These are very dependent on pore sizes and on the potential in the pore. The excess energy and the electrostatic Helmholtz free energy of the ions in the pores are also simulated directly. The electrostatic entropy is found as the difference.     

Ion transport in the simplest single file pore.

A kinetic scheme is developed to describe single-file transport through pores containing up to two ions which may be of different species. The solution for the fluxes in terms of rate constants for entry, exit, and transfer is derived without specific assumptions about symmetry or the voltage and activity dependence of the constants. For a symmetrical pore the relation between the slope conductance at zero applied potential and ion activity can have two distinct regions in which the conductance increases linearly. Zero current or reversal potentials depend on the absolute values of the activities as well as their ratios. The use of this theory to describe the cation fluxes through the pores formed by gramicidin A will be considered in a subsequent paper. Here the model is discussed for a number of more specific assumptions, most extensively the following combination: (1) while entry to a pore is less likely when the pore is already occupied at the opposite end, this entry is still rapid; (2) exit is much more rapid when the pore is occupied by two ions; and (3) transfer from one end to the other of a singly occupied pore is rapid. With these assumptions and for a range of concentrations over which the fluxes are proportional to ion activities, the model predicts a flux ratio exponent nearly equal to 2, blocking by impermeant ions, rectification due to blocking particles on one side only, relief of block by increase in the permeant ion concentration on the opposite side, and anomalous variations of the conductance and zero current potential with mole ratio when the total concentration of the two permeants is held constant.     

Ion transport in the simplest single file pore

A kinetic scheme is developed to describe single-file transport through pores containing up to two ions which may be of different species. The solution for the fluxes in terms of rate constants for entry, exit, and transfer is derived without specific assumptions about symmetry or the voltage and activity dependence of the constants. For a symmetrical pore the relation between the slope conductance at zero applied potential and ion activity can have two distinct regions in which the conductance increases linearly. Zero current or reversal potentials depend on the absolute values of the activities as well as their ratios. The use of this theory to describe the cation fluxes through the pores formed by gramicidin A will be considered in a subsequent paper. Here the model is discussed for a number of more specific assumptions, most extensively the following combination: (1) while entry to a pore is less likely when the pore is already occupied at the opposite end, this entry is still rapid; (2) exit is much more rapid when the pore is occupied by two ions; and (3) transfer from one end to the other of a singly occupied pore is rapid. With these assumptions and for a range of concentrations over which the fluxes are proportional to ion activities, the model predicts a flux ratio exponent nearly equal to 2, blocking by impermeant ions, rectification due to blocking particles on one side only, relief of block by increase in the permeant ion concentration on the opposite side, and anomalous variations of the conductance and zero current potential with mole ratio when the total concentration of the two permeants is held constant.     

Gated pores in the ferritin protein nanocage

Properties of ferritin gated pores control rates of FMNH₂ reduction of ferric iron in hydrated oxide minerals inside the protein nanocage, and are discussed in terms of: (1) the conserved pore gate residues (ion pairs: arginine 72, aspartate 122, and a hydrophobic pair, leucine 110-leucine 134), (2) pore sensitivity to heat at temperatures 30 °C below that of the nanocage itself, and (3) pore sensitivity to physiological changes in urea (1-10 mM). Conditions which alter ferritin pore structure/function in solution, coupled with the high evolutionary conservation of the pore gates, suggest the presence of molecular regulators in vivo that recognize the pore gates and hold them either closed or open, depending on biological iron need. The apparent homology between ferrous ion transport through gated pores in the ferritin nanocage and ion transport through gated pores in ion channel proteins embedded in cell membranes, make studies of water soluble ferritin and the pore gating folding/unfolding a useful model for other gated pores.     

A BEST example of channel structure annotation by molecular simulation

An increasing number of ion channel structures are being determined. This generates a need for computational tools to enable functional annotation of channel structures. However, several studies of ion channel and model pores have indicated that the physical dimensions of a pore are not always a reliable indicator of its conductive status. This is due to the unusual behavior of water within nano-confined spaces, resulting in a phenomenon referred to as “hydrophobic gating”. We have recently demonstrated how simulating the behavior of water within an ion channel pore can be used to predict its conductive status. In this addendum to our study, we apply this method to compare the recently solved structure of a mutant of the bestrophin chloride channel BEST1 with that of the wild-type channel. Our results support the hypothesis of a hydrophobic gate within the narrow neck of BEST1. This provides further validation that this simulation approach provides the basis for an accurate and computationally efficient tool for the functional annotation of ion channel structures.     

Modeling of discrete currents of single ion channels of cell membranes using synthetic nanometer pores in polyethylene terephthalate films

Study of the conductivity of single supernarrow pores (1–15 nm in diameter) formed in thin membranes (10–12 μm in thickness) from polyethylene terephthalate (PETP) has revealed discrete changes in the currents passing through such pores when applied from an external source of potential difference of 200–1000 mV. Based on several characteristics, such discrete currents (discrete conductivity changes) appeared to be identical to the so-called currents of single ionic channels in cell membranes. The supernarrow pores whose properties are described in the present work were obtained by alkaline etching of tracks in thin PETP membranes (a variant of the so-called nuclear filters). On the walls of the pores, carboxyl groups, i.e., negative fixed charges, and their compensating counterion (cation) layer are formed. Upon setting the potential difference onto the PETP membrane, this cation layer is able to transfer current, through a process called surface conductance. In the case of nanometer-sized diameters of the pores, such surface conductance can turn out to be dominating. We have shown that these discrete changes of currents passing through the nanometer pores are associated with metastability of their surface conductance. In the highly cation-selective channels in the cell membranes, there should inevitably exist an area with dominating cation surface conductance and, hence, conductance metastability. Therefore, a new explanation is proposed of the characteristic discreteness of the currents of single cation-specific ionic channels in cell membranes. Such an explanation does not rule out the existence of any other traditional explanation of the discreteness of ion channel currents.     

Simultaneous stochastic sensing of divalent metal ions

Stochastic sensing is an emerging analytical technique that relies upon single-molecule detection. Transmembrane pores, into which binding sites for analytes have been placed by genetic engineering, have been developed as stochastic sensing elements. Reversible occupation of an engineered binding site modulates the ionic current passing through a pore in a transmembrane potential and thereby provides both the concentration of an analyte and, through a characteristic signature, its identity. Here, we show that the concentrations of two or more divalent metal ions in solution can be determined simultaneously with a single sensor element. Further, the sensor element can be permanently calibrated without a detailed understanding of the kinetics of interaction of the metal ions with the engineered pore.     

Ion transport through membrane-spanning nanopores studied by molecular dynamics simulations and continuum electrostatics calculations

Narrow hydrophobic regions are a common feature of biological channels, with possible roles in ion-channel gating. We study the principles that govern ion transport through narrow hydrophobic membrane pores by molecular dynamics simulation of model membranes formed of hexagonally packed carbon nanotubes. We focus on the factors that determine the energetics of ion translocation through such nonpolar nanopores and compare the resulting free-energy barriers for pores with different diameters corresponding to the gating regions in closed and open forms of potassium channels. Our model system also allows us to compare the results from molecular dynamics simulations directly to continuum electrostatics calculations. Both simulations and continuum calculations show that subnanometer wide pores pose a huge free-energy barrier for ions, but a small increase in the pore diameter to approximately 1 nm nearly eliminates that barrier. We also find that in those wider channels the ion mobility is comparable to that in the bulk phase. By calculating local electrostatic potentials, we show that the long range Coulomb interactions of ions are strongly screened in the wide water-filled channels. Whereas continuum calculations capture the overall energetics reasonably well, the local water structure, which is not accounted for in this model, leads to interesting effects such as the preference of hydrated ions to move along the pore wall rather than through the center of the pore.     

Editorial

Abstract

Grand canonical molecular dynamics (GCMD) simulations are used to study the adsorption and desorption of Lennard-Jones nitrogen in three slit pore junction models of microporous graphite. These networks consist of two narrow pores separated by a wider (cavity) pore. We report results for cases where the narrow pore has a width of only two or three molecular diameters. Using the GCMD technique, a novel freezing transition is observed which results in pore blocking in the narrow pores of the network, which are less than 1 nm wide. This freezing results from the adsorption energy barrier at the junction between the narrow and wider pores. This type of pore blocking could account for the apparent increase in pore volume with increasing temperature that has been experimentally observed in microporous graphite systems. For networks in which the narrower pores are somewhat larger, with a width of 1.28 nm, this pore blocking effect is much reduced, and adsorbate molecules enter and fill the central cavity. In such cases, however, desorption is incomplete, some residual adsorbate remaining in the central cavity even at the lowest pressures.     

Submersion respiration in small diving beetles (Dytiscidae)

Some small diving beetles can survive submerged through weeks and months, because they can extract oxygen, dissolved in the water, through respiratory pores in their integument. An air flux from the outside to the inside through the respiratory pores has been demonstrated. All diving beetles capable of such pore respiration are small, but not all small diving beetles have pore respiration. With increasing size, more and more of the surface must be covered by respiratory pores to meet the increasing demand of oxygen. In running water species the pore-respiration mode is regarded as an adaptation to life in current exposed substrates, thus they avoid the risk of being swept away during frequent surface visits. In stagnant water species the pore respiration mode reduces the risk of falling victim to pelagic predators. The submersion tolerant species can switch to surface respiration, e.g. during low oxygen content. The pore respiratory mechanism is believed to be a specialised plastron. The oxygen flux through the scattered, small respiratory pore area may be enhanced by a functional thinning of the boundary layer.     

Pore characteristics of nontypeable Haemophilus influenzae outer membrane protein P5 in planar lipid bilayers

The structure of outer membrane protein P5 of NTHi, a homolog of Escherichia coli OmpA, was investigated by observing its pore characteristics in planar lipid bilayers. Recombinant NTHi P5 was overexpressed in E. coli and purified using ionic detergent, LDS-P5, or nonionic detergent, OG-P5. LDS-P5 and OG-P5 could not be distinguished by their migration on SDS-PAGE gels; however, when incorporated into planar bilayers of DPhPC between symmetric aqueous solutions of 1 M KCl at 22 degrees C, LDS-P5 formed narrow pores (58 +/- 6 pS) with low open probability, whereas OG-P5 formed large pores (1.1 +/- 0.1 nS) with high open probability (0.99). LDS-P5 narrow pores were gradually and irreversibly transformed into large pores, indistinguishable from those formed by OG-P5, at temperatures >or=40 degrees C; the process took 4-6 h at 40 degrees C or 35-45 min at 42 degrees C. Large pores were stable to changes in temperatures; however, large pores were rapidly converted to narrow pores when exposed to LDS at room temperatures, indicating acute sensitivity of this conformer to ionic detergent. These studies suggest that narrow pores are partially denatured forms and support the premise that the native conformation of NTHi P5 is that of a large monomeric pore.     

Carrier-like behaviour from a static but electrically responsive model pore.

Because of the low dielectric constant of most proteins and lipids, the electric field of an ion passing through a narrow pore is long range and will interact with neighbouring ionizable residues of the channel protein. The electrical structure of the channel may thus change transiently in response to an ion passing through the pore. Model calculations then reveal that the ratio of the unidirectional ion fluxes may approach 1 as expected for a carrier or shuttling ionophore rather than the Ussing ratio expected for a pore. Saturation behaviour also becomes carrier-like. Computer simulation is reported showing a continuous variation between pore-like and carrier-like behaviour as the parameters of the system are allowed to change smoothly.     

Voltage-dependent hydration and conduction properties of the hydrophobic pore of the mechanosensitive channel of small conductance

A detailed picture of water and ion properties in small pores is important for understanding the behavior of biological ion channels. Several recent modeling studies have shown that small, hydrophobic pores exclude water and ions even if they are physically large enough to accommodate them, a mechanism called hydrophobic gating. This mechanism has been implicated in the gating of several channels, including the mechanosensitive channel of small conductance (MscS). Although the pore in the crystal structure of MscS is wide and was initially hypothesized to be open, it is lined by hydrophobic residues and may represent a nonconducting state. Molecular dynamics simulations were performed on MscS to determine whether or not the structure can conduct ions. Unlike previous simulations of hydrophobic nanopores, electric fields were applied to this system to model the transmembrane potential, which proved to be important. Although simulations without a potential resulted in a dehydrated, occluded pore, the application of a potential increased the hydration of the pore and resulted in current flow through the channel. The calculated channel conductance was in good agreement with experiment. Therefore, it is likely that the MscS crystal structure is closer to a conducting than a nonconducting state.     

Ion channels in murine nuclei during early development and in fully differentiated adult cells

Summary The nuclear envelope functions as a selective barrier between nucleus and cytoplasm. During cycles of cell division the nuclear envelope repeatedly disassembles and re-associates. Presumably, each cycle re-establishes the functional and structural integrity of the nuclear envelope. After repeated rounds of cell division, as occurs during differentiation, the selectivity and configuration of the envelope may change. We compare the ionic conductance and the nuclear pore density in four types of murine nuclei: germinal vesicles in oocytes, pronuclei in zygotes, nuclei from two-cell blastomeres, and somatic cell nuclei from the liver. A large-conductance ion channel is present in all nuclear envelopes. Liver cell nuclei have a greater number of these channels than those from earlier developmental stages, and they also have a higher density of nuclear pores. In this article we hypothesize an association between the ion channels and the nuclear pores.