Ion movement through gramicidin A channels. Studies on the diffusion-controlled association step

The permeability characteristics of gramicidin A channels are generally considered to reflect accurately the intrinsic properties of the channels themselves; i.e., the aqueous convergence regions are assumed to be negligible barriers for ion movement through the channels. The validity of this assumption has been examined by an analysis of gramicidin A single-channel current-voltage characteristics up to very high potentials (500 mV). At low permeant ion concentrations the currents approach a voltage-independent limiting value, whose magnitude is proportional to the permeant ion concentration. The magnitude of this current is decreased by experimental maneuvers that decrease the aqueous diffusion coefficient of the ions. It is concluded that the magnitude of this limiting current is determined by the diffusive ion movement through the aqueous convergence regions up to the channel entrance. It is further shown that the small-signal (ohmic) permeability properties also reflect the existence of the aqueous diffusion limitation. These results have considerable consequences for the construction of kinetic models for ion movement through gramicidin A channels. It is shown that the simple two-site-three-barrier model commonly used to interpret gramicidin A permeability data may lead to erroneous conclusions, as biionic potentials will be concentration dependent even when the channel is occupied by at most one ion. The aqueous diffusion limitation must be considered explicitly in the analysis of gramicidin A permeability characteristics. Some implications for understanding the properties of ion-conducting channels in biological membranes will be considered.     

Probes of the conduction process of a voltage-gated Cl- channel from Torpedo electroplax

The open-channel conductance properties of a voltage-gated Cl- channel derived from Torpedo californica electroplax and incorporated into planar bilayers were studied by several approaches. In neutral bilayers the channel conductance saturates with Cl- activity according to a rectangular hyperbolic relation with a half-saturation activity of 75 mM and a maximum conductance of 32 pmho. The observation of identical behavior in charged membranes implies that ions permeating the channel do not sense the surface potential of the bulk membrane. The Cl-:Br- permeability ratio, measured under biionic conditions, is independent of salt concentration. SCN- ion reversibly blocks the channel. The voltage dependence of the block implies the existence of two separate blocking sites within the channel: one accessible from the cis side only (the side to which vesicles are added) and the other accessible from the trans side only. The block at each site is competitive with Cl-. The results are consistent with a single-ion Eyring model of the conduction process in which the ion must traverse three kinetic barriers as it permeates the channel and in which the channel can accommodate at most one ion at a time.     

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 explicity 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.     

Release of small transmitters through kiss-and-run fusion pores in rat pancreatic beta cells

Exocytosis of secretory vesicles begins with a fusion pore connecting the vesicle lumen to the extracellular space. This pore may then expand or it may close to recapture the vesicle intact. The contribution of the latter, termed kiss-and-run, to exocytosis of pancreatic beta cell large dense-core vesicles (LDCVs) is controversial. Examination of single vesicle fusion pores demonstrated that rat beta cell LDCVs can undergo exocytosis by rapid pore expansion, by the formation of stable pores, or via small transient kiss-and-run fusion pores. Elevation of cAMP shifted LDCV fusion pore openings to the transient mode. Under this condition, the small fusion pores were sufficient for release of ATP, stored within LDCVs together with insulin. Individual ATP release events occurred coincident with amperometric "stand alone feet" representing kiss-and-run. Therefore, the LDCV kiss-and-run fusion pores allow small transmitter release but likely retain the larger insulin peptide. This may represent a mechanism for selective intraislet signaling.     

The Behaviour of Ions in Narrow Water-Filled Pores

Today, the equilibrium behavior of ions in solution may be predicted with some confidence, essentially because rapid ionic diffusion over small distances ensures homogeneity throughout the solution. Equilibrium concepts such as ionic strength and pH apply. However, when attempting to understand the behavior of ions passing rapidly through narrow pores such as ion channels, no such equilibrium state may be assumed. The passing solution may have been in equilibrium with conditions at the mouth of the pore but will not be in equilibrium with charged molecules on the pore wall. In addition, the water in narrow pores will be partially ordered by contact with the pore walls and will not behave like bulk water.To illustrate this difference, a simple equilibrium calculation of the ion concentrations near a plastic sheet penetrated by narrow pores and containing in its surface partially ionized carboxyl groups is shown to be in good agreement with experiment. However, to predict the non-equilibrium behavior within the narrow pores is much more difficult. To illustrate the difficulty, a Monte Carlo computer model is described which attempts to predict the rapid switching of ion current observed experimentally with these narrow pores.     

A hydrodynamic theory of ion conductance through Ohmic pores

A method of calculating the size of membrane pores lacking strong ionic selectivity is presented. By treating the flow of ions through a small channel as a hydrodynamic phenomenon, the electrical conductance becomes a function of the ratio of ion radius to channel radius. Thus when both the channel conductance and the ion size are known, the radius of the channel may be estimated. The method gives good agreement among radii predicted from conductances of four different alkali cations in alamethicin pores.     

Permeation of internal and external monovalent cations through the catfish cone photoreceptor cGMP-gated channel

The permeation of monovalent cations through the cGMP-gated channel of catfish cone outer segments was examined by measuring permeability and conductance ratios under biionic conditions. For monovalent cations presented on the cytoplasmic side of the channel, the permeability ratios with respect to extracellular Na followed the sequence NH4 > K > Li > Rb = Na > Cs while the conductance ratios at +50 mV followed the sequence Na approximately NH4 > K > Rb > Li = Cs. These patterns are broadly similar to the amphibian rod channel. The symmetry of the channel was tested by presenting the test ion on the extracellular side and using Na as the common reference ion on the cytoplasmic side. Under these biionic conditions, the permeability ratios with respect to Na at the intracellular side followed the sequence NH4 > Li > K > Na > Rb > Cs while the conductance ratios at +50 mV followed the sequence NH4 > K approximately Na > Rb > Li > Cs. Thus, the channel is asymmetric with respect to external and internal cations. Under symmetrical 120 mM ionic conditions, the single-channel conductance at +50 mV ranged from 58 pS in NH4 to 15 pS for Cs and was in the order NH4 > Na > K > Rb > Cs. Unexpectedly, the single-channel current-voltage relation showed sufficient outward rectification to account for the rectification observed in multichannel patches without invoking voltage dependence in gating. The concentration dependence of the reversal potential for K showed that chloride was impermeant. Anomalous mole fraction behavior was not observed, nor, over a limited concentration range, were multiple dissociation constants. An Eyring rate theory model with a single binding site was sufficient to explain these observations.     

Outer-membrane protein PhoE from Escherichia coli forms anion-selective pores in lipid-bilayer membranes

Porin PhoE of the outer membrane of Escherichia coli was isolated and purified. Reconstitution experiments with lipid bilayer membranes showed that this protein formed pores which had a single channel conductance of 210 pS at 0.1 M KCl. The PhoE pores were obviously not voltage-controlled or regulated. In contrast to pores formed by the OmpF porin from E. coli the PhoE channel was found to be anion-selective at neutral pH. Chloride is about three to ten times more permeable through the pore than alkali ions. On the basis of the observed pH dependence of the permeability ratio of anions and cations, this anionic selectivity is explained by the assumption that the PhoE pore contains an excess of fixed positive charges.     

Outer surface modification of synthetic multifunctional pores

The characteristics of pores formed by p-octiphenyl beta-barrels with LWV triads at the outer surface are reported in comparison with the conventional rigid-rod beta-barrels with all-L outer surface. Maintained multifunctionality of tetrameric pores with external LWV triads (inversion of ion selectivity, molecular recognition and transformation) is implicative for intact barrel interior. Increased pore activity supports dominance of high bilayer affinity for W over low affinity for V. Transmembrane p-octiphenyl orientation (from fluorescence depth quenching) supports barrel-stave (rather than toroidal) pores and dominance of transmembrane preference of rigid rods over interfacial preference of W. Destabilization of beta-barrel pores in membranes (from short single-channel lifetimes) and in the media (from 4th-power dependence on monomer concentration) by LWV triads supports dominance of low beta-propensity for W over high beta-propensity for V. The relation between the stability of supramolecular (pre)pores and dependence of activity on monomer concentration is discussed in a more general context.     

The "independence principle" in the processes of water transport.

The processes of membrane transport exhibiting permeability coefficients depending on the species activities do not obey the "independence principle" and are assumed to take place by a mechanism of discrete nature, analyzable by a kinetic formalism. In this article, we study the dependence of the osmotic permeability coefficient on the water activities, from the steady-state analysis of a kinetic model of single-file water transport that simultaneously incorporates the vacancy-mediated and "knock-on" mechanisms into the state diagram. In particular, we study the relation between the near-equilibrium osmotic permeability (Pe) and the equilibrium water activity of the compartments (w). The analysis and numerical calculations performed for a simple case of the model show that, for values of the parameters consistent with experimental data, Pe exhibits only a small variation with w within the physiological range in the majority of the situations considered here. It is not possible to predict, from the study of these simple models, whether more complicated kinetic diagrams of water transport may be characterized by permeability coefficients with a more evident dependence on the water activities. Nevertheless, the results obtained here suggest that, for the case of physiological water pores, the analysis of the kinetic dependence of the permeability coefficients on the water activities may not yield evidence pointing to a discrete nature for the transport process.     

Ionic selectivity of pores formed by the matrix protein (porin) of Escherichia coli.

Incorporation of the matrix protein (porin) from the outer membrane of Escherichia coli into black lipid films results in the formation of ion-permeable pores with a single-pore conductance of the order of 2 nS (in 1 M KCl). Information on the structure of this pore has been obtained by determining the selectivity for various species differing in charge and size. From the permeability of the pore for large organic ions (Tris+, glucosamine+, Hepes-) a minimum pore diameter of 0.8 nm is estimated. At neutral pH the pore is two to four times more permeable for alkali ions than for chloride. On the basis of the observed pH dependence of permeability, this cationic selectivity is explained by the assumption that the pore contains fixed negative charges.     

Symmetry and asymmetry of permeation through toxin-modified Na+ channels.

Single Na+ channels from rat skeletal muscle were inserted into planar lipid bilayers in the presence of either 200 nM batrachotoxin (BTX) or 50 microM veratridine (VT). These toxins, in addition to their ability to shift inactivation of voltage-gated Na+ channels, may be used as probes of ion conduction in these channels. Channels modified by either of the toxins have qualitatively similar selectivity for the alkali cations (Na+ approximately Li+ greater than K+ greater than Rb+ greater than Cs+). Biionic reversal potentials, for example, were concentration independent for all ions studied. Na+/K+ and Na+/Rb+ reversal potentials, however, were dependent on the orientation of the ionic species with respect to the intra- or extracellular face of the channel, whereas Na+/Li+ biionic reversal potentials were not orientation dependent. A simple, four-barrier, three-well, single-ion occupancy model was used to generate current-voltage relationships similar to those observed in symmetrical solutions of Na, K, or Li ions. The barrier profiles for Na and Li ions were symmetric, whereas that for K ions was asymmetric. This suggests the barrier to ion permeation for K ions may be different than that for Na and Li ions. With this model, these hypothetical energy barrier profiles could predict the orientation-dependent reversal potentials observed for Na+/K+ and Na+/Rb+. The energy barrier profiles, however, were not capable of describing biionic Na/Li ion permeation. Together these results support the hypothesis that Na ions have a different rate determining step for ion permeation than that of K and Rb ions.     

Dimensions of the narrow portion of a recombinant NMDA receptor channel.

Glutamate-activated single-channel and ensemble currents were recorded from Xenopus laevis oocytes and HEK 293 cells expressing a recombinant NMDA receptor, assembled from NR1 and NR2A subunits. Cesium was the main charge carrier, and organic cations were used to determine the presence of vestibules of this channel and to estimate its pore diameter. The large organic cations tris-(hydroxymethyl)-aminomethane (Tris), N-methyl-glucamine (NMG), arginine (NMG), arginine (Arg), choline, and tetramethylammonium (TMA), when added in millimolar concentrations to the extracellular or cytoplasmic side, produced a voltage-dependent blockade of single-channel Cs+ currents. These molecules behaved as impermeant ions that only partially traverse the channel from either side. The smaller cations trimethylammonium (TriMA) and dimethylammonium (DMA) produced a small and nearly voltage-independent reduction in current amplitude, suggesting that they are permeant. In biionic experiments with Cs+ as the reference ion, the large blocking cations NMG, Arg, Tris, TMA, choline, hexamethonium (Hme), triethylammonium (TriEA), and tetraethylammonium (TEA) showed no measurable permeability. TriMA and smaller ammonium derivatives were permeant. Both the permeability and single-channel conductance of organic cations, relative to Cs+, decreased as the ion size increased. The results suggest that the NMDA receptor has extracellular and cytoplasmic mouths that can accommodate large cations up to 7.3 A in mean diameter. The narrow portion of the pore is estimated to have a mean diameter of 5.5 A.     

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.     

The multi-ion nature of the pore in Shaker K+ channels.

We have investigated some of the permeation properties of the pore in Shaker K channels. We determined the apparent permeability ratio of K+, Rb+, and NH4+ ions and block of the pore by external Cs+ ions. Shaker channels were expressed with the baculovirus/Sf9 expression system and the channel currents measured with the whole-cell variant of the patch clamp technique. The apparent permeability ratio, PRb/PK, determined in biionic conditions with internal K+, was a function of external Rb+ concentration. A large change in PRb/PK occurred with reversed ionic conditions (internal Rb+ and external K+). These changes in apparent permeability were not due to differences in membrane potential. With internal K+, PNH4/PK was not a function of external NH4+ concentration (at least over the range 50-120 mM). We also investigated block of the pore by external Cs+ ions. At a concentration of 20 mM, Cs+ block had a voltage dependence equivalent to that of an ion with a valence of 0.91; this increased to 1.3 at 40 mM Cs+. We show that a 4-barrier, 3-site permeation model can simulate these and many of the other known properties of ion permeation in Shaker channels.     

Electrostatic modeling of ion pores. II. Effects attributable to the membrane dipole potential.

This paper presents calculations of the shielded dipole potential in the interior of a pore piercing a lipid membrane that is at a potential V0 with respect to the aqueous solution. Except in the case of long narrow pores, there is substantial shielding of the membrane dipole potential. The associated dipole field never extends a significant distance into the aqueous region. The fact that the single-channel conductance of gramicidin B is only twice as large in glyceryl monooleate membranes as in phosphatidyl choline (PC) membranes, even though PC is approximately 120 mV more positive with respect to water, is interpreted in terms of the potential energy profile calculated for a gramicidin-like channel. It is demonstrated that the membrane dipole potential can significantly affect channel conductance only if the pore is narrow and if the peak in the potential energy profile occurs in the pore interior.     

Translocation of RNA-coated gold particles through the nuclear pores of oocytes

In the present study, various sized gold particles coated with tRNA, 5S RNA, or poly(A) were used to localize and characterize the pathways for RNA translocation to the cytoplasm. RNA-coated gold particles were microinjected into the nucleus of Xenopus oocytes. The cells were fixed after 15, 60 min, or 6 h, and the particle distribution was later observed by electron microscopy. Similar results were obtained with all classes of RNA used. After nuclear injection, particles ranging from 20-230 A in diameter were observed within central channels of the nuclear pores and in the cytoplasm immediately adjacent to the pores. Particles of this size would not be expected to diffuse through the pores, suggesting that some form of mediated transport occurred. In addition, it was found that the translocation process is saturable. At least 97% of the pores analyzed appeared to be involved in the translocation process. Gold coated with nonphysiological polynucleotides (poly[I] or poly[dA]) were also translocated. When nuclei were injected with either BSA-, ovalbumin-, polyglutamic acid-, or PVP-coated gold, the particles were essentially excluded from the pores. These results indicate that the accumulation of RNA-gold within the pores and adjacent cytoplasm was not due to non-specific effects. We conclude that the translocation sites for gold particles coated with different classes of RNA are located in the centers of the nuclear pores and that particles at least 230 A in diameter can cross the envelope. Tracer particles injected into the cytoplasm were observed within the nuclear pores in areas near the site of injection. However, only a small percentage of the particles actually entered the nucleus. It was also determined, by performing double injection experiments, that individual pores are bifunctional, that is, capable of transporting both proteins and RNA.     

Permeability of single nuclear pores.

In this first application of optical single transporter recording (OSTR), a recently established technique for optically monitoring the activity of single transporters in membrane patches (Tsch?drich-Rotter and Peters. 1998. J. Microsc. 192:114-125), the passive permeability of the nuclear pore complex (NPC) was measured for a homologous series of hydrophilic probe molecules. Nuclei were isolated from Xenopus oocytes and firmly attached to filters containing small cylindrical pores. Transport through membrane patches spanning filter pores was measured by scanning microphotolysis. Thus the permeability coefficients of single NPCs were determined for fluorescently labeled dextrans of approximately 4, 10, and 20 kDa. Dextrans of >/=40 kDa could not permeate the NPC. The data were consistent with a model in which the NPC contains a single diffusion channel. By application of established theories for the restricted diffusion through small pores, the diffusion channel was approximated as a cylinder with a radius of 4.4-6.1 nm (mean 5. 35 nm). Because the transport rate constant of the single NPC was known, the equivalent length of the channel could be also determined and was found to be 40-50 nm (mean 44.5 nm). The symmetry of the NPC implies that a singular component such as the diffusion channel is located at the center of the NPC. Therefore a common transport pathway apparently mediates both passive and signal-dependent transport. To test this hypothesis, measurements of signal-dependent transport and of the mutual effects signal-dependent and passive transport may exert on each other are in progress.     

General continuum analysis of transport through pores. I. Proof of Onsager's reciprocity postulate for uniform pore.

The nonelectrolyte (Js) and volume (Jv) flux across a membrane is usually described in terms of two equations derived from the theory of irreversible thermodynamics: (see article) where delta c and delta P are the concentration and pressure difference; omega and Lp are the diffuse and hydraulic permeability; and sigma s and sigma v are the reflection coefficients. If Onsager's reciprocity postulate is assumed, it can be shown that signa s and sigma v are equal. This is an important assumption because it allows one to apply the continuum theory relationship between sigma s and the pore radius to experimental measurements of sigma v. In this paper, general continuum expressions for both the Jv (a new result) and Js equation will be derived and the equality of sigma s and sigma v proved. The proof uses only general hydrodynamic results and does not require explicit solutions for the drag coefficients or, for example, the assumption that the solute is in the center of the pore. The proof applys to arbitrarily shaped solutes and any pore whose shape is independent of axial position (uniform). In addition, new expressions for the functional dependence of omega and sigma on the pore radius are derived (including the effect of the particle lying off the pore axis). These expressions differ slightly from earlier results and are probably more accurate.