Selectivity and permeation in calcium release channel of cardiac muscle: alkali metal ions

Current was measured from single open channels of the calcium release channel (CRC) of cardiac sarcoplasmic reticulum (over the range +/-180 mV) in pure and mixed solutions (e.g., biionic conditions) of the alkali metal ions Li+, K+, Na+, Rb+, Cs+, ranging in concentration from 25 mM to 2 M. The current-voltage (I-V) relations were analyzed by an extension of the Poisson-Nernst-Planck (PNP) formulation of electrodiffusion, which includes local chemical interaction described by an offset in chemical potential, which likely reflects the difference in dehydration/solvation/rehydration energies in the entry/exit steps of permeation. The theory fits all of the data with few adjustable parameters: the diffusion coefficient of each ion species, the average effective charge distribution on the wall of the pore, and an offset in chemical potential for lithium and sodium ions. In particular, the theory explains the discrepancy between "selectivities" defined by conductance sequence and "selectivities" determined by the permeability ratios (i.e., reversal potentials) in biionic conditions. The extended PNP formulation seems to offer a successful combined treatment of selectivity and permeation. Conductance selectivity in this channel arises mostly from friction: different species of ions have different diffusion coefficients in the channel. Permeability selectivity of an ion is determined by its electrochemical potential gradient and local chemical interaction with the channel. Neither selectivity (in CRC) seems to involve different electrostatic interaction of different ions with the channel protein, even though the ions have widely varying diameters.     

Ion permeation and glutamate residues linked by Poisson-Nernst-Planck theory in L-type calcium channels.

L-type Ca channels contain a cluster of four charged glutamate residues (EEEE locus), which seem essential for high Ca specificity. To understand how this highly charged structure might produce the currents and selectivity observed in this channel, a theory is needed that relates charge to current. We use an extended Poisson-Nernst-Planck (PNP2) theory to compute (mean) Coulombic interactions and thus to examine the role of the mean field electrostatic interactions in producing current and selectivity. The pore was modeled as a central cylinder with tapered atria; the cylinder (i.e., "pore proper") contained a uniform volume density of fixed charge equivalent to that of one to four carboxyl groups. The pore proper was assigned ion-specific, but spatially uniform, diffusion coefficients and excess chemical potentials. Thus electrostatic selection by valency was computed self-consistently, and selection by other features was also allowed. The five external parameters needed for a system of four ionic species (Na, Ca, Cl, and H) were determined analytically from published measurements of thre limiting conductances and two critical ion concentrations, while treating the pore as a macroscopic ion-exchange system in equilibrium with a uniform bath solution. The extended PNP equations were solved with these parameters, and the predictions were compared to currents measured in a variety of solutions over a range of transmembrane voltages. The extended PNP theory accurately predicted current-voltage relations, anomalous mole fraction effects in the observed current, saturation effects of varied Ca and Na concentrations, and block by protons. Pore geometry, dielectric permittivity, and the number of carboxyl groups had only weak effects. The successful prediction of Ca fluxes in this paper demonstrates that ad hoc electrostatic parameters, multiple discrete binding sites, and logistic assumptions of single-file movement are all unnecessary for the prediction of permeation in Ca channels over a wide range of conditions. Further work is needed, however, to understand the atomic origin of the fixed charge, excess chemical potentials, and diffusion coefficients of the channel. The Appendix uses PNP2 theory to predict ionic currents for published "barrier-and-well" energy profiles of this channel.     

Reinterpreting the Anomalous Mole Fraction Effect: The Ryanodine Receptor Case Study

The origin of the anomalous mole fraction effect (AMFE) in calcium channels is explored with a model of the ryanodine receptor. This model predicted and experiments verified new AMFEs in the cardiac isoform. In mole fraction experiments, conductance is measured in mixtures of ion species X and Y as their relative amounts (mole fractions) vary. This curve can have a minimum (an AMFE). The traditional interpretation of the AMFE is that multiple interacting ions move through the pore in a single file. Mole fraction curves without minima (no AMFEs) are generally interpreted as X displacing Y from the pore in a proportion larger than its bath mole fraction (preferential selectivity). We find that the AMFE is also caused by preferential selectivity of X over Y, if X and Y have similar conductances. This is a prediction applicable to any channel and provides a fundamentally different explanation of the AMFE that does not require single filing or multiple occupancy: preferential selectivity causes the resistances to current flow in the baths, channel vestibules, and selectivity filter to change differently with mole fraction, and produce the AMFE.     

Anomalous mole fraction effect, electrostatics, and binding in ionic channels.

Ionic channels bathed in mixed solutions of two permeant electrolytes often conduct less current than channels bathed in pure solutions of either. For many years, this anomalous mole fraction effect (AMFE) has been thought to occur only in single-file pores containing two or more ions at a time. Most thinking about channels incorporates this view. We show here that the AMFE arises naturally, as an electrostatic consequence of localized ion specific binding, if the average current through a channel is described by a theory (Poisson-Nernst-Planck, PNP) that computes the average electric field from the average concentration of charges in and near the channel. The theory contains only those ion-ion interactions mediated by the mean field, and it does not enforce single filing. The AMFE is predicted by PNP over a wide range of mean concentrations of ions in the channel; for example, it is predicted when (on the average) less, or much less, than one ion is found in the channel's pore. In this treatment, the AMFE arises, in large measure, from a depletion layer produced near a region of ion-specific binding. The small excess concentration of ions in the binding region repels all nearby ions of like charge, thereby creating a depletion layer. The overall conductance of the channel arises in effect from resistors in series, one from the binding region, one from the depletion zone, and one from the unbinding region. The highest value resistor (which occurs in the depletion zone) limits the overall series conductance. Here the AMFE is not the result of single filing or multiple occupancy, and so previous views of permeation need to be revised: the presence of an AMFE does not imply that ions permeate single file through a multiply occupied pore.     

Ion permeation and selectivity of OmpF porin: a theoretical study based on molecular dynamics,Brownian dynamics,and continuum electrodiffusion theory

Three different theoretical approaches are used and compared to refine our understanding of ion permeation through the channel formed by OmpF porin from Escherichia coli. Those approaches are all-atom molecular dynamics (MD) in which ions, solvent, and lipids are represented explicitly, Brownian dynamics (BD) in which ions are represented explicitly, while solvent and lipids are represented as featureless dielectrics, and Poisson-Nernst-Planck (PNP) electrodiffusion theory in which both solvent and local ion concentrations are represented as a continuum. First, the ability of the different theoretical approaches in reproducing the equilibrium average ion density distribution in OmpF porin bathed by a 1M KCl symmetric salt solution is examined. Under those conditions the PNP theory is equivalent to the non-linear Poisson-Boltzmann (PB) theory. Analysis shows that all the three approaches are able to capture the important electrostatic interactions between ions and the charge distribution of the channel that govern ion permeation and selectivity in OmpF. The K(+) and Cl(-) density distributions obtained from the three approaches are very consistent with one another, which suggests that a treatment on the basis of a rigid protein and continuum dielectric solvent is valid in the case of OmpF. Interestingly, both BD and continuum electrostatics reproduce the distinct left-handed twisted ion pathways for K(+) and Cl(-) extending over the length of the pore which were observed previously in MD. Equilibrium BD simulations in the grand canonical ensemble indicate that the channel is very attractive for cations, particularly at low salt concentration. On an average there is 1.55 K(+) inside the pore in 10mM KCl. Remarkably, there is still 0.17 K(+) on average inside the pore even at a concentration as low as 1microM KCl. Secondly, non-equilibrium ion flow through OmpF is calculated using BD and PNP and compared with experimental data. The channel conductance in 0.2M and 1M KCl calculated using BD is in excellent accord with the experimental data. The calculations reproduce the experimentally well-known conductance-concentration relation and also reveal an asymmetry in the channel conductance (a larger conductance is observed under a positive transmembrane potential). Calculations of the channel conductance for three mutants (R168A, R132A, and K16A) in 1M KCl suggest that the asymmetry in the channel conductance arises mostly from the permanent charge distribution of the channel rather than the shape of the pore itself. Lastly, the calculated reversal potential in a tenfold salt gradient (0.1:1M KCl) is 27.4(+/-1.3)mV (BD) and 22.1(+/-0.6)mV (PNP), in excellent accord with the experimental value of 24.3mV. Although most of the results from PNP are qualitatively reasonable, the calculated channel conductance is about 50% higher than that calculated from BD probably because of a lack of some dynamical ion-ion correlations.     

Hybrid MD-Nernst Planck model of α-hemolysin conductance properties

Motivated by experiments in which an applied electric field translocates polynucleotides through an α-hemolysin protein channel causing ionic current transient blockade, a hybrid simulation model is proposed to predict the conductance properties of the open channel. Time scales corresponding to ion permeation processes are reached using the Poisson–Nernst–Planck (PNP) electro-diffusion model in which both solvent and local ion concentrations are represented as a continuum. The diffusion coefficients of the ions (K⁺ and Cl^?) input in the PNP model are, however, calculated from all-atom molecular dynamics (MD). In the MD simulations, a reduced representation of the channel is used. The channel is solvated in a 1?M KCl solution, and an external electric field is applied. The pore specific diffusion coefficients for both ionic species are reduced 5–7 times in comparison to bulk values. Significant statistical variations (17–45%) of the pore-ions diffusivities are observed. Within the statistics, the ionic diffusivities remain invariable for a range of external applied voltages between 30 and 240?mV. In the 2D-PNP calculations, the pore stem is approximated by a smooth cylinder of radius ～9?Å with two constriction blocks where the radius is reduced to ～6?Å. The electrostatic potential includes the contribution from the atomistic charges. The MD-PNP model shows that the atomic charges are responsible for the rectifying behaviour and for the slight anion selectivity of the α-hemolysin pore. Independent of the hierarchy between the anion and cation diffusivities, the anionic contribution to the total ionic current will dominate. The predictions of the MD-PNP model are in good agreement with experimental data and give confidence in the present approach of bridging time scales by combining a microscopic and macroscopic model.     

Anion permeation in an apical membrane chloride channel of a secretory epithelial cell

Single channel currents though apical membrane Cl channels of the secretory epithelial cell line T84 were measured to determine the anionic selectivity and concentration dependence of permeation. The current-voltage relation was rectified with single channel conductance increasing at positive potentials. At 0 mV the single channel conductance was 41 +/- 2 pS. Permeability, determined from reversal potentials, was optimal for anions with diameters between 0.4 and 0.5 nm. Anions of larger diameter had low permeability, consistent with a minimum pore diameter of 0.55 nm. Permeability for anions of similar size was largest for those ions with a more symmetrical charge distribution. Both HCO3 and H2PO4 had lower permeability than the similar-sized symmetrical anions, NO3 and ClO4. The permeability sequence was SCN greater than I approximately NO3 approximately ClO4 greater than Br greater than Cl greater than PF6 greater than HCO3 approximately F much greater than H2PO4. Highly permeant anions had lower relative single channel conductance, consistent with longer times of residence in the channel for these ions. The conductance sequence for anion efflux was NO3 greater than SCN approximately ClO4 greater than Cl approximately I approximately Br greater than PF6 greater than F approximately HCO3 much greater than H2PO4. At high internal concentrations, anions with low permeability and conductance reduced Cl influx consistent with block of the pore. The dependence of current on Cl concentration indicated that Cl can also occupy the channel long enough to limit current flow. Interaction of Cl and SCN within the conduction pathway is supported by the presence of a minimum in the conductance vs. mole fraction relation. These results indicate that this 40-pS Cl channel behaves as a multi-ion pathway in which other permeant anions could alter Cl flow across the apical membrane.     

A simple method for the determination of the pore radius of ion channels in planar lipid bilayer membranes

Abstract A new method of pore size determination is presented. The results of applying this simple method to ion channels formed by staphylococcal α-toxin and its N-terminal fragment as well as to cholera toxin channels are shown. The advantages and the difficulties of this method are discussed. It was found that (i) the mobility of ions in solutions depends only on the percentage of concentration of added non-electrolytes and practically not on their chemical nature (sugars or polyglycols) and molecular size; (ii) the proportional change of both ion channel conductance and bulk solution conductivity by low M . non-electrolytes may be used as an indication of a diffusion mechanism of ion transport through channels; (iii) the slope of the dependence of the ion channel conductance on the bulk conductivity of solutions containing different concentrations of non-electrolyte is a good measure of channel permeability for non-electrolytes.     

Poisson-Nernst-Planck Models of Nonequilibrium Ion Electrodiffusion through a Protegrin Transmembrane Pore

Protegrin peptides are potent antimicrobial agents believed to act against a variety of pathogens by forming nonselective transmembrane pores in the bacterial cell membrane. We have employed 3D Poisson-Nernst-Planck (PNP) calculations to determine the steady-state ion conduction characteristics of such pores at applied voltages in the range of −100 to +100 mV in 0.1 M KCl bath solutions. We have tested a variety of pore structures extracted from molecular dynamics (MD) simulations based on an experimentally proposed octomeric pore structure. The computed single-channel conductance values were in the range of 290–680 pS. Better agreement with the experimental range of 40–360 pS was obtained using structures from the last 40 ns of the MD simulation, where conductance values range from 280 to 430 pS. We observed no significant variation of the conductance with applied voltage in any of the structures that we tested, suggesting that the voltage dependence observed experimentally is a result of voltage-dependent channel formation rather than an inherent feature of the open pore structure. We have found the pore to be highly selective for anions, with anionic to cationic current ratios (I_Cl−/I_K+) on the order of 10³. This is consistent with the highly cationic nature of the pore but surprisingly in disagreement with the experimental finding of only slight anionic selectivity. We have additionally tested the sensitivity of our PNP model to several parameters and found the ion diffusion coefficients to have a significant influence on conductance characteristics. The best agreement with experimental data was obtained using a diffusion coefficient for each ion set to 10% of the bulk literature value everywhere inside the channel, a scaling used by several other studies employing PNP calculations. Overall, this work presents a useful link between previous work focused on the structure of protegrin pores and experimental efforts aimed at investigating their conductance characteristics.     

Ion permeation through the alpha-hemolysin channel: theoretical studies based on Brownian dynamics and Poisson-Nernst-Plank electrodiffusion theory

Identification of the molecular interaction governing ion conduction through biological pores is one of the most important goals of modern electrophysiology. Grand canonical Monte Carlo Brownian dynamics (GCMC/BD) and three-dimensional Poisson-Nernst-Plank (3d-PNP) electrodiffusion algorithms offer powerful and general approaches to study of ion permeation through wide molecular pores. A detailed analysis of ion flows through the staphylococcal alpha-hemolysin channel based on series of simulations at different concentrations and transmembrane potentials is presented. The position-dependent diffusion coefficient is approximated on the basis of a hydrodynamic model. The channel conductance calculated by GCMC/BD is approximately 10% higher than (electrophysiologically measured) experimental values, whereas results from 3d-PNP are always 30-50% larger. Both methods are able to capture all important electrostatic interactions in equilibrium conditions. The asymmetric conductance upon the polarity of the transmembrane potential observed experimentally is reproduced by GCMC/BD and 3d-PNP. The separation of geometrical and energetic influence of the channel on ion conduction reveals that such asymmetries arise from the permanent charge distribution inside the pore. The major determinant of the asymmetry is unbalanced charge in the triad of polar residues D127, D128, and K131. The GCMC/BD or 3d-PNP calculations reproduce also experimental reversal potentials and permeability rations in asymmetric ionic solutions. The weak anionic selectivity of the channel results from the presence of the salt bridge between E111 and K147 in the constriction zone. The calculations also reproduce the experimentally derived dependence of the reversible potential to the direction of the salt gradient. The origin of such effect arises from the asymmetrical distribution of energetic barriers along the channel axis, which modulates the preferential ion passage in different directions.     

The anomalous mole fraction effect in calcium channels: a measure of preferential selectivity

The cause of the anomalous mole fraction effect (AMFE) in calcium-selective ion channels is studied. An AMFE occurs when the conductance through a channel is lower in a mixture of salts than in the pure salts at the same concentration. The textbook interpretation of the AMFE is that multiple ions move through the pore in coordinated, single-file motion. Instead of this, we find that at its most basic level an AMFE reflects a channel's preferential binding selectivity for one ion species over another. The AMFE is explained by considering the charged and uncharged regions of the pore as electrical resistors in series: the AMFE is produced by these regions of high and low ion concentration changing differently with mole fraction due to the preferential ion selectivity. This is demonstrated with simulations of a model L-type calcium channel and a mathematical analysis of a simplistic point-charge model. The particle simulations reproduce the experimental data of two L-type channel AMFEs. Conditions under which an AMFE may be found experimentally are discussed. The resistors-in-series model provides a fundamentally different explanation of the AMFE than the traditional theory and does not require single filing, multiple occupancy, or momentum-correlated ion motion.     

Ionic selectivity, saturation and block in gramicidin A channels: I. Theory for the electrical properties of ion selective channels having two pairs of binding sites and multiple conductance states.

A model for the gramicidin A channel is proposed which extends existing models by adding a specific cationic binding site at each entrance to the channel. The binding of ions to these outer channel sites is assumed to shift the energy levels of the inner sites and barriers and thereby alter the channel conductance. The resulting properties are analyzed theoretically for the simplest case of two inner sites and a single energy barrier. This for-site model (two outer and two inner) predicts that the membrane potential at zero current (Uo) should be a Goldman-Hodgkin-Katz equation with concentration-dependent permeability ratios. The coefficients of the concentration-dependent terms are shown to be related to the peak energy shifts of the barrier and to the binding constants of the outer sites. The thory also predicts the channel conductance in symmetrical solutions to exhibit three limiting behaviors, from which the properties of the outer and inner sites can be characterized. In two-cation symmetrical mixtures the conductance as a function of mole fraction is shown to have a minimum, and the related phenomenon of inhibition and block exerted by one ion on the other is explained explicitly by the theory. These various phenomena, having ion interactions in a multiply occupied channel as a common physical basis, are all related (by the theory) through a set of measurable parameters describing the properties of the system.     

Interaction of Clostridium perfringens iota-toxin with lipid bilayer membranes. Demonstration of channel formation by the activated binding component Ib and channel block by the enzyme component Ia.

The interaction between model lipid membranes and the binding component (Ib) of the ADP-ribosylating iota-toxin of Clostridium perfringens was studied in detail. Ib had to be activated by trypsin to result in channel formation in artificial lipid bilayers. The channels formed readily by Ib had a small single-channel conductance of about 85 picosiemens in 1 m KCl. Channel function was blocked in single-channel and multichannel experiments by the enzymatic component Ia in a pH-dependent manner. The strong Ia-mediated channel block of Ib occurred only when the pH was at least lowered to pH 5.6. The single-channel conductance showed a linear dependence on the bulk aqueous KCl concentration, which indicated that the channel properties were more general than specific. Zero current membrane potential measurements suggested the Ib channel has an approximately 6-fold higher permeability for potassium ions than for chloride. The selectivity ratio changed for salts composed of cations and anions of different mobility in the aqueous phase, again suggesting that Ib formed a water-filled general diffusion pore. Asymmetric addition of activated Ib to lipid bilayer membranes resulted in an asymmetric voltage dependence, indicating its full orientation within the membrane. Titration experiments with chloroquine and different tetraalkylammonium ions suggested that the Ib channel was blocked by these compounds but had only a weak affinity to them. In vivo measurements using Vero cells demonstrate that chloroquine and related molecules also did not efficiently block intoxication of the cells by iota-toxin. The possible role of Ib in the translocation of iota-toxin across the target cell membrane is discussed.     

Anion competition for a volume-regulated current.

We have examined whether the anionic amino acids, glutamate and aspartate, permeate through the same volume-regulated conductance permeant to Cl- ions. Cell swelling was initiated in response to establishing a whole-cell configuration in the presence of a hyposmotic gradient. Volume-regulated anion currents carried by Cl-, glutamate, or aspartate developed with similar time courses and showed similar voltage-dependent inactivation. Permeability ratios (Paa/PCl) calculated from measured reversal potentials were dependent on the mole fraction ratio (MFR) of the permeant anions ([aa]/([aa] + [Cl-])). MFR was varied from 0.00 to 0.97. As the fraction of amino acid increased, Paa/PCl decreased. Current amplitude was similarly dependent on MFR. These results show that the permeation of anionic amino acids and that of Cl- ions are not independent of each other, indicating that the ion channel underlying the volume-regulated conductance can be occupied by more than one ion at a time. Application of Eyring rate theory indicated that the major barrier to Cl- ion permeation is at the intracellular side of the membrane, and that the major barrier to amino acid permeation is at the extracellular side of the membrane. The interactions between these permeant ions may have a physiological modulatory role in volume regulation through a volume-regulated anion conductance.     

The permeability of endplate channels to monovalent and divalent metal cations

The relative permeability of endplate channels to monovalent and divalent metal ions was determined from reversal potentials. Thallium is the most permeant ion with a permeability ratio relative to Na+ of 2.5. The selectivity among alkali metals is weak with a sequence, Cs+ greater than Rb+ greater than K+ greater than Na+ greater than Li+, and permeability ratios of 1.4, 1.3, 1.1, 1.0, and 0.9. The selectivity among divalent ions is also weak, with a sequence for alkaline earths of Mg++ greater than Ca++ greater than Ba++ greater than Sr++. The transition metal ions Mn++, Co++, Ni++, Zn++, and Cd++ are also permeant. Permeability ratios for divalent ions decreased as the concentration of divalent ion was increased in a manner consistent with the negative surface potential theory of Lewis (1979 J. Physiol. (Lond.). 286: 417--445). With 20 mM XCl2 and 85.5 mM glucosamine.HCl in the external solution, the apparent permeability ratios for the alkaline earth cations (X++) are in the range 0.18--0.25. Alkali metal ions see the endplate channel as a water-filled, neutral pore without high-field-strength sites inside. Their permeability sequence is the same as their aqueous mobility sequence. Divalent ions, however, have a permeability sequence almost opposite from their mobility sequence and must experience some interaction with groups in the channel. In addition, the concentrations of monovalent and divalent ions are increased near the channel mouth by a weak negative surface potential.     

Ionic selectivity, saturation, and block in sodium channels. A four- barrier model

Ionic fluxes in Na channels of myelinated axons show ionic competition, block, and deviations from simple flux independence. These phenomena are particularly evident when external Na+ ions are replaced by other permeant or impermeant ions. The observed currents require new flux equations not based on the concepts of free diffusion. A specific permeability model for the Na channel is developed from Eyring rate theory applied to a chain of saturable binding sites. There are four energy barriers in the pore and only one ion is allowed inside at a time. Deviations from independence arise from saturation. The model shows that ionic permeability ratios measured from zero-current potentials can differ from those measured from relative current amplitudes or conductances. The model can be fitted to experiments with various external sodium substitutes by varying only two parameters: For each ion the height of the major energy barrier (the selectivity filter) determines the biionic zero-current potential and the depth of the energy well (binding site) just external to that barrier then determines the current amplitudes. Voltage clamp measurements with myelinated nerve fibers are given showing numerous examples of deviations from independence in ionic fluxes. Strong blocks of ionic currents by guanidinium compounds and Tl+ ions are fitted by binding within the channel with apparent dissociation constants in the range 50-122 mM. A small block with high Na+ concentrations can be fitted by Na+ ion binding with a dissociation constant of 368 mM. The barrier model is given a molecular interpretation that includes stepwise dehydration of the permeating ion as it interacts with an ionized carboxylic acid.     

Multi-ion conduction and selectivity in the high-conductance Ca++-activated K+ channel from skeletal muscle.

Open-channel ion permeation properties were investigated for Ca++-activated K+ (CaK) channels in solutions of K+ and its analogues T1+, Rb+, and NH4+. Single CaK channels were inserted into planar lipid bilayers composed of neutral phospholipids, and open-channel current-voltage (I-V) relations were measured in symmetrical and asymmetrical solutions of each of these individual ions. For all concentrations studied, the zero-voltage conductance falls in the sequence K+ greater than T1+ greater than NH4+ greater than Rb+. The shape of the I-V curve in symmetrical solutions of a single permeant ion is non-ohmic and is species-dependent. The I-V shape is sublinear for K+ and T1+ and superlinear for Rb+ and NH4+. As judged by reversal potentials under bi-ionic conditions with K+ on one side of the bilayer and the test cation on the other, the permeability sequence is T1+ greater than K+ greater than Rb+ greater than NH4+ at 300 mM, which differs from the conductance sequence. Symmetrical mixtures of K+ or NH4+ with Rb+ show a striking anomalous mole fraction behavior, i.e., a minimum in single-channel conductance when the composition of a two-ion mixture is varied at constant total ion concentration. This result is incompatible with present models that consider the CaK channel a single-ion pore. In total, the results show that the CaK channel finely discriminates among K+-like ions, exhibiting different energy profiles among these species, and that several such ions can reside simultaneously within the conduction pathway.     

The cardiac ryanodine receptor: Looking for anomalies in permeation properties

Anomalies in the permeation properties of the cardiac RyR channel reconstituted into bilayer lipid membranes were investigated systematically. We tested the presence of the anomalous mole fraction effect (AMFE) for the ion conductance and the reversal potential with varying mole fractions of two permeant ions, while the total ion concentration was lower, as in previous studies, to avoid the masking effect of the channel pore saturation with ions. Mixtures of Ba²⁺ with other divalents (Ca²⁺, Sr²⁺), of Ca²⁺ with monovalents (Li⁺, Cs⁺), and of Na⁺ with other monovalents (Cs⁺, Li⁺) were used. We revealed a clear anomaly only for the ion conductance measured in the Na⁺-Cs⁺ and Ca²⁺-Li⁺ mixtures as computed by a Poisson-Nernst-Planck/density functional theory (PNP/DFT) model. Furthermore, we found a significant minimum in the concentration dependence of the reversal potential determined under Li⁺/Ca²⁺ bi-ionic conditions. Our study led to new observations that may have important implications for understanding the mechanisms involved in ion handling in the RyR channel pore; furthermore our results could be useful for further validation of ion permeation models developed for the RyR channel.     

Potassium channel of cardiac sarcoplasmic reticulum is a multi-ion channel

We have characterized mechanisms of ionic permeation in the K channel of canine cardiac sarcoplasmic reticulum (SR K channel). Ionic selectivity, as measured by relative permeabilities, followed Eisenman sequence l, a low field strength sequence. Slope conductance measured in symmetrical solutions across the bilayer followed Eisenman sequence V. In all cases, the selectivity characteristics of the prominent subconductance state (O1) were similar to those of the main-state (O2). Further, our studies have revealed that this channel differs in three significant ways from the highly characterized SR K channel of skeletal muscle. First, the ratio of permeabilities Cs+ to K+ was a complex function of ion concentration. Second, the concentration dependence of conductance was not well described by the Michaelis-Menten formalism. Instead, we modeled the observed relations using a more general approach based on classical rate theory. Third, mole fraction experiments (Cs+ with K+) demonstrated a prominent anomalous effect. Certain of our Cs+ data required the Eyring rate theory approach for adequate interpretation. We adopted a symmetrical energy profile incorporating ion-ion interaction and thereby accounted for much of the data. We conclude that the canine cardiac SR K channel is significantly different from that of skeletal muscle, and it may accommodate more than one ion at a time.