How electrolyte shielding influences the electrical potential in transmembrane ion channels.

The electrical potential due to fixed charge distributions is strongly altered in the vicinity of a membrane and notably dependent on aqueous electrolyte concentration. We present an efficient way to solve the nonlinear Poisson-Boltzmann equation applicable to general cylindrically symmetric dielectric geometries. It generalizes Gouy-Chapman theory to systems containing transmembrane channels. The method is applied to three channel systems: gramicidin, gap junction, and porin. We find that for a long, narrow channel such as gramicidin concentration variation has little influence on the electrical image barrier to ion permeation. However, electrolyte shielding reduces the image induced contribution to the energy required for multiple occupancy. In addition, the presence of electrolyte significantly affects the voltage profile due to an applied potential, substantially compressing the electric field to the immediate vicinity of the pore itself. In the large diameter channels, where bulk electrolyte may be assumed to enter the pore, the electrolyte greatly reduces the image barrier to ion permeation. At physiological ionic strengths this barrier is negligible and the channel may be readily multiply occupied. At all ionic strengths considered (l greater than 0.005 M) the image barrier saturates rapidly and is essentially constant more than one channel radius from the entrance to the pore. At lower ionic strengths (l less than 0.016 M) there are noticeable (greater than 20 mV) energy penalties associated with multiple occupancy.     

Partial purification of a potassium channel with low permeability for sodium from tonoplast membranes of Hordeum vulgare cv. Gerbel

Summary A potassium-specific tonoplast channel was identified by reconstitution of tonoplast polypeptides into planar lipid bilayer membranes. Highly purified tonoplast membranes were solubilized in Triton X-100-containing buffer and fractionated by size-exclusion chromatography. The protein fractions were assayed for ion channel activity in a planar bilayer system, and the potassium channel was routinely recovered in specific fractions corresponding to an apparent molecular mass of 80 kDa. In symmetrical electrolyte solutions of 100 mM potassium chloride, the potassium channel had a single-channel conductance of 72 pS. Substates of the channel with conductances of 17, 33 and 52 pS were frequently observed. After identification of the channel in low or high KCl, addition of sodium acetate or sodium chloride caused only insignificant conductance changes. This result suggested that the channel was not or little permeable for sodium or chloride, whereas it had similar single-channel conductance for rubidium and caesium ions as compared with potassium ions. The channel is presumably responsible for the equilibration of potassium between the vacuole and the cytosol. The role of the channel in the physiology of the barley cell under salt stress is discussed.The authors would like to thank U. Heber for many helpful discussions. This work was supported by grants of the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 176, projects B3 and B7) and by the Fonds der Chemischen Industrie.     

Single-channel parameters of gramicidin a,b, and c

The single-channel conductance Λ and the mean channel lifetime τ1 of natural and synthetic gramicidins A, B, and C has been studied. Significant differences in Λ were found between gramicidin A and B; both gramicidins differ only in one amino acid (tryptophan replaced by phenylalinine). The distribution of Λ is narrow in glycerylmonooleate membranes but considerably broader in dioleoyl phosphatidylcholine and dioleoyl phosphatidylethanolamine membranes. The ratio of the single-channel conductances in glycerylmonooleate and dioleoyl phosphatidylcholine membranes is only about two and is considerable smaller than the conductance ratio of nonactin-mediated cation transport. This finding suggests that dipolar potentials at the membrane/solution interface have little influence on the conductance of the gramicidin channel.     

Single-channel parameters of gramicidin A,B, and C.

The single-channel conductance lambda and the mean channel lifetime gamma of natural and synthetic gramicidins A, B, and C has been studied. Significant differences in delta were found between gramicidin A and B; both gramicidins differ only in one amino acid (tryptophan replaced by phenylaline). The distribution of lambda is narrow in glycerylmonooleate membranes but considerably broader in dioleoyl phosphatidylcholine and dioleoyl phosphatidylethanolamine membranes. The ratio of the single-channel conductances in glycerylmonooleate and dioleoyl phosphatidylcholine membranes is only about two and is considerable smaller than the conductance ratio of nonactin-mediated cation transport. This finding suggests that dipolar potentials at the membrane/solution interface have little influence on the conductance of the gramicidin channel.     

Induction of conductance heterogeneity in gramicidin channels

In previous work from our laboratory, 5-10% of the channels formed by [Val1]gramicidin A have conductances that fall outside the narrow range that conventionally has defined the standard gramicidin channel [e.g., see Russell et al. (1986) Biophys. J. 49, 673]. Reports from other laboratories, however, show that up to 50% of [Val1]gramicidin channels have conductances that fall outside the range for standard channels [e.g., see Prasad et al. (1986) Biochemistry 25, 456]. This laboratory-to-laboratory variation in the distribution of gramicidin single-channel conductances suggests that the conductance variants are induced by some environmental factor(s) [Busath et al. (1987) Biophys. J. 51, 79]. In order to test whether extrinsic agents can induce such conductance heterogeneity, we examined the effects of nonionic or zwitterionic detergents upon gramicidin channel behavior. In phospholipid bilayers, detergent addition induces many changes in gramicidin channel behavior: all detergents tested increase the channel appearance rate and average duration; most detergents decrease the conductance of the standard channel; and all but one of the detergents increase the conductance heterogeneity. These results show that the conductance heterogeneity can result from environmental perturbations, thus providing a possible explanation for the laboratory-to-laboratory variation in the heterogeneity of gramicidin channels. In addition, the differential detergent effects suggest possible mechanisms by which detergents can induce the conformational perturbations that result in gramicidin single-channel conductance variations.     

Molecular dynamics - potential of mean force calculations as a tool for understanding ion permeation and selectivity in narrow channels

Ion channels catalyze the permeation of charged molecules across cell membranes and are essential for many vital physiological functions, including nerve and muscle activity. To understand better the mechanisms underlying ion conduction and valence selectivity of narrow ion channels, we have employed free energy techniques to calculate the potential of mean force (PMF) for ion movement through the prototypical gramicidin A channel. Employing modern all-atom molecular dynamics (MD) force fields with umbrella sampling methods that incorporate one hundred 1-2 ns trajectories, we find that it is possible to achieve semi-quantitative agreement with experimental binding and conductance measurements. We also examine the sensitivity of the MD-PMF results to the choice of MD force field and compare PMFs for potassium, calcium and chloride ions to explore the basis for the valence selectivity of this narrow and uncharged ion channel. A large central barrier is observed for both anions and divalent ions, consistent with lack of experimental conductance. Neither anion or divalent cation is seen to be stabilized inside the channel relative to the bulk electrolyte and each leads to large disruptions to the protein and membrane structure when held deep inside the channel. Weak binding of calcium ions outside the channel corresponds to a free energy well that is too shallow to demonstrate channel blocking. Our findings emphasize the success of the MD-PMF approach and the sensitivity of ion energetics to the choice of biomolecular force field.     

Effects of external cesium and rubidium on outward potassium currents in squid axons

We have studied the effects of external cesium and rubidium on potassium conductance of voltage clamped squid axons over a broad range of concentrations of these ions relative to the external potassium concentration. Our primary novel finding concerning cesium is that relatively large concentrations of this ion are able to block a small, but statistically significant fraction of outward potassium current for potentials less than approximately 50 mV positive to reversal potential. This effect is relieved at more positive potentials. We have also found that external rubidium blocks outward current with a qualitatively similar voltage dependence. This effect is more readily apparent than the cesium blockade, occurring even for concentrations less than that of external potassium. Rubidium also has a blocking effect on inward current, which is relieved for potentials more than 20-40 mV negative to reversal, thereby allowing both potassium and rubidium ions to cross the membrane. We have described these results with a single-file diffusion model of ion permeation through potassium channels. The model analysis suggests that both rubidium and cesium ions exert their blocking effects at the innermost site of a two-site channel, and that rubidium competes with potassium ions for entry into the channel more effectively than does cesium under comparable conditions.     

Energy barriers for passage of ions through channels. Exact solution of two electrostatic problems

Exact solutions are given to two electrostatic problems relevant to ion permeation through pores in membranes. The first assesses the importance of the pore forming molecule as a dielectric shield. It is shown on the basis of structural and dielectric considerations alone (neglecting effects attributable to possible charge distribution at the interior surface of the pre-former) that the minimum electrostatic barrier for monovalent ion passage through a gramicidin-like channel is 11 kT. It is further shown that given favorable circumstances, dielectric shielding might dramatically reduce the barrier to ion passage through potassium channels. The second problem considers the error introduced by treating ions as point charges. It is shown that for structureless pores the point charge approximation introduces no meaningful error, even if the ratio of ion radius to pore radius is as great as 0.95.     

A semi-microscopic Monte Carlo study of permeation energetics in a gramicidin-like channel: the origin of cation selectivity.

The influence of a gramicidin-like channel former on ion free energy barriers is studied using Monte Carlo simulation. The model explicitly describes the ion, the water dipoles, and the peptide carbonyls; the remaining degrees of freedom, bulk electrolyte, non-polar lipid and peptide regions, and electronic (high frequency) permittivity, are treated in continuum terms. Contributions of the channel waters and peptide COs are studied both separately and collectively. We found that if constrained to their original orientations, the COs substantially increase the cationic permeation free energy; with or without water present, CO reorientation is crucial for ion-CO interaction to lower cation free energy barriers; the translocation free energy profiles for potassium-, rubidium-, and cesium-like cations exhibit no broad barriers; the lipid-bound peptide interacts more effectively with anions than cations; anionic translocation free energy profiles exhibit well defined maxima. Using experimental data to estimate transfer free energies of ions and water from bulk electrolyte to a non-polar dielectric (continuum lipid), we found reasonable ion permeation profiles; cations bind and permeate, whereas anions cannot enter the channel. Cation selectivity arises because, for ions of the same size and charge, anions bind hydration water more strongly.     

Gramicidin channels that have no tryptophan residues.

In order to understand how aromatic residues modulate the function of membrane-spanning proteins, we examined the role of the four tryptophans in gramicidin A (gA) in determining the average duration and permeability characteristics of membrane-spanning gramicidin channels; the tryptophan residues were replaced by tyrosine (gramicidin T, gT), tyrosine O-benzyl ether [gramicidin T(Bzl), gT(Bzl)], naphthylalanine (gramicidin N, gN), and phenylalanine (gramicidin M enantiomer, gM-). These analogues form channels with durations and conductances that differ some 10- and 16-fold, respectively. The single-channel conductance was invariably decreased by the Trp----Yyy replacement, and the relative conductance alterations were similar in phosphatidylcholine (DPhPC) and monoglyceride (GMO) bilayers. The duration variations exhibited a more complex pattern, which was quite different in the two membrane environments: in DPhPC bilayers, gN channels have an average duration that is approximately 2-fold longer than that of gA channels; in GMO bilayers, the average duration of gN channels is about one-tenth that of gA channels. The sequence-dependent alterations in channel function do not result from alterations in the channels' peptide backbone structure, because heterodimers can form between the different analogues and gramicidine A, and there is no energetic cost associated with heterodimer formation [cf. Durkin, J. T., Koeppe, R. E., II, & Andersen, O. S. (1990) J. Mol. Biol. 211, 221]. The alterations in permeability properties are consistent with the notion that Trp residues alter the energy profile for ion permeation through long-range electrostatic interactions.     

Probing conformational changes of gramicidin ion channels by single-molecule patch-clamp fluorescence microscopy

Complex conformational changes influence and regulate the dynamics of ion channels. Such conformational changes are stochastic and often inhomogeneous, which makes it extremely difficult, if not impossible, to characterize them by ensemble-averaged experiments or by single-channel recordings of the electric current that report the open-closed events but do not specifically probe the associated conformational changes. Here, we report our studies on ion channel conformational changes using a new approach, patch-clamp fluorescence microscopy, which simultaneously combines single-molecule fluorescence spectroscopy and single-channel current recordings to probe the open-closed transitions and the conformational dynamics of individual ion channels. We demonstrate patch-clamp fluorescence microscopy by measuring gramicidin ion channel conformational changes in a lipid bilayer formed at a patch-clamp micropipette tip under a buffer solution. By measuring single-pair fluorescence resonance energy transfer and fluorescence self-quenching from dye-labeled gramicidin channels, we observed that the efficiency of single-pair fluorescence resonance energy transfer and self-quenching is widely distributed, which reflects a broad distribution of conformations. Our results strongly suggest a hitherto undetectable correlation between the multiple conformational states of the gramicidin channel and its closed and open states in a lipid bilayer.     

Tryptophan contributions to the empirical free-energy profile in gramicidin A/M heterodimer channels

Gramicidin A/gramicidin M heterodimer conductances were measured in planar lipid bilayers and found to form two distinguishable populations about halfway between the gramicidin A and gramicidin M homodimer conductances. This implies that the principle difference in the gramicidin A and gramicidin M transport free-energy profiles occurs at the channel center, where it would produce similar effects on the rate-limiting barrier for the two heterodimers. Kinetic analysis based on this and nearly all previously published homodimer conductance data for both gramicidin A and gramicidin M channels confirms this conclusion, indicating that the translocation step is approximately 100-fold slower in gramicidin M homodimers than in gramicidin A homodimers and that first- and second-ion exit-rate constants are higher by factors of 24 and 10, respectively. Assuming that the ratios of rate constants are related to the free-energy difference between gramicidin A and gramicidin M, we construct an effective ion-Trp free-energy interaction profile that has a minimum at the channel center.     

Single-channel studies on linear gramicidins with altered amino acid side chains. Effects of altering the polarity of the side chain at position 1 in gramicidin A.

The modulation of gramicidin A single-channel characteristics by the amino acid side chains was investigated using gramicidin A analogues in which the NH2 terminal valine was chemically replaced by other amino acids. The replacements were chosen such that pairs of analogues would have essentially isosteric side chains of different polarities at position 1 (valine vs. trifluorovaline or hexafluorovaline; norvaline vs. S-methyl-cysteine; and norleucine vs. methionine). Even though the side chains are not in direct contact with the permeating ions, the single-channel conductances for Na+ and Cs+ are markedly affected by the changes in the physico-chemical characteristics of the side chains. The maximum single-channel conductance for Na+ is decreased by as much as 10-fold in channels formed by analogues with polar side chains at position 1 compared with their counterparts with nonpolar side chains, while the Na+ affinity is fairly insensitive to these changes. The relative conductance changes seen with Cs+ were less than those seen with Na+; the ion selectivity of the channels with polar side chains at position 1 was increased. Hybrid channels could form between compounds with a polar side chain at position 1 and either valine gramicidin A or their counterparts with a nonpolar side chain at position 1. The structure of channels formed by the modified gramicidins is thus essentially identical to the structure of channels formed by valine gramicidin A. The polarity of the side chain at position 1 is an important determinant of the permeability characteristics of the gramicidin A channel. We discuss the importance of having structural information when interpreting the functional consequences of site-directed amino acid modifications.     

Modulation of gramicidin A open channel lifetime by ion occupancy.

The hypothesis that the gramicidin A channel stability depends on the level of ion occupancy of the channel was used to derive a mathematical model relating channel lifetime to channel occupancy. Eyring barrier permeation models were examined for their ability to fit the zero-voltage conductance, current-voltage, as well as lifetime data. The simplest permeation model required to explain the major features of the experimental data consists of three barriers and four sites (3B4S) with a maximum of two ions occupying the channel. The average lifetime of the channel was calculated from the barrier model by assuming the closing rate constant to be proportional to the probability of the internal channel sites being empty. The link between permeation and lifetime has as its single parameter the experimentally determined averaged lifetime of gramicidin A channels in the limit of infinitely dilute solutions and has therefore no adjustable parameters. This simple assumption that one or more ions inside the channel completely stabilize the dimer conformation is successful in explaining the experimental data considering the fact that this model for stabilization is independent of ion species and configurational occupancy. The model is used to examine, by comparison with experimental data, the asymmetrical voltage dependence of the lifetime in asymmetrical solutions, the effects of blockers, and the effects of elevated osmotic pressure.     

Test of molecular dynamics force fields in gramicidin A

The force fields commonly used in molecular dynamics simulations of proteins are optimized under bulk conditions. Whether the same force fields can be used in simulations of membrane proteins is not well established, although they are increasingly being used for such purposes. Here we consider ion permeation in the gramicidin A channel as a test of the AMBER force field in a membrane environment. The potentials of mean force for potassium ions are calculated along the channel axis and compared with the one deduced from the experimental conductance data. The calculated result indicates a rather large central barrier similar to those obtained from other force fields, which are incompatible with the conductance data. We suggest that lack of polarizability is the most likely cause of this problem, and, therefore, urge development of polarizable force fields for simulations of membrane proteins.     

Gating and flickery block differentially affected by rubidium in homomeric KCNQ1 and heteromeric KCNQ1/KCNE1 potassium channels

The voltage-gated potassium channel KCNQ1 associates with the small KCNE1 subunit to form the cardiac IKs delayed rectifier potassium current and mutations in both genes can lead to the long QT syndrome. KCNQ1 can form functional homotetrameric channels, however with drastically different biophysical properties compared to heteromeric KCNQ1/KCNE1 channels. We analyzed gating and conductance of these channels expressed in Xenopus oocytes using the two-electrode voltage-clamp and the patch-clamp technique and high extracellular potassium (K) and rubidium (Rb) solutions. Inward tail currents of homomeric KCNQ1 channels are increased about threefold upon substitution of 100 mM potassium with 100 mM rubidium despite a smaller rubidium permeability, suggesting an effect of rubidium on gating. However, the kinetics of tail currents and the steady-state activation curve are only slightly changed in rubidium. Single-channel amplitude at negative voltages was estimated by nonstationary noise analysis, and it was found that rubidium has only a small effect on homomeric channels (1.2-fold increase) when measured at a 5-kHz bandwidth. The apparent single-channel conductance was decreased after filtering the data at lower cutoff frequencies indicative of a relatively fast "flickery/block" process. The relative conductance in rubidium compared to potassium increased at lower cutoff frequencies (about twofold at 10 Hz), suggesting that the main effect of rubidium is to decrease the probability of channel blockage leading to an increase of inward currents without large changes in gating properties. Macroscopic inward tail currents of heteromeric KCNQ1/KCNE1 channels in rubidium are reduced by about twofold and show a pronounced sigmoidal time course that develops with a delay similar to the inactivation process of homomeric KCNQ1, and is indicative of the presence of several open states. The single channel amplitude of heteromers is about twofold smaller in rubidium than in potassium at a bandwidth of 5 kHz. Filtering at lower cutoff frequencies reduces the apparent single-channel conductance, the ratio of the conductance in rubidium versus potassium is, however, independent of the cutoff frequency. Our results suggest the presence of a relatively rapid process (flicker) that can occur almost independently of the gating state. Occupancy by rubidium at negative voltages favors the flicker-open state and slows the flickering rate in homomeric channels, whereas rubidium does not affect the flickering in heteromeric channels. The effects of KCNE1 on the conduction properties are consistent with an interaction of KCNE1 in the outer vestibule of the channel.     

Experimental search for combined AC and DC magnetic field effects on ion channels

The hypothesis that specific combinations of DC and low frequency AC magnetic fields at so-called cyclotron-resonance conditions could affect the transport of ions through ion channels, or alter the kinetics of ion channels (opening and closing rates), has been tested. As a model system, the ion channels formed by gramicidin A incorporated in lipid bilayer membranes were studied. No significant changes in channel conductance, average lifetime, or formation rate as a function of applied fields could be detected over a wide range of frequencies and field strengths. Experiments were carried out to measure the time-resolved single-channel events and the average conductances of many-channel events in the presence of K⁺ and H⁺ ions. The channel blocking effect of Ca⁺⁺ was also studied. © 1993 Wiley-Liss. Inc.