Charge delocalization in proton channels, II: the synthetic LS2 channel and proton selectivity

In this study, the minimalist synthetic LS2 channel is used as a prototype to examine the selectivity of protons over other cations. The free-energy profiles along the transport pathway of LS2 are calculated for three cation species: a realistic delocalized proton (including Grotthuss shuttling)--H(+), a classical (nonshuttling) hydronium--H(3)O(+), and a potassium cation--K(+). The overall barrier for K(+) is approximately twice as large as that for H(+), explaining the >100 times larger maximal ion conductance for the latter, in qualitative agreement with the experimental result. The profile for the classical hydronium is quantitatively intermediate between those of H(+) and K(+) and qualitatively more similar to that of H(+), for which the locations of the peaks are well correlated with the troughs of the pore radius profile. There is a strong correlation between the free-energy profiles and the very different characteristic hydration structures of the three cation species. This work suggests that the passage of various cations through ion channels cannot always be explained by simple electrostatic desolvation considerations.     

Alteration of the pH-dependent ion selectivity of the colicin E1 channel by site-directed mutagenesis.

Colicin E1 is a soluble, bacteriocidal protein that forms voltage-gated channels in planar lipid bilayers. The channel-forming region of the 522-amino acid protein is near the COOH terminus, and contains a 35-amino acid hydrophobic segment which is presumed to be important in interacting with the membrane. We have used site-directed mutagenesis in the region immediately upstream from the hydrophobic segment to construct several functional colicin mutants in which a wild-type residue was replaced with a cysteine. We also replaced the only naturally occurring cysteine in the molecule, Cys-505, with alanine, so that synthetically introduced cysteines could unambiguously serve as targets for chemical modification. All of the replacements reported here (at positions 449, 459, 473, 505, and some combinations) resulted in a channel that had an ion selectivity (K+ versus Cl-) identical to wild type at low pH. At higher pH, however, one of these mutations, which replaced the negatively charged aspartate at position 473 (the upstream boundary of the hydrophobic segment), resulted in a channel that was less cation-selective than was wild type. When the introduced Cys-473 was reacted with iodoacetic acid, which inserted a COOH group close to the position of the missing aspartate COOH, wild-type ion selectivity was restored, suggesting that the greater cation selectivity of the wild-type channel was directly produced by the negative charge at Asp-473. By comparing the ion selectivity of the Cys-473 mutant channel to that of the wild type as a function of the pH on the cis and trans sides of the membrane, it was possible to locate residue 473 close to the cis side. Locating in this manner the positions in the channel of particular residues places important constraints on channel model building.     

A computer simulation study of the hydrated proton in a synthetic proton channel

Classical molecular dynamics simulations using the multistate empirical valence bond model for aqueous proton transport were performed to characterize the hydration structure of an excess proton inside a leucine-serine synthetic ion channel, LS2. For such a nonuniform pore size ion channel, it is found that the Zundel ion (H(5)O(2)(+)) solvation structure is generally more stable in narrow channel regions than in wider channel regions, which is in agreement with a recent study on idealized hydrophobic proton channels. However, considerable diversity in the relative stability of the Zundel to Eigen cation (H(9)O(4)(+)) was observed. Three of the five wide channel regions, one located at the channel's center and the other two located near the channel mouths, are found to show extraordinary preference for the Eigen solvation structure. This implies that proton hopping is inhibited in these regions and therefore suggests that these regions may behave as barriers in the proton conducting pathway inside the channel. The proton solvation is also greatly influenced by the local molecular environment of the protein. In particular, the polar side chains of the Ser residues, which are intimately involved in the solvation structure, can greatly influence proton solvation. However, no preference of the influence by the various Ser side chains was found; they can either promote or prevent the formation of certain solvation structures.     

Channels formed by colicin E1 in planar lipid bilayers are large and exhibit pH-dependent ion selectivity

Summary The E1 subgroup (E1, A, Ib, etc.) of antibacterial toxins called colicins are known to form voltage-dependent channels in planar lipid bilayers. The genes for colicins E1, A and Ib have been cloned and sequenced, making these channels interesting models for the widespread phenomenon of voltage dependence in cellular channels. In this paper we investigate ion selectivity and channel size—properties relevant to model building. Our major finding is that the colicin E1 channel is large, having a diameter ofat least 8 Å at its narrowest point. We established this from measurements of reversal potentials for gradients formed by salts of large cations or large anions. In so doing, we exploited the fact that the colicin channel is permeable to both cations and anions, and its relative selectivity to them is a functions and anions, and its relative selectivity to them is a function of pH. The channel is anion selective (Cl^– over K⁺) in neutral membranes, and the degree of selectivity is highly dependent on pH. In negatively charged membranes, it becomes cation selective at pH's higher than about 5. Experiments with pH gradients cross the membrane suggest that titratable groups both within the channel lumen and near the channel ends affect the selectivity. Individual E1 channels have more than one open conductance state, all displaying comparable ion selectivity. Colicins A and Ib also exhibit pH-dependent ion selectivity, and appear to have even larger lumens than E1.     

Ion selectivity of colicin E1: III. Anion permeability

The antibiotic protein colicin E1 forms ion channels in planar lipid bilayers that are capable of conducting monovalent organic cations having mean diameters of at least 9 Å. Polyvalent organic cations appear to be completely impermeant, regardless of size. All permeant ions, whether large or small, positively or negatively charged, are conducted by this channel at very slow rates. We have examined the permeability of colicin E1 channels to anionic probes having a variety of sizes, shapes, and charge distributions. In contrast to the behavior of cations, polyvalent as well as monovalent organic anions were found to permeate the colicin E1 channel. Inorganic sulfate was able to permeate the channel only when the pH was 4 or less, conditions under which the colicin E1 protein is predominantly in an anion-preferring conformational state. The less selective state(s) of the colicin E1 channel, observed when the pH was 5 or greater, was not permeable to inorganic sulfate. The sulfate salt of the impermeant cation Bis-T6 (N,N,N,N-tetramethyl-1,6-hexanediamine) had no effect on the single channel conductance of colicin E1 channels exposed to solutions containing 1 m NaCl at pH 5. The complete lack of blocking activity by either of these two impermeant ions indicates that both are excluded from the channel lumen. These results are consistent with our hypothesis that there is but a single location in the lumen of the colicin E1 channel where positively charged groups can be effectively hydrated. This site may coincide with the location of the energetic barrier which impedes the movement of anions.The authors wish to thank Dr. F.S. Cohen for making available unpublished data and for helpful comments. This work was supported by National Institutes of Health grant GM 37396 and by the Howard Hughes Medical Institute Undergraduate Biological Sciences Education Initiative (E.R.K.)     

Noncontact dipole effects on channel permeation. III. Anomalous proton conductance effects in gramicidin

Proton transport on water wires, of interest for many problems in membrane biology, is analyzed in side-chain analogs of gramicidin A channels. In symmetrical 0.1 N HCl solutions, fluorination of channel Trp(11), Trp-(13), or Trp(15) side chains is found to inhibit proton transport, and replacement of one or more Trps with Phe enhances proton transport, the opposite of the effects on K(+) transport in lecithin bilayers. The current-voltage relations are superlinear, indicating that some membrane field-dependent process is rate limiting. The interfacial dipole effects are usually assumed to affect the rate of cation translocation across the channel. For proton conductance, however, water reorientation after proton translocation is anticipated to be rate limiting. We propose that the findings reported here are most readily interpreted as the result of dipole-dipole interactions between channel waters and polar side chains or lipid headgroups. In particular, if reorientation of the water column begins with the water nearest the channel exit, this hypothesis explains the negative impact of fluorination and the positive impact of headgroup dipole on proton conductance.     

A very short peptide makes a voltage-dependent ion channel: the critical length of the channel domain of colicin E1

Cleavage of colicin E1 molecules with a variety of proteases or with cyanogen bromide (CNBr) generates COOH-terminal fragments which have channel-forming activity similar to that of intact colicin in planar lipid bilayer membranes. The smallest channel-forming fragment obtained by CNBr cleavage of the wild-type molecule consists of the C-terminal 152 amino acids. By the use of oligonucleotide-directed mutagenesis, we have made nine mutants along this 152 amino acid peptide, in which an amino acid was replaced by methionine in order to create a new CNBr cleavage site. The smallest of the CNBr-cleaved C-terminal fragments with channel-forming activity, in planar bilayer membranes, was generated by cleavage at new Met position 428 and has 94 amino acids, whereas a 75 amino acid peptide produced by cleavage of a new Met at position 447 did not have channel activity. The NH2-terminus of the channel-forming domain of colicin E1 appears therefore to lie between residues 428 and 447. Since, however, the last six C-terminal residues of the colicin can be removed without changing activity, the number of amino acids necessary to form the channel is 88 or less. In addition, the unique Cys residue in colicin E1 was replaced by Gly, and nine mutants were then made with Cys placed at sequential locations along the peptide for eventual use as sulfhydryl attachment sites to determine the local environment of the replaced amino acid. In the course of making 21 mutants, eight charged residues have been replaced by uncharged Met or Cys without changing the biological activity of the intact molecule. It has been proposed previously that the conformation of the colicin E1 channel is a barrel formed from five or six alpha-helices, each having 20 amino acids spanning the membrane and two to four residues making the turn at the boundary of the membrane. Our finding that 88 amino acids can make an active channel, combined with recently reported stoichiometric evidence that the channel is a monomer excludes this model and adds significant constraints which can be used in building a molecular model of the channel.     

Origins of proton transport behavior from selectivity domain mutations of the aquaporin-1 channel

The permeation free-energy profile and maximum ion conductance of proton transport along the channel of three aquaporin-1 (AQP1) mutants (H180A/R195V, H180A, and R195V) are calculated via molecular dynamics simulations and Poisson-Nernst-Planck theory. The proton dynamics was described by the multistate empirical valence bond (MS-EVB) model. The results reveal three major contributions to the overall free-energy barrier for proton transport in AQP1: 1), the bipolar field, 2), the electrostatic repulsion due to the Arg-195 residue, and 3), the dehydration penalty due to the narrow channel pore. The double mutation (H180A/R195V) drastically drops the overall free-energy barrier by roughly 20 kcal/mol via simultaneously relaxing the direct electrostatic interaction (by R195V) and dehydration effect (by H180A).     

Conformation of the closed channel state of colicin A in proteoliposomes: an umbrella model

Colicin A (ColA) is a water-soluble toxin that forms a voltage-gated channel in the cytoplasmic membrane of Escherichia coli. Until now, two models were proposed for the closed channel state: the umbrella model and the penknife model. Mutants of ColA, each containing a single cysteine, were labeled with a nitroxide spin label, reconstituted into liposomes, and studied by electron paramagnetic resonance (EPR) spectroscopy to study the membrane-bound closed channel state. The spin-labeled ColA variants in solution and in liposomes of native E. coli lipid composition were analyzed in terms of the mobility of the nitroxide, its accessibility to paramagnetic reagents, and the polarity of its microenvironment. The EPR data determined for the soluble ColA pore-forming domain are in agreement with its crystal structure. Moreover, the EPR results show that ColA has a conformation in liposomes different from its water-soluble conformation. Residues that belong to helices H8 and H9 are significantly accessible for O₂ but not for nickel-ethylene diamine diacetic acid, indicating their location inside the membrane. In addition, the polarity values determined from the hyperfine tensor component A_zz of residues 176, 181, and 183 (H9) indicate the location of these residues close to the center of the lipid bilayer, supporting a transmembrane orientation of the hydrophobic hairpin. Furthermore, the accessibility and polarity data suggest that the spin-labeled side chains of the amphipathic helices (H1-H7 and H10) are located at the membrane-water interface. Evidence that the conformation of the closed channel state in artificial liposomes depends on lipid composition is given. The EPR results for ColA reconstituted into liposomes of E. coli lipids support the umbrella model for the closed channel state.     

Temperature dependence of proton permeation through a voltage-gated proton channel

Voltage-gated proton channels are found in many different types of cells, where they facilitate proton movement through the membrane. The mechanism of proton permeation through the channel is an issue of long-term interest, but it remains an open question. To address this issue, we examined the temperature dependence of proton permeation. Under whole cell recordings, rapid temperature changes within a few milliseconds were imposed. This method allowed for the measurement of current amplitudes immediately before and after a temperature jump, from which the ratios of these currents (I_ratio) were determined. The use of I_ratio for evaluating the temperature dependence minimized the contributions of factors other than permeation. Temperature jumps of various degrees (ΔT, −15 to 15°C) were applied over a wide temperature range (4–49°C), and the Q₁₀s for the proton currents were evaluated from the I_ratios. Q₁₀ exhibited a high temperature dependence, varying from 2.2 at 10°C to 1.3 at 40°C. This implies that processes with different temperature dependencies underlie the observed Q₁₀. A novel resistivity pulse method revealed that the access resistance with its low temperature dependence predominated in high temperature ranges. The measured temperature dependence of Q₁₀ was decomposed into Q₁₀ of the channel and of the access resistances. Finally, the Q₁₀ for proton permeation through the voltage-gated proton channel itself was calculated and found to vary from 2.8 at 5°C to 2.2 at 45°C, as expected for an activation enthalpy of 64 kJ/mol. The thermodynamic features for proton permeation through proton-selective channels were discussed for the underlying mechanism.     

Ion selectivity of colicin E1: Modulation by pH and membrane composition

Colicin E1 is a plasmid-encoded bacteriocidal protein which, though water soluble when secreted by its host bacterium, spontaneously interacts with planar lipid bilayers to form voltage-gated ion channels. In asolectin bilayers, the preference for anions over cations exhibited by these channels at low pH can be reversed by raising the pH on either side of the membrane. When incorporated into membranes composed of either of the two zwitterionic lipids, bacterial phosphatidylethanolamine and diphytanoyl phosphatidylcholine, colicin E1 channels were nearly ideally anion selective in the limit of low pH and moderately cation selective at the high pH limit. In phosphatidylcholine membranes, however, the response of these channels to changes in pH exhibited a pattern of behavior peculiar to this lipid. If the side of the membrane on which the protein had been introduced (the cis side) was exposed to pH 4.0, all the channels in the bilayer, whether opened or closed, became refractory to further changes in pH. This irreversibility has been interpreted as evidence that the selectivity of colicin E1 is under the control of a pH-sensitive conformational change. Protonation of groups on the cis side of the membrane appear to be essential to the conversion to the anion-selective state. These groups are rendered kinetically inaccessible to the aqueous phase when the transition takes place in phosphatidylcholine membranes.     

Ion selectivity of colicin E1: II. Permeability to organic cations

Channels formed by colicin E1 in planar lipid bilayers have large diameters and conduct both cations and anions. The rates at which ions are transported, however, are relatively slow, and the relative anion-to-cation selectivity is modulated over a wide range by the pH of the bathing solutions. We have examined the permeability of these channels to cationic probes having a variety of sizes, shapes, and charge distributions. All of the monovalent probes were found to be permeant, establishing a minimum diameter at the narrowest part of the pore of approximately 9 A. In contrast to this behavior, all of the polyvalent organic cations were shown to be impermeant. This simple exclusionary rule is interpreted as evidence that, when steric restrictions require partial dehydration of an ion, the structure of the channel is able to provide a substitute electrostatic environment for only one charged group at time.     

Peregrination of the selectivity filter delineates the pore of the human voltage-gated proton channel hHV1

Extraordinary selectivity is crucial to all proton-conducting molecules, including the human voltage-gated proton channel (hH_V1), because the proton concentration is >10⁶ times lower than that of other cations. Here we use “selectivity filter scanning” to elucidate the molecular requirements for proton-specific conduction in hH_V1. Asp¹¹², in the middle of the S1 transmembrane helix, is an essential part of the selectivity filter in wild-type (WT) channels. After neutralizing Asp¹¹² by mutating it to Ala (D112A), we introduced Asp at each position along S1 from 108 to 118, searching for “second site suppressor” activity. Surprisingly, most mutants lacked even the anion conduction exhibited by D112A. Proton-specific conduction was restored only with Asp or Glu at position 116. The D112V/V116D channel strikingly resembled WT in selectivity, kinetics, and ΔpH-dependent gating. The S4 segment of this mutant has similar accessibility to WT in open channels, because R211H/D112V/V116D was inhibited by internally applied Zn²⁺. Asp at position 109 allowed anion permeation in combination with D112A but did not rescue function in the nonconducting D112V mutant, indicating that selectivity is established externally to the constriction at F150. The three positions that permitted conduction all line the pore in our homology model, clearly delineating the conduction pathway. Evidently, a carboxyl group must face the pore directly to enable conduction. Molecular dynamics simulations indicate reorganization of hydrogen bond networks in the external vestibule in D112V/V116D. At both positions where it produces proton selectivity, Asp frequently engages in salt linkage with one or more Arg residues from S4. Surprisingly, mean hydration profiles were similar in proton-selective, anion-permeable, and nonconducting constructs. That the selectivity filter functions in a new location helps to define local environmental features required to produce proton-selective conduction.     

Definitive assignment of proton selectivity and attoampere unitary current to the M2 ion channel protein of influenza A virus

The viral ion channel protein M2 supports the transit of influenza virus and its glycoproteins through acidic compartments of the cell. M2 conducts endosomal protons into the virion to initiate uncoating and, by equilibrating the pH at trans-Golgi membranes, preserves the native conformation of acid-sensitive viral hemagglutinin. The exceptionally low conductance of the M2 channel thwarted resolution of single channels by electrophysiological techniques. Assays of liposome-reconstituted M2 yielded the average unitary channel current of the M2 tetramer--1.2 aA (1.2 x 10(-18) A) at neutral pH and 2.7 to 4.1 aA at pH 5.7--which activates the channel. Extrapolation to physiological temperature predicts 4.8 and 40 aA, respectively, and a unitary conductance of 0.03 versus 0.4 fS. This minute activity, below previous estimates, appears sufficient for virus reproduction, but low enough to avert abortive cytotoxicity. The unitary permeability of M2 was within the range reported for other proton channels. To address the ion selectivity of M2, we exploited the coupling of ionic influx and efflux in sealed liposomes. Metal ion fluxes were monitored by proton counterflow, employing a pH probe 1,000 times more sensitive than available Na+ or K+ probes. Even low-pH-activated M2 did not conduct Na+ and K+. The proton selectivity of M2 was estimated to be at least 3 x 10(6) (over sodium or potassium ions), in agreement with electrophysiological studies. The stringent proton selectivity of M2 suggests that the cytopathology of influenza virus does not involve direct perturbation of cellular sodium or potassium gradients.     

Ion selectivity mechanism in a bacterial pentameric ligand-gated ion channel

The proton-gated ion channel from Gloeobacter violaceus (GLIC) is a prokaryotic homolog of the eukaryotic nicotinic acetylcholine receptor that responds to the binding of neurotransmitter acetylcholine and mediates fast signal transmission. Recent emergence of a high-resolution crystal structure of GLIC captured in a potentially open state allowed detailed, atomic-level insight into ion conduction and selectivity mechanisms in these channels. Herein, we have examined the barriers to ion conduction and origins of ion selectivity in the GLIC channel by the construction of potential-of-mean-force profiles for sodium and chloride ions inside the transmembrane region. Our calculations reveal that the GLIC channel is open for a sodium ion to transport, but presents a ∼11 kcal/mol free energy barrier for a chloride ion. Our collective findings identify three distinct contributions to the observed preference for the permeant ions. First, there is a substantial contribution due to a ring of negatively charged glutamate residues (E-2′) at the narrow intracellular end of the channel. The negative electrostatics of this region and the ability of the glutamate side chains to directly bind cations would strongly favor the passage of sodium ions while hindering translocation of chloride ions. Second, our results imply a significant hydrophobic contribution to selectivity linked to differences in the desolvation penalty for the sodium versus chloride ions in the central hydrophobic region of the pore. This hydrophobic contribution is evidenced by the large free energy barriers experienced by Cl⁻ in the middle of the pore for both GLIC and the E-2′A mutant. Finally, there is a distinct contribution arising from the overall negative electrostatics of the channel.     

Mechanism of proton gating of a urea channel

The size and complexity of many pH-gated channels have frustrated the development of specific structural models. The small acid-activated six-membrane segment urea channel of Helicobacter hepaticus (HhUreI), homologous to the essential UreI of the gastric pathogen Helicobacter pylori, enables identification of all the periplasmic sites of proton gating by site-directed mutagenesis. Exposure to external acidity enhances [(14)C]urea uptake by Xenopus oocytes expressing HhUreI, with half-maximal activity (pH(0.5)) at pH 6.8. A downward shift of pH(0.5) in single site mutants identified four of six protonatable periplasmic residues (His-50 at the boundary of the second transmembrane segment TM2, Glu-56 in the first periplasmic loop, Asp-59 at the boundary of TM3, and His-170 at the boundary of TM6) that affect proton gating. Asp-59 was the only site at which a protonatable residue appeared to be essential for pH gating. Mutation of Glu-110 or Glu-114 in PL2 did not affect the pH(0.5) of gating. A chimera, where the entire periplasmic domain of HhUreI was fused to the membrane domain of Streptococcus salivarius UreI (SsUreI), retained the pH-independent properties of SsUreI. Hence, proton gating of HhUreI likely depends upon the formation of hydrogen bonds by periplasmic residues that in turn produce conformational changes of the transmembrane domain. Further studies on HhUreI may facilitate understanding of other physiologically important pH-responsive channels.     

Altered ion channel conductance and ionic selectivity induced by large imposed membrane potential pulse.

The effects of large magnitude transmembrane potential pulses on voltage-gated Na and K channel behavior in frog skeletal muscle membrane were studied using a modified double vaseline-gap voltage clamp. The effects of electroconformational damage to ionic channels were separated from damage to lipid bilayer (electroporation). A 4 ms transmembrane potential pulse of -600 mV resulted in a reduction of both Na and K channel conductivities. The supraphysiologic pulses also reduced ionic selectivity of the K channels against Na+ ions, resulting in a depolarization of the membrane resting potential. However, TTX and TEA binding effects were unaltered. The kinetics of spontaneous reversal of the electroconformational damage of channel proteins was found to be dependent on the magnitude of imposed membrane potential pulse. These results suggest that muscle and nerve dysfunction after electrical shock may be in part caused by electroconformational damage to voltage-gated ion channels.