Molecular design and synthesis of artificial ion channels based on cyclic peptides containing unnatural amino acids

A series of novel cyclic peptides composed of 3 to 5 dipeptide units with alternating natural-unnatural amino acid units, have been designed and synthesized, employing 5-(N-alkanoylamino)-3-aminobenzoic acid with a long alkanoyl chain as the unnatural amino acid. All cyclic peptides with systematically varying pore size, shape, and lipophilicity are found to form ion channels with a conductance of ca. 9 pS in aqueous KCl (500 mM) upon examination by the voltage clamp method. These peptide channels are cation selective with the permeability ratio P(Cl(-))/P(K(+)) of around 0.17. The ion channels formed by the neutral, cationic, and anionic cyclic peptides containing L-alanine, L-lysine, and L-aspartate, respectively, show the monovalent cation selectivity with the permeability ratio P(Na(+))/P(K(+)) of ca. 0.39. On the basis of structural information provided by voltage-dependent blockade of the single channel current of all the tested peptides by Ca(2+), we inferred that each channel is formed from a dimer of the peptide with its peptide ring constructing the channel entrance and its alkanoyl chains lining across the membrane to build up the channel pore. The experimental results are consistent with an idea that the rate of ion conduction is determined by the nature of the hydrophobic alkanoyl chain region, which is common to all the channels.     

Ion channels from linear and branched bola-amphiphiles

The synthesis and characterization of the ion channel activity of three new bola-amphiphiles is described. These compounds are conceptually derived from a previously reported bis-cyclophane bola-amphiphile through opening of the cyclophanes to acyclic structures and were found to readily form ion channels in planar bilayer membranes as assessed by bilayer clamp single-channel analysis. All three compounds behaved very similarly: the dominant channels formed by all three are Ohmic with specific conductance of 10 +/- 1 pS (NaCl electrolyte) and 39 +/- 1 pS (CsCl electrolyte). Single-ion permeability ratios, determined from dissymmetric electrolyte experiments, showed the selectivity P(Cs(+)) > P(Na(+)) > P(Cl(-)). Less frequently, lower conductance channels were also observed to act independently of the dominant channels. The lifetimes of the dominant channels range from 70 to 280 ms for the three compounds with some very long-lived openings (20-40 s) observed for two of the three. The lower conductance states have shorter lifetimes. This study demonstrates that bis-macrocyclic compounds are not essential for channel formation by bola-amphiphiles, and opens a new class of channel-forming compounds for structure-activity optimization.     

Alamethicin-leucine zipper hybrid peptide: a prototype for the design of artificial receptors and ion channels.

In this report, we describe a novel concept of extramembrane control of channel peptide assembly and the eventual channel current modulation. Alamethicin is a peptide antibiotic, which usually forms ion channels in various association states. By introducing an extramembrane leucine zipper segment (Alm-LeuZ), the association number of alamethicin was effectively controlled to produce a single predominant channel open state. The assembly was estimated to be a tetramer, by comparison of the channel conductance with that of the template-assembled Alm-LeuZ tetramer, which was prepared by the conjugation of a maleimide-functionalized peptide template with cysteine-derivatized Alm-LeuZ segments. Employment of an extramembrane segment of a random conformation provided higher levels of channel conductance. The result exemplified the possibility of channel current control by a conformational switch of the extramembrane segments.     

(De)constructing the ryanodine receptor: modeling ion permeation and selectivity of the calcium release channel

Biological ion channels are proteins that passively conduct ions across membranes that are otherwise impermeable to ions. Here, we present a model of ion permeation and selectivity through a single, open ryanodine receptor (RyR) ion channel. Combining recent mutation data with electrodiffusion of finite-sized ions, the model reproduces the current/voltage curves of cardiac RyR (RyR2) in KCl, LiCl, NaCl, RbCl, CsCl, CaCl(2), MgCl(2), and their mixtures over large concentrations and applied voltage ranges. It also reproduces the reduced K(+) conductances and Ca(2+) selectivity of two skeletal muscle RyR (RyR1) mutants (D4899N and E4900Q). The model suggests that the selectivity filter of RyR contains the negatively charged residue D4899 that dominates the permeation and selectivity properties and gives RyR a DDDD locus similar to the EEEE locus of the L-type calcium channel. In contrast to previously applied barrier models, the current model describes RyR as a multi-ion channel with approximately three monovalent cations in the selectivity filter at all times. Reasons for the contradicting occupancy predictions are discussed. In addition, the model predicted an anomalous mole fraction effect for Na(+)/Cs(+) mixtures, which was later verified by experiment. Combining these results, the binding selectivity of RyR appears to be driven by the same charge/space competition mechanism of other highly charged channels.     

Phospholamban generates cation selective ion channels

Phosholamban (PLN) is involved in the contractility of cardiac muscles by regulating the intracellular calcium concentration (Ca(2+)(cyt)) of cardiac myocytes. This occurs via a modulation of the sarco-/endoplasmic CaATPase (SERCA). In spite of high-resolution structures the molecular mode of PLN action is yet not known. In the present paper we readdress the question whether PLN proteins can generate ion channel activity. Reconstitution of PLN in planar lipid bilayers reveals single channel fluctuations, which are characterized by two conductance levels, long open/closed dwell times, moderate selectivity between monovalent cations and no perceivable Ca(2+) permeability. The PLN generated channel activity could be inhibited by a PLN antibody (abPLN) implying that the channel activity is indeed due to the inherent channel function of the PLN protein.     

Further characterization of cation channels present in the chicken red blood cell membrane

In this paper, we provide an update on cation channels in nucleated chicken erythrocytes. Patch-clamp techniques were used to further characterize the two different types of cation channels present in the membrane of chicken red blood. In the whole-cell mode, with Ringer in the bath and internal K+ saline in the pipette solution, the membrane conductance was generated by cationic currents, since the reversal potential was shifted toward cations equilibrium when the impermeant cation NMDG was substituted to small cations. The membrane conductance could be increased by application of mechanical deformation or by the addition of agonists of the cAMP-dependent pathway. At the unitary level, two different types of cationic channels were revealed and could account for the cationic conductance observed in whole-cell configuration. One of them belongs to the family of stretch-activated cationic channel showing changes in activity under conditions of membrane deformation, whereas the second one belongs to the family of the cAMP activated cationic channels. These two channels could be distinguished according to their unitary conductances and drug sensitivities. The stretch-activated channel was sensitive to Gd(3+) and the cAMP-dependent channel was sensitive to flufenamic acid. Possible role of these channels in cell volume regulation process is discussed.     

Ion channels in the human red blood cell membrane: their further investigation and physiological relevance.

Using the patch-clamp technique, two different ion channels have been characterized further in the human red blood cell (RBC) membrane. We demonstrate that the non-selective cation channel (NSC) is permeable to Ca(2+) and can be activated by prostaglandin E(2) (PGE(2)). Therefore, the physiological role of this channel could be, together with the Ca(2+)-activated K(+) channel, the participation in the process of blood clot formation. We give also evidence that another channel in the RBC membrane, so far assumed to be a small conductance anion channel, is more likely to be a proton or a hydroxyl ion channel.     

Structure-based prediction of the conductance properties of ion channels

The HOLE procedure allows the prediction of the absolute conductance of an ion channel model from its structure. The original prediction method uses an empirically corrected Ohmic method. It is most successful, with predictions being reliable to within a factor of two. A new modification of the procedure is presented in which the self-diffusion coefficients of water molecules from molecular dynamics simulation are used to replace the empirical correction factor. A "prediction" of the conductance for the porin OmpF by the new method is made and shown to be very close to the experimental value. HOLE also allows the prediction of the effect that the addition of non-electrolyte polymers will have on channel conductance. The method has great potential to yield structural information from data provided by single channel recordings but needs further validation by making measurements on channels of known structure. Preliminary results are given of single channel records establishing the effects of non-electrolytes on the conductance of gramicidin D channels. As an example of the potential uses of the procedure application is made to examine the oligomerization of alpha-toxin (alpha-hemolysin) channels. A model for the alpha-toxin hexamer, based on the crystal structure for the heptamer, is generated using molecular mechanics methods. The compatibility of the structures with single channel conductance data is assessed using HOLE.     

AC conductance of transmembrane protein channels. The number of ionized residue mobile counterions at infinite dilution

Simultaneous measurements of the AC and DC conductances of alpha-hemolysin (alphaHL) ion channels and outer membrane protein F (OmpF) porins in dilute ionic solutions is described. AC conductance measurements were performed by applying a 10 mV rms AC voltage across a suspended planar bilayer of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine in the absence and presence of the protein and detecting the AC current response using phase-sensitive lock-in techniques. The conductances of individual alphaHL channels and OmpF porins were measured in symmetric KCl solutions containing between 5 and 1000 mM KCl. The AC and DC conductances of each protein were in agreement for all solution conditions, demonstrating the reliability of the AC method in single-channel recordings. Linear plots of conductance versus bulk KCl concentration for both proteins extrapolate to significant nonzero conductances (0.150 +/- 0.050 nS and 0.028 +/- 0.008 nS for OmpF and alphaHL, respectively) at infinite KCl dilution. The infinite dilution conductances are ascribed to mobile counterions of the ionizable residues within the protein lumens. A method of analyzing the plots of conductance vs KCl concentration is introduced that allows the determination of the concentration of mobile counterions associated with ionizable groups without knowledge of either the protein geometry or the ion mobilities. At neutral pH, an equivalent of 3 mobile counterions (K+ or Cl-) is estimated to contribute to the conductivity of the alphaHL channel.     

Spatial distribution of ion channel activity in biological membranes: the role of noise

A new approach is proposed to model a collective ion channel dynamics. We have assumed that ion channels create a two-component spatio-temporal interaction field. Every channel at its current spatial location in membrane contributes permanently to this field with its state (open or closed) and coupling strength to other channels. This field is described by a reaction-diffusion equation, the transition of ion channel from closed to open state (and vice versa) is described by a master equation, and migration of channels in membrane is described by a set of Langevin equations coupled by the interaction field. Within this model, we have investigated critical conditions for spatial distribution of ion channel activity.     

Mechanism of the solid state reaction NaCl + KF → NaF + KCl

The mechanism of the solid state displacement reaction NaCl + KF → NaF + KCl was investigated, employing diffusion couples (single crystals), in air at 550°C.The product layer obtained was formed with NaF and (Na, K)Cl solid solution.From cation concentration profiles, photometrically determined, for the NaClz.sfnc;KF and NaCl|KCl systems after annealing at 550°C, and from X-ray diffraction analysis on product layer surfaces for the NaCl|KF system, it was possible to state that the overall process is governed by a cation-counterdiffusion mechanism.A comparison between the diffusion coefficient evaluated through the rate constant and that calculated by means of the Boltzmann-Matano analysis for the system NaClKCl, allows one to deduce that the cations Na⁺ and K⁺ are transported in the (Na, K)Cl solid solution.     

Bis-Alkylureido Imidazole Artificial Water Channels

Nature creates aquaporins to effectively transport water, rejecting all ions including protons. Aquaporins (AQPs) has brought inspiration for the development of Artificial Water Channels (AWCs). Imidazole-quartet (I-quartet) was the first AWC that enabled to self-assemble a tubular backbone for rapid water and proton permeation with total ion rejection. Here, we report the discovery of bis-alkylureido imidazole compounds, which outperform the I-quartets by exhibiting ≈3 times higher net and single channel permeabilities (10⁷ H₂O/s/channel) and a ≈2–3 times lower proton conductance. The higher water conductance regime is associated to the high partition of more hydrophobic bis-alkylureido channels in the membrane and to their pore sizes, experiencing larger fluctuations, leading to an increase in the number of water molecules in the channel, with decreasing H-bonding connectivity. This new class of AWCs will open new pathways toward scalable membranes with enhanced water transport performances.     

Transmembrane ion channels constructed of cholic acid derivatives.

A new class of supramolecular transmembrane ion channels was prepared by linking two amphiphilic cholic acid methyl ethers through biscarbamate bonds to afford bis(7,12-dimethyl-24-carboxy-3-cholanyl)-N,N'-xylylene dicarbamate 2 and bis[7,12-dimethyl-24-(N,N,N-trimethylethanaminium-2-carboxylate)-3-cholanyl]-N,N'-xylylene dicarbamate dichloride 3. When incorporated into a planar bilayer membrane, both compounds showed stable (lasting 10 ms to 10 s) single ion channel currents. Only limited numbers of relatively small conductances were characterized for these channels (5-20 pS for 2 and 5-10 pS for 3, 10 and 17 pS for 2, and 9 pS for 3 in particular). Both channels were cation selective, and permeability ratios of potassium cation to chloride anion were 17 and 7.9 for 2 and 3, respectively, reflecting the difference in ionic species of the headgroup. Both channels 2 and 3 showed significant potassium selectivity over sodium by a factor of 3.1 and 3.2, respectively. No Li(+) currents were observed for 2, showing sharp discrimination between Na(+) or K(+).     

Activity coefficients of DL-valine in aqueous solutions of KCl at 25°C. Measurement with ion selective electrodes and modelling

Electrochemical cells with two ion selective electrodes, a cation and an anion ion selective electrode, versus a double junction reference electrode were used to measure the activity coefficients of DL-valine at 298.15 K, up to 0.5 molality, in aqueous solutions of KCl up to 1.0 molality. The results obtained in this work are compared with those reported before for the activity coefficients of DL-valine in aqueous solutions of NaCl. The experimental data were correlated using the model proposed previously by Khoshkbarchi and Vera for the activity coefficients of amino acids in aqueous electrolytes solutions.     

Photomodification of Cardiac Membrane: Chaotic Currents and High Conductance States in Isolated Patches

We have demonstrated previously that photomodification permeabilizes cardiac cells as evidenced by activation of a whole-cell leak current. In this paper we report that photomodification induces in cell-attached and inside-out cardiac membrane patches a chaotic current. Unlike current recordings from many protein ion channels that show stepwise amplitude changes associated with open and closed states of the channel, the chaotic current consists of variable amplitude spike-like transitions. The amplitudes of these spikes can vary from tenths to tens of picoamperes at a constant transmembrane potential. We provide evidence that the chaotic current is transmembrane rather than trans-seal and has a voltage dependency expected for current flow through nonspecific conductance pathways. Photomodification can also induce high conductance states (greater than 500 pS) in cell-attached and inside-out patches. We present evidence that the high conductance state is also not related to seal breakdown. Our results suggest that both the chaotic current activity in and high conductance state of photo-modified cardiac membrane patches result from the opening of many small conductance, nonspecific pathways through the membrane.     

Effect of channel block on the collective spiking activity of coupled stochastic Hodgkin-Huxley neurons

Toxins, such as tetraethylammonium (TEA) and tetrodotoxin (TTX), can make potassium or sodium ion channels poisoned, respectively, and hence reduce the number of working ion channels and lead to the diminishment of conductance. In this paper, we have studied by numerical simulations the effects of sodium and potassium ion channel poisoning on the collective spiking activity of an array of coupled stochastic Hodgkin-Huxley (HH) neurons. It is found for a given number of neurons sodium or potas- sium ion channel block can either enhance or reduce the collective spiking regularity, depending on the membrane patch size. For a given smaller or larger patch size, potassium and sodium ion channel block can reduce or enhance the collective spiking regularity, but they have different patch size ranges for the transformation. This result shows that sodium or potassium ion channel block might have dif- ferent effects on the collective spiking activity in coupled HH neurons from the effects for a single neuron, which represents the interplay among the diminishment of maximal conductance and the in- crease of channel noise strength due to the channel blocks, as well as the bi-directional coupling be- tween the neurons.     

Synthetic ion channel activity documented by electrophysiological methods in living cells

Hydraphiles are synthetic ion channels that use crown ethers as entry portals and that span phospholipid bilayer membranes. Proton and sodium cation transport by these compounds has been demonstrated in liposomes and planar bilayers. In the present work, whole cell patch clamp experiments show that hydraphiles integrate into the membranes of human embryonic kidney (HEK 293) cells and significantly increase membrane conductance. The altered membrane permeability is reversible, and the cells under study remain vital during the experiment. Control compounds that are too short (C(8)-benzyl channel) to span the bilayer or are inactive owing to a deficiency in the central relay do not induce similar conductance increases. Control experiments confirm that the inactive channel analogues do not show nonspecific effects such as activation of native channels. These studies show that the combination of structural features that have been designed into the hydraphiles afford true, albeit simple, channel function in live cells.     

Lipid packing stress and polypeptide aggregation: alamethicin channel probed by proton titration of lipid charge

Lipid membranes are not passive, neutral scaffolds to hold membrane proteins. In order to examine the influence of lipid packing energetics on ion channel expression, we study the relative probabilities of alamethicin channel formation in dioleoylphosphatidylserine (DOPS) bilayers as a function of pH. The rationale for this strategy is our earlier finding that the higher-conductance states, corresponding to larger polypeptide aggregates, are more likely to occur in the presence of lipids prone to hexagonal HII-phase formation (specifically DOPE), than in the presence of lamellar L alpha-forming lipids (DOPC). In low ionic strength NaCl solutions at neutral pH, the open channel in DOPS membranes spends most of its time in states of lower conductance and resembles alamethicin channels in DOPC; at lower pH, where the lipid polar groups are neutralized, the channel probability distribution resembles that in DOPE. X-Ray diffraction studies on DOPS show a progressive decrease in the intrinsic curvature of the constituent monolayers as well as a decreased probability of HII-phase formation when the charged lipid fraction is increased. We explore how proton titration of DOPS affects lipid packing energetics, and how these energetics couple titration to channel formation.     

Molecular dynamics simulation links conformation of a pore-flanking region to hyperekplexia-related dysfunction of the inhibitory glycine receptor

Inhibitory glycine receptors mediate rapid synaptic inhibition in mammalian spinal cord and brainstem. The previously identified hyperekplexia mutation GLRA1(P250T), located within the intracellular TM1-2 loop of the GlyR alpha1 subunit, results in altered receptor activation and desensitization. Here, elementary steps of ion channel function of alpha1(250) mutants were resolved and shown to correlate with hydropathy and molar volume of residue alpha1(250). Single-channel recordings and rapid activation kinetic studies using laser pulse photolysis showed reduced conductance but similar open probability of alpha1(P250T) mutant channels. Molecular dynamics simulation of a helix-turn-helix motif representing the intracellular TM1-2 domain revealed alterations in backbone conformation, indicating an increased flexibility in these mutants that paralleled changes in elementary steps of channel function. Thus, the architecture of the TM1-2 loop is a critical determinant of ion channel conductance and receptor desensitization.