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Selectivity filter is a gate in ion channels which are responsible for the selection and fast conduction of particular ions across the membrane (with high throughput rates of $10^8$ ions/sec and a high 1:$10^4$ discrimination rate between ions). It is made of four strands as the backbone, and each strand is composed of sequences of five amino acids connected by peptide units H-N-C=O in which the main molecules in the backbone that interact with ions in the filter are carbonyl (C=O) groups that mimic the transient interactions of ion with binding sites during ion conduction. It has been suggested that quantum coherence and possible emergence of resonances in the backbone carbonyl groups may play a role in mediating ion conduction and selectivity in the filter. Here, we investigate the influence of noise and disorder on the efficiency of excitation energy transfer (EET) in a linear harmonic chain of $N=5$ sites with dipole-dipole couplings as a simple model for one P-loop strand of the selectivity filter backbone in biological ion channels. We include noise and disorder inherent in real biological systems by including spatial disorder in the chain, and random noise within a weak coupling quantum master equation approach. Our results show that disorder in the backbone considerably reduces EET, but the addition of noise helps to recover high EET for a wide range of system parameters. Our analysis may help for better understanding of the coordination of ions in the filter as well as the fast and efficient functioning of the selectivity filters in ion channels., 13 pages, 7 Figures + 2 Figures for SI

Ionic channels form pores in biomembranes. These pores are large macromolecular structures. Due to thermal fluctuations of countless degrees-of-freedom of the biomembrane material, the actual form of the pores is permanently subject to modification. Furthermore, the arrival of an ion at the binding site can change this form by repolarizing the surrounding aminoacids. In any case the variations of the pore structure are stochastic. In this paper, we discuss the effect of such modifications on the channel conductivity. Applying a simple kinetic description, we show that stochastic variations in channel properties can significantly alter the ionic current, even leading to its substantial increase or decrease for the specific matching of some time-scales of the system.

The properties of the malaria parasite-induced permeability pathways in the host red blood cell have been a major area of interest particularly in the context of whether the pathways are host- or parasite-derived. In the present study, the whole-cell configuration of the patch-clamp technique has been used to show that, compared with normal cells, chicken red blood cells infected by Plasmodium gallinaceum exhibited a 5–40-fold larger membrane conductance, which could be further increased up to 100-fold by raising intracellular Ca2+ levels. The increased conductance was not due to pathways with novel electrophysiological properties. Rather, the parasite increased the activity of endogenous 24 pS stretch-activated non-selective cationic (NSC) and 62 pS calcium-activated NSC channels, and, in some cases, of endogenous 255 pS anionic channels.

Amphotericin B is an antimycotic agent that has been studied for a long time, both because of its pharmacological action and the interest in understanding how this ionic channel works. It has been proposed that the channel is formed by a barrel of monomers, and that the presence of sterol is needed for the formation of such a barrel. As a matter of fact this need of a sterol has been used as a guiding idea in attemps to design derivatives more efficient in the discrimination of the cholesterol containing membranes, as compared to the ergosterol containing ones, henceforth diminishing the unwanted side effects in its pharmacological use. In this work we show that unitary channels that appear in a cholesterol containing membrane also appear when this membrane is free of cholesterol. We prove this to be the case for two membranes, a biological one, asolectin, and a synthetic one, DMPC. We then advance the idea that the role of sterols in the formation of the amphotericin B channel is related to the effects they have on the structure of the membrane itself, rather than to a direct involvement in the channel formation. We further look into the effect that different cholesterol concentrations in the membrane produce on the single channel properties. fl 1998 Elsevier Science B.V. All rights reserved.

In this letter, we study the effect of cation charge on anion selectivity in the pore using grand canonical Monte Carlo simulations. The mechanism of anion selectivity inside nanopores was found to be primarily a consequence of the screening of negative charges by the cations. In the case of monovalent cations, screening was not very effective and anions were rejected. We found an ‘off-state’ at high pH and an ‘on-state’ at low pH. When there are divalent cations, screening is good and there is no rejection of the anion. The concentration of anions at high pH is similar to that at low pH. The system is always in an ‘on-state’. Trivalent cations show an inverse selectivity mechanism: at high pH the concentration is higher than at low pH, i.e., the pore is in the ‘on-state’ at high pH and in the ‘off-state’ at low pH., Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas

Photosensitized inactivation of ionic channels formed by gramicidin in the planar bilayer lipid membrane (BLM) has been studied upon exposure of the BLM to single flashes of visible light in the presence of tetrasulphonated aluminium phthalocyanine. The gramicidin photoinactivation is inhibited by the addition of unsaturated phospholipids to the membrane-forming solution as well as by the addition of azide to the bathing solution, consistent with involvement of singlet oxygen. The characteristic time of the photoinactivation (T) does not change markedly under these conditions. Moreover, T remains nearly constant upon alteration of the flash energy and the photosensitizer concentration. The value of T appears to be sensitive to the gramicidin concentration and to the factors affecting the open time of the gramicidin channels, namely the temperature and the solvent used in the membrane-forming solution. The photoinactivation is not observed with covalent gramicidin dimers. The equations derived from the model of Bamberg and Laeuger (J. Membrane Biol. (1973) 11, 177–194), describing the relaxation of the gramicidin-induced conductance after a sudden distortion of the dimer-monomer equilibrium, are shown to explain consistently the time course of the photoinactivation provided that the damage of the gramicidin molecules leads to deviation from the equilibrium.

The effect of phthalocyanines, the potent photodynamic sensitizers, on the electric properties of the bilayer lipid membrane (BLM) is studied. It is shown, that tetrasulfonated, as well as trisulfonated, aluminium phthalocyanine do not alter the conductance of BLM, but elicit certain changes in the boundary potential difference, which points in favor of dye adsorption on BLM. Under the conditions of intense visible light irradiation, the phthalocyanines cause an increase in the conductance, resulting in the irreversible breakdown of BLM, formed from soy bean phosphati-dylcholine, but fail to change the conductance of BLM, formed from diphytanoilphosphatidylcholine. The phthalocyanine-sensitized inactivation of gramicidin channels incorporated into BLM is observed under the conditions of weak visible light irradiation using an He-Ne laser. The photodynamic blockage of model ionic channels is considerably suppressed after oxygen depletion. The phenomenon consists of a marked reduction of a number of open channels, probably due to photomodification of tryptophan residues, essential for gramicidin functioning. The mechanism of the channel inactivation, involving the photosensitized reaction of the II type, and the relevance to the interaction of sensitizers with biomembranes, is discussed.

A cytolytic toxin produced by G. vaginalis was incorporated in artificial membranes and giant liposomes. The toxin formed ionic channels when incorporated in lipid bilayers. The electrical properties of such channels were studied. Current records revealed a unitary conductance of 126 pS (in symmetrical 150 mM KCl). The open state probability of the cytolysin formed channels was a function of the applied membrane potential. The permeability ratio of cations to anions was estimated to be 6.5.

Membrane potential measurements using a fluorescent dye indicated that two specific toxins active against Spodoptera frugiperda larvae (CryIC and CryID) cause immediate permeability changes in midgut epithelial brush border membrane vesicles (BBMV). The initial response and the sustained permeability change are cationic, notvery K+ selective, and occur at in vivo lethal doses (nM). The toxin response has a different ion selectivity and is more sensitive to Ba2+ than the intrinsic cation permeability of BBMV. Experiments incorporating BBMV into planar lipid bilayers (PLB) demonstrated that these vesicles contain cation channels (31, 47 and 76 pS). A 2–40 fold conductance increase was induced by nM concentrations of toxin in PLB containing BBMV. Cationic single channel transitions of 50, 106, 360 and 752 pS were resolved. Thus, Bacillus thuringiensis δ-endotoxins induce an increase in cation membrane permeability involving ion channels in BBMV-containing functional receptors.

Synthetic peptides with amino acid sequences corresponding to predicted transmembrane segments of tetanus toxin were used as probes to identify a channel-forming motif. A peptide denoted TeTx II, with sequence GVVLLLEYIPEITLPVIAALSIA, forms cation-selective channels when reconstituted in planar lipid bilayers. The single channel conductance in 0.5 M NaCl or KCl is 28 ± 3 and 24 ± 2 pS, respectively. In contrast, a peptide with sequence NFIGALETTGVVLLLEYIPEIT, denoted as TeTx I. or a peptide with the same amino acid composition as TeTx II but with a randomized sequence, do not form channels. Conformational energy calculations show that a bundle of four amphipathic α-helices is a plausible structural motif underlying observable pore properties. The identified functional module may account for the channel-forming activity of both tetanus toxin and the homologous botulinum toxin A.

The channel pore of the nicotinic acetylcholine receptor (AChR) has been investigated by analysing single-channel conductances of systematically mutated Torpedo receptors expressed in Xenopus oocytes. The mutations mainly alter the size and polarity of uncharged polar amino acid residues of the acetylcholine receptor subunits positioned between the cytoplasmic ring and the extracellular ring. From the results obtained, we conclude that a ring of uncharged polar residues comprising threonine 244 of the alpha-subunit (alpha T244), beta S250, gamma T253 and delta S258 (referred to as the central ring) and the anionic intermediate ring, which are adjacent to each other in the assumed alpha-helical configuration of the M2-containing transmembrane segment, together form a narrow channel constriction of short length, located close to the cytoplasmic side of the membrane. Our results also suggest that individual subunits, particularly the gamma-subunit, are asymmetrically positioned at the channel constriction.

Nicotinic cholinergic receptors are membrane proteins composed of five subunits organized around a central aqueous pore. A pentameric channel protein, T5M2 delta, that emulates the presumed pore-forming structure of this receptor was generated by assembling five helix-forming peptide modules at the lysine epsilon-amino groups of the 11-residue template [K*AK*KK*PGK*EK*G], where * indicates attachment sites. Helical modules represent the sequence of the M2 segment of the Torpedo californica acetylcholine receptor (AChR) delta subunit; M2 segments are considered involved in pore-lining. Purified T5M2 delta migrates in SDS-PAGE with an apparent M(r) approximately 14,000, concordant with a protein of 126 residues. T5M2 delta forms cation-selective channels when reconstituted in planar lipid bilayers. The single channel conductance in symmetric 0.5 M KCl is 40 pS. This value approximates the 45 pS single channel conductance characteristic of authentic purified Torpedo AChR, recorded under otherwise identical conditions. These results, together with conformational energy calculations, support the notion that a bundle of five amphipathic alpha-helices is a plausible structural motif underlying the inner bundle that forms the pore of the pentameric AChR channel.

The present work concerns mechanisms of ionic conductivity of photosynthetic membranes. It is shown that reconstitution of vesicles of photosynthetic membranes (chromatophores) of purple bacteria Rhodospirillum rubrum into a planar bilayer lipid membrane leads to fluctuations of current showing the existence of a channel with a predominant conductance of approximately 230 pS in the presence of 100 mM KCl. Measurements under the conditions of KCl gradient prove that this channel is cation selective (PK/PCl = 7.2). Voltage inactivation of the channel is demonstrated which is prevented by treatment with trypsin.

Protegrin 1 (PG-1) is a naturally occurring cationic antimicrobial peptide that is 18 residues long, has an aminated carboxy terminus and contains two disulphide bridges. Here, we investigated the antimicrobial activity of PG-1 and three linear analogues. Then, the membrane permeabilisation induced by these peptides was studied upon Xenopus laevis oocytes by electrophysiological methods. From the results obtained, we concluded that protegrin is able to form anion channels. Moreover, it seems clear that the presence of disulphide bridges is a prerequisite for the pore formation at the membrane level and not for the antimicrobial activity.

The ability of three primary amphipathic Cell-Penetrating Peptides (CPPs) CH3-CO-GALFLGFLGAAGSTMGAWSQPKKKRKV-NH-CH2-CH2-SH, CH3-CO-GALFLAFLAAALS LMGLWSQPKKKRKV-NH-CH2-CH2-SH, and CH3-CO-KETWWETWWTEWSQPKKKRKV-NH-CH2-CH2-SH called Pbeta, Palpha and Pep-1, respectively, to promote pore formation is examined both in Xenopus oocytes and artificial planar lipid bilayers. A good correlation between pore formation and their structural properties, especially their conformational versatility, was established. This work shows that the cell-penetrating peptides Pbeta and Pep-1 are able to induce formation of transmembrane pores in artificial bilayers and that these pores are most likely at the basis of their ability to facilitate intracellular delivery of therapeutics. In addition, their behaviour provides some information concerning the positioning of the peptides with respect to the membrane and confirms the role of the membrane potential in the translocation process.

Patch-clamp measurements were made on osteoblast-like cells isolated from embryonic chick calvaria. Cell-attached-patch measurements revealed two types of high conductance (100-250 pS) channels, which rapidly activated upon 50-100 mV depolarization. One type showed sustained and the other transient activation over a 10-sec period of depolarization. The single-channel conductances of these channel types were about 100 or 250 pS, depending on whether the pipettes were filled with a low K+ (3 mM) or high K+ (143 mM) saline, respectively. The different reversal potentials under these conditions were consistent with at least K+ conduction. Whole-cell measurements revealed the existence of two types of outward rectifying conductances. The first type conducts K+ ions and activates within 20-200 msec (depending on the stimulus) upon depolarizing voltage steps from less than -60 mV to greater than -30 mV. It inactivates almost completely with a time constant of 2-3 sec. Recovery from inactivation is biphasic with an initial rapid phase (1-2 sec) followed by a slow phase (greater than 20 sec). The second whole-cell conductance activates at positive membrane potentials of greater than +50 mV. It also rapidly turns on upon depolarizing voltage steps. Activation may partly disappear at the higher voltages. Its single channels of 140 pS conductance were identified in the whole cell and did conduct K+ ions but were not highly Cl- or Na+ selective. The results show that osteoblasts may express various types of voltage controlled ionic channels. We predict a role for such channels in mineral metabolism of bone tissue and its control by osteoblasts.