Biophysical characterization of Vpu from HIV-1 suggests a channel-pore dualism

Vpu from HIV-1 is an 81 amino acid type I integral membrane protein which consists of a cytoplasmic and a transmembrane (TM) domain. The TM domain is known to alter membrane permeability for ions and substrates when inserted into artificial membranes. Peptides corresponding to the TM domain of Vpu (Vpu(1-32)) and mutant peptides (Vpu(1-32)-W23L, Vpu(1-32)-R31V, Vpu(1-32)-S24L) have been synthesized and reconstituted into artificial lipid bilayers. All peptides show channel activity with a main conductance level of around 20 pS. Vpu(1-32)-W23L has a considerable flickering pattern in the recordings and longer open times than Vpu(1-32). Whilst recordings for Vpu(1-32)-R31V are almost indistinguishable from those of the WT peptide, recordings for Vpu(1-32)-S24L do not exhibit any noticeable channel activity. Recordings of WT peptide and Vpu(1-32)-W23L indicate Michaelis-Menten behavior when the salt concentration is increased. Both peptide channels follow the Eisenman series I, indicative for a weak ion channel with almost pore like characteristics.     

Towards a mechanism of function of the viral ion channel Vpu from HIV-1

Vpu, an integral membrane protein encoded in HIV-1, is implicated in the release of new virus particles from infected cells, presumably mediated by ion channel activity of homo-oligomeric Vpu bundles. Reconstitution of both full length Vpu(1-81) and a short, the transmembrane (TM) domain comprising peptide Vpu(1-32) into bilayers under a constant electric field results in an asymmetric orientation of those channels. For both cases, channel activity with similar kinetics is observed. Channels can open and remain open within a broad series of conductance states even if a small or no electric potential is applied. The mean open time for Vpu peptide channels is voltage-independent. The rate of channel opening shows a biphasic voltage activation, implicating that the gating is influenced by the interaction of the dipole moments of the TM helices with an electric field.     

Molecular Dynamics Simulations Reveal the HIV-1 Vpu Transmembrane Protein to Form Stable Pentamers

The human immunodeficiency virus type I (HIV-1) Vpu protein is 81 residues long and has two cytoplasmic and one transmembrane (TM) helical domains. The TM domain oligomerizes to form a monovalent cation selective ion channel and facilitates viral release from host cells. Exactly how many TM domains oligomerize to form the pore is still not understood, with experimental studies indicating the existence of a variety of oligomerization states. In this study, molecular dynamics (MD) simulations were performed to investigate the propensity of the Vpu TM domain to exist in tetrameric, pentameric, and hexameric forms. Starting with an idealized α-helical representation of the TM domain, a thorough search for the possible orientations of the monomer units within each oligomeric form was carried out using replica-exchange MD simulations in an implicit membrane environment. Extensive simulations in a fully hydrated lipid bilayer environment on representative structures obtained from the above approach showed the pentamer to be the most stable oligomeric state, with interhelical van der Waals interactions being critical for stability of the pentamer. Atomic details of the factors responsible for stable pentamer structures are presented. The structural features of the pentamer models are consistent with existing experimental information on the ion channel activity, existence of a kink around the Ile17, and the location of tetherin binding residues. Ser23 is proposed to play an important role in ion channel activity of Vpu and possibly in virus propagation.     

Modular design of Cys-loop ligand-gated ion channels: functional 5-HT3 and GABA rho1 receptors lacking the large cytoplasmic M3M4 loop

Cys-loop receptor neurotransmitter-gated ion channels are pentameric assemblies of subunits that contain three domains: extracellular, transmembrane, and intracellular. The extracellular domain forms the agonist binding site. The transmembrane domain forms the ion channel. The cytoplasmic domain is involved in trafficking, localization, and modulation by cytoplasmic second messenger systems but its role in channel assembly and function is poorly understood and little is known about its structure. The intracellular domain is formed by the large (>100 residues) loop between the alpha-helical M3 and M4 transmembrane segments. Putative prokaryotic Cys-loop homologues lack a large M3M4 loop. We replaced the complete M3M4 loop (115 amino acids) in the 5-hydroxytryptamine type 3A (5-HT(3A)) subunit with a heptapeptide from the prokaryotic homologue from Gloeobacter violaceus. The macroscopic electrophysiological and pharmacological characteristics of the homomeric 5-HT(3A)-glvM3M4 receptors were comparable to 5-HT(3A) wild type. The channels remained cation-selective but the 5-HT(3A)-glvM3M4 single channel conductance was 43.5 pS as compared with the subpicosiemens wild-type conductance. Coexpression of hRIC-3, a protein that modulates expression of 5-HT(3) and acetylcholine receptors, significantly attenuated 5-HT-induced currents with wild-type 5-HT(3A) but not 5-HT(3A)-glvM3M4 receptors. A similar deletion of the M3M4 loop in the anion-selective GABA-rho1 receptor yielded functional, GABA-activated, anion-selective channels. These results imply that the M3M4 loop is not essential for receptor assembly and function and suggest that the cytoplasmic domain may fold as an independent module from the transmembrane and extracellular domains.     

Structure-function correlates of Vpu,a membrane protein of HIV-1

Vpu, a membrane protein from human immunodeficiency virus-1, folds into two distinct structural domains with different biological activities: a transmembrane (TM) helical domain involved in the budding of new virions from infected cells, and a cytoplasmic domain encompassing two amphipathic helices, which is implicated in CD4 degradation. The molecular mechanism by which Vpu facilitates virion budding is not clear. This activity of Vpu requires an intact TM helical domain. And it is known that oligomerization of the VPU TM domain results in the formation of sequence-specific, cation-selective channels. It has been shown that the channel activity of Vpu is confined to the TM domain, and that the cytoplasmic helices regulate the lifetime of the Vpu channel in the conductive state. Structure-function correlates based on the convergence of information about the channel activity of Vpu reconstituted in lipid bilayers and on its 3-D structure in membranes by a combination of solution and solid-state nuclear magnetic resonance spectroscopy may provide valuable insights to understand the role of Vpu in the pathogenesis of AIDS and for drug design aimed to block channel activity.     

The two halves of CFTR form a dual-pore ion channel

The cystic fibrosis transmembrane conductance regulator (CFTR) exhibits two conductance states, 9 picosiemens (pS) and 3 pS. To investigate the origin of these two distinct conductance states, we measured the single-channel activity of three truncated forms of CFTR. These include: TNR, which contains the first transmembrane domain, the first nucleotide binding domain, and the R domain; RT2N2, which contains the R domain, the second transmembrane domain, and the second nucleotide-binding domain; and T2N2, which contains only the second transmembrane domain and the second nucleotide-binding domain. The results show that TNR exhibits only the large conductance of 9.2 pS, whereas RT2N2 and T2N2 exhibit only the small conductance (3.8-4.0 pS). Co-expression of TNR with T2N2 resulted in a mixed pattern of two conductance states, which is similar to that observed in wild-type CFTR. In further studies, a "dual-R mutant," R334W and R347P in the transmembrane segment 6 of the first half of CFTR, severely impaired the large conductance channel without affecting the small conductance channel. The ion selectivity and gating behavior of the two conductance channels are different regardless of whether they are measured in wild-type CFTR or in truncated CFTRs. The ion selectivity of the large conductance channel is Br(-) > Cl(-) > I(-), whereas the ion selectivity of the small conductance channel is Br(-) = Cl(-) = I(-). The open probability (P(o)) of the large conductance is about 4-fold higher than that of the small conductance. Transition from closed to open states of the small conductance is not dependent upon the open or closed states of the large conductance. The independent behaviors of the two conductances in CFTR strongly suggest that CFTR may have two distinct pores. Thus, like ClC0, CFTR is likely to be a double-barreled ion channel, with the first half of CFTR forming the large conductance and the second half forming the small conductance.     

Towards a Mechanism of Function of the Viral Ion Channel Vpu from HIV-1

Abstract

Vpu, an integral membrane protein encoded in HIV-1, is implicated in the release of new virus particles from infected cells, presumably mediated by ion channel activity of homo- oligomeric Vpu bundles.

Reconstitution of both full length Vpu_1–81 and a short, the transmembrane (TM) domain comprising peptide Vpu_1-32 into bilayers under a constant electric field results in an asymmetric orientation of those channels. For both cases, channel activity with similar kinetics is observed. Channels can open and remain open within a broad series of conductance states even if a small or no electric potential is applied.

The mean open time for Vpu peptide channels is voltage-independent. The rate of channel opening shows a biphasic voltage activation, implicating that the gating is influenced by the interaction of the dipole moments of the TM helices with an electric field.     

Atomistic Detailed Mechanism and Weak Cation-Conducting Activity of HIV-1 Vpu Revealed by Free Energy Calculations

The viral protein U (Vpu) encoded by HIV-1 has been shown to assist in the detachment of virion particles from infected cells. Vpu forms cation-specific ion channels in host cells, and has been proposed as a potential drug target. An understanding of the mechanism of ion transport through Vpu is desirable, but remains limited because of the unavailability of an experimental structure of the channel. Using a structure of the pentameric form of Vpu – modeled and validated based on available experimental data – umbrella sampling molecular dynamics simulations (cumulative simulation time of more than 0.4 µs) were employed to elucidate the energetics and the molecular mechanism of ion transport in Vpu. Free energy profiles corresponding to the permeation of Na⁺ and K⁺ were found to be similar to each other indicating lack of ion selection, consistent with previous experimental studies. The Ser23 residue is shown to enhance ion transport via two mechanisms: creating a weak binding site, and increasing the effective hydrophilic length of the channel, both of which have previously been hypothesized in experiments. A two-dimensional free energy landscape has been computed to model multiple ion permeation, based on which a mechanism for ion conduction is proposed. It is shown that only one ion can pass through the channel at a time. This, along with a stretch of hydrophobic residues in the transmembrane domain of Vpu, explains the slow kinetics of ion conduction. The results are consistent with previous conductance studies that showed Vpu to be a weakly conducting ion channel.     

Purification and functional reconstitution of N- and C-halves of the MscL channel

MscL is a mechanosensitive channel gated by membrane tension in the lipid bilayer alone. Its structure, known from x-ray crystallography, indicates that it is a homopentamer. Each subunit comprises two transmembrane segments TM1 and TM2 connected by a periplasmic loop. The closed pore is lined by five TM1 helices. We expressed in Escherichia coli and purified two halves of the protein, each containing one of the transmembrane segments. Their electrophysiological activity was studied by the patch-clamp recording upon reconstitution in artificial liposomes. The TM2 moiety had no electrophysiological activity, whereas the TM1 half formed channels, which were not affected by membrane tension and varied in conductance between 50 and 350 pS in 100 mM KCl. Coreconstitution of the two halves of MscL however, yielded mechanosensitive channels having the same conductance as the native MscL (1500 pS), but exhibiting increased sensitivity to pressure. Our results confirm the current view on the functional role of TM1 and TM2 helices in the MscL gating and emphasize the importance of helix-helix interactions for the assembly and functional properties of the channel protein. In addition, the results indicate a crucial role of the periplasmic loop for the channel mechanosensitivity.     

Design, synthesis and functional characterization of a pentameric channel protein that mimics the presumed pore structure of the nicotinic cholinergic receptor

Nicotinic cholinergic receptors are membrane proteins composed of five subunits organized around a central aqueous pore. A pentameric channel protein, T₅M2δ, that emulates the presumed pore-forming structure of this receptor was generated by assembling five helix-forming peptide modules at the lysine ε-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) δ subunit; M2 segments are considered involved in pore-lining. Purified T₅M2δ migrates in SDS-PAGE with an apparent M_r˜14,000, concordant with a protein of 126 residues. T₅M2δ forms cation-selective channels when reconstituted in planar lipid bilayers. The single channel conductance in symmetric 0.5 M K.C1 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 a-helices is a plausible structural motif underlying the inner bundle that forms the pore of the pentameric AChR channel.     

Structure,Dynamics and Implied Gating Mechanism of a Human Cyclic Nucleotide-Gated Channel

Cyclic nucleotide-gated (CNG) ion channels are nonselective cation channels, essential for visual and olfactory sensory transduction. Although the channels include voltage-sensor domains (VSDs), their conductance is thought to be independent of the membrane potential, and their gating regulated by cytosolic cyclic nucleotide–binding domains. Mutations in these channels result in severe, degenerative retinal diseases, which remain untreatable. The lack of structural information on CNG channels has prevented mechanistic understanding of disease-causing mutations, precluded structure-based drug design, and hampered in silico investigation of the gating mechanism. To address this, we built a 3D model of the cone tetrameric CNG channel, based on homology to two distinct templates with known structures: the transmembrane (TM) domain of a bacterial channel, and the cyclic nucleotide-binding domain of the mouse HCN2 channel. Since the TM-domain template had low sequence-similarity to the TM domains of the CNG channels, and to reconcile conflicts between the two templates, we developed a novel, hybrid approach, combining homology modeling with evolutionary coupling constraints. Next, we used elastic network analysis of the model structure to investigate global motions of the channel and to elucidate its gating mechanism. We found the following: (i) In the main mode of motion, the TM and cytosolic domains counter-rotated around the membrane normal. We related this motion to gating, a proposition that is supported by previous experimental data, and by comparison to the known gating mechanism of the bacterial KirBac channel. (ii) The VSDs could facilitate gating (supplementing the pore gate), explaining their presence in such ‘voltage-insensitive’ channels. (iii) Our elastic network model analysis of the CNGA3 channel supports a modular model of allosteric gating, according to which protein domains are quasi-independent: they can move independently, but are coupled to each other allosterically.     

Conformation and ion channel properties of a five-helix Bundle protein.

The primary amphipathic peptide Ac-Met-Gly-Leu-Gly-Leu-Trp-Leu-Leu-Val-Leu10-Ala-Ala-Ala-Leu-Gln-Gly-Ala-Lys-Lys-Lys20-Arg-Lys-Val-NH-CH2-CH2-SH called SPM was able to induce formation of ion channels into planar lipid bilayers with main conductance values of 75 and 950 pS in 1 M KCl. The 75 pS value can be attributed to an aggregate composed of five monomers since the corresponding five-unit bundle (5-SPM) also presented a 70 pS channels under the same conditions. The upper 950 pS level would be generated by a hexameric aggregate. Ion channels induced by both SPM and its pentameric bundle are slightly cation selective but not voltage-dependent. The structural studies showed that the SPM and 5-SPM possess mainly an alpha-helical structure (approximately 40%) and are strongly embedded in the bilayer. This behaviour and the strong hydrophobic interactions occurring between helices in the bundle induce a strong stabilization of 5-SPM in the bilayer and would be responsible for the stepwise current fluctuations observed during the incorporation of 5-SPM into the membrane.     

Bundles consisting of extended transmembrane segments of Vpu from HIV-1: computer simulations and conductance measurements

Part of the genome of the human immunodeficiency virus type 1 (HIV-1) encodes for a short membrane protein Vpu, which has a length of 81 amino acids. It has two functional roles: (i) to downregulate CD4 and (ii) to support particle release. These roles are attributed to two distinct domains of the peptide, the cytoplasmic and transmembrane (TM) domains, respectively. It has been suggested that the enhanced particle release function is linked to the ion channel activity of Vpu, with a slight preference for cations over anions. To allow ion flux across the membrane Vpu would be required to assemble in homooligomers to form functional water-filled pores. In this study molecular dynamics simulations are used to address the role of particular amino acids in 4, 5, and 6 TM helix bundle structures. The helices (Vpu(6-33)) are extended to include hydrophilic residues such as Glu, Tyr, and Arg (EYR motif). Our simulations indicate that this motif destabilizes the bundles at their C-terminal ends. The arginines point into the pore to form a positive charged ring that could act as a putative selectivity filter. The helices of the bundles adopt slightly higher average tilt angles with decreasing number of helices. We also suggest that the helices are kinked. Conductance measurements on a peptide (Vpu(1-32)) reconstituted into lipid membranes show that the peptide forms ion channels with several conductance levels.     

A central role for the T1 domain in voltage-gated potassium channel formation and function

To interpret the recent atomic structures of the Kv (voltage-dependent potassium) channel T1 domain in a functional context, we must understand both how the T1 domain is integrated into the full-length functional channel protein and what functional roles the T1 domain governs. The T1 domain clearly plays a role in restricting Kv channel subunit heteromultimerization. However, the importance of T1 tetramerization for the assembly and retention of quarternary structure within full-length channels has remained controversial. Here we describe a set of mutations that disrupt both T1 assembly and the formation of functional channels and show that these mutations produce elevated levels of the subunit monomer that becomes subject to degradation within the cell. In addition, our experiments reveal that the T1 domain lends stability to the full-length channel structure, because channels lacking the T1 containing N terminus are more easily denatured to monomers. The integration of the T1 domain ultrastructure into the full-length channel was probed by proteolytic mapping with immobilized trypsin. Trypsin cleavage yields an N-terminal fragment that is further digested to a tetrameric domain, which remains reactive with antisera to T1, and that is similar in size to the T1 domain used for crystallographic studies. The trypsin-sensitive linkages retaining the T1 domain are cleaved somewhat slowly over hours. Therefore, they seem to be intermediate in trypsin resistance between the rapidly cleaved extracellular linker between the first and second transmembrane domains, and the highly resistant T1 core, and are likely to be partially structured or contain dynamic structure. Our experiments suggest that tetrameric atomic models obtained for the T1 domain do reflect a structure that the T1 domain sequence forms early in channel assembly to drive subunit protein tetramerization and that this structure is retained as an integrated stabilizing structural element within the full-length functional channel.     

Conductance of recombinant GABA (A) channels is increased in cells co-expressing GABA(A) receptor-associated protein

High conductance gamma-aminobutyric acid type A (GABA(A)) channels (>40 picosiemens (pS)) have been reported in some studies on GABA(A) channels in situ but not in others, whereas recombinant GABA(A) channels do not appear to display conductances above 40 pS. Furthermore, the conductance of some native GABA(A) channels can be increased by diazepam or pentobarbital, which are effects not reported for expressed GABA(A) channels. GABARAP, a protein associated with native GABA(A) channels, has been reported to cause clustering of GABA(A) receptors and changes in channel kinetics. We have recorded single channel currents activated by GABA in L929 cells expressing alpha(1), beta(1), and gamma(2S) subunits of human GABA(A) receptors. Channel conductance was never higher than 40 pS and was not significantly increased by diazepam or pentobarbital, although open probability was increased. In contrast, in cells expressing the same three subunits together with GABARAP, channel conductance could be significantly higher than 40 pS, and channel conductance was increased by diazepam and pentobarbital. GABARAP caused clustering of receptors in L929 cells, and we suggest that there may be interactions between subunits of clustered GABA(A) receptors that make them open co-operatively to give high conductance "channels." Recombinant channels may require the influence of GABARAP and perhaps other intracellular proteins to adopt a fuller repertoire of properties of native channels.     

Amiloride derivatives block ion channel activity and enhancement of virus-like particle budding caused by HIV-1 protein Vpu

The Vpu protein of human immunodeficiency virus type 1 forms cation-selective ion channels and enhances the process of virion budding and release. Mutagenesis studies have shown that the N-terminal transmembrane domain primarily controls both of these activities. Here we report that the Vpu ion channel is inhibited by the amiloride derivatives 5-(N,N-hexamethylene)amiloride and 5-(N,N-dimethyl)amiloride but not by amiloride itself, nor by amantadine. Hexamethyleneamiloride also inhibits budding of virus-like particles from HeLa cells expressing HIV-1 Gag and Vpu proteins. These results confirm the link between Vpu ion channel activity and the budding process and also suggest that amiloride derivatives might have useful anti-HIV-1 properties.     

Mechanosensitive channels of bacteria and archaea share a common ancestral origin

The ubiquity of mechanosensitive (MS) ion channels set off a search for their functional homologues in archaea, the third domain of life. A new MS channel was identified in the archaeon Methanococcus jannaschii by using the TM1 transmembrane domain of the bacterial MS channel of large conductance, MscL, as a genetic probe to search the archaeal genomic database for MS channel homologues. The hypothetical protein MJ0170 (MscMJ) was found to harbor two MscL-like TM1 structural motifs and showed a high degree of se quence and secondary structure conservation with MscS (YggB) homologues. The alignment of sequences of MscL, MscS and MscMJ homologues further revealed that bacterial and archaeal channels form a phylogenetic tree composed of three main branches and share a common ancestral origin. This suggests the evolution of prokaryotic MS channels via gene duplication of a MscL-like progenitor gene followed by divergence, fur ther indicating that the common ancestor of the prokaryotic MS channels most likely resembled MscL. When expressed in E. coli and functionally examined by the patch clamp, the MscMJ protein behaved as a MS channel with a conductance of 270 pS in 200 mM KCl and a cation selectivity (PK/PC]) of approximately 6. The structural and functional homologue of MscMJ, MscMJLR, was identified as a second type of MS channel in M. jannaschii. The channel has a conductance of approximately 2 nS, rectifies with voltage and shares cation selectivity with MscMJ. The stoichiometry of both types of MS channels revealed that the free energy of activation, deltaG0 approximately 7kT, obtained for MscMJ matches the one calculated for MscS, deltaG0 approximately 5kT, whereas the free energy of activation approximately deltaG0 approximately 18kT of MscMJLR resembles more the deltaG0 = 14-19kT reported for MscL. The presence of two types of MS channels discovered in the cell envelope of M. jannaschii indicates that multiplicity of MS channels in prokaryotes is a necessary element for their survival in the habitats frequently challenged by sudden changes in osmolarity. Further functional and phylogenetic study of MS channels from all three domains of the universal phylogenetic tree may help to understand the evolution and common biophysical principles that govern mechanosensory transduction.     

NMR structure of the transmembrane domain of the n-acetylcholine receptor β2 subunit

Nicotinic acetylcholine receptors (nAChRs) are involved in fast synaptic transmission in the central and peripheral nervous system. Among the many different types of subunits in nAChRs, the β2 subunit often combines with the α4 subunit to form α4β2 pentameric channels, the most abundant subtype of nAChRs in the brain. Besides computational predictions, there is limited experimental data available on the structure of the β2 subunit. Using high-resolution NMR spectroscopy, we solved the structure of the entire transmembrane domain (TM1234) of the β2 subunit. We found that TM1234 formed a four-helix bundle in the absence of the extracellular and intracellular domains. The structure exhibited many similarities to those previously determined for the Torpedo nAChR and the bacterial ion channel GLIC. We also assessed the influence of the fourth transmembrane helix (TM4) on the rest of the domain. Although secondary structures and tertiary arrangements were similar, the addition of TM4 caused dramatic changes in TM3 dynamics and subtle changes in TM1 and TM2. Taken together, this study suggests that the structures of the transmembrane domains of these proteins are largely shaped by determinants inherent in their sequence, but their dynamics may be sensitive to modulation by tertiary and quaternary contacts.     

Design of a functional calcium channel protein: inferences about an ion channel-forming motif derived from the primary structure of voltage-gated calcium channels.

To identify sequence-specific motifs associated with the formation of an ionic pore, we systematically evaluated the channel-forming activity of synthetic peptides with sequence of predicted transmembrane segments of the voltage-gated calcium channel. The amino acid sequence of voltage-gated, dihydropyridine (DHP)-sensitive calcium channels suggests the presence in each of four homologous repeats (I-IV) of six segments (S1-S6) predicted to form membrane-spanning, alpha-helical structures. Only peptides representing amphipathic segments S2 or S3 form channels in lipid bilayers. To generate a functional calcium channel based on a four-helix bundle motif, four-helix bundle proteins representing IVS2 (T4CaIVS2) or IVS3 (T4CaIVS3) were synthesized. Both proteins form cation-selective channels, but with distinct characteristics: the single-channel conductance in 50 mM BaCl2 is 3 pS and 10 pS. For T4CaIVS3, the conductance saturates with increasing concentration of divalent cation. The dissociation constants for Ba2+, Ca2+, and Sr2+ are 13.6 mM, 17.7 mM, and 15.0 mM, respectively. The conductance of T4CaIVS2 does not saturate up to 150 mM salt. Whereas T4CaIVS3 is blocked by microM Ca2+ and Cd2+, T4CaIVS2 is not blocked by divalent cations. Only T4CaIVS3 is modulated by enantiomers of the DHP derivative BayK 8644, demonstrating sequence requirement for specific drug action. Thus, only T4CaIVS3 exhibits pore properties characteristic also of authentic calcium channels. The designed functional calcium channel may provide insights into fundamental mechanisms of ionic permeation and drug action, information that may in turn further our understanding of molecular determinants underlying authentic pore structures.     

Bis[(benzo-15-crown-5)-15-yl methyl] pimelate forms ion channels in planar lipid bilayer: a novel model ion channel

Some crown ethers translocate cations across the liposomal membrane either by a carrier mechanism or by forming ion channels. We report formation of ion channels in lipid bilayer membranes by bis[(benzo-15-crown-5)-15-yl methyl] pimelate, a crown ether known to form ion inclusion complexes with alkali metal cations. The channels have characteristic long openings lasting several seconds and a low conductance (4 pS in 500 mM KCl and 2.5 pS in 500 mM NaCl). A model of the crown ether channel formed by stacking of four monomers is proposed. A large database of structural information on crown ethers and their ion inclusion complexes as well as large family of crown ethers with a variety of substitutions in the ring are commercially available. Thus the crown ether channel is an attractive model system to study the role of various chemical moieties in ion conduction which may provide deeper insight into understanding the mechanism(s) of selectivity, ion transport, etc. in biological ion channels.