Channel Conductance Levels Research Articles

Heating-enabled formation of droplet interface bilayers using Escherichia coli total lipid extract.

Droplet interface bilayers (DIBs) serve as a convenient platform to study interactions between synthetic lipid membranes and proteins. However, a majority of DIBs have been assembled using a single lipid type, diphytanoylphosphatidylcholine (DPhPC). The work described herein establishes a new method to assemble DIBs using total lipid extract from Escherichia coli (eTLE); it is found that incubating oil-submerged aqueous droplets containing eTLE liposomes at a temperature above the gel-fluid phase transition temperature (Tg) promotes monolayer self-assembly that does not occur below Tg. Once monolayers are properly assembled via heating, droplets can be directly connected or cooled below Tg and then connected to initiate bilayer formation. This outcome contrasts immediate droplet coalescence observed upon contact between nonheated eTLE-infused droplets. Specific capacitance measurements confirm that the interface between droplets containing eTLE lipids is a lipid bilayer with thickness of 29.6 Å at 25 °C in hexadecane. We observe that bilayers formed from eTLE or DPhPC survive cooling and heating between 25 and 50 °C and demonstrate gigaohm (GΩ) membrane resistances at all temperatures tested. Additionally, we study the insertion of alamethicin peptides into both eTLE and DPhPC membranes to understand how lipid composition, temperature, and membrane phase influence ion channel formation. Like in DPhPC bilayers, alamethicin peptides in eTLE exhibit discrete, voltage-dependent gating characterized by multiple open channel conductance levels, though at significantly lower applied voltages. Cyclic voltammetry measurements of macroscopic channel currents confirm that the voltage-dependent conductance of alamethicin channels in eTLE bilayers occurs at lower voltages than in DPhPC bilayers at equivalent peptide concentrations. This result suggests that eTLE membranes, via composition, fluidity, or the presence of subdomains, offer an environment that enhances alamethicin insertion. For both membrane compositions, increasing temperature reduces the lifetimes of single channel gating events and increases the voltage required to cause an exponential increase in channel current. However, the fact that alamethicin insertion in eTLE exhibits significantly greater sensitivity to temperature changes through its Tg suggests that membrane phase plays an important role in channel formation. These effects are much less severe in DPhPC, where heating from 25 to 50 °C does not induce a phase change. The described technique for heating-assisted monolayer formation permits the use of other high transition temperature lipids in aqueous droplets for DIB formation, thereby increasing the types of lipids that can be considered for assembling model membranes.

Construction of a Ca2+-Gated Artificial Channel by Fusing Alamethicin with a Calmodulin-Derived Extramembrane Segment

Using native chemical ligation, we constructed a Ca(2+)-gated fusion channel protein consisting of alamethicin and the C-terminal domain of calmodulin. At pH 5.4 and in the absence of Ca(2+), this fusion protein yielded a burst-like channel current with no discrete channel conductance levels. However, Ca(2+) significantly lengthened the specific channel open state and increased the mean channel current, while Mg(2+) produced no significant changes in the channel current. On the basis of 8-anilinonaphthalene-1-sulfonic acid (ANS) fluorescent measurement, Ca(2+)-stimulated gating may be related to an increased surface hydrophobicity of the extramembrane segment of the fusion protein.

TARP‐associated AMPA receptors display an increased maximum channel conductance and multiple kinetically distinct open states

Fast excitatory synaptic transmission in the CNS is mediated mainly by AMPA-type glutamate receptors (AMPARs), whose biophysical properties are dramatically modulated by the presence of transmembrane AMPAR regulatory proteins (TARPs). To help construct a kinetic model that will realistically describe native AMPAR/TARP function, we have examined the single-channel properties of homomeric GluA1 AMPARs in combination with the TARPs, γ-2, γ-4 and γ-5. In a saturating concentration of agonist, each of these AMPAR/TARP combinations gave rise to single-channel currents with multiple conductance levels that appeared intrinsic to the receptor-channel complex, and showed long-lived subconductance states. The open time and burst length distributions of the receptor complexes displayed multiple dwell-time components. In the case of γ-2- and γ-4-associated receptors, these distributions included a long-lived component lasting tens of milliseconds that was absent from both GluA1 alone and γ-5-associated receptors. The open time distributions for each conductance level required two dwell-time components, indicating that at each conductance level the channel occupies a minimum of two kinetically distinct open states. We have explored how these data place novel constraints on possible kinetic models of TARP-associated AMPARs that may be used to define AMPAR-mediated synaptic transmission.

Neuromodulation independently determines correlated channel expression and conductance levels in motor neurons of the stomatogastric ganglion

Neuronal identity depends on the regulated expression of numerous molecular components, especially ionic channels, which determine the electrical signature of a neuron. Such regulation depends on at least two key factors, activity itself and neuromodulatory input. Neuronal electrical activity can modify the expression of ionic currents in homeostatic or nonhomeostatic fashion. Neuromodulators typically modify activity by regulating the properties or expression levels of subsets of ionic channels. In the stomatogastric system of crustaceans, both types of regulation have been demonstrated. Furthermore, the regulation of the coordinated expression of ionic currents and the channels that carry these currents has been recently reported in diverse neuronal systems, with neuromodulators not only controlling the absolute levels of ionic current expression but also, over long periods of time, appearing to modify their correlated expression. We hypothesize that neuromodulators may regulate the correlated expression of ion channels at multiple levels and in a cell-type-dependent fashion. We report that in two identified neuronal types, three ionic currents are linearly correlated in a pairwise manner, suggesting their coexpression or direct interactions, under normal neuromodulatory conditions. In each cell, some currents remain correlated after neuromodulatory input is removed, whereas the correlations between the other pairs are either lost or altered. Interestingly, in each cell, a different suite of currents change their correlation. At the transcript level we observe distinct alterations in correlations between channel mRNA amounts, including one of the cell types lacking a correlation under normal neuromodulatory conditions and then gaining the correlation when neuromodulators are removed. Synaptic activity does not appear to contribute, with one possible exception, to the correlated expression of either ionic currents or of the transcripts that code for the respective channels. We conclude that neuromodulators regulate the correlated expression of ion channels at both the transcript and the protein levels.

Functional Effects of Cornichon Proteins on Homomeric Glua1 AMPAR Single-Channels

Fast excitatory synaptic transmission in the brain is mediated mainly by AMPA receptors (AMPARs). AMPAR subunits associate with auxiliary transmembrane AMPAR regulatory proteins (TARPs) that have well established roles in modulating AMPAR trafficking, kinetics, and pharmacology. It has recently been proposed that the trafficking and kinetics of native AMPARs are also modulated by members of the cornichon family of proteins (Schwenk et al, 2009). There is also recent evidence that cornichons act only as intracellular trafficking chaperones for AMPARs in neurons (Shi et al, 2010). To determine the influence of cornichons on AMPAR single-channel properties we co-expressed GluA1 and cornichon 3 (CNIH3) in tsA201 cells and examined AMPARs in outside-out patches. The weighted mean single-channel conductance was increased in a dose-dependent manner when increasing amounts of CNIH3 cDNA were included in the transfection mix. Up to three open channel conductance levels were detected, with the lower conductance levels being more prevalent in cells with a low CNIH3/GluA1 cDNA ratio, and higher level conductance levels predominating in cells with a high CNIH3/ GluA1 cDNA ratio. CNIH3 also increased the burst duration of GluA1 single-channels. Fast application of glutamate onto excised patches indicated that CNIH3 slowed the rate of desensitization. As our data indicated that AMPAR channel properties were markedly altered by cornichons, we considered whether these proteins were expressed at the surface of cells where such modulation may be functionally important. Positive cell surface labeling of cornichons was not seen in neurons (see also Shi et al. 2010) but was detected in glial cells which express AMPARs. Supported by the Wellcome Trust. Schwenk, J. et al. (2009). Science, 323, 1313-9. Shi, Y., et al. (2010). Proc Natl Acad Sci USA, 107, 16315-9.

Activation of Recombinant Rat GluN1/gluN2D NMDA Receptors

N-methyl-D-aspartate (NMDA) receptors are members of a class of ionotropic glutamate receptors and mediate slow, Ca2+-permeable synaptic transmission. Four separate GluN2 subunit genes (N2A-D) have been identified, which give rise to many of the observed differences in functional properties of the NMDA receptors, including conductance levels, open probability, and deactivation time course upon removal of agonists. A number of macroscopic and single channel properties of NMDA receptors can be grouped according to functional similarities. To study the distinctions between N2A and N2D-containing NMDA receptors, we have conducted single channel voltage-clamp recordings of N1/N2A and N1/N2D receptors to compare channel properties such as open probability and conductance. N1/N2A receptors have a higher channel conductance level (69 + 0.5 pS) and open probability (0.48 + 0.05) than N1/N2D receptors, which have a lower main conductance level and prominent subconductance level (55 + 2.3 pS and 33 + 1.4 pS) and a low open probability (0.015 + 0.004). One explanation of this difference in open probability is that rates constants describing activation steps are slower for NR1/N2D than for N1/N2A receptors, thereby reducing the probability of activating the receptor. However, our whole cell voltage clamp recordings indicate N1/N2D show a surprisingly rapid rise time (6.7 + 0.49 ms), similar to N1/N2A (8.5 + 0.50 ms). To understand how these two receptors with such strikingly different open probabilities can activate at a similar rate, we have fitted models of NMDA receptor activation to our single channel recordings. Preliminary analysis of our data has identified several rate constants describing pre-gating activation steps for N1/N2A that are more than 10-fold faster than in N1/N2D, suggesting that it may be possible to identify pre-gating steps responsible for the distinct characteristics of the N1/N2A and N1/N2D NMDA receptors.

Multiple levels of the single L-type Ca2+ channel conductance in adult mammalian ventricular myocytes

In cardiac muscle, Ca2+ entry through the L-type Ca2+ channel during excitation triggers Ca2+ release from sarcoplasmic reticulum (SR) via the ryanodine receptor, causing muscle contraction. This Ca2+ induced Ca2+ release (CICR) is locally controlled by openings of the single L-type Ca2+ channel that triggers the opening of ryanodine receptors located within the dyadic junction. The unitary current through the single Ca2+ channel determines the open probability of the ryanodine receptors, and hence the efficacy of CICR. Here, we report findings of multiple conductance levels of L-type Ca2+ channels in freshly isolated rat ventricular myocytes. When 10mM Ca2+ was used in the pipette solution as the charge carrier in the cell-attached configuration, the most frequently occurring conductance levels are 6.9pS and 2.9pS. Three distinct conductance levels were also observed, although infrequently, in the same cell, corresponding to unitary currents of 0.33pA, 0.24pA, and 0.17pA upon depolarization to −10mV. In conclusion, our data demonstrate the existence of multiple single L-type Ca2+ channel conductance levels in the adult mammalian ventricular myocytes with Ca2+ as the charge carrier. The multiple conductance levels present heterogeneity in the Ca2+ trigger signal strength at local dyadic junctions. The physiological significance of this heterogeneity on affecting the efficacy of CICR in the cardiac muscle is discussed.

Electromagnetic field (EMF) effects on channel activity of nanopore OmpF protein

In this study, the effects of nonionizing electromagnetic fields (EMF; 925 MHz) on the OmpF porin channel have been characterized at the single-channel level. Channel activity was recorded in real time by the voltage clamp method. Our results showed an increase in the frequency of channel gating and voltage sensitivity. The effects of EMF lasted for several milliseconds after the field source was terminated. However, the conductance levels of channels did not change significantly. Thermal effects of EMF on single-channel properties are a possible cause, based on theoretical evaluation of results that were comparable to those seen in conventional experiments at different temperatures. We conclude that EMF affects both the dynamics and conformation of the channel, either directly by affecting critical amino acid side-chain arrangement, or indirectly, via the electrolyte or the lipid membrane.

Functional Characteristics of alpha3beta3gamma2 and alpha1beta2gamma2 GABA-A receptors

The GABAA receptor is a member of the cys-loop family of ligand gated ion channels and the major mediator of neuronal inhibition. Each receptor is a pentameric protein complex composed of homologous subunits, the combination of which gives rise to numerous GABAA receptor subtypes. Within the thalamus, two synaptic GABAA receptor isoforms predominate; the α1β2γ2 channels, which are found at the synapses of relay neurons, and the α3β3γ2 channels that are located at synapses in the reticular nucleus. We endeavoured to characterize the kinetic properties to these two GABAA receptors. Our preliminary data suggest that both receptors open to a single channel conductance level of 26 pS, irrespective of the GABA concentration used to elicit activity. α3β3γ2 GABAA receptors, however, exhibit longer active periods (bursts) at saturating (5 mM) and low (2 μM) concentrations of GABA compared to α1β2γ2 GABAA receptors. The mean burst length and intra-burst open probability (Po) for the α3β3γ2 channels was 136.7 ± 16.8 ms and 0.84 ± 0.05 (n = 4), respectively and for the α1β2γ2 channels was 87.4 ± 12.0 ms and 0.82 ± 0.02 (n = 3), respectively, when exposed to 5 mM GABA. At 2 μM GABA, the mean burst length and Po for α3β3γ2 channels was 25.8 ± 4.7 ms and 0.78 ± 0.03 (n = 3), respectively and for α1β2γ2 channels was 8.4 ± 1.9 ms and 0.73 ± 0.02 (n = 3), respectively. These measurements are consistent with the reported slower deactivation phase of ensemble, α3β3γ2 mediated synaptic currents and suggest that GABA has a longer occupancy at α3β3γ2 channels than at α1β2γ2 channels.

Regulation Of The Single Channel Conductance Of K2p10.1 (Trek2) By The Amino-terminus

K2P2.1 (TREK-1) and K2P10.1 (TREK-2), when expressed in mammalian cell lines, show several single channel conductance levels. Recent studies show that the N-terminus of TREKs controls the unitary conductance levels via alternative translation initiation mechanism that produces isoforms with long and short N-termini. For TREK-2, the isoform with the full length N-terminus (residues 1-69) has a low conductance level, whereas that with the short one (residues 55-69 or 67-69) has a large conductance level. The role of the N-terminus and the putative slide helix region in the control of the unitary conductance was studied further using deletion and substitution TREK-2 mutants. Deletion of the N-terminus up to residue 36 (out of 69 residues) had no effect on conductance levels. Further deletions up to residues 40 (∼150-pS at −40 mV), 44 (∼90-pS), and 49 (>30-pS) produced channels with levels that were different from the levels observed in the wild type TREK-2. A mutation within the putative slide helix region (residues 47-55) to render it non-helical resulted in formation of mainly the low conductance channel, and greatly reduced the open probability. These TREK-2 mutants also inhibited the alternative translation initiation. These results suggest that the distal region of the N-terminus (residues 37-55) including the putative helical region (residues 37-54) controls the unitary conductance of TREK-2 in an unpredictable way. The data also indicate that the putative helical region is important for the control of channel activity.

The mechanism of channel formation by alamethicin as viewed by molecular dynamics simulations.

Alamethicin is a 20-residue channel-forming peptide that forms a stable amphipathic alpha-helix in membrane and membrane-mimetic environments. This helix contains a kink induced by a central Gly-X-X-Pro sequence motif. Alamethicin channels are activated by a cis positive transbilayer voltage. Channel activation is suggested to correspond to voltage-induced insertion of alamethicin helices in the bilayer. Alamethicin forms multi-conductance channels in lipid bilayers. These channels are formed by parallel bundles of transmembrane helices surrounding a central pore. A change in the number of helices per bundle switches the single channel conductance level. Molecular dynamics simulations of alamethicin in a number of different environments have been used to explore its channel-forming properties. These simulations include: (i) alamethicin in solution in water and in methanol; (ii) a single alamethicin helix at the surface of a phosphatidylcholine bilayer; (iii) single alamethicin helices spanning a phosphatidylcholine bilayer; and (iv) channels formed by bundles of 5, 6, 7 or 8 alamethicin helices spanning a phosphatidylcholine bilayer. The total simulation time is c. 30 ns. Thus, these simulations provide a set of dynamic snapshots of a possible mechanism of channel formation by this peptide.

Maurocalcine interacts with the cardiac ryanodine receptor without inducing channel modification

We have previously shown that MCa (maurocalcine), a toxin from the venom of the scorpion Maurus palmatus, binds to RyR1 (type 1 ryanodine receptor) and induces strong modifications of its gating behaviour. In the present study, we investigated the ability of MCa to bind to and modify the gating process of cardiac RyR2. By performing pull-down experiments we show that MCa interacts directly with RyR2 with an apparent affinity of 150 nM. By expressing different domains of RyR2 in vitro, we show that MCa binds to two domains of RyR2, which are homologous with those previously identified on RyR1. The effect of MCa binding to RyR2 was then evaluated by three different approaches: (i) [(3)H]ryanodine binding experiments, showing a very weak effect of MCa (up to 1 muM), (ii) Ca(2+) release measurements from cardiac sarcoplasmic reticulum vesicles, showing that MCa up to 1 muM is unable to induce Ca(2+) release, and (iii) single-channel recordings, showing that MCa has no effect on the open probability or on the RyR2 channel conductance level. Long-lasting opening events of RyR2 were observed in the presence of MCa only when the ionic current direction was opposite to the physiological direction, i.e. from the cytoplasmic face of RyR2 to its luminal face. Therefore, despite the conserved MCa binding ability of RyR1 and RyR2, functional studies show that, in contrast with what is observed with RyR1, MCa does not affect the gating properties of RyR2. These results highlight a different role of the MCa-binding domains in the gating process of RyR1 and RyR2.

Molecular and Functional Characterization of the Melastatin-related Cation Channel TRPM3

Proteins of the mammalian TRP (transient receptor potential) family form a heterogenous group of cation channels important for cellular Ca2+ signaling and homeostasis. Here we present the full-length sequence of TRPM3, a member of the melastatin-like subfamily (TRPM) of TRP channels. TRPM3 expression was found in human kidney and brain. HEK293 cells transiently transfected with TRPM3 showed a constitutive Ca2+ and Mn2+ entry. Whole-cell patch clamp experiments confirmed the spontaneous activity of TRPM3 and revealed permeability ratios PCa/PNa of 1.57 and PNa/PCs of 0.75. In cell-attached patches, spontaneous inward and outward currents were observed. At negative membrane potentials and in the presence of either 140 mm Cs+, 140 mm Na+, or 100 mm Ca2+ in the pipette solution, the single channel conductance levels were 133, 83, and 65 pS, respectively. The Ca2+ entry in TRPM3-expressing HEK293 cells increased during treatment with hypotonic extracellular solution. The reduction of extracellular osmolarity was accompanied by cell swelling, suggesting volume-regulated activity of TRPM3. From its function and expression in human kidney, we propose a role of TRPM3 in renal Ca2+ homeostasis.

Stretch-sensitive switching among different channel sublevels of an endothelial cation channel

A mechanosensitive Ca 2+-permeable cation channel was recorded by patch clamp in isolated rat aortic endothelial cells. A low level of channel activity could be observed after seal formation. The channel displayed some inward rectification and had a conductance for inward current of approx. 32 pS in Ca 2+-free pipette and bath solutions. Negative suction of −10 to −20 mmHg increased the probability of the channel being open. When the negative pressure in the pipette was raised to −35 to −45 mmHg, the channel underwent an abrupt transition to a large conductance substate that was interrupted occasionally by two other low conductance levels. Under this condition, the overwhelming majority of openings and closings were between a main level of 83 pS and the closed level. Compared to the 32 pS substate, the 83 pS large conductance substate had shorter mean open and closed times. The two channel substates had similar ionic selectivity and both were sensitive to the inhibition of cGMP and protein kinase G. This is the first demonstration showing that mechanostress can change the single channel conductance level of an ion channel in eukaryotic cells.

Concentration-dependent substate behavior of native AMPA receptors.

AMPA-type glutamate receptors mediate most excitatory postsynaptic currents (EPSCs) at central synapses, and their conductance determines in part the size of EPSCs. The conductance of a recombinant AMPA receptor depends on the number of agonist molecules bound to the channel. Here we tested whether native AMPA and kainate receptors show this behavior in outside-out patches from neurons in situ by measuring conductance levels of single channels over a wide range of agonist concentrations. We found that the conductance of AMPA, but not kainate, receptors depended strongly on agonist concentration. Our results suggest that alterations in the glutamate concentration in the synaptic cleft may change the apparent unitary conductance of postsynaptic AMPA receptors.

Functional changes of N-methyl-d-aspartic acid and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate channels in gerbil hippocampal CA1, in relation to postischemic enhancement of glutamate receptor-mediated responses

Glutamate receptor-mediated responses have been reported to be enhanced in the postischemic CA1 pyramidal neurons before the appearance of delayed neuronal death, and the enhancement has been thought to be one of crucial factors leading postischemic CA1 pyramidal neurons to irreversible neuronal injury. In the present study, we examined what changes in functional properties of N-methyl-d-aspartic acid (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) channels are responsible for the enhanced postischemic glutamate receptor-mediated responses. Gerbils were subjected to 5-min ischemia to induce the enhancement of glutamate receptor-mediated responses and the hippocampal slices were prepared 3 h after ischemia. Single channel activities evoked by NMDA and AMPA were recorded from outside-out patches excised from the postischemic CA1 pyramidal neurons. The main conductance levels of NMDA and AMPA channels in the postischemic CA1 pyramidal neurons were not significantly different from those in control CA1 pyramidal neurons. The mean open time and the open-state probability of NMDA and AMPA channels significantly increased in the postischemic CA1 pyramidal neurons (NMDA channels: mean open time, 1.4-fold increase; open-state probability, 1.5-fold increase) (AMPA channels: mean open time, 1.3-fold increase; open-state probability, 1.8-fold increase). These findings indicate that the increases in the mean open time and the open-state probability of NMDA and AMPA channels are responsible for the enhancement of postischemic NMDA and non-NMDA receptor-mediated responses.

The startle disease mutation Q266H, in the second transmembrane domain of the human glycine receptor, impairs channel gating.

Hyperekplexia (startle disease) results from mutations in the glycine receptor chloride channel that disrupt inhibitory synaptic transmission. The Q266H missense mutation is the only hyperekplexia mutation located in the transmembrane domains of the receptor. Using recombinant expression and patch-clamping techniques, we have investigated the functional properties of this mutation. The ability of glycine and taurine to open the channel was reduced in the mutated channel, as shown by a 6-fold shift in the concentration-response curve for both agonists. This was not accompanied by similar changes in agonist displacement of strychnine binding, suggesting that the mutation affects functions subsequent to ligand binding. Taurine was also converted to a weak partial agonist and antagonized the actions of glycine, consistent with changes in its channel gating efficacy. Because the Q266H mutation is within the pore-forming second transmembrane domain, we tested for a direct interaction with permeating ions. No change in either the cation/anion selectivity ratio or in single channel conductance levels was observed. No differential effects of Zn++, pH, and diethylpyrocarbonate were observed, implying that the histidine side chain is not exposed to the channel lumen. Single-channel recordings revealed a significant reduction in open times in the mutant receptors, at both high and low agonist concentrations, consistent with the open state of the channel being less stable. This study demonstrates that residues within the second transmembrane domain of ligand-gated ion channel receptors, even those whose side chains do not directly interact with permeating ions, can affect the kinetics of channel gating.

PH-modulation of chloride channels from the sarcoplasmic reticulum of skeletal muscle.

The understanding of the role of cytoplasmic pH in modulating sarcoplasmic reticulum (SR) ion channels involved in Ca2+ regulation is important for the understanding of the function of normal and adversely affected muscles. The dependency of the SR small chloride (SCl) channel from rabbit skeletal muscle on cytoplasmic pH (pHcis) and luminal pH (pHtrans) was investigated using the lipid bilayer-vesicle fusion technique. Low pHcis 6.75-4.28 modifies the operational mode of this multiconductance channel (conductance levels between 5 and 75 pS). At pHcis 7.26-7.37 the channel mode is dominated by the conductance and kinetics of the main conductance state (65-75 pS) whereas at low pHcis 6.75-4.28 the channel mode is dominated by the conductance and kinetics of subconductance states (5-40 pS). Similarly, low pHtrans 4.07, but not pHtrans 6.28, modified the activity of SCl channels. The effects of low pHcis are pronounced at 10(-3) and 10(-4) M [Ca2+]cis but are not apparent at 10(-5) M [Ca2+]cis, where the subconductances of the channel are already prominent. Low pHcis-induced mode shift in the SCl channel activity is due to modification of the channel proteins that cause the uncoupling of the subconductance states. The results in this study suggest that low pHcis can modify the functional properties of the skeletal SR ion channels and hence contribute, at least partly, to the malfunction in the contraction-relaxation mechanism in skeletal muscle under low cytoplasmic pH levels.

Alamethicin channels in a membrane: molecular dynamics simulations.

Alamethicin (Alm) is a 20 residue peptide which forms a kinked alpha-helix in membrane and membrane-mimetic environments. Ion channels formed by intramembraneous aggregates of Alm are thought to be formed by bundles of approximately parallel Alm helices surrounding a central bilayer pore. Different channel conductance levels correspond to different numbers of helices per bundle, ranging from N = 5 to N > 8. Calculation of the predicted pKA values of the ring of Glu18 sidechains at the C-terminal mouth of the pore suggests that at neutral pH most or all of these sidechains will remain protonated. Nanosecond molecular dynamics (MD) simulations of N = 5, 6, 7 and 8 bundles of Alm helices in a POPC bilayer have been run, corresponding to a total simulation time of 4 ns. These simulations explore the stability and conformational dynamics of these helix bundle channels when embedded in a full phospholipid bilayer in an aqueous environment. The structural and dynamic properties of water in these model channels are examined. As in earlier in vacuo simulations (J. Breed, R. Sankararamakrishnan, I. D. Kerr and M. S. P. Sansom, Biophys. J., 1996, 70, 1643) the dipole moments of water molecules within the pores are aligned antiparallel to the helix dipoles. This helps to contribute to the stability of the helix bundles.

Ion channels formed by HIV-1 Vpu: a modelling and simulation study

Vpu is an oligomeric integral membrane protein encoded by HIV-1 which forms ion channels, each subunit of which contains a single transmembrane helix. Models of Vpu channels formed by bundles of N=4, 5 or 6 transmembrane helices have been developed by restrained molecular dynamics and refined by 100 ps simulations with water molecules within the pore. Pore radius profiles and conductance predictions suggest that the N=5 model corresponds to the predominant channel conductance level of the channel. Potential energy profiles for translation of Na + or Cl − ions along the Vpu N=5 pore are consistent with the weak cation selectivity of Vpu channels.