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Coarse-grained protein models approximate the first-principle physical potentials. Among those modeling approaches, the relative entropy framework yields promising and physically sound results, in which a mapping from the target protein structure and dynamics to a model is defined and subsequently adjusted by an entropy minimization of the model parameters. Minimization of the relative entropy is equivalent to maximization of the likelihood of reproduction of (configurational ensemble) observations by the model. In this study, we extend the relative entropy minimization procedure beyond parameter fitting by a second optimization level, which identifies the optimal mapping to a (dimension-reduced) topology. We consider anisotropic network models of a diverse set of ion channels and assess our findings by comparison to experimental results. Electronic supplementary material The online version of this article (10.1007/s00249-020-01480-7) contains supplementary material, which is available to authorized users.

Most potassium channels have two main gate locations, hosting an inner gate at the cytosolic entrance and a filter gate in the selectivity filter; the function of these gates is in many channels coupled. To obtain exclusive insights into the molecular mechanisms that determine opening and closing of the filter gate, we use a combination of single-channel recordings and gating analysis in the minimal viral channel Kcv

Modulating the activity of ion channels by blockers yields information on both the mode of drug action and on the biophysics of ion transport. Here we investigate the interplay between ions in the selectivity filter (SF) of K+ channels and the release kinetics of the blocker tetrapropylammonium in the model channel KcvNTS. A quantitative expression calculates blocker release rate constants directly from voltage-dependent ion occupation probabilities in the SF. The latter are obtained by a kinetic model of single-channel currents recorded in the absence of the blocker. The resulting model contains only two adjustable parameters of ion-blocker interaction and holds for both symmetric and asymmetric ionic conditions. This data-derived model is corroborated by 3D reference interaction site model (3D RISM) calculations on several model systems, which show that the K+ occupation probability is unaffected by the blocker, a direct consequence of the strength of the ion-carbonyl attraction in the SF, independent of the specific protein background. Hence, KcvNTS channel blocker release kinetics can be reduced to a small number of system-specific parameters. The pore-independent asymmetric interplay between K+ and blocker ions potentially allows for generalizing these results to similar potassium channels.

Winterstein et al. use the natural diversity of small viral K+ channels to address the impact of phospholipid bilayer composition on channel function. Single-channel recordings reveal a stimulating effect of anionic phospholipids on channel conductance and protein-specific effects on gating., It has become increasingly apparent that the lipid composition of cell membranes affects the function of transmembrane proteins such as ion channels. Here, we leverage the structural and functional diversity of small viral K+ channels to systematically examine the impact of bilayer composition on the pore module of single K+ channels. In vitro–synthesized channels were reconstituted into phosphatidylcholine bilayers ± cholesterol or anionic phospholipids (aPLs). Single-channel recordings revealed that a saturating concentration of 30% cholesterol had only minor and protein-specific effects on unitary conductance and gating. This indicates that channels have effective strategies for avoiding structural impacts of hydrophobic mismatches between proteins and the surrounding bilayer. In all seven channels tested, aPLs augmented the unitary conductance, suggesting that this is a general effect of negatively charged phospholipids on channel function. For one channel, we determined an effective half-maximal concentration of 15% phosphatidylserine, a value within the physiological range of aPL concentrations. The different sensitivity of two channel proteins to aPLs could be explained by the presence/absence of cationic amino acids at the interface between the lipid headgroups and the transmembrane domains. aPLs also affected gating in some channels, indicating that conductance and gating are uncoupled phenomena and that the impact of aPLs on gating is protein specific. In two channels, the latter can be explained by the altered orientation of the pore-lining transmembrane helix that prevents flipping of a phenylalanine side chain into the ion permeation pathway for long channel closings. Experiments with asymmetrical bilayers showed that this effect is leaflet specific and most effective in the inner leaflet, in which aPLs are normally present in plasma membranes. The data underscore a general positive effect of aPLs on the conductance of K+ channels and a potential interaction of their negative headgroup with cationic amino acids in their vicinity.

The miniature channel, Kcv, is a structural equivalent of the pore of all K+ channels. Here, we follow up on a previous observation that a largely voltage-insensitive channel can be converted into a slow activating inward rectifier after extending the outer transmembrane domain by one Ala. This gain of rectification can be rationalized by dynamic salt bridges at the cytosolic entrance to the channel; opening is favored by voltage-sensitive formation of salt bridges and counteracted by their disruption. Such latent voltage sensitivity in the pore could be relevant for the understanding of voltage gating in complex Kv channels.

Transport processes through ion-channel proteins, protein pores, or solid-state nanopores are traditionally recorded with commercial patch-clamp amplifiers. The bandwidth of these systems is typically limited to 10 kHz by signal-to-noise-ratio (SNR) considerations associated with these measurement platforms. At high bandwidth, the input-referred current noise in these systems dominates, determined by the input-referred voltage noise of the transimpedance amplifier applied across the capacitance at the input of the amplifier. This capacitance arises from several sources: the parasitic capacitance of the amplifier itself; the capacitance of the lipid bilayer harboring the ion channel protein (or the membrane used to form the solid-state nanopore); and the capacitance from the interconnections between the electronics and the membrane. Here, we review state-of-the-art applications of high-bandwidth conductance recordings of both ion channels and solid-state nanopores. These approaches involve tightly integrating measurement electronics fabricated in complementary metal-oxide semiconductors (CMOS) technology with lipid bilayer or solid-state membranes. SNR improvements associated with this tight integration push the limits of measurement bandwidths, in some cases in excess of 10 MHz. Recent case studies demonstrate the utility of these approaches for DNA sequencing and ion-channel recordings. In the latter case, studies with extended bandwidth have shown the potential for providing new insights into structure-function relations of these ion-channel proteins as the temporal resolutions of functional recordings matches time scales achievable with state-of-the-art molecular dynamics simulations.

The calculation of flux equations or current-voltage relationships in reaction kinetic models with a high number of states can be very cumbersome. Here, a recipe based on an arrow scheme is presented, which yields a straightforward access to the minimum form of the flux equations and the occupation probability of the involved states in cyclic and linear reaction schemes. This is extremely simple for cyclic schemes without branches. If branches are involved, the effort of setting up the equations is a little bit higher. However, also here a straightforward recipe making use of so-called reserve factors is provided for implementing the branches into the cyclic scheme, thus enabling also a simple treatment of such cases.

A main ingredient for the understanding of structure/function correlates of ion channels is the quantitative description of single-channel gating and conductance. However, a wealth of information provided from fast current fluctuations beyond the temporal resolution of the recording system is often ignored, even though it is close to the time window accessible to molecular dynamics simulations. This kind of current fluctuations provide a special technical challenge, because individual opening/closing or blocking/unblocking events cannot be resolved, and the resulting averaging over undetected events decreases the single-channel current. Here, I briefly summarize the history of fast-current fluctuation analysis and focus on the so-called "beta distributions." This tool exploits characteristics of current fluctuation-induced excess noise on the current amplitude histograms to reconstruct the true single-channel current and kinetic parameters. A guideline for the analysis and recent applications demonstrate that a construction of theoretical beta distributions by Markov Model simulations offers maximum flexibility as compared to analytical solutions.

We investigated the insertion of small potassium (K+) channel proteins (Kcv(MA-1D) and Kcv(NTS)) into model membranes and the lipid-protein structural interference, combining neutron reflectometry and electrophysiology. Neutron reflectometry experiments showed how the transverse structure and mechanical properties of the bilayer were modified, upon insertion of the proteins in single model-membranes, either supported on solid substrate or floating. Parallel electrophysiology experiments were performed on the same channels reconstituted in free-standing planar lipid bilayers, of both typical composition and matched to the neutron reflectometry experiment, assessing their electrical features. Functional and structural results converge in detecting that the proteins, conical in shape, insert with a directionality, cytosolic side first. Our work addresses the powerful combination of the two experimental approaches. We show here that membrane structure spectroscopy and ion channel electrophysiology can become synergistic tools in the analysis of structural-functional properties of biomimetic complex environment.

Single-channel recordings are widely used to explore functional properties of ion channels. Typically, such recordings are performed at bandwidths of less than 10 kHz because of signal-to-noise considerations, limiting the temporal resolution available for studying fast gating dynamics to greater than 100 µs. Here we present experimental methods that directly integrate suspended lipid bilayers with high-bandwidth, low-noise transimpedance amplifiers based on complementary metal-oxide-semiconductor (CMOS) integrated circuits (IC) technology to achieve bandwidths in excess of 500 kHz and microsecond temporal resolution. We use this CMOS-integrated bilayer system to study the type 1 ryanodine receptor (RyR1), a Ca2+-activated intracellular Ca2+-release channel located on the sarcoplasmic reticulum. We are able to distinguish multiple closed states not evident with lower bandwidth recordings, suggesting the presence of an additional Ca2+ binding site, distinct from the site responsible for activation. An extended beta distribution analysis of our high-bandwidth data can be used to infer closed state flicker events as fast as 35 ns. These events are in the range of single-file ion translocations.

[Image: see text] Gating of ion channels is based on structural transitions between open and closed states. To uncover the chemical basis of individual gates, we performed a comparative experimental and computational analysis between two K(+) channels, Kcv(S) and Kcv(NTS). These small viral encoded K(+) channel proteins, with a monomer size of only 82 amino acids, resemble the pore module of all complex K(+) channels in terms of structure and function. Even though both proteins share about 90% amino acid sequence identity, they exhibit different open probabilities with ca. 90% in Kcv(NTS) and 40% in Kcv(S). Single channel analysis, mutational studies and molecular dynamics simulations show that the difference in open probability is caused by one long closed state in Kcv(S). This state is structurally created in the tetrameric channel by a transient, Ser mediated, intrahelical hydrogen bond. The resulting kink in the inner transmembrane domain swings the aromatic rings from downstream Phes in the cavity of the channel, which blocks ion flux. The frequent occurrence of Ser or Thr based helical kinks in membrane proteins suggests that a similar mechanism could also occur in the gating of other ion channels.

Lipid bilayers provide many benefits for ion channel recordings, such as control of membrane composition and transport molecules. However, they suffer from high membrane capacitance limiting the bandwidth and impeding analysis of fast gating. This can be overcome by fitting the deviations of the open channel noise from the baseline noise by extended beta distributions. We demonstrate this analysis step-by-step by applying it to the example of viral K(+) channels (Kcv), from the choice of the gating model through the fitting process, validation of the results, and what kinds of results can be obtained. These voltage sensor-less channels show profoundly voltage-dependent gating with dwell times in the closed state of about 50 μs. Mutations assign it to the selectivity filter. This article is protected by copyright. All rights reserved.

The modular architecture of voltage-gated K+ (Kv) channels suggests that they resulted from the fusion of a voltage-sensing domain (VSD) to a pore module. Here, we show that the VSD of Ciona intestinalis phosphatase (Ci-VSP) fused to the viral channel Kcv creates KvSynth1, a functional voltage-gated, outwardly rectifying K+ channel. KvSynth1 displays the summed features of its individual components: pore properties of Kcv (selectivity and filter gating) and voltage dependence of Ci-VSP (V1/2 = +56 mV; z of ∼1), including the depolarization-induced mode shift. The degree of outward rectification of the channel is critically dependent on the length of the linker more than on its amino acid composition. This highlights a mechanistic role of the linker in transmitting the movement of the sensor to the pore and shows that electromechanical coupling can occur without coevolution of the two domains.

Single-channel current-voltage (IV) curves of human large-conductance, voltage- and Ca(2+)-activated K(+) (BK) channels are quite linear in 150 mM KCl. In the presence of Ca(2+) and/or Mg(2+), they show a negative slope conductance at high positive potentials. This is generally explained by a Ca(2+)/Mg(2+) block as by Geng et al. (2013. J. Gen. Physiol. http://dx.doi.org/10.1085/jgp.201210955) in this issue. Here, we basically support this finding but add a refinement: the analysis of the open-channel noise by means of β distributions reveals what would be found if measurements were done with an amplifier of sufficient temporal resolution (10 MHz), namely that the block by 2.5 mM Ca(2+) and 2.5 mM Mg(2+) per se would only cause a saturating curve up to +160 mV. Further bending down requires the involvement of a second process related to flickering in the microsecond range. This flickering is hardly affected by the presence or absence of Ca(2+)/Mg(2+). In contrast to the experiments reported here, previous experiments in BK channels (Schroeder and Hansen. 2007. J. Gen. Physiol. http://dx.doi.org/10.1085/jgp.200709802) showed saturating IV curves already in the absence of Ca(2+)/Mg(2+). The reason for this discrepancy could not be identified so far. However, the flickering component was very similar in the old and new experiments, regardless of the occurrence of noncanonical IV curves.

The miniature channel, Kcv, is a structural equivalent of the pore of all K

One major determinant of the efficacy of antibiotics on gram-negative bacteria is the passage through the outer membrane. During transport of the fluoroquinolone enrofloxacin through the trimeric outer membrane protein OmpF of Escherichia coli, the antibiotic interacts with two binding sites within the pore, thus partially blocking the ionic current. The modulation of one affinity site by Mg(2+) reveals further details of binding sites and binding kinetics. At positive membrane potentials, the slow blocking events induced by enrofloxacin in Mg(2+)-free media are converted to flickery sojourns at the highest apparent current level (all three pores flickering). This indicates weaker binding in the presence of Mg(2+). Analysis of the resulting amplitude histograms with β distributions revealed the rate constants of blocking (k(OB)) and unblocking (k(BO)) in the range of 1,000 to 120,000 s(-1). As expected for a bimolecular reaction, k(OB) was proportional to blocker concentration and k(BO) independent of it. k(OB) was approximately three times lower for enrofloxacin coming from the cis side than from the trans side. The block was not complete, leading to a residual conductivity of the blocked state being ∼25% of that of the open state. Interpretation of the results has led to the following model: fast flickering as caused by interaction of Mg(2+) and enrofloxacin is related to the binding site at the trans side, whereas the cis site mediates slow blocking events which are also found without Mg(2+). The difference in the accessibility of the binding sites also explains the dependency of k(OB) on the side of enrofloxacin addition and yields a means of determining the most plausible orientation of OmpF in the bilayer. The voltage dependence suggests that the dipole of the antibiotic has to be adequately oriented to facilitate binding.

Some algal viruses contain genes that encode proteins with the hallmarks of K(+) channels. One feature of these proteins is that they are less than 100 amino acids in size, which make them truly minimal for a K(+) channel protein. That is, they consist of only the pore module present in more complex K(+) channels. The combination of miniature size and the functional robustness of the viral K(+) channels make them ideal model systems for studying how K(+) channels work. Here we summarize recent structure/function correlates from these channels, which provide insight into functional properties such as gating, pharmacology and sorting in cells.

Fast gating of ion channels with rate constants higher than the corner frequency of the recording set-up can be evaluated by fitting so-called beta distributions to measured amplitude histograms. Up to now, this was preferentially done for O-C Markov sub-models with one open and one closed state. Here, a fit of the amplitude histograms from MaxiK (BK) single-channel records was achieved with a five-state model with two open and three closed states including three open-close transitions with rate constants higher than the corner frequency (20 kHz) of the inevitable low-pass filter of the recording system. The numerical values of the rate constants of these transitions enabled a nearly one-to-one relationship between typical regions of the histograms and the reactions in the Markov model. These characteristic features are the width of the peak at the apparent single-channel current, the side slopes at the open and at the closed peak, and the depth of the valley between the two peaks. However, the simplex routine alone was incapable of finding the solution but could do so if guided by hand along a suggested strategy.

Microsecond gating of ion channels can be evaluated by fitting beta distributions to amplitude histograms of measured time series. The shape of these histograms is determined not only by the rate constants of the gating process (in relation to the filter frequency) but also by baseline noise and shot noise, resulting from the stochastic nature of ion flow. Under normal temporal resolution, the small shot noise can be ignored. This simplification may no longer be legitimate when rate constants reach the range above 1 mus(-1). Here, the influence of shot noise is studied by means of simulated time series for several values of single-channel current of the fully open state and baseline noise. Under realistic optimal conditions (16 pA current, 1 pA noise, 50 kHz bandwidth), ignoring the shot noise leads to an underestimation of the rate constants above 1 mus(-1) by a factor of about 2.5. However, in that range, the scatter of the evaluated rate constants is at least of the same magnitude, obscuring the systematic error. The incorporation of shot noise into the analysis will become more important when amplifiers with significantly reduced noise become available.

Kcv from the chlorella virus PBCV-1 is a viral protein that forms a tetrameric, functional K+ channel in heterologous systems. Kcv can serve as a model system to study and manipulate basic properties of the K+ channel pore because its minimalistic structure (94 amino acids) produces basic features of ion channels, such as selectivity, gating, and sensitivity to blockers. We present a characterization of Kcv properties at the single-channel level. In symmetric 100 mM K+, single-channel conductance is 114 ± 11 pS. Two different voltage-dependent mechanisms are responsible for the gating of Kcv. “Fast” gating, analyzed by β distributions, is responsible for the negative slope conductance in the single-channel current–voltage curve at extreme potentials, like in MaxiK potassium channels, and can be explained by depletion-aggravated instability of the filter region. The presence of a “slow” gating is revealed by the very low (in the order of 1–4%) mean open probability that is voltage dependent and underlies the time-dependent component of the macroscopic current.

Patch clamp experiments on single MaxiK channels expressed in HEK293 cells were performed with a high temporal resolution (50-kHz filter) in symmetrical solutions with 50, 150, or 400 mM KCl and 2.5 mM CaCl2 and 2.5 mM MgCl2. At membrane potentials >+100 mV, the single-channel current showed a negative slope resistance, concomitantly with a flickery block, which was not influenced by Ca2+ or Mg2+. The analysis of the amplitude histograms by beta distributions revealed that current in this voltage range was reduced by two effects: rate limitation at the cytosolic side of the pore and gating with rate constants 10–20-fold higher than the cutoff frequency of the filter (i.e., dwell times in the microsecond range). The data were analyzed in terms of a model that postulates a coupling between both effects; if the voltage over the selectivity filter withdraws ions from the cavity at a higher rate than that of refilling from the cytosol, the selectivity filter becomes instable because of ion depletion, and current is interrupted by the resulting flickering. The fit of the IV curves revealed a characteristic voltage of 35 mV. In contrast, the voltage dependence of the gating factor R, i.e., the ratio between true and apparent single-channel current, could be fitted by exponentials with a characteristic voltage of 60 mV, suggesting that only part of the transmembrane potential is felt by the flux through the selectivity filter.

Ionizing radiation is a universal tool in tumor therapy but may also cause secondary cancers or cell invasiveness. These negative side effects could be causally related to the human-intermediate-conductance Ca2+-activated-K+-channel (hIK), which is activated by X-ray irradiation and affects cell proliferation and migration. To analyze the signaling cascade downstream of ionizing radiation we use genetically encoded reporters for H2O2 (HyPer) and for the dominant redox-buffer glutathione (Grx1-roGFP2) to monitor with high spatial and temporal resolution, radiation-triggered excursions of H2O2 in A549 and HEK293 cells. The data show that challenging cells with ≥1 Gy X-rays or with UV-A laser micro-irradiation causes a rapid rise of H2O2 in the nucleus and in the cytosol. This rise, which is determined by the rate of H2O2 production and glutathione-buffering, is sufficient for triggering a signaling cascade that involves an elevation of cytosolic Ca2+ and eventually an activation of hIK channels.

Two-dimensional (2D) dwell-time analysis of time series of single-channel patch-clamp current was improved by employing a Hinkley detector for jump detection, introducing a genetic fit algorithm, replacing maximum likelihood by a least square criterion, averaging over a field of 9 or 25 bins in the 2D plane and normalizing per measuring time, not per events. Using simulated time series for the generation of the "theoretical" 2D histograms from assumed Markov models enabled the incorporation of the measured filter response and noise. The effects of these improvements were tested with respect to the temporal resolution, accuracy of the determination of the rate constants of the Markov model, sensitivity to noise and requirement of open time and length of the time series. The 2D fit was better than the classical hidden Markov model (HMM) fit in all tested fields. The temporal resolution of the two most efficient algorithms, the 2D fit and the subsequent HMM/beta fit, enabled the determination of rate constants 10 times faster than the corner frequency of the low-pass filter. The 2D fit was much less sensitive to noise. The requirement of computing time is a problem of the 2D fit (100 times that of the HMM fit) but can now be handled by personal computers. The studies revealed a fringe benefit of 2D analysis: it can reveal the "true" single-channel current when the filter has reduced the apparent current level by averaging over undetected fast gating.

Single-channel current seems to be one of the most obvious characteristics of ion transport. But in some cases, its determination is more complex than anticipated at first glance. Problems arise from fast gating in time series of patch-clamp current, which can lead to a reduced apparent (measured) single-channel current. Reduction is caused by undetected averaging over closed and open intervals in the anti-aliasing filter. Here it is shown that fitting the measured amplitude histograms by Beta distributions is an efficient tool of reconstructing the true current level from measured data. This approach becomes even more powerful when it is applied to amplitude distributions-per-level. Simulated time series are employed to show that the error sum is a good guideline for finding the correct current level. Furthermore, they show that a Markov model smaller than the one used for gating analysis can be used for current determination (mostly O-C, i.e., open-closed). This increases the reliability of the Beta fit. The knowledge of the true current level is not only important for the understanding of the biophysical properties of the channel. It is also a prerequisite for the correct determination of the rate constants of gating. The approach is applied to measured data. The examples reveal the limits of the analysis imposed by the signal-to-noise ratio and the shape of the amplitude distribution. One application shows that the negative slope of the I-V curve of the human MaxiK channel expressed in HEK293 cells is caused by fast gating.

cAMP mediates autonomic regulation of heart rate by means of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which underlie the pacemaker current I-f. cAMP binding to the C-terminal cyclic nucleotide binding domain enhances HCN open probability through a conformational change that reaches the pore via the C-linker. Using structural and functional analysis, we identified a binding pocket in the C-linker of HCN4. Cyclic dinucleotides, an emerging class of second messengers in mammals, bind the C-linker pocket (CLP) and antagonize cAMP regulation of the channel. Accordingly, cyclic dinucleotides prevent cAMP regulation of I-f in sinoatrial node myocytes, reducing heart rate by 30%. Occupancy of the CLP hence constitutes an efficient mechanism to hinder beta-adrenergic stimulation on I-f. Our results highlight the regulative role of the C-linker and identify a potential drug target in HCN4. Furthermore, these data extend the signaling scope of cyclic dinucleotides in mammals beyond their first reported role in innate immune system.

Transport activity through the mutant D44A of the M2 proton channel from influenza virus A was measured in excised inside-out macro-patches of Xenopus laevis oocytes at cytosolic pH values of 5.5, 7.5 and 8.2. The current-voltage relationships reveal some peculiarities: 1. "Transinhibition", i.e., instead of an increase of unidirectional outward current with increasing cytosolic H(+) concentration, a decrease of unidirectional inward current was found. 2. Strong inward rectification. 3. Exponential rise of current with negative potentials. In order to interpret these findings in molecular terms, different kinetic models have been tested. The transinhibition basically results from a strong binding of H(+) to a site in the pore, presumably His37. This assumption alone already provides inward rectification and exponential rise of the IV curves. However, it results in poor global fits of the IV curves, i.e., good fits were only obtained for cytosolic pH of 8.2, but not for 7.5. Assuming an additional transport step as e.g. caused by a constriction zone at Val27 resulted in a negligible improvement. In contrast, good global fits for cytosolic pH of 7.5 and 8.2 were immediately obtained with a cyclic model. A "recycling step" implies that the protein undergoes conformational changes (assigned to Trp41 and Val27) during transport which have to be reset before the next proton can be transported. The global fit failed at the low currents at pHcyt = 5.5, as expected from the interference of putative transport of other ions besides H(+). Alternatively, a regulatory effect of acidic cytosolic pH may be assumed which strongly modifies the rate constants of the transport cycle.

The channel proteins from Chlorella viruses are miniature versions of K+ channels. They are the structural equivalents of the pore module of complex K+ channels from eukaryotes and they include the selectivity filter, the cavity and gates. The channel KcvNTS is with only 82 amino acids particularly small and molecular dynamics simulations suggest that the channel is quasi fully embedded in a di-myristoylphosphatidylcholin (C14) lipid bilayer [1]. This close interaction between the channel protein and its lipid environment offers the possibility to examine lipid/protein interaction for a channel pore in a systematic manner. For this purpose we reconstituted the purified channel protein in lipid bilayers with different chain lengths, phospholipid head groups and in the absence and presence of detergents or cholesterol. The analysis of single channel activity shows that the membrane composition affects channel gating but not the unitary conductance. Most interesting is a voltage dependency of the channel, which is introduced by the presence of cholesterol, detergent or bilayers with long chain length.Reference1. Braun, C.J., et al., Viral potassium channels as a robust model system for studies of membrane-protein interaction. Biochim Biophys Acta, 2013.

The current of the minimal viral K(+) channel Kcv(PCBV-1) heterologously expressed in Xenopus oocytes is strongly inhibited by reactive oxygen species (ROS) like H(2)O(2). Possible targets for the ROS effect are two cysteines (C53 and C79) and four methionines (M1, M15, M23, and M26). The C53A/C79A and M23L/M26L double mutations maintained the same ROS sensitivity as the wild type. However, M15L as a single mutant or in combination with C53A/C79A and/or M23L/M26L caused a complete loss of sensitivity to H(2)O(2). These results indicate a prominent role of M15 at the cytosolic end of the outer transmembrane helix for gating of Kcv(PCBV-1). Furthermore, even though the channel lacks a canonical voltage sensor, it exhibits a weak voltage sensitivity, resulting in a slight activation in the millisecond range after a voltage step to negative potentials. The M15L mutation inverts this kinetics into an inactivation, underlining the critical role of this residue for gating. The negative slope of the I-V curves of M15L is the same as in the wild type, indicating that the selectivity filter is not involved.

Conventional electronics typically limit the available temporal resolution for single ion-channel measurements, making fast channel gating events (

Patch clamp experiments on single MaxiK channels expressed in HEK293 cells were performed at high temporal resolution (50-kHz filter) in asymmetrical solutions containing 0, 25, 50, or 150 mM Tl+ on the luminal or cytosolic side with [K+] + [Tl+] = 150 mM and 150 mM K+ on the other side. Outward current in the presence of cytosolic Tl+ did not show fast gating behavior that was significantly different from that in the absence of Tl+. With luminal Tl+ and at membrane potentials more negative than -40 mV, the single-channel current showed a negative slope resistance concomitantly with a flickery block, resulting in an artificially reduced apparent single-channel current I(app). The analysis of the amplitude histograms by beta distributions enabled the estimation of the true single-channel current and the determination of the rate constants of a simple two-state O-C Markov model for the gating in the bursts. The voltage dependence of the gating ratio R = I(true)/I(app) = (k(CO) + k(OC))/k(CO) could be described by exponential functions with different characteristic voltages above or below 50 mM Tl(+). The true single-channel current I(true) decreased with Tl+ concentrations up to 50 mM and stayed constant thereafter. Different models were considered. The most likely ones related the exponential increase of the gating ratio to ion depletion at the luminal side of the selectivity filter, whereas the influence of [Tl+] on the characteristic voltage of these exponential functions and of the value of I(true) were determined by [Tl+] at the inner side of the selectivity filter or in the cavity.

Gating of ion channels is based on structural transitions between open and closed states. To uncover the chemical basis of individual gates, we performed a comparative experimental and computational analysis between two K+ channels, Kcv(S) and Kcv(NTS). These small viral encoded K+ channel proteins, with a monomer size of only 82 amino acids, resemble the pore module of all complex K+ channels in terms of structure and function. Even though both proteins share about 90% amino acid sequence identity, they exhibit different open probabilities with ca. 90% in Kcv(NTS) and 40% in Kcv(S). Single channel analysis, mutational studies and molecular dynamics simulations show that the difference in open probability is caused by one long closed state in Kcv(S). This state is structurally created in the tetrameric channel by a transient, Ser mediated, intrahelical hydrogen bond. The resulting kink in the inner transmembrane domain swings the aromatic rings from downstream Phes in the cavity of the channel, which blocks ion flux. The frequent occurrence of Ser or Thr based helical kinks in membrane proteins suggests that a similar mechanism could also occur in the gating of other ion channels.

The viral channel Kcv(NTS) belongs to the smallest K+ channels known so far. A monomer of a functional homotetramer contains only 82 amino acids. As a consequence of the small size the protein is almost fully submerged into the membrane. This suggests that the channel is presumably sensitive to its lipid environment. Here we perform a comparative analysis for the function of the channel protein embedded in three different membrane environments. 1. Single-channel currents of Kcv(NTS) were recorded with the patch clamp method on the plasma membrane of HEK293 cells. 2. They were also measured after reconstitution of recombinant channel protein into classical planar lipid bilayers and 3. into horizontal bilayers derived from giant unilamellar vesicles (GUVs). The recombinant channel protein was either expressed and purified from Pichia pastoris or from a cell-free expression system; for the latter a new approach with nanolipoprotein particles was used. The data show that single-channel activity can be recorded under all experimental conditions. The main functional features of the channel like a large single-channel conductance (80 pS), high open-probability (>50%) and the approximate duration of open and closed dwell times are maintained in all experimental systems. An apparent difference between the approaches was only observed with respect to the unitary conductance, which was ca. 35% lower in HEK293 cells than in the other systems. The reason for this might be explained by the fact that the channel is tagged by GFP when expressed in HEK293 cells. Collectively the data demonstrate that the small viral channel exhibits a robust function in different experimental systems. This justifies an extrapolation of functional data from these systems to the potential performance of the channel in the virus/host interaction. This article is part of a Special Issue entitled: Viral Membrane Proteins-Channels for Cellular Networking. (C) 2013 Elsevier B.V. All rights reserved.

Phycodnaviruses are dsDNA viruses, which infect algae. Their large genomes encode many gene products, like small K(+) channels, with homologs in prokaryotes and eukaryotes. Screening for K(+) channels revealed their abundance in viruses from fresh-water habitats. Recent sequencing of viruses from marine algae or from salt water in Antarctica revealed sequences with the predicted characteristics of K(+) channels but with some unexpected features. Two genes encode either 78 or 79 amino acid proteins, which are the smallest known K(+) channels. Also of interest is an unusual sequence in the canonical α-helixes in K(+) channels. Structural prediction algorithms indicate that the new channels have the conserved α-helix folds but the algorithms failed to identify the expected transmembrane domains flanking the K(+) channel pores. In spite of these unexpected properties electophysiological studies confirmed that the new proteins are functional K(+) channels.

Many potassium channels show voltage-dependent gating without a dedicated voltage sensor domain. This is not fully understood yet, but often explained by voltage-induced changes of ion occupation in the five distinct K+ binding sites in the selectivity filter. To better understand this mechanism of filter gating we measured the single-channel current and the rate constant of sub-millisecond channel closure of the viral K+ channel KcvNTS for a wide range of voltages and symmetric and asymmetric K+ concentrations in planar lipid membranes. A model-based analysis employed a global fit of all experimental data, i.e., using a common set of parameters for current and channel closure under all conditions. Three different established models of ion permeation and various relationships between ion occupation and gating were tested. Only one of the models described the data adequately. It revealed that the most extracellular binding site (S0) in the selectivity filter functions as the voltage sensor for the rate constant of channel closure. The ion occupation outside of S0 modulates its dependence on K+ concentration. The analysis uncovers an important role of changes in protein flexibility in mediating the effect from the sensor to the gate.