KcsA Research Articles

Structural Determinants of Specific Lipid Binding to Potassium Channels

We have investigated specific lipid binding to the pore domain of potassium channels KcsA and chimeric KcsA-Kv1.3 on the structural and functional level using extensive coarse-grained and atomistic molecular dynamics simulations, solid-state NMR, and single channel measurements. We show that, while KcsA activity is critically modulated by the specific and cooperative binding of anionic nonannular lipids close to the channel's selectivity filter, the influence of nonannular lipid binding on KcsA-Kv1.3 is much reduced. The diminished impact of specific lipid binding on KcsA-Kv1.3 results from a point-mutation at the corresponding nonannular lipid binding site leading to a salt-bridge between adjacent KcsA-Kv1.3 subunits, which is conserved in many voltage-gated potassium channels and prevents strong nonannular lipid binding to the pore domain. Our findings elucidate how protein-lipid and protein-protein interactions modulate K(+) channel activity. The combination of MD, NMR, and functional studies as shown here may help to dissect the structural and dynamical processes that are critical for the functioning of larger membrane proteins, including Kv channels in a membrane setting.

Influence of interionic interactions on functional state and blocker binding of voltage-gated potassium channels

A mechanism of ion conduction of a voltagegated potassium channel KcsA was investigated in fullatomic approximation at a trajectory length of 100 ns using the Lomonosov supercomputer. Methods of molecular dynamics were employed. A structure of the KcsA channel in the open state obtained by Xray structure analysis (PDB ID 3fb7) was used. Free energy profiles of the KcsA pore occupied with either one or three potassium ions were calculated. It was shown that, under physiological conditions, ions pass through the channel pore cooperatively and the mechanism most probably includes three ions permeating in concert. Interactions of the mammalian voltagegated channel Kv 1.2 with neurotoxin were investigated. It was dem� onstrated that the effect of interionic interactions on binding of a blocker is rather insufficient.

The Open Gate Structure of the Membrane-Embedded KcsA Potassium Channel Viewed From the Cytoplasmic Side

Crystallographic studies of channel proteins have provided insight into the molecular mechanisms of ion channels, even though these structures are obtained in the absence of the membrane and some structural portions have remained unsolved. Here we report the gating structure of the membrane-embedded KcsA potassium channel using atomic force microscopy (AFM). The activation gate of the KcsA channel is located on the intracellular side, and the cytoplasmic domain was truncated to clear the view of this location. Once opened, the individual subunits in the tetramer were resolved with the pore open at the center. Furthermore, AFM was able to capture the previously unsolved bulge helix at the entrance. A molecular dynamics simulation revealed that the bulge helices fluctuated dramatically at the open entryway. This dynamic behavior was observed as vigorous open-channel noise in the single-channel current recordings. The role of the bulge helices in the open gate structure is discussed.

Targeted molecular dynamics (TMD) of the full-length KcsA potassium channel: on the role of the cytoplasmic domain in the opening process

Some recent papers clearly indicate that the cytoplasmic domain of KcsA plays a role in pH sensing. We have performed, for the first time, a targeted molecular dynamics (TMD) simulation of the opening of full-length KcsA at pH 4 and pH 7, with a special interest for the cytoplasmic domain. Association energy calculations show a stabilization at pH 7 confirming that the protonation of some amino-acids at pH 4 in this domain plays a role in the opening process. A careful analysis of the pH dependent charges borne by residues in the cytoplasmic domain and their interactions confirms some literature experimental data and permits to give further insight into the role played by some of them in the opening process.

Purification and Characterization of the Tetrameric Potassium Channel Kcsa in “Native Nanodiscs”

We explored a new discovery in membrane research of amphipathic copolymers of styrene and maleic acid (SMA) that have the unique potential to directly extract proteins from membranes in the form of “native nanodiscs” without the need for detergent [1,2]. E-coli cells overexpressing a His-tagged version of the potassium channel KcsA were incubated with SMA and the conditions were optimized for extraction of the protein. After solubilization of the membrane, we found that KcsA indeed could be purified on a Ni2+-NTA column in the form of nano-sized discs, as was confirmed by negative stain transmission electron microscopy (TEM) experiments. SDS-PAGE analysis showed that the protein in these nanodiscs is present as a tetramer, running at the same position as when subjected to SDS-PAGE from its native membrane or after purification in detergent. Presently we are comparing the stability of this tetramer in the nanodiscs with that of purified KcsA in n-dodecyl β-D-maltoside (DDM) micelles and KcsA in native membranes. This is done by comparing the effects of heat-incubation and exposure to small alcohols using gel shift assays. Furthermore, to gain information about possible preferential lipid interactions of KcsA, the lipid composition of the purified, KcsA containing nanodiscs is being analyzed and compared to that of the total lipid composition in E-coli. Results of these studies will be presented. 1) Knowles TJ, Finka R, Smith C, Lin Y-P, Dafforn T, Overduin M (2009) J Am Chem Soc 131, 7484-7485 2) Orwich-Rydmark M, Lovett JE, Lindholm L, Graziadei A, Hicks M, Watts A (2012) Nanoletters 12, 4687-4692

The Inactivated Bacterial Potassium Channel Conducts Water

The potassium channel KcsA was reported to conduct water at an extraordinarily high rate, which implied a 20-fold increase in mobility of water molecules inside the channel as compared to bulk water [1]. A conformational state of the channel that is permeable to water, but closed to ions would have resulted in an underestimation of channel number, and thus, in an overestimation of water mobility. Here we tested whether the recently described C-type inactivated state of KcsA may represent such a conformation. Therefore, we reconstituted a KcsA mutant in which the inactivation was suppressed with an E71A mutation in the pore helix. KcsA labeling with a fluorescent dye allowed determination of the number of reconstituted channels per vesicle by fluorescence correlation spectroscopy. It was equal to the number of open channels, as revealed by fusing part of these vesicles with planar lipid bilayers. We subjected the remaining part of these vesicles to stopped flow experiments. This approach allowed unitary water permeability calculation of the non-inactivating KcsA variant. It is roughly an order of magnitude smaller than the single channel permeability of the wild type channel, suggesting that the open but inactivated potassium channel is a selective water channel.[1] Saparov, S. M., and P. Pohl. 2004. Beyond the diffusion limit: Water flow through the empty bacterial potassium channel. Proc. Natl. Acad. Sci. U. S. A. 101: 4805-4809.

2SCA-01 カリウムイオンチャネルKcsAのゲート開閉とリンクした脂質膜中での集合・離散ダイナミクス(2SCA 最新イオンチャネル1分子科学 : 素過程から疾患克服まで,シンポジウム,日本生物物理学会第51回年会(2013年度))

2SCA-01 カリウムイオンチャネルKcsAのゲート開閉とリンクした脂質膜中での集合・離散ダイナミクス(2SCA 最新イオンチャネル1分子科学 : 素過程から疾患克服まで,シンポジウム,日本生物物理学会第51回年会(2013年度))

The Sensing Sites for the Membrane Inner Lipids Regulate the Activation Gating of the KcsA Potassium Channel

The activity of the ion channels are influenced by membrane lipid compositions and specific lipid molecules are indispensable for maintaining channel activities. For the KcsA potassium channel, the presence of anionic phospholipids such as phosphatidylglycerol (PG) is prerequisite for the channel activity. Previously we demonstrated by means of single-channel current recordings in the asymmetric lipid bilayer that the PG molecule on the inner leaflet, rather than the outer leaflet, renders the KcsA channel highly active. The fluorescent method revealed that the helix-bundle gate is kept open in the PG liposome but not much in the liposomes made of neutral or cationic phospholipids. To elucidate the underlying mechanism of the interaction between anionic lipids on the inner leaflet and the activation gate, charge-neutralizing mutations to positively charged residues were introduced. Several amino acid residues lying at the inner boundary of the membrane were found to be sensitive to the PG effect on the gating. Mechanism underlying lipid-mediated regulation of the activation gating of the KcsA channel will be discussed.

Discovery and Characterization of a Novel Toxin from Dendroaspis Angusticeps, Named TX7335, with an Activating Effect on the Potassium Channel KscA

Due to their important role in essential physiological processes such as the propagation of the nerve signal or regulation of the heartbeat, potassium channels are common targets for animal toxins. These toxins provide valuable tools for the study of ion channel function and have potential as lead compounds for drug development. Most toxins affecting potassium channels act as pore blockers, thus inhibiting potassium flow. Using a direct pull-down toxin binding assay with immobilized channel (the bacterial potassium channel KcsA) and crude Dendroaspis angusticeps venom, we identified a novel toxin binder of KcsA, which we called Tx7335.

Single-channel electrophysiology of cell-free expressed ion channels by direct incorporation in lipid bilayers

Single-channel electrophysiology with lipid bilayer systems requires ion channel expression, purification from cell culture, and reconstitution in proteoliposomes for delivery to a planar bilayer. Here we demonstrate that single-channel current measurements of the potassium channels KcsA and hERGS5-S6 can be obtained by direct insertion in interdroplet lipid bilayers from microliters of a cell-free expression medium.

Activation Gating of KcsA: New Insights into Cooperativity and Energy Landscape from Essential Dynamics Simulations

Recent crystal structures of the bacterial potassium channel KcsA have shed light on the intermediate and open conformation of the channel (Cuello et al. 2010). Although crystal structures provide excellent insights into different conformations of proteins, they feature only snapshots of dynamical proteins. Therefore, the transition steps and the mechanisms of activation gating are still unknown. In order to elucidate the activation gating dynamics KcsA, we applied essential dynamics (ED) simulations. The successful simulation of activation gating allowed us to calculate the underlying energy landscape by umbrella sampling. The energy profile revealed three energy wells that are separated by two barriers. We identified structural rearrangements of F114 which correspond the first energy barrier. The change in rotameric state of F114 is crucial for initial pore opening. Regarding the second energy barrier, we observed large conformational changes of the TM2 helices which represent the main final activation gate opening. Cooperativity of activation gating was studied by applying our ED protocol to different number of subunits (SUs). Our simulations showed that the movement of a single SU is not sufficient to open the activation gate. But by moving three SUs by ED simulations, the activation gate opened to the same extent as in the four SUs ED simulation protocol. These finding is in line with fluorescence detection studies, which showed that the SUs act cooperatively during gating (Blunck et al. 2008).

Dynamics of Conformational Transitions during KcsA Gating from EPR and Solution NMR Experiments

Despite its apparent simplicity, the potassium channel KcsA possesses a relatively complex gating cycle. Previous studies show conformational changes at the inner gate with an increase in distance from the central axis of symmetry when activated. These changes, coupled to rearrangements at the selectivity filter, are hypothesized to underlie activation, inactivation and modal gating. Defining the dynamic properties of the conformational wave linking the inner gate and selectivity filter is a critical step in understanding the mechanism of gating and permeation in K+ channels.Here, we have used CW and DEER Electron Paramagnetic Resonance to measure the opening of the inner gate and NMR to investigate the dynamics of the backbone of KcsA in a pH dependent manner. KcsA G116C was spin-labeled and reconstituted into liposomes or DDM micelles for EPR studies. NMR studies used wild type KcsA Δ125 in micelles (from pH7 to pH3 in 0.5 unit increments) with 2H/15N labeling and a TROSY pulse sequence. Pilot experiments investigating dynamics used a CPMG pulse sequence at pH7 and pH3.The extent of conformational dynamics from the inner gate ranged from 10.0 A (closed, pH7) to 23.7 A (open, pH3) with pH activation. Distance changes were abrupt near the pKa of activation (pH 3.9). Importantly, an observed increase in the width of the population distribution, and hence dynamics, was also observed at this point. Preliminary NMR experiments confirm that this region possesses distinct chemical shifts from a closed versus an open, inactivated state.Ongoing experiments target the process of backbone dynamics at different regions of KcsA. New pulse sequence schemes and kinetic gating mutants of KcsA are being studied to isolate the multiple dynamics components that define KcsA modal gating.

Towards Detergent Free Solubilization of Membrane Proteins into Nanodiscs: A Biophysical Study on the Interaction between Styrene Maleic Acid (SMA) Copolymers and Synthetic Phospholipid Vesicles

Recently, it has been discovered that SMA copolymers are able to solubilize membrane proteins in the form of styrene maleic acid lipid particles (SMALPs) without the use of destabilizing detergents [1,2]. In order to understand this unique property of SMA we studied the solubilization of multilamellar vesicles (MLVs) of synthetic saturated phospholipids by SMA as function of acyl chain length and temperature. The rate of solublization was monitored by transmission experiments. It was found that SMA polymers are excellent phospholipid membrane solubilizers, that are able to solubilize below, at and above gel to crystalline liquid phase temperature, with most efficient solubilization close to and at the gel to liquid-crystalline phase transition temperature (Tm). The formation of ∼10 nm diameter SMALPs, also referred to as lipid-nanodisks was verified by negative stain-transmission electron microscopy (TEM) and dynamic light scattering (DLS). Comparison with the recombinant apolipoprotein MSP1D1 showed that SMA is a significantly more potent membrane solubilizer than MSP1D1. Based on these and other results we developed a model for the mode of action of SMA that will be presented here.Our study contributes to the fundamental knowledge about the molecular mode of action of SMA, which is essential to develop general methods to succesfully extract membrane proteins directly from their native environment in the form of lipid-nanodiscs. As proof of principle that this is indeed possible, we succeeded in purifying the potassium channel KcsA from the inner membrane of E-coli using the SMA technology. These results and some of its implications will be presented in an accompanying poster.1. Knowles et al., 2009, JACS, 131, 7484-7485.2. Orwick et al., 2012, Angew.Chem., 51,1-6

Amphipathic antenna of an inward rectifier K + channel responds to changes in the inner membrane leaflet

Membrane lipids modulate the function of membrane proteins. In the case of ion channels, they bias the gating equilibrium, although the underlying mechanism has remained elusive. Here we demonstrate that the N-terminal segment (M0) of the KcsA potassium channel mediates the effect of changes in the lipid milieu on channel gating. The M0 segment is a membrane-anchored amphipathic helix, bearing positively charged residues. In asymmetric membranes, the M0 helix senses the presence of negatively charged phospholipids on the inner leaflet. Upon gating, the M0 helix revolves around the axis of the helix on the membrane surface, inducing the positively charged residues to interact with the negative head groups of the lipids so as to stabilize the open conformation (i.e., the "roll-and-stabilize model"). The M0 helix is thus a charge-sensitive "antenna," capturing temporary changes in lipid composition in the fluidic membrane. This unique type of sensory device may be shared by various types of membrane proteins.

ATR-FTIR Spectroscopy Revealing the Different Vibrational Modes of the Selectivity Filter Interacting with K+ and Na+ in the Open and Collapsed Conformations of the KcsA Potassium Channel

The potassium channel is highly selective for K(+) over Na(+), and the selectivity filter binds multiple dehydrated K(+) ions upon permeation. Here, we applied attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy to extract ion-binding-induced signals of the KcsA potassium channel at neutral pH. Shifts in the peak of the amide-I signal towards lower vibrational frequencies were observed as K(+) was replaced with Na(+). These ion species-specific shifts deduced the selectivity filter as the source of the signal, which was supported by the spectra of a mutant for the selectivity filter (Y78F). The difference FTIR spectra between the solution containing various concentrations of K(+) and that containing pure Na(+) demonstrated two types of peak shifts of the amide-I vibration in response to the K(+) concentration. These signals represent the binding of K(+) ions to the different sites in the selectivity filter with different dissociation constants (KD = 9 or 18 mM).

Detergent-labile, supramolecular assemblies of KcsA: Relative abundance and interactions involved

In this work, we illustrate the ability of the prokaryotic potassium channel KcsA to assemble into a variety of supramolecular clusters of defined sizes containing the tetrameric KcsA as the repeating unit. Such clusters, particularly the larger ones, are markedly detergent-labile and thus, disassemble readily upon exposure to the detergents commonly used in protein purification or conventional electrophoresis analysis. This is a reversible process, as cluster re-assembly occurs upon detergent removal and without the need of added membrane lipids. Interestingly, the dimeric ensemble between two tetrameric KcsA molecules are quite resistant to detergent disassembly to individual KcsA tetramers and along with the latter, are likely the basic building blocks through which the larger clusters are organized.As to the proteins domains involved in clustering, we have observed disassembly of KcsA clusters by SDS-like alkyl sulfates. As these amphiphiles bind to inter-subunit, “non-annular” sites on the protein, these observations suggest that such sites also mediate channel–channel interactions leading to cluster assembly.

Characterizing the Fatty Acid Binding Site in the Cavity of Potassium Channel KcsA

We show that interactions of fatty acids with the central cavity of potassium channel KcsA can be characterized using the fluorescence probe 11-dansylaminoundecanoic acid (Dauda). The fluorescence emission spectrum of Dauda bound to KcsA in bilayers of dioleoylphosphatidylcholine contains three components, which can be attributed to KcsA-bound and lipid-bound Dauda together with unbound Dauda. The binding of Dauda to KcsA was characterized by a dissociation constant of 0.47 ± 0.10 μM with 0.94 ± 0.06 binding site per KcsA tetramer. Displacement of KcsA-bound Dauda by the tetrabutylammonium (TBA) ion confirmed that the Dauda binding site was in the central cavity of KcsA. Dissociation constants for a range of fatty acids were determined by displacement of Dauda: binding of fatty acids increased in strength with an increasing chain length from C14 to C20 but then decreased in strength from C20 to C22. Increasing the number of double bonds in the chain from one to four had little effect on binding, dissociation constants for oleic acid and arachidonic acid, for example, being 2.9 ± 0.2 and 3.0 ± 0.4 μM, respectively. Binding of TBA to KcsA was very slow, whereas binding of Dauda was fast, suggesting that TBA can enter the cavity only through an open channel whereas Dauda can bind to the closed channel, presumably entering the cavity via the lipid bilayer.

Protonation state of E71 in KcsA and its role for channel collapse and inactivation

The prototypical prokaryotic potassium channel KcsA alters its pore depending on the ambient potassium; at high potassium, it exists in a conductive form, and at low potassium, it collapses into a nonconductive structure with reduced ion occupancy. We present solid-state NMR studies of KcsA in which we test the hypothesis that an important channel-inactivation process, known as C-type inactivation, proceeds via a state similar to this collapsed state. We test this using an inactivation-resistant mutant E71A, and show that E71A is unable to collapse its pore at both low potassium and low pH, suggesting that the collapsed state is structurally similar to the inactivated state. We also show that E71A has a disordered selectivity filter. Using site-specific K(+) titrations, we detect a local change at E71 that is coupled to channel collapse at low K(+). To gain more insight into this change, we site specifically measure the chemical shift tensors of the side-chain carboxyls of E71 and its hydrogen bond partner D80, and use the tensors to assign protonation states to E71 and D80 at high K(+) and neutral pH. Our measurements show that E71 is protonated at pH 7.5 and must have an unusually perturbed pK(a) (> 7.5) suggesting that the change at E71 is a structural rearrangement rather than a protonation event. The results offer new mechanistic insights into why the widely used mutant KcsA-E71A does not inactivate and establish the ambient K(+) level as a means to populate the inactivated state of KcsA in a controlled way.

Effects of Lipid Structure on the State of Aggregation of Potassium Channel KcsA

The state of aggregation of potassium channel KcsA was determined as a function of lipid:protein molar ratio in bilayer membranes of the zwitterionic lipid phosphatidylcholine (PC) and of the anionic lipid phosphatidylglycerol (PG). EPR (electron paramagnetic resonance) with spin-labeled phospholipids was used to determine the number of motionally restricted lipids per KcsA tetramer. Unexpectedly, this number decreased with a decreasing lipid:KcsA tetramer molar ratio in the range of 88:1 to 30:1, consistent with sharing of annular lipid shells and KcsA-KcsA contact at high mole fractions of protein. Fluorescence quenching experiments with brominated phospholipids showed a decrease in fluorescence quenching at low lipid:KcsA tetramer mole ratios, also consistent with KcsA-KcsA contact at high mole fractions of protein. The effects of low mole ratios of lipid seen in EPR and fluorescence quenching experiments were more marked in bilayers of PC than in bilayers of PG, suggesting stronger association of PG than PC with KcsA. This was confirmed by direct measurement of lipid association constants using spin-labeled phospholipids, showing higher association constants for all anionic lipids than for PC. The results show that the probability of contacts between KcsA tetramers will be very low at lipid:protein molar ratios that are typical of native biological membranes.

Multiple Binding Sites for Fatty Acids on the Potassium Channel KcsA

Interactions of fatty acids with the potassium channel KcsA were studied using Trp fluorescence quenching and electron paramagnetic resonance (EPR) techniques. The brominated analogue of oleic acid was shown to bind to annular sites on KcsA and to the nonannular sites at each protein–protein interface in the homotetrameric structure with binding constants relative to dioleoylphosphatidylcholine of 0.67 ± 0.04 and 0.87 ± 0.08, respectively. Mutation of the two Arg residues close to the nonannular binding sites had no effect on fatty acid binding. EPR studies with a spin-labeled analogue of stearic acid detected a high-affinity binding site for the fatty acid with strong immobilization. Fluorescence quenching studies with the spin-labeled analogue showed that the binding site detected in the EPR experiments could not be one of the annular or nonannular binding sites. Instead, it is proposed that the EPR studies detect binding to the central hydrophobic cavity of the channel, with a binding constant in the range of ∼0.1–1 μM.