KvAP Channel Research Articles

Preferential electrostatic interactions of phosphatidic acid with arginines.

Phosphatidic acid (PA) is an anionic lipid that preferentially interacts with proteins in a diverse set of cellular processes such as transport, apoptosis, and neurotransmission. One such interaction is that of the PA lipids with the proteins of voltage-sensitive ion channels. In comparison to several other similarly charged anionic lipids, PA lipids exhibit much stronger interactions. Intrigued and motivated by this finding, we sought out to gain deeper understanding into the electrostatic interactions of anionic lipids with charged proteins. Using the voltage sensor domain (VSD) of the KvAP channel as a model system, we performed long-timescale atomistic simulations to analyze the interactions of POPA, POPG, and POPI lipids with arginines (ARGs). Our simulations reveal two mechanisms. First, POPA is able to interact not only with surface ARGs but is able to snorkel and interact with a buried arginine. POPG and POPI lipids on the other hand show weak interactions even with both the surface and buried ARGs. Second, deprotonated POPA with -2 charge is able to break the salt-bridge connection between VSD protein segments and establish its own electrostatic bond with the ARG. Based on these findings, we propose a headgroup size hypothesis for preferential solvation of proteins by charged lipids. These findings may be valuable in understanding how PA lipids could be modulating kinematics of transmembrane proteins in cellular membranes.

Protein reconstitution into giant unilamellar vesicles from native nanodiscs.

The reconstitution of purified membrane proteins into lipid bilayers represents a tedious challenge as it relies mainly on empirically optimized purification protocols tailored to the target protein, including laborious detergent selection. The situation is even more complex when the protein's function or structural stability depends on its lipid environment. Using nanodisc-forming amphiphilic polymers helps circumnavigate these problems by allowing the extracted protein to stay in its native lipid environment. Yet, thus far, nanodiscs are of limited use for transport studies due to the problematic protein transfer to vesicles or planar bilayers. Here we exploited the recently introduced glycopolymer Glyco-DIBMA to purify integral membrane proteins such as the voltage-gated potassium ion channel KvAP, the voltage-gated sodium ion channel NavMs, the bacterial translocon complex SecYEG, the eukaryotic serotonin-gated cation channel 5Ht3A, as well as the phospholipid translocating scramblase TMEM16. We used fluorescence correlation spectroscopy to quantify the abundance of the labeled proteins in nanodiscs and optimize protein transfer into giant unilamellar vesicles (GUVs). We thus could achieve protein densities of roughly one copy per diffraction-limited focal area or ∼5400 copies in a GUV with a diameter of ∼20 µm. Such protein density is sufficient for visualizing the reconstituted proteins in vesicular membranes by conventional fluorescence microscopy. The proteins retain functionality, as our GUV influx and efflux studies demonstrated. This project was supported by a grant from the FWF (Austrian Science Fund): P34826.

Exploring voltage sensing in protein-translocating channel SecYEG.

The conserved protein translocation channel SecYEG facilitates protein transport across as well as integration into the membrane. To maintain ion gradients across the membrane, essential for many cellular processes, it is of utmost importance that the Sec channel remains impermeable to these ions despite its bulky substrates. It has been shown via electrophysiological experiments that physiological levels of transmembrane voltage allow the channel to stay impermeable. Yet, the substrate-bound SecYEG becomes leaky once the absolute value of the transmembrane voltage drops below a certain threshold [1,2]. We proposed that the dipole moment of helix 2b is responsible for voltage sensing and that other complex parts must also contribute [3]. Molecular dynamics simulations suggested the involvement of the 5th helix dipole. Replacing S188 on its cytoplasmatic terminus allowed reverting its electrostatic contribution. Here, we measured the kinetics of voltage-gated closings of the thus mutated bacterial SecYEG translocon, reconstituted into planar bilayers. The translocon openings were triggered by the binding of ribosome nascent chain complexes harboring the voltage sensor domain of the voltage-gated potassium channel KvAP. S188E exhibited an increased voltage sensitivity, whereas S188R partially de-sensitized the translocon. Our results confirm the earlier hypothesis, in which the dipole moment of multiple TM-helices serves as SecYEG's voltage sensor. Support of the Austrian Science Fund (FWF), grant P34584 to PP, is gratefully acknowledged.

Investigation of lipid/protein interactions in trifluoroethanol-water mixtures proposes the strategy for the refolding of helical transmembrane domains.

Membrane proteins are one of the keystone objects in molecular biology, but their structural studies often require an extensive search for an appropriate membrane-like environment and an efficient refolding protocol for a recombinant protein. Isotropic bicelles are a convenient membrane mimetic used in structural studies of membrane proteins. Helical membrane domains are often transferred into bicelles from trifluoroethanol-water mixtures. However, the protocols for such a refolding are empirical and the process itself is still not understood in detail. In search of the optimal refolding approaches for helical membrane proteins, we studied here how membrane proteins, lipids, and detergents interact with each other at various trifluoroethanol-water ratios. Using high-resolution NMR spectroscopy and dynamic light scattering, we determined the key states of the listed compounds in the trifluoroethanol/water mixture, found the factors that could be critical for the efficiency of refolding, and proposed several most optimal protocols. These protocols were developed on the transmembrane domain of neurotrophin receptor TrkA and tested on two model helical membrane domains-transmembrane of Toll-like receptor TLR9 and voltage-sensing domain of a potassium channel KvAP.

A mutation in the S6 segment of the KvAP channel changes the secondary structure and alters ion channel activity in a lipid bilayer membrane.

The peptide segment S6 is known to form the inner lining of the voltage-gated K+ channel KvAP (potassium channel of archaea-bacterium, Aeropyrum pernix). In our previous work, it has been demonstrated that S6 itself can form an ion channel on a bilayer lipid membrane (BLM). In the present work, the role of a specific amino acid sequence 'LIG' in determining the secondary structure of S6 has been investigated. For this purpose, 22-residue synthetic peptides named S6-Wild (S6W) and S6-Mutant (S6M) were used. Sequences of these peptides are similar except that the two amino acids isoleucine and glycine of the wild peptide interchanged in the mutant peptide. Channel forming capabilities of both the peptides were checked electro-physiologically on BLM composed of DPhPC and cholesterol. Bilayer electrophysiological experiments showed that the conductance of S6M is higher than that of S6W. Significant differences in the current versus voltage (I-V) plot, open probability, and gating characteristics were observed. Interestingly, two sub-types of channels, S6M Type 1 and Type 2, were identified in S6M differing in conductances and open probability patterns. Circular dichroism (CD) spectroscopy indicated that the secondary structures of the two peptides are different in phosphatidyl choline/asolectin liposomes and 1% SDS detergent. Reduced helicity of S6M was also noticed in membrane mimetic liposomes and 1% SDS detergent micelles. These results are interpreted in view of the difference in hydrophobicity of the two amino acids, isoleucine and glycine. It is concluded that the 'LIG' stretch regulates the structure and pore-forming ability of the S6 peptide.

Spontaneous local membrane curvature induced by transmembrane proteins

The (local) curvature of cellular membranes acts as a driving force for the targeting of membrane-associated proteins to specific membrane domains, as well as a sorting mechanism for transmembrane proteins, e.g., by accumulation in regions of matching spontaneous curvature. The latter measure was previously experimentally employed to study the curvature induced by the potassium channel KvAP and by aquaporin AQP0. However, the direction of the reported spontaneous curvature levels as well as the molecular driving forces governing the membrane curvature induced by these integral transmembrane proteins could not be addressed experimentally.Here, using both coarse-grained and atomistic molecular dynamics (MD) simulations, we report induced spontaneous curvature values for the homologous potassium channel Kv 1.2/2.1 Chimera (KvChim) and AQP0 embedded in unrestrained lipid bicelles that are in very good agreement with experiment. Importantly, the direction of curvature could be directly assessed from our simulations: KvChim induces a strong positive membrane curvature (≈0.036 nm−1) whereas AQP0 causes a comparably small negative curvature (≈−0.019 nm−1).Analyses of protein-lipid interactions within the bicelle revealed that the potassium channel shapes the surrounding membrane via structural determinants. Differences in shape of the protein-lipid interface of the voltage-gating domains between the extracellular and cytosolic membrane leaflets induce membrane stress and thereby promote a protein-proximal membrane curvature. In contrast, the water pore AQP0 displayed a high structural stability and an only faint effect on the surrounding membrane environment that is connected to its wedge-like shape.

The Voltage-Dependent Deactivation of the KvAP Channel Involves the Breakage of Its S4 Helix.

Voltage-gated potassium channels (Kv) allow ion permeation upon changes of the membrane electrostatic potential (Vm). Each subunit of these tetrameric channels is composed of six transmembrane helices, of which the anti-parallel helix bundle S1-S4 constitutes the voltage-sensor domain (VSD) and S5-S6 forms the pore domain. Here, using 82 molecular dynamics (MD) simulations involving 266 replicated VSDs, we report novel responses of the archaebacterial potassium channel KvAP to membrane polarization. We show that the S4 α-helix, which is straight in the experimental crystal structure solved under depolarized conditions (Vm ∼ 0), breaks into two segments when the cell membrane is hyperpolarized (Vm << 0), and reversibly forms a single straight helix following depolarization (Vm = 0). The outermost segment of S4 translates along the normal to the membrane, bringing new perspective to previously paradoxical accessibility experiments that were initially thought to imply the displacement of the whole VSD across the membrane. The novel model is applied through steered and unbiased MD simulations to the recently solved whole structure of KvAP. The simulations show that the resting state involves a re-orientation of the S5 α-helix by ∼ 5–6 degrees in respect to the normal of the bilayer, which could result in the constriction and closure of the selectivity filter. Our findings support the idea that the breakage of S4 under (hyper)polarization is a general feature of Kv channels with a non-swapped topology.

Cryo-EM structure of the KvAP channel reveals a non-domain-swapped voltage sensor topology.

Conductance in voltage-gated ion channels is regulated by membrane voltage through structural domains known as voltage sensors. A single structural class of voltage sensor domain exists, but two different modes of voltage sensor attachment to the pore occur in nature: domain-swapped and non-domain-swapped. Since the more thoroughly studied Kv1-7, Nav and Cav channels have domain-swapped voltage sensors, much less is known about non-domain-swapped voltage-gated ion channels. In this paper, using cryo-EM, we show that KvAP from Aeropyrum pernix has non-domain-swapped voltage sensors as well as other unusual features. The new structure, together with previous functional data, suggests that KvAP and the Shaker channel, to which KvAP is most often compared, probably undergo rather different voltage-dependent conformational changes when they open.

A Shared Mechanism for the Folding of Voltage-Gated K+ Channels

In this study, we probe the folding of KvAP, a voltage-gated K+ (Kv) channel. The KvAP channel, though of archaebacterial origin, is structurally and functionally similar to eukaryotic Kv channels. An advantage of the KvAP channel is that it can be folded in vitro from an extensively unfolded state and the folding can be controlled by temperature. We utilize these properties of the KvAP channel to separately study the membrane insertion and the tetramerization stages during folding. We use two quantitative assays: a Cys PEGylation assay to monitor membrane insertion and a cross-linking assay to monitor tetramerization. We show that during folding the KvAP polypeptide is rapidly inserted into the lipid bilayer with a "native-like" topology. We identify a segment at the C-terminus that is important for multimerization of the KvAP channel. We show that this C-terminal domain forms a dimer, which raises the possibility that the tetramerization of the KvAP channel proceeds through a dimer of dimers pathway. Our studies show that the in vitro folding of the KvAP channel mirrors aspects of the cellular assembly pathway for voltage-gated K+ channels and therefore suggest that evolutionarily distinct Kv channels share a common folding pathway. The pathway for the folding and assembly of a Kv channel is of central importance as defects in this pathway have been implicated in the etiology of several disease states. Our studies indicate that the KvAP channel provides an experimentally tractable system for elucidating the folding mechanism of Kv channels.

Effects of Low Intensity Focused Ultrasound on Liposomes Containing Channel proteins

The ability to reversibly and non-invasively modulate region-specific brain activity in vivo suggests Low Intensity Focused Ultrasound (LIFU) as potential therapeutics for neurological dysfunctions such as epilepsy and Parkinson’s disease. While in vivo studies provide evidence of the bioeffects of LIFU on neuronal activity, they merely hint at potential mechanisms but do not fully explain how this technology achieves these effects. One potential hypothesis is that LIFU produces local membrane depolarization by mechanically perturbing the neuronal cell membrane, or activating channels or other proteins embedded in the membrane. Proteins that sense mechanical perturbations of the membrane, such as those gated by membrane tension, are prime candidates for activating in response to LIFU and thus leading to the neurological responses that have been measured. Here we use the bacterial mechanosensitive channel MscL, which has been purified and reconstituted in liposomes, to determine how LIFU may affect the activation of this membrane-tension gated channel. Two bacterial voltage-gated channels, KvAP and NaK2K F92A channels were also studied. Surprisingly, the results suggest that ultrasound modulation and membrane perturbation does not induce channel gating, but rather induces pore formation at the membrane protein-lipid interface. However, in vesicles with high MscL mechanosensitive channel concentrations, apparent decreases in pore formation are observed, suggesting that this membrane-tension-sensitive protein may serve to increase the elasticity of the membrane, presumably because of expansion of the channel in the plane of the membrane independent of channel gating.

Combining Electrical and Optical Measurements to Reveal the Structure-Function Relationship of Voltage-Gated Potassium Channels

Although the structure of the voltage-gated potassium channel KvAP has been resolved more than a decade ago, there is still controversy about their precise gating mechanism. The discrepancies among proposed models result in part from differences in experimental techniques: electrical measurements of channels in lipid membranes to study their function versus determination of the structure of channels purified using detergents. To be able to combine functional and structural measurements, we developed a second-generation membrane interferometer in which both electrophysiology measurements and high-resolution fluorescence microscopy imaging can be performed on ion channels reconstituted in a lipid membrane, a much advanced design over the original (Ganesan et al., Proc. Natl. Acad. Sci. 106, 2008). In the membrane interferometer, a freestanding bilayer is formed over a micropore that is positioned above a reflective mirror. The mirror allows the use of Fluorescence Interference Contrast (FLIC) and Variable Incidence Angle-FLIC (VIA-FLIC) microscopy, two surface characterization techniques that precisely locate the height of fluorescent objects relative to the mirror with nanometer resolution. KvAP overexpressed in E. coli is detergent-free extracted using polymer nanodiscs. We show that functional KvAP can be reconstituted in black lipid membranes and freestanding lipid bilayers on the membrane interferometer. No differences in gating properties were found between KvAP extracted by polymer nanodiscs versus detergents. We label the channels with a fluorescent tag at different positions at the S3 or S4 strand. Our goal is to correlate the change in position of the S3 and S4 strands measured using FLIC microscopy with single-channel electrical measurements to reveal the structure-function relationship of KvAP. Progress towards this goal will be described.

Lipid-Dependent Gating of Kv Channels and Excitability Change of Cerebellar Purkinje Neurons in an NPC1 Model Mouse

Recent studies of Kv channels in different membrane systems with altered ratios between phospholipids and nonphospholipids support our lipid-dependent gating hypothesis that the nonphospholipids favor the conformational switch of the voltage sensor domains of a Kv channel to a resting state. To enable more precise control of lipid composition in an artificial membrane and avoid possible solvent contamination or phase separation of lipids in bilayer membranes, we constructed a bead-supported unilamellar membrane (bSUM) and used it to define a more quantitative relationship between nonphospholipid content and change in the gating property of a voltage-gated potassium channel. In the bSUMs, we were able to show for the first time that a small amount of cholesterol (∼10 molar %) in a phospholipid membrane in a fluidic phase exerts a strong inhibitory effect on a KvAP channel, suggesting that the Kv channels might be highly sensitive to change in membrane cholesterol content. To test this prediction in a physiological system where lipid metabolic defects cause significant neurological disorders, we analyzed the neuronal excitability in a mouse model for the Niemann-Pick disease type C, an NPC1-I1061T knockin mice. Systematic comparison of five different groups of cerebellar Purkinje neurons from both knockin mice and control animals revealed a significant decrease in the firing frequency of action potentials in the tonic-burst firing Purkinje neurons of the NPC1 mice, which appears to be consistent with the predicted cholesterol effects on Kv channels. Detailed studies of the gating properties in these Purkinje neurons are being conducted to understand the underlying mechanism. Our data support a potential connection among cholesterol content in cell membranes, cholesterol-dependent gating of voltage-gated K channels and change in neuronal excitability in CNS neurons.

The topogenic function of S4 promotes membrane insertion of the voltage-sensor domain in the KvAP channel.

Voltage-dependent K+ (KV) channels control K+ permeability in response to shifts in the membrane potential. Voltage sensing in KV channels is mediated by the positively charged transmembrane domain S4. The best-characterized KV channel, KvAP, lacks the distinct hydrophilic region corresponding to the S3-S4 extracellular loop that is found in other K+ channels. In the present study, we evaluated the topogenic properties of the transmembrane regions within the voltage-sensing domain in KvAP. S3 had low membrane insertion activity, whereas S4 possessed a unique type-I signal anchor (SA-I) function, which enabled it to insert into the membrane by itself. S4 was also found to function as a stop-transfer signal for retention in the membrane. The length and structural nature of the extracellular S3-S4 loop affected the membrane insertion of S3 and S4, suggesting that S3 membrane insertion was dependent on S4. Replacement of charged residues within the transmembrane regions with residues of opposite charge revealed that Asp72 in S2 and Glu93 in S3 contributed to membrane insertion of S3 and S4, and increased the stability of S4 in the membrane. These results indicate that the SA-I function of S4, unique among K+ channels studied to date, promotes the insertion of S3 into the membrane, and that the charged residues essential for voltage sensing contribute to the membrane-insertion of the voltage sensor domain in KvAP.

Molecular basis of the interaction between gating modifier spider toxins and the voltage sensor of voltage-gated ion channels.

Voltage-sensor domains (VSDs) are modular transmembrane domains of voltage-gated ion channels that respond to changes in membrane potential by undergoing conformational changes that are coupled to gating of the ion-conducting pore. Most spider-venom peptides function as gating modifiers by binding to the VSDs of voltage-gated channels and trapping them in a closed or open state. To understand the molecular basis underlying this mode of action, we used nuclear magnetic resonance to delineate the atomic details of the interaction between the VSD of the voltage-gated potassium channel KvAP and the spider-venom peptide VSTx1. Our data reveal that the toxin interacts with residues in an aqueous cleft formed between the extracellular S1-S2 and S3-S4 loops of the VSD whilst maintaining lipid interactions in the gaps formed between the S1-S4 and S2-S3 helices. The resulting network of interactions increases the energetic barrier to the conformational changes required for channel gating, and we propose that this is the mechanism by which gating modifier toxins inhibit voltage-gated ion channels.

Open channel current noise analysis of S6 peptides from KvAP channel on bilayer lipid membrane shows bimodal power law scaling

Open channel current noise in synthetic peptide S6 of KvAP channel was investigated in a voltage clamp experiment on bilayer lipid membrane (BLM). It was observed that the power spectral density (PSD) of the component frequencies follows power law with different slopes in different frequency ranges. In order to know the origin of the slopes PSD analysis was done with signal filtering. It was found that the first slope in the noise profile follows 1/f pattern which exists at lower frequencies and has high amplitude current noise, while the second slope corresponds to 1/f2−3 pattern which exists at higher frequencies with low amplitude current noise. In addition, white noise was observed at very large frequencies. It was concluded that the plausible reason for the multiple power-law scaling is the existence of different modes of non-equilibrium ion transport through the S6 channel.

KvAP Gating States Probed by Electron Spin-Echo Envelope Modulation (ESEEM) Spectroscopy

During a gating event, the voltage gated potassium channel KvAP undergoes a conformational change in the voltage sensor (helices 1-4), causing the opening of the pore domain (helices 5-6). Throughout this conformational change, the extracellular portion of the pore remains relatively immobile. However, the environment surrounding this part of the pore domain may be altered through changes in lipid packing or tertiary contacts within the channel. In this study, model membranes were used to stabilize KvAP in different gating states and the environment surrounding the pore were probed with ESEEM both in the presence and absence of the voltage sensing domain.

Structural basis for the inhibition of voltage-dependent K+ channel by gating modifier toxin

Voltage-dependent K+ (Kv) channels play crucial roles in nerve and muscle action potentials. Voltage-sensing domains (VSDs) of Kv channels sense changes in the transmembrane potential, regulating the K+-permeability across the membrane. Gating modifier toxins, which have been used for the functional analyses of Kv channels, inhibit Kv channels by binding to VSD. However, the structural basis for the inhibition remains elusive. Here, fluorescence and NMR analyses of the interaction between VSD derived from KvAP channel and its gating modifier toxin, VSTx1, indicate that VSTx1 recognizes VSD under depolarized condition. We identified the VSD-binding residues of VSTx1 and their proximal residues of VSD by the cross-saturation (CS) and amino acid selective CS experiments, which enabled to build a docking model of the complex. These results provide structural basis for the specific binding and inhibition of Kv channels by gating modifier toxins.

Curvature-dependent protein-lipid bilayer interaction and cell mechanosensitivity.

Cells respond to applied external forces through different mechanosensitive processes, with many of them based on the interaction between membrane-embedded proteins and their lipid environments. This interaction can depend on membrane curvature at the location of such proteins. Here we elucidate the general characteristics of a macroscopically defined protein-lipid bilayer interaction based on a mismatch between the shape of the protein surface and the surrounding membrane curvature. It is shown how the parameters of this interaction are related to the experimentally determined distribution of membrane-embedded proteins between highly curved tubular and flat membrane regions of a giant phospholipid vesicle. The results obtained for such distribution of potassium channel KvAP are presented as an example. Possible participation of the curvature-dependent protein-lipid bilayer interaction in mechanosensitive processes is indicated.

Sorting of integral membrane proteins mediated by curvature-dependent protein-lipid bilayer interaction.

Cell membrane proteins, both bound and integral, are known to preferentially accumulate at membrane locations with curvatures favorable to their shape. This is mainly due to the curvature dependent interaction between membrane proteins and their lipid environment. Here, we analyze the effects of the protein-lipid bilayer interaction energy due to mismatch between the protein shape and the principal curvatures of the surrounding bilayer. The role of different macroscopic parameters that define the interaction energy term is elucidated in relation to recent experiment in which the lateral distribution of a membrane embedded protein potassium channel KvAP is measured on a giant unilamellar lipid vesicle (reservoir) and a narrow tubular extension - a tether - kept at constant length. The dependence of the sorting ratio, defined as the ratio between the areal density of the protein on the tether and on the vesicle, on the inverse tether radius is influenced by the strength of the interaction, the intrinsic shape of the membrane embedded protein, and its abundance in the reservoir. It is described how the values of these constants can be extracted from experiments. The intrinsic principal curvatures of a protein are related to the tether radius at which the sorting ratio attains its maximum value. The estimate of the principal intrinsic curvature of the protein KvAP, obtained by comparing the experimental and theoretical sorting behavior, is consistent with the available information on its structure.

Protein Spatial Distribution Depends on Membrane Curvature

In cells, membranes are highly bent in many places in order to fulfill different functions. This is the case of membrane trafficking, which is accompanied by the formation of tubes or vesicles with diameter below 100 nm that transport proteins and lipids throughout the cell. Moreover, during vesicle budding, curvature at the neck becomes even higher before final scission. Structures at the cell periphery, such as lamellipodia’ edge or filopodia used for migration and environment sensing, also exhibit high membrane curvatures. Many peripheral or integral proteins have an intrinsic shape that produces membrane bending or can deform membrane upon binding. In membrane physics, this property is described by a phenomenological parameter, the protein spontaneous curvature Cp, which represents the capability of the protein to produce membrane spontaneous bending. It is expected that proteins that deform membranes can reciprocally be enriched in curved areas. We have addressed the question of the role of membrane curvature in protein sorting both experimentally and theoretically. We have tested the sorting hypothesis using in vitro membrane system (GUV) and different proteins with positive Cp (BAR-domain), negative Cp (I-BAR-domain) and with integral proteins reconstituted in the GUVs (a potassium channel KvAP). Membrane nanotubes with a controlled diameter (15-500 nm) are pulled out of the GUV with optical tweezers; the relative protein sorting is measured as a function of curvature (inverse of tube radius) from fluorescence measurements with a confocal microscope. We show that our mechanical model based on spontaneous curvature induction is in good agreement with these experiments. All together, this demonstrates that membrane shape is an important parameter that might drive the lateral distribution proteins in membranes, independently of more specific sorting mechanisms.