How Does KCNE1 Regulate the Kv7.1 Potassium Channel? Model-Structure, Mutations, and Dynamics of the Kv7.1-KCNE1 Complex
2012; 20 (8): 1343-1352
Targeting the voltage sensor of Kv7.2 voltage-gated K+ channels with a new gating-modifier
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2010; 107 (35): 15637-15642
The voltage-gated potassium channel Kv7.1 and its auxiliary subunit KCNE1 are expressed in the heart and give rise to the major repolarization current. The interaction of Kv7.1 with the single transmembrane helix of KCNE1 considerably slows channel activation and deactivation, raises single-channel conductance, and prevents slow voltage-dependent inactivation. We built a Kv7.1-KCNE1 model-structure. The model-structure agrees with previous disulfide mapping studies and enables us to derive molecular interpretations of electrophysiological recordings that we obtained for two KCNE1 mutations. An elastic network analysis of Kv7.1 fluctuations in the presence and absence of KCNE1 suggests a mechanistic perspective on the known effects of KCNE1 on Kv7.1 function: slow deactivation is attributed to the low mobility of the voltage-sensor domains upon KCNE1 binding, abolishment of voltage-dependent inactivation could result from decreased fluctuations in the external vestibule, and amalgamation of the fluctuations in the pore region is associated with enhanced ion conductivity.
View details for DOI 10.1016/j.str.2012.05.016
View details for Web of Science ID 000307419200010
View details for PubMedID 22771213
Monte Carlo simulations of peptide-membrane interactions with the MCPep web server.
Nucleic acids research
2012; 40 (Web Server issue): W358-63
The pore and gate regions of voltage-gated cation channels have been often targeted with drugs acting as channel modulators. In contrast, the voltage-sensing domain (VSD) was practically not exploited for therapeutic purposes, although it is the target of various toxins. We recently designed unique diphenylamine carboxylates that are powerful Kv7.2 voltage-gated K(+) channel openers or blockers. Here we show that a unique Kv7.2 channel opener, NH29, acts as a nontoxin gating modifier. NH29 increases Kv7.2 currents, thereby producing a hyperpolarizing shift of the activation curve and slowing both activation and deactivation kinetics. In neurons, the opener depresses evoked spike discharges. NH29 dampens hippocampal glutamate and GABA release, thereby inhibiting excitatory and inhibitory postsynaptic currents. Mutagenesis and modeling data suggest that in Kv7.2, NH29 docks to the external groove formed by the interface of helices S1, S2, and S4 in a way that stabilizes the interaction between two conserved charged residues in S2 and S4, known to interact electrostatically, in the open state of Kv channels. Results indicate that NH29 may operate via a voltage-sensor trapping mechanism similar to that suggested for scorpion and sea-anemone toxins. Reflecting the promiscuous nature of the VSD, NH29 is also a potent blocker of TRPV1 channels, a feature similar to that of tarantula toxins. Our data provide a structural framework for designing unique gating-modifiers targeted to the VSD of voltage-gated cation channels and used for the treatment of hyperexcitability disorders.
View details for DOI 10.1073/pnas.0911294107
View details for Web of Science ID 000281468500062
View details for PubMedID 20713704
Membrane integration of a mitochondrial signal-anchored protein does not require additional proteinaceous factors
2012; 442: 381-389
The MCPep server (http://bental.tau.ac.il/MCPep/) is designed for non-experts wishing to perform Monte Carlo (MC) simulations of helical peptides in association with lipid membranes. MCPep is a web implementation of a previously developed MC simulation model. The model has been tested on a variety of peptides and protein fragments. The simulations successfully reproduced available empirical data and provided new molecular insights, such as the preferred locations of peptides in the membrane and the contribution of individual amino acids to membrane association. MCPep simulates the peptide in the aqueous phase and membrane environments, both described implicitly. In the former, the peptide is subjected solely to internal conformational changes, and in the latter, each MC cycle includes additional external rigid body rotational and translational motions to allow the peptide to change its location in the membrane. The server can explore the interaction of helical peptides of any amino-acid composition with membranes of various lipid compositions. Given the peptide's sequence or structure and the natural width and surface charge of the membrane, MCPep reports the main determinants of peptide-membrane interactions, e.g. average location and orientation in the membrane, free energy of membrane association and the peptide's helical content. Snapshots of example simulations are also provided.
View details for DOI 10.1093/nar/gks577
View details for PubMedID 22695797
W-Band pulse EPR distance measurements in peptides using Gd3+-dipicolinic acid derivatives as spin labels
PHYSICAL CHEMISTRY CHEMICAL PHYSICS
2011; 13 (22): 10771-10780
The MOM (mitochondrial outer membrane) contains SA (signal-anchored) proteins that bear at their N-terminus a single hydrophobic segment that serves as both a mitochondrial targeting signal and an anchor at the membrane. These proteins, like the vast majority of mitochondrial proteins, are encoded in the nucleus and have to be imported into the organelle. Currently, the mechanisms by which they are targeted to and inserted into the OM (outer membrane) are unclear. To shed light on these issues, we employed a recombinant version of the SA protein OM45 and a synthetic peptide corresponding to its signal-anchor segment. Both forms are associated with isolated mitochondria independently of cytosolic factors. Interaction with mitochondria was diminished when a mutated form of the signal-anchor was employed. We demonstrate that the signal-anchor peptide acquires an ?-helical structure in a lipid environment and adopted a TM (transmembrane) topology within artificial lipid bilayers. Moreover, the peptide's affinity to artificial membranes with OM-like lipid composition was much higher than that of membranes with ER (endoplasmic reticulum)-like lipid composition. Collectively, our results suggest that SA proteins are specifically inserted into the MOM by a process that is not dependent on additional proteins, but is rather facilitated by the distinct lipid composition of this membrane.
View details for DOI 10.1042/BJ20111363
View details for Web of Science ID 000301038400014
View details for PubMedID 22142226
Membrane Interactions of Novicidin, a Novel Antimicrobial Peptide: Phosphatidylglycerol Promotes Bilayer Insertion
JOURNAL OF PHYSICAL CHEMISTRY B
2010; 114 (34): 11053-11060
We present high field DEER (double electron-electron resonance) distance measurements using Gd(3+) (S = 7/2) spin labels for probing peptides' conformations in solution. The motivation for using Gd(3+) spin labels as an alternative for the standard nitroxide spin labels is the sensitivity improvement they offer because of their very intense EPR signal at high magnetic fields. Gd(3+) was coordinated by dipicolinic acid derivative (4MMDPA) tags that were covalently attached to two cysteine thiol groups. Cysteines were introduced in positions 15 and 27 of the peptide melittin and then two types of spin labeled melittins were prepared, one labeled with two nitroxide spin labels and the other with two 4MMDPA-Gd(3+) labels. Both types were subjected to W-band (95 GHz, 3.5 T) DEER measurements. For the Gd(3+) labeled peptide we explored the effect of the solution molar ratio of Gd(3+) and the labeled peptide, the temperature, and the maximum dipolar evolution time T on the DEER modulation depth. We found that the optimization of the [Gd(3+)]/[Tag] ratio is crucial because excess Gd(3+) masked the DEER effect and too little Gd(3+) resulted in the formation of Gd(3+)-tag(2) complexes, generating peptide dimers. In addition, we observed that the DEER modulation depth is sensitive to spectral diffusion processes even at Gd(3+) concentrations as low as 0.2 mM and therefore experimental conditions should be chosen to minimize it as it decreases the DEER effect. Finally, the distance between the two Gd(3+) ions, 3.4 nm, was found to be longer by 1.2 nm than the distance between the two nitroxides. The origin and implications of this difference are discussed.
View details for DOI 10.1039/c1cp00011j
View details for Web of Science ID 000290994900041
View details for PubMedID 21552622
Interaction of an Antimicrobial Peptide with Membranes: Experiments and Simulations with NKCS
JOURNAL OF PHYSICAL CHEMISTRY B
2010; 114 (12): 4230-4237
Novicidin is an antimicrobial peptide derived from ovispirin, a cationic peptide which originated from the ovine cathelicidin SMAP-29. Novicidin, however, has been designed to minimize the cytotoxic properties of SMAP-29 and ovisipirin toward achieving potential therapeutic applications. We present an analysis of membrane interactions and lipid bilayer penetration of novicidin, using an array of biophysical techniques and biomimetic membrane assemblies, complemented by Monte Carlo (MC) simulations. The data indicate that novicidin interacts minimally with zwitterionic bilayers, accounting for its low hemolytic activity. Negatively charged phosphatidylglycerol, on the other hand, plays a significant role in initiating membrane binding of novicidin, and promotes peptide insertion into the interface between the lipid headgroups and the acyl chains. The significant insertion into bilayers containing negative phospholipids might explain the enhanced antibacterial properties of novicidin. Overall, this study highlights two distinct outcomes for membrane interactions of novicidin, and points to a combination between electrostatic attraction to the lipid/water interface and penetration into the subsurface lipid headgroups region as important determinants for the biological activity of novicidin.
View details for DOI 10.1021/jp1052248
View details for Web of Science ID 000281128700016
View details for PubMedID 20690652
A Combined Pulse EPR and Monte Carlo Simulation Study Provides Molecular Insight on Peptide-Membrane Interactions
JOURNAL OF PHYSICAL CHEMISTRY B
2009; 113 (38): 12687-12695
We used Monte Carlo simulations and biophysical measurements to study the interaction of NKCS, a derivative of the antimicrobial peptide NK-2, with a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) membrane. The simulations showed that NKCS adsorbed on the membrane surface and the dominant conformation featured two amphipathic helices connected by a hinge region. We designed two mutants in the hinge to investigate the interplay between helicity and membrane affinity. Simulations with a Leu-to-Pro substitution showed that the helicity and membrane affinity of the mutant (NKCS-[LP]) decreased. Two Ala residues were added to NKCS to produce a sequence that is compatible with a continuous amphipathic helix structure (NKCS-[AA]), and the simulations showed that the mutant adsorbed on the membrane surface with a particularly high affinity. The circular dichroism spectra of the three peptides also showed that NKCS-[LP] is the least helical and NKCS-[AA] is the most. However, the activity of the peptides, determined in terms of their antimicrobial potency and influence on the temperature of the transition of the lipid to hexagonal phase, displayed a complex behavior: NKCS-[LP] was the least potent and had the smallest influence on the transition temperature, and NKCS was the most potent and had the largest effect on the temperature.
View details for DOI 10.1021/jp909154y
View details for Web of Science ID 000275855800017
View details for PubMedID 20201501
Effects of purified endogenous inhibitor of the Na+/Ca2+ exchanger on ouabain-induced arrhythmias in the atria and ventricle strips of guinea pig
EUROPEAN JOURNAL OF PHARMACOLOGY
2006; 553 (1-3): 196-204
We present a new approach to obtain details on the distribution and average structure and locations of membrane-associated peptides. The approach combines (i) pulse double electron-electron resonance (DEER) to determine intramolecular distances between residues in spin labeled peptides, (ii) electron spin echo envelope modulation (ESEEM) experiments to measure water exposure and the direct interaction of spin labeled peptides with deuterium nuclei on the phospholipid molecules, and (iii) Monte Carlo (MC) simulations to derive the peptide-membrane populations, energetics, and average conformation of the native peptide and mutants mimicking the spin labeling. To demonstrate the approach, we investigated the membrane-bound and solution state of the well-known antimicrobial peptide melittin, used as a model system. A good agreement was obtained between the experimental results and the MC simulations regarding the distribution of distances between the labeled amino acids, the side chain mobility, and the peptide's orientation. A good agreement in the extent of membrane penetration of amino acids in the peptide core was obtained as well, but the EPR data reported a somewhat deeper membrane penetration of the termini compared to the simulations. Overall, melittin adsorbed on the membrane surface, in a monomeric state, as an amphipatic helix with its hydrophobic residues in the hydrocarbon region of the membrane and its charged and polar residues in the lipid headgroup region.
View details for DOI 10.1021/jp905129b
View details for Web of Science ID 000269747300011
View details for PubMedID 19725508
Previous studies demonstrated that the purified endogenous inhibitor (NCX(IF)) of the cardiac Na(+)/Ca(2+) exchanger (NCX1) has the capacity to modulate cardiac muscle contractility. Here, we tested the effects of purified NCX(IF) on arrhythmias induced by ouabain in the atria and ventricle strips of guinea pig. For the sake of comparison NCX(IF) was compared to lidocaine and KB-R7943. In the ventricle strip, NCX(IF) ( approximately 10 U/ml) results in rapid, complete and stable inhibition of ouabain-induced arrhythmias (the inhibition of arrhythmia is not followed by revival of irregular contractions). Under similar experimental conditions the atria strips require somewhat higher doses of NCX(IF) (25-50 U/ml) for complete suppression of arrhythmia. In the atria strip, NCX(IF) (10-25 U/ml) increases the threshold dose (1 microM) of ouabain for arrhythmia onset 2.2+/-0.5-fold (n=5, p<0.05) as well as prolongs the lag-phase for arrhythmia appearance 4.0+/-0.5-fold (n=5, p<0.01). The lag period for arrhythmia onset was also lengthened (2.0+/-0.4-fold) by NCX(IF) in the ventricle strips (n=6, p<0.002). At low frequency of pacing (1 Hz), all three tested substances, lidocaine, KB-R7943, and NCX(IF) can effectively suppress the ouabain-induced arrhythmia. However, at higher frequency (2 Hz), lidocaine is ineffective in suppressing arrhythmia, whereas KB-R7943 becomes pro-arrhythmic. In contrast to reference drugs, NCX(IF) retains its anti-arrhythmic capacity at high frequencies, either in the atria (n=6, p<0.01) or ventricle (n=5, p<0.05) strips. In conclusion, NCX(IF) results in rapid, effective and stable suppression of arrhythmia both in the atria and ventricle preparations under conditions at which the reference drugs become ineffective.
View details for DOI 10.1016/j.ejphar.2006.09.014
View details for Web of Science ID 000242772600027
View details for PubMedID 17078946