S1 S4 Research Articles

Molecular Dynamics Investigation of the ω-Current in the Kv1.2 Voltage Sensor Domains

Voltage sensor domains (VSD) are transmembrane proteins that respond to changes in membrane voltage and modulate the activity of ion channels, enzymes, or in the case of proton channels allow permeation of protons across the cell membrane. VSDs consist of four transmembrane segments, S1–S4, forming an antiparallel helical bundle. The S4 segment contains several positively charged residues, mainly arginines, located at every third position along the helix. In the voltage-gated Shaker K+ channel, the mutation of the first arginine of S4 to a smaller uncharged amino acid allows permeation of cations through the VSD. These currents, known as ω-currents, pass through the VSD and are distinct from K+ currents passing through the main ion conduction pore. Here we report molecular dynamics simulations of the ω-current in the resting-state conformation for Kv1.2 and for four of its mutants. The four tested mutants exhibit various degrees of conductivity for K+ and Cl− ions, with a slight selectivity for K+ over Cl−. Analysis of the ion permeation pathway, in the case of a highly conductive mutant, reveals a negatively charged constriction region near the center of the membrane that might act as a selectivity filter to prevent permeation of anions through the pore. The residues R1 in S4 and E1 in S2 are located at the narrowest region of the ω-pore for the resting state conformation of the VSD, in agreement with experiments showing that the largest increase in current is produced by the double mutation E1D and R1S.

The core domain as the force sensor of the yeast mechanosensitive TRP channel

Stretch-activated conductances are commonly encountered in careful electric recordings. Those of known proteins (TRP, MscL, MscS, K2p, Kv, etc.) all share a core, which houses the ion pathway and the gate, but no recognizable force-sensing domain. Like animal TRPs, the yeast TRPY1 is polymodal, activated by stretch force, Ca2+, etc. To test whether its S5–S6 core senses the stretch force, we tried to uncouple it from the peripheral domains by strategic peptide insertions to block the covalent core–periphery interactions. Insertion of long unstructured peptides should distort, if not disrupt, protein structures that transmit force. Such insertions between S6 and the C-terminal tail largely removed Ca2+ activation, showing their effectiveness. However, such insertions as well as those between S5 and the N-terminal region, which includes S1–S4, did not significantly alter mechanosensitivity. Even insertions at both locations flanking the S5–S6 core did not much alter mechanosensitivity. Tryptophan scanning mutations in S5 were also constructed to perturb possible noncovalent core–periphery contacts. The testable tryptophan mutations also have little or no effects on mechanosensitivity. Boltzmann fits of the wild-type force–response curves agree with a structural homology model for a stretch-induced core expansion of ∼2 nm2 upon opening. We hypothesize that membrane tension pulls on S5–S6, expanding the core and opening the TRPY1 gate. The core being the major force sensor offers the simplest, though not the only, explanation of why so many channels of disparate designs are mechanically sensitive. Compared with the bacterial MscL, TRPY1 is much less sensitive to force, befitting a polymodal channel that relies on multiple stimuli.

Commentary: The response process validity of a script concordance test item

While the Script Concordance Test (SCT) item format has generated considerable researchthat has reflected positively on its reliability (Charlin et al. 1998) and ease of construction(Fournier et al. 2008), a recent review suggests the response process validity of the SCT isweakly supported (Lubarsky et al. 2011). The Gagnon et al. (2011) paper published in thisissue of Advances in Health Sciences Education suggests that the validity of an SCT scoreas a measure of clinical reasoning hinges on the degree to which aggregate scoring conveysa concordance between experts’ and examinees’ scripts. However, the assumption that anexaminee’s solution process reflects their clinical scripts has not been confirmed. Since thevalidity of an SCT score derives from the congruence between current score interpretationsand a logical analysis of the task presented by the item, a sound validity argument mustconsider what the SCT task is actually asking the examinee to do. While it is obviouslyimportant to consider the underlying cognitive processes used to respond to the item, aneven more fundamental prerequisite is to logically decompose the SCT task into itscomponent parts. By breaking down the SCT problem into its logical steps, it will be easierto understand the cognitive demands of an SCT item.Figure 1 presents a flow diagram of the four task stimuli (S1–S4) and the three cal-culation steps (P1, P2, {P2–P1}) required by an SCT-based question. Since Gagnon et al.(2011) already present an excellent example and description of the SCT item format, hereit will be sufficient to simply note that an SCT first presents a clinical scenario (S1) to theexaminee and then suggests a hypothesis (S2). Next, the item presents a single discrete newpiece of information (S3), and then asks how that new clinical information impacts theprobability of the suggested hypothesis (S4). As Fig. 1 indicates, after both S2 and S3 theexaminee is implicitly asked to make a probabilistic calculation (P1 & P2 respectively) inorder to answer the formal question (S4). The first probability (P1) represents the likeli-hood of the hypothesis (S2) given the clinical scenario (S1). The second probability (P2) isthe likelihood of the hypothesis (S2) given both the scenario (S1) and the new piece of

Elucidation of the Molecular Basis of Selective Recognition Uncovers the Interaction Site for the Core Domain of Scorpion α-Toxins on Sodium Channels

Neurotoxin receptor site-3 at voltage-gated Na(+) channels is recognized by various peptide toxin inhibitors of channel inactivation. Despite extensive studies of the effects of these toxins, their mode of interaction with the channel remained to be described at the molecular level. To identify channel constituents that interact with the toxins, we exploited the opposing preferences of LqhαIT and Lqh2 scorpion α-toxins for insect and mammalian brain Na(+) channels. Construction of the DIV/S1-S2, DIV/S3-S4, DI/S5-SS1, and DI/SS2-S6 external loops of the rat brain rNa(v)1.2a channel (highly sensitive to Lqh2) in the background of the Drosophila DmNa(v)1 channel (highly sensitive to LqhαIT), and examination of toxin activity on the channel chimera expressed in Xenopus oocytes revealed a substantial decrease in LqhαIT effect, whereas Lqh2 was as effective as at rNa(v)1.2a. Further substitutions of individual loops and specific residues followed by examination of gain or loss in Lqh2 and LqhαIT activities highlighted the importance of DI/S5-S6 (pore module) and the C-terminal region of DIV/S3 (gating module) of rNa(v)1.2a for Lqh2 action and selectivity. In contrast, a single substitution of Glu-1613 to Asp at DIV/S3-S4 converted rNa(v)1.2a to high sensitivity toward LqhαIT. Comparison of depolarization-driven dissociation of Lqh2 and mutant derivatives off their binding site at rNa(v)1.2a mutant channels has suggested that the toxin core domain interacts with the gating module of DIV. These results constitute the first step in better understanding of the way scorpion α-toxins interact with voltage-gated Na(+)-channels at the molecular level.

Differential expression of allene oxide synthase (AOS), and jasmonate relationship with ethylene biosynthesis in seed and mesocarp of developing peach fruit

To better understand the physiological role of jasmonates (JAs) during fruit development and ripening, and relationships with ethylene, the time course of transcript accumulation of the key enzyme in JA biosynthesis, allene oxide synthase (AOS), was analyzed during S1–S4 fruit developmental stages, until ripening, in the mesocarp and seed of peach ( Prunus persica var. laevis Gray), cv. ‘Stark Red Gold’. In the mesocarp, transcript amounts of PpAOS1 were the highest in S1–S2, then decreased to almost undetectable levels at the transition S3/S4, to rise slightly at harvest. In parallel, JA concentration was measured in the mesocarp during development; maximum jasmonic acid accumulation was observed in S1, the lowest levels in S2 and S3 and a new rise in S4. In the seed, an AOS1 transcript was detected whose accumulation differed in the embryo as compared to cotyledons during seed growth. The reciprocal JA/ethylene relationships were investigated in fruit at different physiological stages by exogenously supplying methyl jasmonate (MJ). The effects of treatments with 0.88 mM MJ on transcript levels of PpAOS1 and ethylene biosynthetic genes PpACS1, PpACO1 and PpACO2 were evaluated in the mesocarp and seed of detached nectarines at an early (S1) and late (S4) developmental stage. Results show that JA biosynthesis appears to be developmentally regulated in seed and mesocarp of nectarine fruit and that the response to MJ depends upon the fruit physiological stage.

Common Molecular Determinants of Tarantula Huwentoxin-IV Inhibition of Na+ Channel Voltage Sensors in Domains II and IV

The voltage sensors of domains II and IV of sodium channels are important determinants of activation and inactivation, respectively. Animal toxins that alter electrophysiological excitability of muscles and neurons often modify sodium channel activation by selectively interacting with domain II and inactivation by selectively interacting with domain IV. This suggests that there may be substantial differences between the toxin-binding sites in these two important domains. Here we explore the ability of the tarantula huwentoxin-IV (HWTX-IV) to inhibit the activity of the domain II and IV voltage sensors. HWTX-IV is specific for domain II, and we identify five residues in the S1-S2 (Glu-753) and S3-S4 (Glu-811, Leu-814, Asp-816, and Glu-818) regions of domain II that are crucial for inhibition of activation by HWTX-IV. These data indicate that a single residue in the S3-S4 linker (Glu-818 in hNav1.7) is crucial for allowing HWTX-IV to interact with the other key residues and trap the voltage sensor in the closed configuration. Mutagenesis analysis indicates that the five corresponding residues in domain IV are all critical for endowing HWTX-IV with the ability to inhibit fast inactivation. Our data suggest that the toxin-binding motif in domain II is conserved in domain IV. Increasing our understanding of the molecular determinants of toxin interactions with voltage-gated sodium channels may permit development of enhanced isoform-specific voltage-gating modifiers.

Profiling the Structural Determinants for the Selectivity of Representative Factor-Xa and Thrombin Inhibitors Using Combined Ligand-Based and Structure-Based Approaches

The current study deciphers the combined ligand- and structure-based computational insights to profile structural determinants for the selectivity of representative diverse classes of FXa-selective and thrombin-selective as well as dual FXa-thrombin high affinity inhibitors. The thrombin-exclusive insertion 60-loop (D-pocket) was observed to be one of the most notable recognition sites for the known thrombin-selective inhibitors. Based on the topological comparison of four common active-site pockets (S1-S4) of FXa and thrombin, the greater structural disparity was observed in the S4-pocket, which was more symmetrical (U-shaped) in FXa as compared to thrombin mainly due to the presence of L99 and I174 residues in latter in place of Y99 and F174 respectively in former protease. The S2 pocket forming partial roof at the entry of 12 Å deep S1-pocket, with two extended β-sheets running antiparallel to each other by undergoing U-turn (∼180̊), has two conserved glycine residues forming H-bonds with the bound ligand for governing ligand binding affinity. The docking, scoring, and binding pose comparison of the representative high-affinity and selective inhibitors into the active sites of FXa and thrombin revealed critical residues (S214, Y99, W60D) mediating selectivity through direct- and long-range electrostatic interactions. Interestingly, most of the thrombin-selective inhibitors attained S-shaped conformation in thrombin, while FXa-selective inhibitors attained L-shaped conformations in FXa. The role of residue at 99th position of FXa and thrombin toward governing protease selectivity was further substantiated using molecular dynamics simulations on the wild-type and mutated Y99L FXa bound to thrombin-selective inhibitor 2. Furthermore, predictive CoMFA (FXa q² = 0.814; thrombin q² = 0.667) and CoMSIA (FXa q² = 0.807; thrombin q² = 0.624) models were developed and validated (FXa r²(test) = 0.823; thrombin r(2)(test) = 0.816) to feature molecular determinants of ligand binding affinity using the docking-based conformational alignments (DBCA) of 141 (88(train)+53(test)) and 39 (27(train)+11(test)) nonamidine class of potent FXa (0.004 ≤ K(i) (nM) ≤ 4700) and thrombin (0.001 ≤ K(i) (nM) ≤ 940) inhibitors, respectively. Interestingly, the ligand-based insights well corroborated with the structure-based insights in terms of the role of steric, electrostatic, and hydrophobic parameters for governing the selectivity for the two proteases. The new computational insights presented in this study are expected to be valuable for understanding and designing potent and selective antithrombotic agents.

A Synthetic S6 Segment Derived from KvAP Channel Self-assembles, Permeabilizes Lipid Vesicles, and Exhibits Ion Channel Activity in Bilayer Lipid Membrane

KvAP is a voltage-gated tetrameric K(+) channel with six transmembrane (S1-S6) segments in each monomer from the archaeon Aeropyrum pernix. The objective of the present investigation was to understand the plausible role of the S6 segment, which has been proposed to form the inner lining of the pore, in the membrane assembly and functional properties of KvAP channel. For this purpose, a 22-residue peptide, corresponding to the S6 transmembrane segment of KvAP (amino acids 218-239), and a scrambled peptide (S6-SCR) with rearrangement of only hydrophobic amino acids but without changing its composition were synthesized and characterized structurally and functionally. Although both peptides bound to the negatively charged phosphatidylcholine/phosphatidylglycerol model membrane with comparable affinity, significant differences were observed between these peptides in their localization, self-assembly, and aggregation properties onto this membrane. S6-SCR also exhibited reduced helical structures in SDS micelles and phosphatidylcholine/phosphatidylglycerol lipid vesicles as compared with the S6 peptide. Furthermore, the S6 peptide showed significant membrane-permeabilizing capability as evidenced by the release of calcein from the calcein-entrapped lipid vesicles, whereas S6-SCR showed much weaker efficacy. Interestingly, although the S6 peptide showed ion channel activity in the bilayer lipid membrane, despite having the same amino acid composition, S6-SCR was significantly inactive. The results demonstrated sequence-specific structural and functional properties of the S6 wild type peptide. The selected S6 segment is probably an important structural element that could play an important role in the membrane interaction, membrane assembly, and functional property of the KvAP channel.

A Novel Current Pathway Parallel to the Central Pore in a Mutant Voltage-gated Potassium Channel

Voltage-gated potassium channels are proteins composed of four subunits consisting of six membrane-spanning segments S1-S6, with S4 as the voltage sensor. The region between S5 and S6 forms the potassium-selective ion-conducting central α-pore. Recent studies showed that mutations in the voltage sensor of the Shaker channel could disclose another ion permeation pathway through the voltage-sensing domain (S1-S4) of the channel, the ω-pore. In our studies we used the voltage-gated hKv1.3 channel, and the insertion of a cysteine at position V388C (Shaker position 438) generated a current through the α-pore in high potassium outside and an inward current at hyperpolarizing potentials carried by different cations like Na(+), Li(+), Cs(+), and NH(4)(+). The observed inward current looked similar to the ω-current described for the R1C/S Shaker mutant channel and was not affected by some pore blockers like charybdotoxin and tetraethylammonium but was inhibited by a phenylalkylamine blocker (verapamil) that acts from the intracellular side. Therefore, we hypothesize that the hKv1.3_V388C mutation in the P-region generated a channel with two ion-conducting pathways. One, the α-pore allowing K(+) flux in the presence of K(+), and the second pathway, the σ-pore, functionally similar but physically distinct from the ω-pathway. The entry of this new pathway (σ-pore) is presumably located at the backside of Y395 (Shaker position 445), proceeds parallel to the α-pore in the S6-S6 interface gap, ending between S5 and S6 at the intracellular side of one α-subunit, and is blocked by verapamil.

Preparation and evaluation of the 1st international standard for the quantitation of HIV-2 RNA in plasma

An international standard for the quantitation of HIV-2 RNA in plasma samples was developed. A collaborative study involving 29 laboratories from 15 countries was carried out in order to evaluate HIV-2 RNA candidate materials for use with nucleic acid-based tests (NATs). Candidate reference standards consisted of duplicate copies of two HIV-2 genotype A viruses, HIV-2 CAM2 and HIV-2 ROD and were coded S1–S4. Each laboratory assayed all four candidates on at least three separate occasions and data were collated and analysed at NIBSC. Of the data sets returned the majority were from qualitative assays. All assays detected both candidate standards with the exception of one commercial assay, the Nuclisens Easy Q, which was designed primarily for HIV-1 detection which did not detect HIV-2 CAM2 but showed good detection of HIV-2 ROD. This highlighted possible cross reactivity with HIV-2 ROD with some NAT primer/probe combinations; as a result the HIV-2 CAM2 material was established as the 1st international standard for HIV-2 RNA with an assigned unitage of 1000 International Units (IU) per ampoule and is available upon request from the National Institute for Biological Standardisation and Control (NIBSC) ( www.nibsc.ac.uk).

Absorption and photovoltaic properties of organic solar cell sensitizers containing fluorene unit as conjunction bridge

A novel series of organic dyes containing a fluorene unit as the conjunction bridge (S1–S4) have been designed and synthesized for a potential application in dye-sensitized solar cells (DSSCs). Their absorption spectra, electrochemical and photovoltaic properties have been investigated in detail. The tuning of the HOMO and LUMO energy levels can be conveniently realized by alternating the donor moiety. As demonstrated, the indoline unit can exhibit a stronger electron donating ability, realizing a broader absorption spectrum when coated onto TiO2. The incorporation of octyl-substituted fluorene is highly beneficial to prevent close π–π aggregation, thus favorably suppressing charge recombination and intermolecular interaction. In both the HOMO and LUMO orbitals, the electron density located at the conjunction bridge (fluorene unit) is quite low, suggesting that the incorporated fluorene unit plays quite a small role in the electron delocalization between the donor and acceptor units due to the twist conformation. The overall conversion efficiencies (η) of DSSCs based on these dyes are in the range of 3.04 to 4.26% and highly dependent upon their donor moiety. Among these dyes, S3, which utilizes a methyl-substituted indoline moiety as an electron donor, has shown a maximum monochromatic incident photon to current efficiency (IPCE) of almost 90%, with a relatively high open circuit voltage (Voc) of 727 mV.

Introduction to genetic analysis workshop 17 summaries

Genetic Analysis Workshop 17 (GAW17) was held on October 13-16, 2010, in Boston, Massachusetts. The focus of GAW17 was on methods and challenges of the analysis of next-generation sequence data using a mini-exome data set. Two different study designs were simulated, unrelated individuals and large families, each with the same sample size. GAW17 was attended by 274 participants from 19 countries. The 166 contributions were organized into 15 discussion groups of 5-13 articles each. This supplement to Genetic Epidemiology provides further details on GAW17, and primary findings are highlighted in the individual group summary articles. In addition, the supplement includes two methodological articles (one on statistical approaches for collapsing methods and one on machine learning methods) to give an overview of the methods that many of the workshop participants used. We conclude the supplement with a discussion of critical aspects in the analysis of next-generation sequencing data.

A phylogenetic analysis ofPfeifferaand the reinstatement ofLymanbensoniaas an independently evolved lineage of epiphyticCactaceaewithin a new tribeLymanbensonieae

Abstract Korotkova N., Zabel L., Quandt D. & Barthlott W.: A phylogenetic analysis of Pfeiffera and the reinstatement of Lymanbensonia as an independently evolved lineage of epiphytic Cactaceae within a new tribe Lymanbensonieae. — Willdenowia 40: 151–172, S1–S4. — Online ISSN 1868–6397; © 2010 BGBM Berlin-Dahlem. doi:10.3372/wi.40.40201 (available via http://dx.doi.org/) Pfeiffera is a genus of epiphytic, terrestrial and epilithic cacti. Its acceptance, circumscription and closest relatives have been debated. In the context of a phylogenetic survey of epiphytic cacti, we have studied relationships in Pfeiffera, sampling eight of nine species and using sequence data from three group II introns (trnK, rpl16, trnG), four intergenic spacers (psbA-trnH, trnQ-rpsl6, rps3-rpl16, trnS-trnG) and the rapidly evolving gene matK of the plastid genome. Phylogenetic analyses revealed Pfeiffera to be polyphyletic, comprising two unrelated lineages, both highly supported. One clade includes the type species, P. ianthoth...

Modification of hERG1 channel gating by Cd2+.

Each of the four subunits in a voltage-gated potassium channel has a voltage sensor domain (VSD) that is formed by four transmembrane helical segments (S1–S4). In response to changes in membrane potential, intramembrane displacement of basic residues in S4 produces a gating current. As S4 moves through the membrane, its basic residues also form sequential electrostatic interactions with acidic residues in immobile regions of the S2 and S3 segments. Transition metal cations interact with these same acidic residues and modify channel gating. In human ether-á-go-go–related gene type 1 (hERG1) channels, Cd2+ coordinated by D456 and D460 in S2 and D509 in S3 induces a positive shift in the voltage dependence of activation of ionic currents. Here, we characterize the effects of Cd2+ on hERG1 gating currents in Xenopus oocytes using the cut-open Vaseline gap technique. Cd2+ shifted the half-point (V1/2) for the voltage dependence of the OFF gating charge–voltage (QOFF-V) relationship with an EC50 of 171 µM; at 0.3 mM, V1/2 was shifted by +50 mV. Cd2+ also induced an as of yet unrecognized small outward current (ICd-out) upon repolarization in a concentration- and voltage-dependent manner. We propose that Cd2+ and Arg residues in the S4 segment compete for interaction with acidic residues in S2 and S3 segments, and that the initial inward movement of S4 associated with membrane repolarization displaces Cd2+ in an outward direction to produce ICd-out. Co2+, Zn2+, and La3+ at concentrations that caused ∼+35-mV shifts in the QOFF-V relationship did not induce a current similar to ICd-out, suggesting that the binding site for these cations or their competition with basic residues in S4 differs from Cd2+. New Markov models of hERG1 channels were developed that describe gating currents as a noncooperative two-phase process of the VSD and can account for changes in these currents caused by extracellular Cd2+.

Coupling between Residues on S4 and S1 Defines the Voltage-Sensor Resting Conformation in NaChBac

The voltage sensor is a four-transmembrane helix bundle (S1–S4) that couples changes in membrane potential to conformational alterations in voltage-gated ion channels leading to pore opening and ion conductance. Although the structure of the voltage sensor in activated potassium channels is available, the conformation of the voltage sensor at rest is still obscure, limiting our understanding of the voltage-sensing mechanism. By employing a heterologously expressed Bacillus halodurans sodium channel (NaChBac), we defined constraints that affect the positioning and depolarization-induced outward motion of the S4 segment. We compared macroscopic currents mediated by NaChBac and mutants in which E43 on the S1 segment and the two outermost arginines (R1 and R2) on S4 were substituted. Neutralization of the negatively charged E43 (E43C) had a significant effect on channel gating. A double-mutant cycle analysis of E43 and R1 or R2 suggested changes in pairing during channel activation, implying that the interaction of E43 with R1 stabilizes the voltage sensor in its closed/available state, whereas interaction of E43 with R2 stabilizes the channel open/unavailable state. These constraints on S4 dynamics that define its stepwise movement upon channel activation and positioning at rest are novel, to the best of our knowledge, and compatible with the helical-screw and electrostatic models of S4 motion.

Amino acid composition, molecular weight distribution and antioxidant activity of protein hydrolysates of soy sauce lees

The proteins of soy sauce lees (SSLP) were hydrolysed by Alcalase in the presence of ultrasound or traditional water bath to obtain hydrolysates S2–S6. The analysis of protein content indicated that enzymatic hydrolysis could significantly improve the extraction efficiency of proteins. By determination of molecular weight distribution, >10 and 5–10 KDa fractions of native SSLP (S1) decreased during hydrolysis, whilst 3–5 KDa fraction increased. Gradual increases of free, total and antioxidant amino acids were observed for S1–S4, and the differences between S4 and S6 were slight. Tyrosine was the major free amino acid of S1–S6, whilst glutamic acid had the highest amount in total amino acid composition. S2–S6 showed stronger DPPH radical scavenging activities in a dose-dependent manner than S1. All the results suggested that ultrasound treatment showed an inhibition behaviour on the enzymatic hydrolysis of SSLP.

Identification of a Thiol/Disulfide Redox Switch in the Human BK Channel That Controls Its Affinity for Heme and CO

Heme is a required prosthetic group in many electron transfer proteins and redox enzymes. The human BK channel, which is a large-conductance Ca(2+) and voltage-activated K(+) channel, is involved in the hypoxic response in the carotid body. The BK channel has been shown to bind and undergo inhibition by heme and activation by CO. Furthermore, evidence suggests that human heme oxygenase-2 (HO2) acts as an oxygen sensor and CO donor that can form a protein complex with the BK channel. Here we describe a thiol/disulfide redox switch in the human BK channel and biochemical experiments of heme, CO, and HO2 binding to a 134-residue region within the cytoplasmic domain of the channel. This region, called the heme binding domain (HBD) forms a linker segment between two Ca(2+)-sensing domains (called RCK1 and RCK2) of the BK channel. The HBD includes a CXXCH motif in which histidine serves as the axial heme ligand and the two cysteine residues can form a reversible thiol/disulfide redox switch that regulates affinity of the HBD for heme. The reduced dithiol state binds heme (K(d) = 210 nm) 14-fold more tightly than the oxidized disulfide state. Furthermore, the HBD is shown to tightly bind CO (K(d) = 50 nm) with the Cys residues in the CXXCH motif regulating affinity of the HBD for CO. This HBD is also shown to interact with heme oxygenase-2. We propose that the thiol/disulfide switch in the HBD is a mechanism by which activity of the BK channel can respond quickly and reversibly to changes in the redox state of the cell, especially as it switches between hypoxic and normoxic conditions.

A Twist on Potassium Channel Gating

The selectivity filter of potassium channels, the narrowest segment of the pore, permits only potassium ions to diffuse through the pore. Clarke et al. (2010) now present 11 structures of an inwardly rectifying potassium channel, providing evidence that the selectivity filter functions in channel gating and conformational changes in the cytoplasmic domains correlate with pore opening.

Single or double? Think zinc!

Single or double? Think zinc!

State-dependent electrostatic interactions of S4 arginines with E1 in S2 during Kv7.1 activation

The voltage-sensing domain of voltage-gated channels is comprised of four transmembrane helices (S1–S4), with conserved positively charged residues in S4 moving across the membrane in response to changes in transmembrane voltage. Although it has been shown that positive charges in S4 interact with negative countercharges in S2 and S3 to facilitate protein maturation, how these electrostatic interactions participate in channel gating remains unclear. We studied a mutation in Kv7.1 (also known as KCNQ1 or KvLQT1) channels associated with long QT syndrome (E1K in S2) and found that reversal of the charge at E1 eliminates macroscopic current without inhibiting protein trafficking to the membrane. Pairing E1R with individual charge reversal mutations of arginines in S4 (R1–R4) can restore current, demonstrating that R1–R4 interact with E1. After mutating E1 to cysteine, we probed E1C with charged methanethiosulfonate (MTS) reagents. MTS reagents could not modify E1C in the absence of KCNE1. With KCNE1, (2-sulfonatoethyl) MTS (MTSES)− could modify E1C, but [2-(trimethylammonium)ethyl] MTS (MTSET)+ could not, confirming the presence of a positively charged environment around E1C that allows approach by MTSES− but repels MTSET+. We could change the local electrostatic environment of E1C by making charge reversal and/or neutralization mutations of R1 and R4, such that MTSET+ modified these constructs depending on activation states of the voltage sensor. Our results confirm the interaction between E1 and the fourth arginine in S4 (R4) predicted from open-state crystal structures of Kv channels and reveal an E1–R1 interaction in the resting state. Thus, E1 engages in electrostatic interactions with arginines in S4 sequentially during the gating movement of S4. These electrostatic interactions contribute energetically to voltage-dependent gating and are important in setting the limits for S4 movement.