State-dependent Interactions Research Articles

Effects of novel subtype selective M-current activators on spinal reflexes in vitro: Comparison with retigabine

The activation of Kv7 channels and the resulting M-current is a powerful mechanism to control neuronal excitability with profound effects in pain pathways. Despite the lack of specific data on the expression and role of these channels in nociceptive processing, much attention has been paid at exploring their potential value as targets for analgesia. Here we have characterized the spinal actions of two novel subunit selective Kv7 activators, ICA-069673 and ML213, and compared their effects to those of retigabine that acts with similar affinity on all neuronal Kv7 channels.Spinal reflexes were recorded in a mouse spinal cord in vitro preparation to allow the testing of the compounds on native spinal pathways at known concentrations. As retigabine, novel compounds depressed spinal segmental transmission with particularly strong effects on wind up, showing an adequate pro-analgesic profile. ML213 presented the highest potency. In contrast to retigabine, the effects of ICA-069673 and ML213 were blocked by XE-991 even at the highest concentrations used, suggesting specific effect on Kv7 channels. In addition, the effects of ICA-069673 on repetitive stimulation are consistent with a mode of action involving state or activity dependent interaction with the channels.Compared to retigabine, novel Kv7 openers maintain strong depressant effects on spinal nociceptive transmission showing an improved specificity on Kv7 channels. The differential effects obtained with these Kv7 openers may indicate the existence of several Kv7 conformations in spinal circuits.

Two-state Bogoliubov theory of a molecular Bose gas

We present an analytic Bogoliubov description of a BEC of polar molecules trapped in a quasi-2D geometry and interacting via internal state-dependent dipole-dipole interactions. We derive the mean-field ground-state energy functional, and we derive analytic expressions for the dispersion relations, Bogoliubov amplitudes, and dynamic structure factors. This method can be applied to any homogeneous, two-component system with linear coupling, and direct, momentum-dependent interactions. The properties of the mean-field ground state, including polarization and stability, are investigated, and we identify three distinct instabilities: a density-wave rotonization that occurs when the gas is fully polarized, a spin-wave rotonization that occurs near zero polarization, and a mixed instability at intermediate fields. These instabilities are clarified by means of the real-space density-density correlation functions, which characterize the spontaneous fluctuations of the ground state, and the momentum-space structure factors, which characterize the response of the system to external perturbations. We find that the gas is susceptible to both density-wave and spin-wave response in the polarized limit but only a spin-wave response in the zero-polarization limit. These results are relevant for experiments with rigid rotor molecules such as RbCs, $\Lambda$-doublet molecules such as ThO that have an anomalously small zero-field splitting, and doublet-$\Sigma$ molecules such as SrF where two low-lying opposite-parity states can be tuned to zero splitting by an external magnetic field.

Thermodynamic property prediction for high molecular weight molecules based on their constituent family

A method for predicting the critical temperature of high molecular weight molecules is proposed. The method is based on a modification of the functional form of the Joback method and the use of constituent functional group trends. To demonstrate the predictive capability of the method several pure fluids and their binary mixtures with CO2 are studied. In particular, diglyme, triglyme and tetraglyme whose molecular weights range from 134 to 222, are shown to reveal excellent predictions between 3 and 9% deviation for saturated liquid densities. Application to vapor–liquid phase equilibria for CO2-glyme systems exhibit 4–8% deviation in saturated liquid densities and 1% for vapor densities. For the system of oleic acid + CO2, pure component parameters from the method allow the correlation of the vapor pressure of oleic acid and the solubility of H2 in oleic acid with a BWR type of equation of state and temperature dependent interaction parameters. Solubility of CO2 in triolein is also well correlated. The critical temperature of a polyalkylene glycol with a molecular weight of 1100 is predicted to be 842 K, whereas the prediction by of the Joback method predicts 8541 K, and so cannot be used in equation of state calculations. The pure component properties predicted with the new functional form of the Joback method also allow reliable correlation of CO2-polyalkylene glycol vapor–liquid equilibria.

Activity-dependent depression of neuronal sodium channels by the general anaesthetic isoflurane

The mechanisms by which volatile anaesthetics such as isoflurane alter neuronal function are poorly understood, in particular their presynaptic mechanisms. Presynaptic voltage-gated sodium channels (Na(v)) have been implicated as a target for anaesthetic inhibition of neurotransmitter release. We hypothesize that state-dependent interactions of isoflurane with Na(v) lead to increased inhibition of Na(+) current (I(Na)) during periods of high-frequency neuronal activity. The electrophysiological effects of isoflurane, at concentrations equivalent to those used clinically, were measured on recombinant brain-type Na(v)1.2 expressed in ND7/23 neuroblastoma cells and on endogenous Na(v) in isolated rat neurohypophysial nerve terminals. Rate constants determined from experiments on the recombinant channel were used in a simple model of Na(v) gating. At resting membrane potentials, isoflurane depressed peak I(Na) and shifted steady-state inactivation in a hyperpolarizing direction. After membrane depolarization, isoflurane accelerated entry (τ(control)=0.36 [0.03] ms compared with τ(isoflurane)=0.33 [0.05] ms, P<0.05) and slowed recovery (τ(control)=6.9 [1.1] ms compared with τ(isoflurane)=9.0 [1.9] ms, P<0.005) from apparent fast inactivation, resulting in enhanced depression of I(Na), during high-frequency stimulation of both recombinant and endogenous nerve terminal Na(v). A simple model of Na(v) gating involving stabilisation of fast inactivation, accounts for this novel form of activity-dependent block. Isoflurane stabilises the fast-inactivated state of neuronal Na(v) leading to greater depression of I(Na) during high-frequency stimulation, consistent with enhanced inhibition of fast firing neurones.

Phosphorylation state-dependent interaction between AKAP7δ/γ and phospholamban increases phospholamban phosphorylation

Changes in heart rate and contractility in response to sympathetic stimulation occur via activation of cAMP dependent protein kinase A (PKA), leading to phosphorylation of numerous substrates that alter Ca2+ cycling. Phosphorylation of these substrates is coordinated by A-kinase anchoring proteins (AKAPs), which recruit PKA to specific substrates [1]. Phosphorylation of the PKA substrate phospholamban (PLB) is a critical determinant of Ca2+ re-entry into the sarcoplasmic reticulum and is coordinated by AKAP7δ/γ [2,3]. Here, we further these findings by showing that phosphorylation of PLB requires interaction with AKAP7δ/γ and that this interaction occurs only when PLB is unphosphorylated. Additionally, we find that two mutants of PLB (R9C and Δ14), which are associated with dilated cardiomyopathy in humans, prevent association with AKAP7δ/γ and display reduced phosphorylation in vitro. This finding implicates the AKAP7δ/γ–PLB interaction in the pathology of the disease phenotype. Further exploration of the AKAP7δ/γ–PLB association demonstrated a phosphorylation state-dependence of the interaction. Computational modeling revealed that this mode of interaction allows for small amounts of AKAP and PKA (100–200nM) to regulate the phosphorylation of large quantities of PLB (50μM). Our results confirm that AKAP7γ/δ binding to PLB is important for phosphorylation of PLB, and describe a novel phosphorylation state-dependent binding mechanism that explains how phosphorylation of highly abundant PKA substrates can be regulated by AKAPs present at ~100–200 fold lower concentrations.

The Mechanism of KCNE1 Modulation of KCNQ1 Channels

The IKs current controls action potential duration in the heart, and abnormal function of this current causes cardiac arrhythmias. The IKs current is carried by the voltage activated KCNQ1 potassium channel associated with KCNE1 β-subunits. 18 years of study have shown that KCNE1 drastically modulates every characteristic of KCNQ1, such that it would appear as if KCNQ1 and KCNQ1+ KCNE1 were completely unrelated channels. However, no coherent mechanism has been provided that can explain all these drastic changes, which are essential for the physiological role of IKs. Here we show that KCNE1 alters the state-dependent interactions (coupling) between the voltage-sensing and pore-gate domains of KCNQ1 and that this sole mechanism is sufficient to explain all these changes. Contrary to conventional belief that the voltage-sensing domain must reach the fully-activated state before promoting pore-opening, we found that the KCNQ1 channels can open when the voltage-sensing domain is at intermediate and fully-activated states. Importantly, the intermediate-open and activated-open channels differ in voltage-dependence, ion-permeation, pharmacology and dependence on PIP2, a cofactor for coupling between the voltage-sensing and pore-gate domains. By changing the coupling, KCNE1 prevents the intermediate-open state and changes the properties of the activated-open state, thereby bringing about the characteristics of the IKs current. These results indicate that, during voltage-dependent ion channel gating, every-state of the voltage-sensing domain along its activation pathway is coupled to the conformation of the pore domain through a unique set of protein-protein and protein-lipid interactions. These interactions determine both the open-probability and the open-pore properties.

Domain-domain interactions determine the gating, permeation, pharmacology, and subunit modulation of the IKs ion channel.

Voltage-gated ion channels generate electrical currents that control muscle contraction, encode neuronal information, and trigger hormonal release. Tissue-specific expression of accessory (β) subunits causes these channels to generate currents with distinct properties. In the heart, KCNQ1 voltage-gated potassium channels coassemble with KCNE1 β-subunits to generate the IKs current (Barhanin et al., 1996; Sanguinetti et al., 1996), an important current for maintenance of stable heart rhythms. KCNE1 significantly modulates the gating, permeation, and pharmacology of KCNQ1 (Wrobel et al., 2012; Sun et al., 2012; Abbott, 2014). These changes are essential for the physiological role of IKs (Silva and Rudy, 2005); however, after 18 years of study, no coherent mechanism explaining how KCNE1 affects KCNQ1 has emerged. Here we provide evidence of such a mechanism, whereby, KCNE1 alters the state-dependent interactions that functionally couple the voltage-sensing domains (VSDs) to the pore.

Collective behavior of mobile agents with state-dependent interactions

In this paper, we develop a novel self-propelled particle model to describe the emergent behavior of a group of mobile agents. Each agent coordinates with its neighbors through a local force accounting for velocity alignment and collision avoidance. The interactions between agents are governed by path loss influence and state-dependent rules, which results in topology changes as well as discontinuities in the local forces. By using differential inclusion technique and algebraic graph theory, we show that collective behavior emerges while collisions between agents can be avoided, if the interaction topology is jointly connected. A trade-off between the path loss influence and connectivity condition to guarantee the collective behavior is discovered and discussed. Numerical simulations are given to validate the theoretical results.

Small-scale environmental variation influences whether coral-dwelling fish promote or impede coral growth.

Mutualistic symbioses are ubiquitous in nature and facilitate high biodiversity and productivity of ecosystems by enhancing the efficiency of energy and nutrient use within ecological communities. For example, small groups of fish that inhabit coral colonies in reef ecosystems potentially enhance coral growth through defense from coral predators, aeration of coral tissue and nutrient provisioning. This study examines whether the prevalence and consequences of fish-coral interactions vary among sites with different environmental conditions in a coral reef lagoon, using the humbug damselfish Dascyllus aruanus and its preferred coral host Pocillopora damicornis as a study system. Using a field experiment, we tested the site-specific effects of D. aruanus on coral growth, and show that the cost-benefit ratio for corals hosting fish varies with local environmental variation. Results of this study also demonstrate that fish prefer to inhabit coral colonies with particular branch-spacing characteristics, and that the local abundance of D. aruanus influences the proportion of coral colonies within a site that are occupied by fish rather than increasing the number of fish per colony. We also show that corals consistently benefit from hosting D. aruanus via defense from predation by corallivorous butterflyfish, regardless of local environmental conditions. These findings highlight the need to consider the potential for multiple scale- and state-dependent interaction effects when examining the ecology of fish-coral associations. We suggest that fluctuating cost-benefit ratios for species interactions may contribute to the maintenance of different colony phenotypes within coral populations.

State-dependent blocker interactions with the CFTR chloride channel: implications for gating the pore

Chloride permeation through the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel is subject to voltage-dependent open-channel block by a diverse range of cytoplasmic anions. However, in most cases the ability of these blocking substances to influence the pore opening and closing process has not been reported. In the present work, patch clamp recording was used to investigate the state-dependent block of CFTR by cytoplasmic Pt(NO2)4(2-) ions. Two major effects of Pt(NO2)4(2-) were identified. First, this anion caused fast, voltage-dependent block of open channels, leading to an apparent decrease in single-channel current amplitude. Secondly, Pt(NO2)4(2-) also decreased channel open probability due to an increase in interburst closed times. Interestingly, mutations in the pore that weakened (K95Q) or strengthened (I344K, V345K) interactions with Pt(NO2)4(2-) altered blocker effects both on Cl(-) permeation and on channel gating, suggesting that both these effects are a consequence of Pt(NO2)4(2-) interaction with a single site within the pore. Experiments at reduced extracellular Cl(-) concentration hinted that Pt(NO2)4(2-) may have a third effect, possibly increasing channel activity by interfering with channel closure. These results suggest that Pt(NO2)4(2-) can enter from the cytoplasm into the pore inner vestibule of both open and closed CFTR channels, and that Pt(NO2)4(2-) bound in the inner vestibule blocks Cl(-) permeation as well as interfering with channel opening and, perhaps, channel closure. Implications for the location of the channel gate in the pore, and the operation of this gate, are discussed.

Inter-Subunit Interactions across the Upper Voltage Sensing-Pore Domain Interface Contribute to the Concerted Pore Opening Transition of Kv Channels

The tight electro-mechanical coupling between the voltage-sensing and pore domains of Kv channels lies at the heart of their fundamental roles in electrical signaling. Structural data have identified two voltage sensor pore inter-domain interaction surfaces, thus providing a framework to explain the molecular basis for the tight coupling of these domains. While the contribution of the intra-subunit lower domain interface to the electro-mechanical coupling that underlies channel opening is relatively well understood, the contribution of the inter-subunit upper interface to channel gating is not yet clear. Relying on energy perturbation and thermodynamic coupling analyses of tandem-dimeric Shaker Kv channels, we show that mutation of upper interface residues from both sides of the voltage sensor-pore domain interface stabilizes the closed channel state. These mutations, however, do not affect slow inactivation gating. We, moreover, find that upper interface residues form a network of state-dependent interactions that stabilize the open channel state. Finally, we note that the observed residue interaction network does not change during slow inactivation gating. The upper voltage sensing-pore interaction surface thus only undergoes conformational rearrangements during channel activation gating. We suggest that inter-subunit interactions across the upper domain interface mediate allosteric communication between channel subunits that contributes to the concerted nature of the late pore opening transition of Kv channels.

A system dynamics model integrating physiology and biochemical regulation predicts extent of crassulacean acid metabolism (CAM) phases

A system dynamics (SD) approach was taken to model crassulacean acid metabolism (CAM) expression from measured biochemical and physiological constants. SD emphasizes state-dependent feedback interaction to describe the emergent properties of a complex system. These mechanisms maintain biological systems with homeostatic limits on a temporal basis. Previous empirical studies on CAM have correlated biological constants (e.g. enzyme kinetic parameters) with expression over the CAM diel cycle. The SD model integrates these constants within the architecture of the CAM 'system'. This allowed quantitative causal connections to be established between biological inputs and the four distinct phases of CAM delineated by gas exchange and malic acid accumulation traits. Regulation at flow junctions (e.g. stomatal and mesophyll conductance, and malic acid transport across the tonoplast) that are subject to feedback control (e.g. stomatal aperture, malic acid inhibition of phosphoenolpyruvate carboxylase, and enzyme kinetics) was simulated. Simulated expression for the leaf-succulent Kalanchoë daigremontiana and more succulent tissues of Agave tequilana showed strong correlation with measured gas exchange and malic acid accumulation (R(2) =0.912 and 0.937, respectively, for K. daigremontiana and R(2) =0.928 and 0.942, respectively, for A. tequilana). Sensitivity analyses were conducted to quantitatively identify determinants of diel CO2 uptake. The transition in CAM expression from low to high volume/area tissues (elimination of phase II-IV carbon-uptake signatures) was achieved largely by the manipulation three input parameters.

Activation state-dependent interaction between Gαq subunits and the Fhit tumor suppressor

BackgroundThe FHIT tumor suppressor gene is arguably the most commonly altered gene in cancer since it is inactivated in about 60% of human tumors. The Fhit protein is a member of the ubiquitous histidine triad proteins which hydrolyze dinucleoside polyphosphates such as Ap3A. Despite the fact that Fhit functions as a tumor suppressor, the pathway through which Fhit inhibits growth of cancer cells remains largely unknown. Phosphorylation by Src tyrosine kinases provides a linkage between Fhit and growth factor signaling. Since many G proteins can regulate cell proliferation through multiple signaling components including Src, we explored the relationship between Gα subunits and Fhit.ResultsSeveral members of the Gαq subfamily (Gα16, Gα14, and Gαq) were found to co-immunoprecipitate with Fhit in their GTP-bound active state in HEK293 cells. The binding of activated Gαq members to Fhit appeared to be direct and was detectable in native DLD-1 colon carcinoma cells. The use of Gα16/z chimeras further enabled the mapping of the Fhit-interacting domain to the α2-β4 region of Gα16. However, Gαq/Fhit did not affect either Ap3A binding and hydrolysis by Fhit, or the ability of Gαq/16 to regulate downstream effectors including phospholipase Cβ, Ras, ERK, STAT3, and IKK. Functional mutants of Fhit including the H96D, Y114F, L25W and L25W/I10W showed comparable abilities to associate with Gαq. Despite the lack of functional regulation of Gq signaling by Fhit, stimulation of Gq-coupled receptors in HEK293 and H1299 cells stably overexpressing Fhit led to reduced cell proliferation, as opposed to an enhanced cell proliferation typically seen with parental cells.ConclusionsActivated Gαq members interact with Fhit through their α2-β4 region which may result in enhancement of the growth inhibitory effect of Fhit, thus providing a possible avenue for G protein-coupled receptors to modulate tumor suppression.

Predicting the thermodynamics by using state-dependent interactions

We reconsider the structure-based route to coarse graining in which the coarse-grained model is defined in such a way to reproduce some distribution functions of the original system as accurately as possible. We consider standard expressions for pressure and chemical potential applied to this family of coarse-grained models with density-dependent interactions and show that they only provide approximations to the pressure and chemical potential of the underlying original system. These approximations are then carefully compared in two cases: we consider a generic microscopic system in the low-density regime and polymer solutions under good-solvent conditions. Moreover, we show that the state-dependent potentials depend on the ensemble in which they have been derived. Therefore, care must be used in applying canonical state-dependent potentials to predict phase lines, which is typically performed in other ensembles.

Involvement of Separate Regions of the NAV Channel DIII-IV Linker Revealed through State Dependent Trapping with Genetically Encoded Photochemistry

Fast inactivation in voltage-gated sodium channels results from rapid and reversible conformational changes in the cytoplasmic linker between domains III and IV (DIII-IV) and their putative receptor in the pore region of the channel. Introduced or acquired mutations in this complex lead to defective channel inactivation, yet the precise structural basis for these interactions has not yet been fully described. Using the photocrosslinking (PC) unnatural amino acid p-benzoyl-L-phenyl alanine (pBpa) it is possible to trap close structural interactions (∼3 A) during transient conformational rearrangements thus mapping structural reorganisations associated with different states. The rates at which crosslinking reactions occur can report on the affinity of such interactions. We have incorporated the pBpa in mammalian cells at the putative inactivation peptide (IFMT) motif of the DIII-IV linker and demonstrated state dependent trapping of the transient conformations associated with fast inactivation, with different phenotypes and rates of trapping revealing distinct environmental changes at each site. Incorporation of pBpa at two tyrosine residues, Y1494 and Y1495 of NaV1.5, which have previously been implicated in the coupling of activation and inactivation and to interact with Calmodulin produced channels with normal gating and robust expression. Interestingly, the observed state dependent trapping upon irradiation with UV light during patch clamp experiments was functionally distinct from the state-dependent cross-linking interactions observed within the IFM locus. Thus, trapping at these sites suggests a more extensive involvement of the DIII-DIV linker in fast inactivation gating beyond the canonical IFMT motif.

Mean field model for ferromagnetic nanowire arrays based on a mechanical analogy

Nanowire arrays have typical distributions of coercive and interaction fields, as revealed in experiments by means of the first-order reversal curve (FORC) method. In an axial applied field, each nanowire is subjected to a state dependent interaction field created by all wires from the array, which strongly influences the switching fields. A mean field model based on Preisach–Krasnosel’skii–Pokrovskii (PKP) hysterons is able to explain the interplay between interactions and critical fields in nanowire arrays and the subtle way in which switching fields are emphasized in FORC diagrams.

Stochastic dynamic causal modelling of fMRI data: Should we care about neural noise?

Dynamic causal modelling (DCM) was introduced to study the effective connectivity among brain regions using neuroimaging data. Until recently, DCM relied on deterministic models of distributed neuronal responses to external perturbation (e.g., sensory stimulation or task demands). However, accounting for stochastic fluctuations in neuronal activity and their interaction with task-specific processes may be of particular importance for studying state-dependent interactions. Furthermore, allowing for random neuronal fluctuations may render DCM more robust to model misspecification and finesse problems with network identification. In this article, we examine stochastic dynamic causal models (sDCM) in relation to their deterministic counterparts (dDCM) and highlight questions that can only be addressed with sDCM. We also compare the network identification performance of deterministic and stochastic DCM, using Monte Carlo simulations and an empirical case study of absence epilepsy. For example, our results demonstrate that stochastic DCM can exploit the modelling of neural noise to discriminate between direct and mediated connections. We conclude with a discussion of the added value and limitations of sDCM, in relation to its deterministic homologue.

Inference of Signs of Interaction Effects in Simultaneous Games With Incomplete Information

This paper studies the inference of interaction effects in discrete simultaneous games with incomplete information. We propose a test for the signs of state-dependent interaction effects that does not require parametric specifications of players' payoffs, the distributions of their private signals, or the equilibrium selection mechanism. The test relies on the commonly invoked assumption that players' private signals are independent conditional on observed states. The procedure is valid in (but does not rely on) the presence of multiple equilibria in the data-generating process (DGP). As a by-product, we propose a formal test for multiple equilibria in the DGP. We also implement the test using data on radio programming of commercial breaks in the United States, and infer stations' incentives to synchronize their commercial breaks. Our results support the earlier finding by Sweeting (2009) that stations have stronger incentives to coordinate and air commercials at the same time during rush hours and in smaller markets.

Phosphorylation State-Dependent Interactions of Hepadnavirus Core Protein with Host Factors

Dynamic phosphorylation and dephosphorylation of the hepadnavirus core protein C-terminal domain (CTD) are required for multiple steps of the viral life cycle. It remains unknown how the CTD phosphorylation state may modulate core protein functions but phosphorylation state-dependent viral or host interactions may play a role. In an attempt to identify host factors that may interact differentially with the core protein depending on its CTD phosphorylation state, pulldown assays were performed using the CTD of the duck hepatitis B virus (DHBV) and human hepatitis B virus (HBV) core protein, either with wild type (WT) sequences or with alanine or aspartic acid substitutions at the phosphorylation sites. Two host proteins, B23 and I2PP2A, were found to interact preferentially with the alanine-substituted CTD. Furthermore, the WT CTD became competent to interact with the host proteins upon dephosphorylation. Intriguingly, the binding site on the DHBV CTD for both B23 and I2PP2A was mapped to a region upstream of the phosphorylation sites even though B23 or I2PP2A binding to this site was clearly modulated by the phosphorylation state of the downstream and non-overlapping sequences. Together, these results demonstrate a novel mode of phosphorylation-regulated protein-protein interaction and provide new insights into virus-host interactions.

Structural basis for gating charge movement in the voltage sensor of a sodium channel

Voltage-dependent gating of ion channels is essential for electrical signaling in excitable cells, but the structural basis for voltage sensor function is unknown. We constructed high-resolution structural models of resting, intermediate, and activated states of the voltage-sensing domain of the bacterial sodium channel NaChBac using the Rosetta modeling method, crystal structures of related channels, and experimental data showing state-dependent interactions between the gating charge-carrying arginines in the S4 segment and negatively charged residues in neighboring transmembrane segments. The resulting structural models illustrate a network of ionic and hydrogen-bonding interactions that are made sequentially by the gating charges as they move out under the influence of the electric field. The S4 segment slides 6-8 Å outward through a narrow groove formed by the S1, S2, and S3 segments, rotates ∼30°, and tilts sideways at a pivot point formed by a highly conserved hydrophobic region near the middle of the voltage sensor. The S4 segment has a 3(10)-helical conformation in the narrow inner gating pore, which allows linear movement of the gating charges across the inner one-half of the membrane. Conformational changes of the intracellular one-half of S4 during activation are rigidly coupled to lateral movement of the S4-S5 linker, which could induce movement of the S5 and S6 segments and open the intracellular gate of the pore. We confirmed the validity of these structural models by comparing with a high-resolution structure of a NaChBac homolog and showing predicted molecular interactions of hydrophobic residues in the S4 segment in disulfide-locking studies.