Radiation Hormesis in Barley Manifests as Changes in Growth Dynamics Coordinated with the Expression of PM19L-like, CML31-like, and AOS2-like.

The stimulation of growth and development of crops using ionising radiation (radiation hormesis) has been reported by many research groups. However, specific genes contributing to the radiation stimulation of plant growth are largely unknown. In this work, we studied the impact of the low-dose γ-irradiation of barley seeds on the growth dynamics and gene expression of eight barley cultivars in a greenhouse experiment. Our findings confirmed that candidate genes of the radiation growth stimulation, previously established in barley seedlings (PM19L-like, CML31-like, and AOS2-like), are significant in radiation hormesis throughout ontogeny. In γ-stimulated cultivars, the expression of these genes was aligned with the growth dynamics, yield parameters, and physiological conditions of plants. We identified contrasting cultivars for future gene editing and found that the γ-stimulated cultivar possessed some specific abiotic stress-responsive elements in the promotors of candidate genes, possibly revealing a new level of radiation hormesis effect execution. These results can be used in creating new productive barley cultivars, ecological toxicology of radionuclides, and eustress biology studies.

Caveolae-associated cAMP/Ca2+-mediated mechano-chemical signal transduction in mouse atrial myocytes.

Caveolae are tiny invaginations in the sarcolemma that buffer extra membrane and contribute to mechanical regulation of cellular function. While the role of caveolae in membrane mechanosensation has been studied predominantly in non-cardiomyocyte cells, caveolae contribution to cardiac mechanotransduction remains elusive. Here, we studied the role of caveolae in the regulation of Ca2+ signaling in atrial cardiomyocytes. In Langendorff-perfused mouse hearts, atrial pressure/volume overload stretched atrial myocytes and decreased caveolae density. In isolated cells, caveolae were disrupted through hypotonic challenge that induced a temporal (<10 min) augmentation of Ca2+ transients and caused a rise in Ca2+ spark activity. Similar changes in Ca2+ signaling were observed after chemical (methyl-ß-cyclodextrin) and genetic ablation of caveolae in cardiac-specific conditional caveolin-3 knock-out mice. Acute disruption of caveolae, both mechanical and chemical, led to the elevation of cAMP level in the cell interior, and cAMP-mediated augmentation of protein kinase A (PKA)-phosphorylated ryanodine receptors (at Ser2030 and Ser2808). Caveolae-mediated stimulatory effects on Ca2+ signaling were abolished via inhibition of cAMP production by adenyl cyclase antagonists MDL12330 and SQ22536, or reduction of PKA activity by H-89. A compartmentalized mathematical model of mouse atrial myocytes linked the observed changes to a microdomain-specific decrease in phosphodiesterase activity, which disrupted cAMP signaling and augmented PKA activity. Our findings add a new dimension to cardiac mechanobiology and highlight caveolae-associated cAMP/PKA-mediated phosphorylation of Ca2+ handling proteins as a novel component of mechano-chemical feedback in atrial myocytes.

A compartmentalized mathematical model of the ß1- and ß2-adrenergic signaling systems in ventricular myocytes from mouse in heart failure.

Mouse models of heart failure are extensively used to research human cardiovascular diseases. In particular, one of the most common is the mouse model of heart failure resulting from transverse aortic constriction (TAC). Despite this, there are no comprehensive compartmentalized mathematical models that describe the complex behavior of the action potential, [Ca2+]i transients, and their regulation by ß1- and ß2-adrenergic signaling systems in failing mouse myocytes. In this paper, we develop a novel compartmentalized mathematical model of failing mouse ventricular myocytes after TAC procedure. The model describes well the cell geometry, action potentials, [Ca2+]i transients, and ß1- and ß2-adrenergic signaling in the failing cells. Simulation results obtained with the failing cell model are compared with those from the normal ventricular myocytes. Exploration of the model reveals the sarcoplasmic reticulum Ca2+ load mechanisms in failing ventricular myocytes. We also show a larger susceptibility of the failing myocytes to early and delayed afterdepolarizations and to a proarrhythmic behavior of Ca2+ dynamics upon stimulation with isoproterenol. The mechanisms of the proarrhythmic behavior suppression are investigated and sensitivity analysis is performed. The developed model can explain the existing experimental data on failing mouse ventricular myocytes and make experimentally testable predictions of a failing myocyte's behavior.

Role of asymmetry and external noise in the development and synchronization of oscillations in the analog Hopfield neural networks with time delay.

The analog Hopfield neural network with time delay and random connections has been studied for its similarities in activity to human electroencephalogram and its usefulness in other areas of the applied sciences such as speech recognition, image analysis, and electrocardiogram modeling. Our goal here is to understand the mechanisms that affect the rhythmic activity in the neural network and how the addition of a Gaussian noise contributes to the network behavior. The neural network studied is composed of ten identical neurons. We investigated the excitatory and inhibitory networks with symmetric (square matrix) and asymmetric (triangular matrix) connections. The differential equations that model the network are solved numerically using the stochastic second-order Runge-Kutta method. Without noise, the neural networks with symmetric and asymmetric matrices possessed different synchronization properties: fully connected networks were synchronized both in time and in amplitude, while asymmetric networks were synchronized in time only. Saturation outputs of the excitatory neural networks do not depend on the time delay, whereas saturation oscillation amplitudes of inhibitory networks increase with the time delay until the steady state. The addition of the Gaussian noise is shown to significantly amplify small-amplitude oscillations, dramatically accelerates the rate of amplitude growth to saturation, and changes synchronization properties of the neural network outputs.

Outcomes of deep brain stimulation surgery for substance use disorder: a systematic review.

Long has the standard of care for substance use disorder (SUD) been pharmacotherapy, psychotherapy, or rehabilitation with varying success. Deep brain stimulation (DBS) may have a beneficial reduction in the addiction-reward pathway. Recent studies have found reduced relapse and improvements in quality of life following DBS stimulation of the nucleus accumbens. We aim to identify positive outcomes and adverse effects to assess the viability of DBS as a treatment of addiction. A PubMed search following PRISMA guidelines was conducted to identify the entirety of reports reporting DBS as a treatment for SUD. Outcomes were extracted from the literature to be summarized, and a review of the quality of publications was also performed. From 2305 publications, 14 studies were found to fit the inclusion criteria published between 2007 and 2019. All studies targeted the nucleus accumbens (NAc) and remission rates at 6 months, 1 year, 2 years, and more than 6 years were 61% (20/33), 53% (17/32), 43% (14/30), and 50% (3/6), respectively. Not all studies detailed the stimulation settings or coordinates. The most common adverse effect across studies was a weight change of at least 2 kg. DBS shows potential as a long-term treatment of SUD in refractory patients. Further studies with controlled double-blind paradigms are needed for evaluation of the efficacy and safety of this treatment. Future studies should also investigate other brain regions for stimulation and optimal device stimulation parameters.

Mathematical model for ß1-adrenergic regulation of the mouse ventricular myocyte contraction.

The ß1-adrenergic regulation of cardiac myocyte contraction plays an important role in regulating heart function. Activation of this system leads to an increased heart rate and stronger myocyte contraction. However, chronic stimulation of the ß1-adrenergic signaling system can lead to cardiac hypertrophy and heart failure. To understand the mechanisms of action of ß1-adrenoceptors, a mathematical model of cardiac myocyte contraction that includes the ß1-adrenergic system was developed and studied. The model was able to simulate major experimental protocols for measurements of steady-state force-calcium relationships, cross-bridge release rate and force development rate, force-velocity relationship, and force redevelopment rate. It also reproduced quite well frequency and isoproterenol dependencies for intracellular Ca2+ concentration ([Ca2+]i) transients, total contraction force, and sarcomere shortening. The mathematical model suggested the mechanisms of increased contraction force and myocyte shortening on stimulation of ß1-adrenergic receptors is due to phosphorylation of troponin I and myosin-binding protein C and increased [Ca2+]i transient resulting from activation of the ß1-adrenergic signaling system. The model was used to simulate work-loop contractions and estimate the power during the cardiac cycle as well as the effects of 4-aminopyridine and tedisamil on the myocyte contraction. The developed mathematical model can be used further for simulations of contraction of ventricular myocytes from genetically modified mice and myocytes from mice with chronic cardiac diseases.NEW & NOTEWORTHY A new mathematical model of mouse ventricular myocyte contraction that includes the ß1-adrenergic system was developed. The model simulated major experimental protocols for myocyte contraction and predicted the effects of 4-aminopyridine and tedisamil on the myocyte contraction. The model also allowed for simulations of work-loop contractions and estimation of the power during the cardiac cycle.

A compartmentalized mathematical model of mouse atrial myocytes.

Various experimental mouse models are extensively used to research human diseases, including atrial fibrillation, the most common cardiac rhythm disorder. Despite this, there are no comprehensive mathematical models that describe the complex behavior of the action potential and [Ca2+]i transients in mouse atrial myocytes. Here, we develop a novel compartmentalized mathematical model of mouse atrial myocytes that combines the action potential, [Ca2+]i dynamics, and ß-adrenergic signaling cascade for a subpopulation of right atrial myocytes with developed transverse-axial tubule system. The model consists of three compartments related to ß-adrenergic signaling (caveolae, extracaveolae, and cytosol) and employs local control of Ca2+ release. It also simulates ionic mechanisms of action potential generation and describes atrial-specific Ca2+ handling as well as frequency dependences of the action potential and [Ca2+]i transients. The model showed that the T-type Ca2+ current significantly affects the later stage of the action potential, with little effect on [Ca2+]i transients. The block of the small-conductance Ca2+-activated K+ current leads to a prolongation of the action potential at high intracellular Ca2+. Simulation results obtained from the atrial model cells were compared with those from ventricular myocytes. The developed model represents a useful tool to study complex electrical properties in the mouse atria and could be applied to enhance the understanding of atrial physiology and arrhythmogenesis.NEW & NOTEWORTHY A new compartmentalized mathematical model of mouse right atrial myocytes was developed. The model simulated action potential and Ca2+ dynamics at baseline and after stimulation of the ß-adrenergic signaling system. Simulations showed that the T-type Ca2+ current markedly prolonged the later stage of atrial action potential repolarization, with a minor effect on [Ca2+]i transients. The small-conductance Ca2+-activated K+ current block resulted in prolongation of the action potential only at the relatively high intracellular Ca2+.

A Mathematical Model of the Human Cardiac Na+ Channel.

Sodium ion channel is a membrane protein that plays an important role in excitable cells, as it is responsible for the initiation of action potentials. Understanding the electrical characteristics of sodium channels is essential in predicting their behavior under different physiological conditions. We investigated several Markov models for the human cardiac sodium channel NaV1.5 to derive a minimal mathematical model that describes the reported experimental data obtained using major voltage clamp protocols. We obtained simulation results for peak current-voltage relationships, the voltage dependence of normalized ion channel conductance, steady-state inactivation, activation and deactivation kinetics, fast and slow inactivation kinetics, and recovery from inactivation kinetics. Good agreement with the experimental data provides us with the mechanisms of the fast and slow inactivation of the human sodium channel and the coupling of its inactivation states to the closed and open states in the activation pathway.

Mathematical modeling physiological effects of the overexpression of ß2-adrenoceptors in mouse ventricular myocytes.

Transgenic (TG) mice overexpressing ß2-adrenergic receptors (ß2-ARs) demonstrate enhanced myocardial function, which manifests in increased basal adenylyl cyclase activity, enhanced atrial contractility, and increased left ventricular function in vivo. To gain insights into the mechanisms of these effects, we developed a comprehensive mathematical model of the mouse ventricular myocyte overexpressing ß2-ARs. We found that most of the ß2-ARs are active in control conditions in TG mice. The simulations describe the dynamics of major signaling molecules in different subcellular compartments, increased basal adenylyl cyclase activity, modifications of action potential shape and duration, and the effects on L-type Ca2+ current and intracellular Ca2+ concentration ([Ca2+]i) transients upon stimulation of ß2-ARs in control, after the application of pertussis toxin, upon stimulation with a specific ß2-AR agonist zinterol, and upon stimulation with zinterol in the presence of pertussis toxin. The model also describes the effects of the ß2-AR inverse agonist ICI-118,551 on adenylyl cyclase activity, action potential, and [Ca2+]i transients. The simulation results were compared with experimental data obtained in ventricular myocytes from TG mice overexpressing ß2-ARs and with simulation data on wild-type mice. In conclusion, a new comprehensive mathematical model was developed that describes multiple experimental data on TG mice overexpressing ß2-ARs and can be used to test numerous hypotheses. As an example, using the developed model, we proved the hypothesis of the major contribution of L-type Ca2+ current to the changes in the action potential and [Ca2+]i transient upon stimulation of ß2-ARs with zinterol. NEW & NOTEWORTHY We developed a new mathematical model for transgenic mouse ventricular myocytes overexpressing ß2-adrenoceptors that describes the experimental findings in transgenic mice. The model reveals mechanisms of the differential effects of stimulation of ß2-adrenoceptors in wild-type and transgenic mice overexpressing ß2-adrenoceptors.

Distinct physiological effects of ß1- and ß2-adrenoceptors in mouse ventricular myocytes: insights from a compartmentalized mathematical model.

The ß1- and ß2-adrenergic signaling systems play different roles in the functioning of cardiac cells. Experimental data show that the activation of the ß1-adrenergic signaling system produces significant inotropic, lusitropic, and chronotropic effects in the heart, whereas the effects of the ß2-adrenergic signaling system is less apparent. In this paper, a comprehensive compartmentalized experimentally based mathematical model of the combined ß1- and ß2-adrenergic signaling systems in mouse ventricular myocytes is developed to simulate the experimental findings and make testable predictions of the behavior of the cardiac cells under different physiological conditions. Simulations describe the dynamics of major signaling molecules in different subcellular compartments; kinetics and magnitudes of phosphorylation of ion channels, transporters, and Ca2+ handling proteins; modifications of action potential shape and duration; and [Ca2+]i and [Na+]i dynamics upon stimulation of ß1- and ß2-adrenergic receptors (ß1- and ß2-ARs). The model reveals physiological conditions when ß2-ARs do not produce significant physiological effects and when their effects can be measured experimentally. Simulations demonstrated that stimulation of ß2-ARs with isoproterenol caused a marked increase in the magnitude of the L-type Ca2+ current, [Ca2+]i transient, and phosphorylation of phospholamban only upon additional application of pertussis toxin or inhibition of phosphodiesterases of type 3 and 4. The model also made testable predictions of the changes in magnitudes of [Ca2+]i and [Na+]i fluxes, the rate of decay of [Na+]i concentration upon both combined and separate stimulation of ß1- and ß2-ARs, and the contribution of phosphorylation of PKA targets to the changes in the action potential and [Ca2+]i transient.

Simulation of the effects of moderate stimulation/inhibition of the ß1-adrenergic signaling system and its components in mouse ventricular myocytes.

The ß1-adrenergic signaling system is one of the most important protein signaling systems in cardiac cells. It regulates cardiac action potential duration, intracellular Ca(2+) concentration ([Ca(2+)]i) transients, and contraction force. In this paper, a comprehensive experimentally based mathematical model of the ß1-adrenergic signaling system for mouse ventricular myocytes is explored to simulate the effects of moderate stimulations of ß1-adrenergic receptors (ß1-ARs) on the action potential, Ca(2+) and Na(+) dynamics, as well as the effects of inhibition of protein kinase A (PKA) and phosphodiesterase of type 4 (PDE4). Simulation results show that the action potential prolongations reach saturating values at relatively small concentrations of isoproterenol (∼0.01 µM), while the [Ca(2+)]i transient amplitude saturates at significantly larger concentrations (∼0.1-1.0 µM). The differences in the response of Ca(2+) and Na(+) fluxes to moderate stimulation of ß1-ARs are also observed. Sensitivity analysis of the mathematical model is performed and the model limitations are discussed. The investigated model reproduces most of the experimentally observed effects of moderate stimulation of ß1-ARs, PKA, and PDE4 inhibition on the L-type Ca(2+) current, [Ca(2+)]i transients, and the sarcoplasmic reticulum Ca(2+) load and makes testable predictions for the action potential duration and [Ca(2+)]i transients as functions of isoproterenol concentration.

Microtubule-associated protein tau in bovine retinal photoreceptor rod outer segments: comparison with brain tau.

Recent studies have suggested a possible involvement of abnormal tau in some retinal degenerative diseases. The common view in these studies is that these retinal diseases share the mechanism of tau-mediated degenerative diseases in brain and that information about these brain diseases may be directly applied to explain these retinal diseases. Here we collectively examine this view by revealing three basic characteristics of tau in the rod outer segment (ROS) of bovine retinal photoreceptors, i.e., its isoforms, its phosphorylation mode and its interaction with microtubules, and by comparing them with those of brain tau. We find that ROS contains at least four isoforms: three are identical to those in brain and one is unique in ROS. All ROS isoforms, like brain isoforms, are modified with multiple phosphate molecules; however, ROS isoforms show their own specific phosphorylation pattern, and these phosphorylation patterns appear not to be identical to those of brain tau. Interestingly, some ROS isoforms, under the normal conditions, are phosphorylated at the sites identical to those in Alzheimer's patient isoforms. Surprisingly, a large portion of ROS isoforms tightly associates with a membranous component(s) other than microtubules, and this association is independent of their phosphorylation states. These observations strongly suggest that tau plays various roles in ROS and that some of these functions may not be comparable to those of brain tau. We believe that knowledge about tau in the entire retinal network and/or its individual cells are also essential for elucidation of tau-mediated retinal diseases, if any.

A polar barrier to transcription can be circumvented by remodeler-induced nucleosome translocation.

Many eukaryotic genes are regulated at the level of transcript elongation. Nucleosomes are likely targets for this regulation. Previously, we have shown that nucleosomes formed on very strong positioning sequences (601 and 603), present a high, orientation-dependent barrier to transcription by RNA polymerase II in vitro. The existence of this polar barrier correlates with the interaction of a 16-bp polar barrier signal (PBS) with the promoter-distal histone H3-H4 dimer. Here, we show that the polar barrier is relieved by ISW2, an ATP-dependent chromatin remodeler, which translocates the nucleosome over a short distance, such that the PBS no longer interacts with the distal H3-H4 dimer, although it remains within the nucleosome. In vivo, insertion of the 603 positioning sequence into the yeast CUP1 gene results in a modest reduction in transcription, but this reduction is orientation-independent, indicating that the polar barrier can be circumvented. However, the 603-nucleosome is present at the expected position in only a small fraction of cells. Thus, the polar barrier is probably non-functional in vivo because the nucleosome is not positioned appropriately, presumably due to nucleosome sliding activities. We suggest that interactions between PBSs and chromatin remodelers might have significant regulatory potential.

Differential gene expression in chronically irradiated herbaceous species from the Chernobyl exclusion zone.

PURPOSE: Transcriptional activity of genes related to ionizing radiation responses in chronically irradiated plant populations at radioactively contaminated territories can be a cost-effective and precise approach for stress response evaluation. However, there are limits to studying non-model plants in field conditions. The work studies the transcriptional activity of candidate genes of adaptation to chronic radiation exposure in plant populations from radioactively contaminated territories of the Chernobyl. MATERIALS AND METHODS: In this work, we studied plant species with different sensitivity to acute irradiation: Trifolium repens L., Taraxacum officinale Wigg., and Dactylis glomerata L., sampled in the Chernobyl exclusion zone. The differential expression of several candidate genes of adaptation to chronic radiation exposure in the leaves of these species was analyzed, including homologs of Arabidopsis thaliana genes SLAC1, APX1, GPX2, CAB1, NTRB, PP2-B11, RBOH-F, HY5, SnRK2.4, PDS1, CIPK20, SIP1, PIP1, TIP1. RESULTS AND CONCLUSIONS: All studied species were characterized by upregulation of the CAB1 homolog, encoding chlorophyll a/b binding protein, at radioactively contaminated plots. An increase in the expression of genes associated with water and hydrogen peroxide transport, intensity of photosynthesis, and stress responses (homolog of aquaporin TIP1 for T. repens; homologs of aquaporin PIP1 and transcription factor HY5 for D. glomerata; homolog of CBL-interacting serine/threonine protein kinase CIPK20 for T. officinale) was revealed. The methodological approach for studying gene expression in non-model plant species is described, which may allow large-scale screening studies of candidate genes in various plant species abundant in radioactively contaminated areas.

Comparative analysis of epigenetic variability in two pine species exposed to chronic radiation in the Chernobyl and Fukushima affected zones.

Comparative analysis of epigenetic variability in two pine species affected as a result of the Chernobyl and Fukushima accidents is presented. The absorbed dose rate within the affected Chernobyl sites varies over a wider range (1.5-24.6 µGy/h) than within the Fukushima sites (3.5-6.5 µGy/h). It was shown that chronic irradiation can change the level of whole genome methylation in pine populations, but in different ways. The genomes of Japanese red pines are hypomethylated, and the degree of methylation and hydroxymethylation decreases with an increase in the level of radiation exposure. In contrast, the percentages of genome methylation and hydroxymethylation in Scots pine populations exceed the reference levels. The observed discrepancy in the patterns of genome-wide DNA methylation can be attributed partly to the design of the study (differences in the climate, radiation dose, age and species of the pines) which could affect the results. In the frame of IRAP analysis, a larger number of different bands was observed in the Chernobyl populations compared to the Japanese populations. Both the Japanese and Chernobyl populations are characterized by significant genetic variability. However, the main part of this variability is observed within populations. The dendrograms, based on presence/absence of IRAP fragments and Nei's genetic distances, revealed subdivisions of the Chernobyl and Japanese populations according to the level of radioactive contamination. Analysis of the results presented will improve our understanding of the mechanisms underlying the responses of pine trees to chronic radiation exposure.

Mathematical modeling mechanisms of arrhythmias in transgenic mouse heart overexpressing TNF-α.

Transgenic mice overexpressing tumor necrosis factor-α (TNF-α mice) possess many of the features of human heart failure, such as dilated cardiomyopathy, impaired Ca(2+) handling, arrhythmias, and decreased survival. Although TNF-α mice have been studied extensively with a number of experimental methods, the mechanisms of heart failure are not completely understood. We created a mathematical model that reproduced experimentally observed changes in the action potential (AP) and Ca(2+) handling of isolated TNF-α mice ventricular myocytes. To study the contribution of the differences in ion currents, AP, Ca(2+) handling, and intercellular coupling to the development of arrhythmias in TNF-α mice, we further created several multicellular model tissues with combinations of wild-type (WT)/reduced gap junction conductance, WT/prolonged AP, and WT/decreased Na(+) current (I(Na)) amplitude. All model tissues were examined for susceptibility to Ca(2+) alternans, AP propagation block, and reentry. Our modeling results demonstrated that, similar to experimental data in TNF-α mice, Ca(2+) alternans in TNF-α tissues developed at longer basic cycle lengths. The greater susceptibility to Ca(2+) alternans was attributed to the prolonged AP, resulting in larger inactivation of I(Na), and to the decreased SR Ca(2+) uptake and corresponding smaller SR Ca(2+) load. Simulations demonstrated that AP prolongation induces an increased susceptibility to AP propagation block. Programmed stimulation of the model tissues with a premature impulse showed that reduced gap junction conduction increased the vulnerable window for initiation reentry, supporting the idea that reduced intercellular coupling is the major factor for reentrant arrhythmias in TNF-α mice.

A model of the interaction between N-type and C-type inactivation in Kv1.4 channels.

Kv1.4 channels are Shaker-related voltage-gated potassium channels with two distinct inactivation mechanisms. Fast N-type inactivation operates by a ball-and-chain mechanism. Slower C-type inactivation is not so well defined, but involves intracellular and extracellular conformational changes of the channel. We studied the interaction between inactivation mechanisms using two-electrode voltage-clamp of Kv1.4 and Kv1.4ΔN (amino acids 2-146 deleted to remove N-type inactivation) heterologously expressed in Xenopus oocytes. We manipulated C-type inactivation by introducing a lysine-tyrosine point mutation (K532Y, equivalent to Shaker T449Y) that diminishes C-type inactivation. We used experimental data to develop a comprehensive computer model of Kv1.4 channels to determine the interaction between activation and N- and C-type inactivation mechanisms needed to replicate the experimental data. C-type inactivation began at lower voltage preactivated states, whereas N-type inactivation was coupled directly to the open state. A model with distinct N- and C-type inactivated states was not able to reproduce experimental data, and direct transitions between N- and C-type inactivated states were required, i.e., there is coupling between N- and C-type inactivated states. C-type inactivation is the rate-limiting step determining recovery from inactivation, so understanding C-type inactivation, and how it is coupled to N-type inactivation, is critical in understanding how channels act to repetitive stimulation.

Heteropoda toxin 2 interaction with Kv4.3 and Kv4.1 reveals differences in gating modification.

Kv4 (Shal) potassium channels are responsible for the transient outward K(+) currents in mammalian hearts and central nervous systems. Heteropoda toxin 2 (HpTx2) is an inhibitor cysteine knot peptide toxin specific for Kv4 channels that inhibits gating of Kv4.3 in the voltage-dependent manner typical for this type of toxin. HpTx2 interacts with four independent binding sites containing two conserved hydrophobic amino acids in the S3b transmembrane segments of Kv4.3 and the closely related Kv4.1. Despite these similarities, HpTx2 interaction with Kv4.1 is considerably less voltage-dependent, has smaller shifts in the voltage-dependences of conductance and steady-state inactivation, and a 3-fold higher K(d) value. Swapping four nonconserved amino acids in S3b between the two channels exchanges the phenotypic response to HpTx2. To understand these differences in gating modification, we constructed Markov models of Kv4.3 and Kv4.1 activation gating in the presence of HpTx2. Both models feature a series of voltage-dependent steps leading to a final voltage-independent transition to the open state and closely replicate the experimental data. Interaction with HpTx2 increases the energy barrier for channel opening by slowing activation and accelerating deactivation. The greater degree of voltage-dependence in Kv4.3 occurs because it is the voltage-dependent transitions that are most affected by HpTx2; in contrast, it is the voltage-independent step in Kv4.1 that is most affected by the presence of toxin. These data demonstrate the basis for subtype-specificity of HpTx2 and point the way to a general model of gating modifier toxin interaction with voltage-gated ion channels.

Transmural heterogeneity of repolarization and Ca2+ handling in a model of mouse ventricular tissue.

Mouse hearts have a diversity of action potentials (APs) generated by the cardiac myocytes from different regions. Recent evidence shows that cells from the epicardial and endocardial regions of the mouse ventricle have a diversity in Ca(2+) handling properties as well as K(+) current expression. To examine the mechanisms of AP generation, propagation, and stability in transmurally heterogeneous tissue, we developed a comprehensive model of the mouse cardiac cells from the epicardial and endocardial regions of the heart. Our computer model simulates the following differences between epicardial and endocardial myocytes: 1) AP duration is longer in endocardial and shorter in epicardial myocytes, 2) diastolic and systolic intracellular Ca(2+) concentration and intracellular Ca(2+) concentration transients are higher in paced endocardial and lower in epicardial myocytes, 3) Ca(2+) release rate is about two times larger in endocardial than in epicardial myocytes, and 4) Na(+)/Ca(2+) exchanger rate is greater in epicardial than in endocardial myocytes. Isolated epicardial cells showed a higher threshold for stability of AP generation but more complex patterns of AP duration at fast pacing rates. AP propagation velocities in the model of two-dimensional tissue are close to those measured experimentally. Simulations show that heterogeneity of repolarization and Ca(2+) handling are sustained across the mouse ventricular wall. Stability analysis of AP propagation in the two-dimensional model showed the generation of Ca(2+) alternans and more complex transmurally heterogeneous irregular structures of repolarization and intracellular Ca(2+) transients at fast pacing rates.

Involvement of rhodopsin and ATP in the activation of membranous guanylate cyclase in retinal photoreceptor outer segments (ROS-GC) by GC-activating proteins (GCAPs): a new model for ROS-GC activation and its link to retinal diseases.

Membranous guanylate cyclase in retinal photoreceptor outer segments (ROS-GC), a key enzyme for the recovery of photoreceptors to the dark state, has a topology identical to and cytoplasmic domains homologous to those of peptide-regulated GCs. However, under the prevailing concept, its activation mechanism is significantly different from those of peptide-regulated GCs: GC-activating proteins (GCAPs) function as the sole activator of ROS-GC in a Ca(2+)-sensitive manner, and neither reception of an outside signal by the extracellular domain (ECD) nor ATP binding to the kinase homology domain (KHD) is required for its activation. We have recently shown that ATP pre-binding to the KHD in ROS-GC drastically enhances its GCAP-stimulated activity, and that rhodopsin illumination, as the outside signal, is required for the ATP pre-binding. These results indicate that illuminated rhodopsin is involved in ROS-GC activation in two ways: to initiate ATP binding to ROS-GC for preparation of its activation and to reduce [Ca(2+)] through activation of cGMP phosphodiesterase. These two signal pathways are activated in a parallel and proportional manner and finally converge for strong activation of ROS-GC by Ca(2+)-free GCAPs. These results also suggest that the ECD receives the signal for ATP binding from illuminated rhodopsin. The ECD is projected into the intradiscal space, i.e., an intradiscal domain(s) of rhodopsin is also involved in the signal transfer. Many retinal disease-linked mutations are found in these intradiscal domains; however, their consequences are often unclear. This model will also provide novel insights into causal relationship between these mutations and certain retinal diseases.