Simulating complex ion channel kinetics with IonChannelLab

In silico simulation based on Markov chains is a powerful way to describe and predict the activity of many transport proteins including ion channels. However, modeling and simulation using realistic models of voltage- or ligand-gated ion channels exposed to a wide range of experimental conditions require building complex kinetic schemes and solving complicated differential equations. To circumvent these problems, we developed IonChannelLab a software tool that includes a user-friendly Graphical User Interface and a simulation library. This program supports channels with Ohmic or Goldman-Hodgkin-Katz behavior and can simulate the time-course of ionic and gating currents, single channel behavior and steady-state conditions. The program allows the simulation of experiments where voltage, ligand and ionic concentration are varied independently or simultaneously.     

Simulating complex ion channel kinetics with IonChannelLab

In silico simulation based on Markov chains is a powerful way to describe and predict the activity of many transport proteins including ion channels. However, modeling and simulation using realistic models of voltage- or ligand-gated ion channels exposed to a wide range of experimental conditions require building complex kinetic schemes and solving complicated differential equations. To circumvent these problems, we developed IonChannelLab a software tool that includes a user-friendly Graphical User Interface and a simulation library. This program supports channels with Ohmic or Goldman-Hodgkin-Katz behavior and can simulate the time-course of ionic and gating currents, single channel behavior and steady-state conditions. The program allows the simulation of experiments where voltage, ligand and ionic concentration are varied independently or simultaneously.     

Developmental gene network analysis

The developmental process is controlled by the information processing functions executed by the cis-elements that regulate the expression of the participating genes. A model of the network of cis-regulatory interactions that underlies the specification of the endomesoderm of the sea urchin embryo is analyzed here. Although not all the relevant interactions have yet been uncovered, the model shows how the information processing functions executed by the cis-regulatory elements involved can control essential functions of the specification process, such as transforming the localization of maternal factors into a domain-specific program of gene expression; refining the specification pattern; and stabilizing states of specification. The analysis suggests that the progressivity of the developmental process is also controlled by the cis-regulatory interactions unraveled by the network model. Given that evolution occurs by changing the program for development of the body plan, we illustrate the potential of developmental gene network analysis in understanding the process by which morphological features are maintained and diversify. Comparison of the network of cis-regulatory interactions with a portion of that underlying the specification of the endomesoderm of the starfish illustrates how the similarities and differences provide insights into how the programs for development work and how they evolve.     

Noise effects on spike propagation in the stochastic Hodgkin-Huxley models

Effects of membrane current noise on spike propagation along a nerve fiber are studied. Additive current noise and channel noise are considered by using stochastic versions of the Hodgkin-Huxley model. The results of computer simulation show that the membrane noise causes considerable variation of the propagation time of a spike (thus changes in interspike intervals) for a small unmyelinated fiber of radius 0.1 approximately 1 micron.     

An Object-Oriented Modeling and Simulation Environment for Reactive Systems Development

An environment to support the modeling, analysis, simulation, and development of state transition models, SMOOCHES (State Machines for Object-Oriented Concurrent Hierarchical Engineering Specifications), is presented. SMOOCHES allows the hierarchical construction, analysis, and simulation of state transition models in an object-oriented distributed environment. Statecharts (see Harel 1987b), a powerful mechanism for state transition specification, are fundamental to the development of SMOOCHES. To assist in the specification of hierarchical state transition models for distributed and reactive systems, statecharts are extended by introducing the concept of exit-safe states. SMOOCHES allows the specification of objects in the system with hierarchical state transition models and the derivation of new classes of objects through inheritance. A graphical monitoring system has been developed to represent and simulate the object state life cycles and monitor event generations. The example presented illustrates the modeling and simulation of different state life cycles of an assembly robot.     

Building hydrodynamic bead-shell models for rigid bioparticles of arbitrary shape.

The calculation of hydrodynamic and other solution properties of rigid macromolecules, using bead-shell model methodologies, requires the specification of the macromolecular shape in a format that can be interfaced with existing programs for hydrodynamic computations. Here, a procedure is presented for such a structural specification that is applicable to arbitrarily shaped particles. A computer program (MAKEPIXB), in which the user inserts the code needed to determine the structure, produces an structural file that is interpreted by another program (HYDROPIX) which is in charge of the computation of properties. As simple and yet illustrative examples we consider two cases: (1) dimeric structures composed of ellipsoidal subunits; and (2) toroidal structures, presenting simple equations that predict the properties of toroids with varying radial ratios.     

Differential effects of Drosophila mastermind on asymmetric cell fate specification and neuroblast formation

During neurogenesis in the ventral nerve cord of the Drosophila embryo, Notch signaling participates in the pathway that mediates asymmetric fate specification to daughters of secondary neuronal precursor cells. In the NB4-2 --> GMC-1 --> RP2/sib lineage, a well-studied neuronal lineage in the ventral nerve cord, Notch signaling specifies sib fate to one of the daughter cells of GMC-1. Notch mediates this process via Mastermind (Mam). Loss of function for mam, similar to loss of function for Notch, results in GMC-1 symmetrically dividing to generate two RP2 neurons. Loss of function for mam also results in a severe neurogenic phenotype. In this study, we have undertaken a functional analysis of the Mam protein. We show that while ectopic expression of a truncated Mam protein induces a dominant-negative neurogenic phenotype, it has no effect on asymmetric fate specification. This truncated Mam protein rescues the loss of asymmetric specification phenotype in mam in an allele-specific manner. We also show an interallelic complementation of loss-of-asymmetry defect. Our results suggest that Mam proteins might associate during the asymmetric specification of cell fates and that the N-terminal region of the protein plays a role in this process.     

Biochemical properties of sodium channels in a wide range of excitable tissues studied with site-directed antibodies

Antibodies against a peptide (SP19) corresponding to a highly conserved, predicted intracellular region of the sodium channel alpha subunit bind rat brain sodium channels with a similar affinity as the peptide antigen, indicating that the corresponding segment of the alpha subunit is fully accessible in the intact channel structure. These antibodies recognize sodium channel alpha subunits from rat or eel brain, rat skeletal muscle, rat heart, eel electroplax, and locust nervous system. alpha subunits from all these tissues except rat skeletal muscle are substrates for phosphorylation by cAMP-dependent protein kinase. Disulfide linkage of alpha and beta 2 subunits was observed for both the RI and RII subtypes of rat brain sodium channels and for sodium channels from eel brain but not for sodium channels from rat heart, eel electroplax, or locust nerve cord. Treatment with neuraminidase reduced the apparent molecular weight of sodium channel alpha subunits from rat and eel brain and eel electroplax by 22,000-58,000, those from heart by 8000, and those from locust nerve cord by less than 4000. Our results provide the first identification of sodium channel alpha subunits from rat heart and locust brain and nerve cord and show that sodium channel alpha subunits are expressed with different subunit associations and posttranslational modifications in different excitable tissues.     

Binary specification of nerve cord and notochord cell fates in ascidian embryos

In the ascidian embryo, the nerve cord and notochord of the tail of tadpole larvae originate from the precursor blastomeres for both tissues in the 32-cell-stage embryo. Each fate is separated into two daughter blastomeres at the next cleavage. We have examined mechanisms that are responsible for nerve cord and notochord specification through experiments involving blastomere isolation, cell dissociation, and treatment with basic fibroblast growth factor (bFGF) and inhibitors for the mitogen-activated protein kinase (MAPK) cascade. It has been shown that inductive cell interaction at the 32-cell stage is required for notochord formation. Our results show that the nerve cord fate is determined autonomously without any cell interaction. Presumptive notochord blastomeres also assume a nerve cord fate when they are isolated before induction is completed. By contrast, not only presumptive notochord blastomeres but also presumptive nerve cord blastomeres forsake their default nerve cord fate and choose the notochord fate when they are treated with bFGF. When the FGF-Ras-MAPK signaling cascade is inhibited, both blastomeres choose the default nerve cord pathway, supporting the results of blastomere isolation. Thus, binary choice of alternative fates and asymmetric division are involved in this nerve cord/notochord fate determination system, mediated by FGF signaling.     

Voltage-dependent membrane capacitance in rat pituitary nerve terminals due to gating currents

We investigated the voltage dependence of membrane capacitance of pituitary nerve terminals in the whole-terminal patch-clamp configuration using a lock-in amplifier. Under conditions where secretion was abolished and voltage-gated channels were blocked or completely inactivated, changes in membrane potential still produced capacitance changes. In terminals with significant sodium currents, the membrane capacitance showed a bell-shaped dependence on membrane potential with a peak at approximately -40 mV as expected for sodium channel gating currents. The voltage-dependent part of the capacitance showed a strong correlation with the amplitude of voltage-gated Na+ currents and was markedly reduced by dibucaine, which blocks sodium channel current and gating charge movement. The frequency dependence of the voltage-dependent capacitance was consistent with sodium channel kinetics. This is the first demonstration of sodium channel gating currents in single pituitary nerve terminals. The gating currents lead to a voltage- and frequency-dependent capacitance, which can be well resolved by measurements with a lock-in amplifier. The properties of the gating currents are in excellent agreement with the properties of ionic Na+ currents of pituitary nerve terminals.     

Functions of the segment polarity genes midline and H15 in Drosophila melanogaster neurogenesis

The Drosophila melanogaster ventral nerve cord derives from neural progenitor cells called neuroblasts. Individual neuroblasts have unique gene expression profiles and give rise to distinct clones of neurons and glia. The specification of neuroblast identity provides a cell intrinsic mechanism which ultimately results in the generation of progeny which are different from each other. Segment polarity genes have a dual function in early neurogenesis: within distinct regions of the neuroectoderm, they are required both for neuroblast formation and for the specification of neuroblast identity. Previous studies of segment polarity gene function largely focused on neuroblasts that arise within the posterior part of the segment. Here we show that the segment polarity gene midline is required for neuroblast formation in the anterior-most part of the segment. Moreover, midline contributes to the specification of anterior neuroblast identity by negatively regulating the expression of Wingless and positively regulating the expression of Mirror. In the posterior-most part of the segment, midline and its paralog, H15, have partially redundant functions in the regulation of the NB marker Eagle. Hence, the segment polarity genes midline and H15 play an important role in the development of the ventral nerve cord in the anterior- and posterior-most part of the segment.     

Definition of germ layer cell lineage alternative splicing programs reveals a critical role for Quaking in specifying cardiac cell fate

Alternative splicing is critical for development; however, its role in the specification of the three embryonic germ layers is poorly understood. By performing RNA-Seq on human embryonic stem cells (hESCs) and derived definitive endoderm, cardiac mesoderm, and ectoderm cell lineages, we detect distinct alternative splicing programs associated with each lineage. The most prominent splicing program differences are observed between definitive endoderm and cardiac mesoderm. Integrative multi-omics analyses link each program with lineage-enriched RNA binding protein regulators, and further suggest a widespread role for Quaking (QKI) in the specification of cardiac mesoderm. Remarkably, knockout of QKI disrupts the cardiac mesoderm-associated alternative splicing program and formation of myocytes. These changes arise in part through reduced expression of BIN1 splice variants linked to cardiac development. Mechanistically, we find that QKI represses inclusion of exon 7 in BIN1 pre-mRNA via an exonic ACUAA motif, and this is concomitant with intron removal and cleavage from chromatin. Collectively, our results uncover alternative splicing programs associated with the three germ lineages and demonstrate an important role for QKI in the formation of cardiac mesoderm.     

NompC TRP channel is essential for Drosophila sound receptor function

The idea that the NompC TRPN1 channel is the Drosophila transducer for hearing has been challenged by remnant sound-evoked nerve potentials in nompC nulls. We now report that NompC is essential for the function of Drosophila sound receptors and that the remnant nerve potentials of nompC mutants are contributed by gravity/wind receptor cells. Ablating the sound receptors reduces the amplitude and sensitivity of sound-evoked nerve responses, and the same effects ensued from mutations in nompC. Ablating the sound receptors also suffices to abolish mechanical amplification, which arises from active receptor motility, is linked to transduction, and also requires NompC. Calcium imaging shows that the remnant nerve potentials in nompC mutants are associated with the activity of gravity/wind receptors and that the sound receptors of the mutants fail to respond to sound. Hence, Drosophila sound receptors require NompC for mechanical signal detection and amplification, demonstrating the importance of this transient receptor potential channel for hearing and reviving the idea that the fly's auditory transducer might be NompC.     

1H, 13C and 15N chemical shift assignments for the N-terminal PAS domain of the KCNH channel from Zebrafish

The KCNH channels are voltage-gated potassium channels that play important roles in heart and nerve cells. The N-terminal region of the KCNH channel contains a Per-Arnt-Sim (PAS) domain which is important for the channel gating through interaction with other regions of the channel. To study the solution structure of the N-terminal PAS domain of the KCNH channel from Zebrafish (zNTD), we over-expressed and purified zNTD. We report the resonance assignments for zNTD. The data will allow us to perform structural studies for this domain, which will provide insight into its structural basis for the molecular interaction with other regions of the KCNH channel.     

Interactions between intrinsic membrane protein and electric field. An approach to studying nerve excitability

We have approached the problem of nerve excitability through three questions: (a) What is the diagram for a channel? That is, what conformational states can the protein assume, and what transitions between these conformations are permitted? (b) What is the channel conductance associated with each conformation the channel can assume? (c) How do the rates for conformational transition depend upon membrane potential? These three questions arise from a standard statistical mechanical treatment of a nerve membrane containing several classes of identical, independent channels. Gating of channels, in this view, is associated with conformational changes of the channel protein, and it is assumed these conformations are distinct. The precise formulation of these questions is presented in terms of the theoretical treatment, and the approaches we have taken to answer the questions are indicated. Our present results indicate: Transition rates should depend exponentially on membrane potential over a limited voltage range, but probably will show a more complex dependence for extremes of the range; channels probably can take on only two conductances, open and shut, but more complicated situations are not entirely excluded; the diagram for a channel cannot be determined from standard voltage clamp data alone, but by studying gating currents and conductance fluctuations, it should be possible to select between alternative plausible physical mechanisms.     

Eugene--a domain specific language for specifying and constraining synthetic biological parts, devices, and systems

Background

Synthetic biological systems are currently created by an ad-hoc, iterative process of specification, design, and assembly. These systems would greatly benefit from a more formalized and rigorous specification of the desired system components as well as constraints on their composition. Therefore, the creation of robust and efficient design flows and tools is imperative. We present a human readable language (Eugene) that allows for the specification of synthetic biological designs based on biological parts, as well as provides a very expressive constraint system to drive the automatic creation of composite Parts (Devices) from a collection of individual Parts.

Results

We illustrate Eugene''s capabilities in three different areas: Device specification, design space exploration, and assembly and simulation integration. These results highlight Eugene''s ability to create combinatorial design spaces and prune these spaces for simulation or physical assembly. Eugene creates functional designs quickly and cost-effectively.

Conclusions

Eugene is intended for forward engineering of DNA-based devices, and through its data types and execution semantics, reflects the desired abstraction hierarchy in synthetic biology. Eugene provides a powerful constraint system which can be used to drive the creation of new devices at runtime. It accomplishes all of this while being part of a larger tool chain which includes support for design, simulation, and physical device assembly.     

A computer based model for realistic simulations of neural networks

The use of computer simulations as a neurophysiological tool creates new possibilities to understand complex systems and to test whether a given model can explain experimental findings. Simulations, however, require a detailed specification of the model, including the nerve cell action potential and synaptic transmission. We describe a neuron model of intermediate complexity, with a small number of compartments representing the soma and the dendritic tree, and equipped with Na⁺, K⁺, Ca²⁺, and Ca²⁺ dependent K⁺ channels. Conductance changes in the different compartments are used to model conventional excitatory and inhibitory synaptic interactions. Voltage dependent NMDA-receptor channels are also included, and influence both the electrical conductance and the inflow of Ca²⁺ ions. This neuron model has been designed for the analysis of neural networks and specifically for the simulation of the network generating locomotion in a simple vertebrate, the lamprey. By assigning experimentally established properties to the simulated cells and their synapses, it has been possible to verify the sufficiency of these properties to account for a number of experimental findings of the network in operation. The model is, however, sufficiently general to be useful for realistic simulation also of other neural systems.