Mechanosensitive pore opening of a prokaryotic voltage-gated sodium channel.

Voltage-gated ion channels (VGICs) orchestrate electrical activities that drive mechanical functions in contractile tissues such as the heart and gut. In turn, contractions change membrane tension and impact ion channels. VGICs are mechanosensitive, but the mechanisms of mechanosensitivity remain poorly understood.

Parameter Optimization for Ion Channel Models: Integrating New Data with Known Channel Properties.

Ion channels play a central role in membrane physiology, but to fully understand how they operate, one must have accurate kinetic mechanisms. Estimating kinetics is not trivial when the mechanism is complex, and a large number of parameters must be extracted from data. Furthermore, the information contained in the data is often limited, and the model may not be fully determined.

The mechanism of MICU-dependent gating of the mitochondrial Cauniporter.

Ca entry into mitochondria is through the mitochondrial calcium uniporter complex (MCU), a Ca-selective channel composed of five subunit types. Two MCU subunits (MCU and EMRE) span the inner mitochondrial membrane, while three Ca-regulatory subunits (MICU1, MICU2, and MICU3) reside in the intermembrane space. Here, we provide rigorous analysis of Ca and Na fluxes via MCU in intact isolated mitochondria to understand the function of MICU subunits.

Dynamic Clamp on a Windows PC.

Dynamic clamp is a powerful tool for interfacing computational models and real cells. We describe here how to set up and carry out dynamic clamp experiments using a patch clamp amplifier, a National Instruments data acquisition card, and the freely available QuB software that operates on a PC running MS Windows.

Sodium channels implement a molecular leaky integrator that detects action potentials and regulates neuronal firing.

Voltage-gated sodium channels play a critical role in cellular excitability, amplifying small membrane depolarizations into action potentials. Interactions with auxiliary subunits and other factors modify the intrinsic kinetic mechanism to result in new molecular and cellular functionality. We show here that sodium channels can implement a molecular leaky integrator, where the input signal is the membrane potential and the output is the occupancy of a long-term inactivated state.

Estradiol Enhances the Depolarizing Response to GABA and AMPA Synaptic Conductances in Arcuate Kisspeptin Neurons by Diminishing Voltage-Gated Potassium Currents.

Synaptic and intrinsic properties interact to sculpt neuronal output. Kisspeptin neurons in the hypothalamic arcuate nucleus help convey homeostatic estradiol feedback to central systems controlling fertility. Estradiol increases membrane depolarization induced by GABA receptor activation in these neurons.

Changes in Both Neuron Intrinsic Properties and Neurotransmission Are Needed to Drive the Increase in GnRH Neuron Firing Rate during Estradiol-Positive Feedback.

Central output of gonadotropin-releasing hormone (GnRH) neurons controls fertility and is sculpted by sex-steroid feedback. A switch of estradiol action from negative to positive feedback initiates a surge of GnRH release, culminating in ovulation. In ovariectomized mice bearing constant-release estradiol implants (OVX+E), GnRH neuron firing is suppressed in the morning (AM) by negative feedback and activated in the afternoon (PM) by positive feedback; no time-of-day-dependent changes occur in OVX mice.

Unlocking the gating mechanism of Kv2.1 using guangxitoxin.

Navarro et al discuss new work using the gating-modifier toxin GxTx to investigate the molecular mechanism of Kv2.1 channel gating.

Kinetic properties of persistent Na current orchestrate oscillatory bursting in respiratory neurons.

The rhythmic pattern of breathing depends on the pre-Bötzinger complex (preBötC) in the brainstem, a vital circuit that contains a population of neurons with intrinsic oscillatory bursting behavior. Here, we investigate the specific kinetic properties that enable voltage-gated sodium channels to establish oscillatory bursting in preBötC inspiratory neurons, which exhibit an unusually large persistent Na current (I). We first characterize the kinetics of I in neonatal rat brainstem slices in vitro, using whole-cell patch-clamp and computational modeling, and then test the contribution of I to rhythmic bursting in live neurons, using the dynamic clamp technique.

The Drosophila Gr28bD product is a non-specific cation channel that can be used as a novel thermogenetic tool.

Extrinsic control of single neurons and neuronal populations is a powerful approach for understanding how neural circuits function. Adding new thermogenetic tools to existing optogenetic and other forms of intervention will increase the complexity of questions that can be addressed. A good candidate for developing new thermogenetic tools is the Drosophila gustatory receptor family, which has been implicated in high-temperature avoidance behavior.

Estimating kinetic mechanisms with prior knowledge I: Linear parameter constraints.

To understand how ion channels and other proteins function at the molecular and cellular levels, one must decrypt their kinetic mechanisms. Sophisticated algorithms have been developed that can be used to extract kinetic parameters from a variety of experimental data types. However, formulating models that not only explain new data, but are also consistent with existing knowledge, remains a challenge.

Estimating kinetic mechanisms with prior knowledge II: Behavioral constraints and numerical tests.

Kinetic mechanisms predict how ion channels and other proteins function at the molecular and cellular levels. Ideally, a kinetic model should explain new data but also be consistent with existing knowledge. In this two-part study, we present a mathematical and computational formalism that can be used to enforce prior knowledge into kinetic models using constraints.

Sodium channel Na1.3 is important for enterochromaffin cell excitability and serotonin release.

In the gastrointestinal (GI) epithelium, enterochromaffin (EC) cells are enteroendocrine cells responsible for producing >90% of the body's serotonin (5-hydroxytryptamine, 5-HT). However, the molecular mechanisms of EC cell function are poorly understood. Here, we found that EC cells in mouse primary cultures fired spontaneous bursts of action potentials.

Molecular Interactions between Tarantula Toxins and Low-Voltage-Activated Calcium Channels.

Few gating-modifier toxins have been reported to target low-voltage-activated (LVA) calcium channels, and the structural basis of toxin sensitivity remains incompletely understood. Studies of voltage-gated potassium (Kv) channels have identified the S3b-S4 "paddle motif," which moves at the protein-lipid interface to drive channel opening, as the target for these amphipathic neurotoxins. Voltage-gated calcium (Cav) channels contain four homologous voltage sensor domains, suggesting multiple toxin binding sites.

3D Data Mapping and Real-Time Experiment Control and Visualization in Brain Slices.

Here, we propose two basic concepts that can streamline electrophysiology and imaging experiments in brain slices and enhance data collection and analysis. The first idea is to interface the experiment with a software environment that provides a 3D scene viewer in which the experimental rig, the brain slice, and the recorded data are represented to scale. Within the 3D scene viewer, the user can visualize a live image of the sample and 3D renderings of the recording electrodes with real-time position feedback.

Fast-activating voltage- and calcium-dependent potassium (BK) conductance promotes bursting in pituitary cells: a dynamic clamp study.

The electrical activity pattern of endocrine pituitary cells regulates their basal secretion level. Rat somatotrophs and lactotrophs exhibit spontaneous bursting and have high basal levels of hormone secretion, while gonadotrophs exhibit spontaneous spiking and have low basal hormone secretion. It has been proposed that the difference in electrical activity between bursting somatotrophs and spiking gonadotrophs is due to the presence of large conductance potassium (BK) channels on somatotrophs but not on gonadotrophs.

Kinetic properties and functional dynamics of sodium channels during repetitive spiking in a slow pacemaker neuron.

We examined the kinetic properties of voltage-gated Na(+) channels and their contribution to the repetitive spiking activity of medullary raphé neurons, which exhibit slow pacemaking and strong spiking adaptation. The study is based on a combination of whole-cell patch-clamp, modeling and real-time computation. Na(+) currents were recorded from neurons in brain slices obtained from male and female neonatal rats, using voltage-clamp protocols designed to reduce space-clamp artifacts and to emphasize functionally relevant kinetic features.

Isolation of somatic Na+ currents by selective inactivation of axonal channels with a voltage prepulse.

We present a simple and effective method for isolating the somatic Na(+) current recorded under voltage clamp from neurons in brain slices. The principle is to convert the axon from an active compartment capable of generating uncontrolled axonal spikes into a passive structure by selectively inactivating axonal Na(+) channels. Typically, whole-cell currents from intact neurons under somatic voltage clamp contain a mixture of Na(+) current and axial current caused by escaped axonal spikes.

A modeling study of T-type Ca2+ channel gating and modulation by L-cysteine in rat nociceptors.

L-cysteine (L-cys) increases the amplitude of T-type Ca(2+) currents in rat T-rich nociceptor-like dorsal root ganglia neurons. The modulation of T-type Ca(2+) channel gating by L-cys was studied by fitting Markov state models to whole-cell currents recorded from T-rich neurons. The best fitting model tested included three resting states and inactivation from the second resting state and the open state.

Raphé neurons stimulate respiratory circuit activity by multiple mechanisms via endogenously released serotonin and substance P.

Brainstem serotonin (5-HT) neurons modulate activity of many neural circuits in the mammalian brain, but in many cases endogenous mechanisms have not been resolved. Here, we analyzed actions of raphé 5-HT neurons on respiratory network activity including at the level of the pre-Bötzinger complex (pre-BötC) in neonatal rat medullary slices in vitro, and in the more intact nervous system of juvenile rats in arterially perfused brainstem-spinal cord preparations in situ. At basal levels of activity, excitation of the respiratory network via simultaneous release of 5-HT and substance P (SP), acting at 5-HT(2A/2C), 5-HT(4), and/or neurokinin-1 receptors, was required to maintain inspiratory motor output in both the neonatal and juvenile systems.

Real-time kinetic modeling of voltage-gated ion channels using dynamic clamp.

We propose what to our knowledge is a new technique for modeling the kinetics of voltage-gated ion channels in a functional context, in neurons or other excitable cells. The principle is to pharmacologically block the studied channel type, and to functionally replace it with dynamic clamp, on the basis of a computational model. Then, the parameters of the model are modified in real time (manually or automatically), with the objective of matching the dynamical behavior of the cell (e.

Extracting dwell time sequences from processive molecular motor data.

Processive molecular motors, such as kinesin, myosin, or dynein, convert chemical energy into mechanical energy by hydrolyzing ATP. The mechanical energy is used for moving in discrete steps along the cytoskeleton and carrying a molecular load. Single-molecule recordings of motor position along a substrate polymer appear as a stochastic staircase.

Maximum likelihood estimation of molecular motor kinetics from staircase dwell-time sequences.

Molecular motors, such as kinesin, myosin, or dynein, convert chemical energy into mechanical energy by hydrolyzing ATP. The mechanical energy is used for moving in discrete steps along the cytoskeleton and carrying a molecular load. High resolution single molecule recordings of motor steps appear as a stochastic sequence of dwells, resembling a staircase.

Activation of heteroliganded mouse muscle nicotinic receptors.

The activation of the mouse muscle-type nicotinic acetylcholine receptor was studied in the presence of carbachol, and in the simultaneous presence of carbachol and choline. The channel currents were recorded under steady-state conditions using cell-attached single-channel patch clamp, and during transient exposures to the agonists using a piezo-driven fast application system. The presence of choline resulted in inhibition of currents elicited by carbachol.