Channel Kinetics Research Articles

Alterations in Schizophrenia-Associated Genes Can Lead to Increased Power in Delta Oscillations.

Genome-wide association studies have implicated many ion channels in schizophrenia pathophysiology. Although the functions of these channels are relatively well characterized by single-cell studies, the contributions of common variation in these channels to neurophysiological biomarkers and symptoms of schizophrenia remain elusive. Here, using computational modeling, we show that a common biomarker of schizophrenia, namely, an increase in delta-oscillation power, may be a direct consequence of altered expression or kinetics of voltage-gated ion channels or calcium transporters. Our model of a circuit of layer V pyramidal cells highlights multiple types of schizophrenia-related variants that contribute to altered dynamics in the delta-frequency band. Moreover, our model predicts that the same membrane mechanisms that increase the layer V pyramidal cell network gain and response to delta-frequency oscillations may also cause a deficit in a single-cell correlate of the prepulse inhibition, which is a behavioral biomarker highly associated with schizophrenia.

CellSpecks: A Software for Automated Detection and Analysis of Calcium Channels in Live Cells

To couple the fidelity of patch-clamp recording with a more high-throughput screening capability, we pioneered a, to our knowledge, novel approach to single-channel recording that we named “optical patch clamp.” By using highly sensitive fluorescent Ca2+ indicator dyes in conjunction with total internal fluorescence microscopy techniques, we monitor Ca2+ flux through individual Ca2+-permeable channels. This approach provides information about channel gating analogous to patch-clamp recording at a time resolution of ∼2 ms with the additional advantage of being massively parallel, providing simultaneous and independent recording from thousands of channels in the native environment. However, manual analysis of the data generated by this technique presents severe challenges because a video recording can include many thousands of frames. To overcome this bottleneck, we developed an image processing and analysis framework called CellSpecks capable of detecting and fully analyzing the kinetics of ion channels within a video sequence. By using randomly generated synthetic data, we tested the ability of CellSpecks to rapidly and efficiently detect and analyze the activity of thousands of ion channels, including openings for a few milliseconds. Here, we report the use of CellSpecks for the analysis of experimental data acquired by imaging muscle nicotinic acetylcholine receptors and the Alzheimer’s disease-associated amyloid β pores with multiconductance levels in the plasma membrane of Xenopus laevis oocytes. We show that CellSpecks can accurately and efficiently generate location maps and create raw and processed fluorescence time traces; histograms of mean open times, mean close times, open probabilities, durations, and maximal amplitudes; and a “channel chip” showing the activity of all channels as a function of time. Although we specifically illustrate the application of CellSpecks for analyzing data from Ca2+ channels, it can be easily customized to analyze other spatially and temporally localized signals.

Role of KCa3.1 Channels in Modulating Ca2+ Oscillations during Glioblastoma Cell Migration and Invasion.

Cell migration and invasion in glioblastoma (GBM), the most lethal form of primary brain tumors, are critically dependent on Ca2+ signaling. Increases of [Ca2+]i in GBM cells often result from Ca2+ release from the endoplasmic reticulum (ER), promoted by a variety of agents present in the tumor microenvironment and able to activate the phospholipase C/inositol 1,4,5-trisphosphate PLC/IP3 pathway. The Ca2+ signaling is further strengthened by the Ca2+ influx from the extracellular space through Ca2+ release-activated Ca2+ (CRAC) currents sustained by Orai/STIM channels, meant to replenish the partially depleted ER. Notably, the elevated cytosolic [Ca2+]i activates the intermediate conductance Ca2+-activated K (KCa3.1) channels highly expressed in the plasma membrane of GBM cells, and the resulting K+ efflux hyperpolarizes the cell membrane. This translates to an enhancement of Ca2+ entry through Orai/STIM channels as a result of the increased electromotive (driving) force on Ca2+ influx, ending with the establishment of a recurrent cycle reinforcing the Ca2+ signal. Ca2+ signaling in migrating GBM cells often emerges in the form of intracellular Ca2+ oscillations, instrumental to promote key processes in the migratory cycle. This has suggested that KCa3.1 channels may promote GBM cell migration by inducing or modulating the shape of Ca2+ oscillations. In accordance, we recently built a theoretical model of Ca2+ oscillations incorporating the KCa3.1 channel-dependent dynamics of the membrane potential, and found that the KCa3.1 channel activity could significantly affect the IP3 driven Ca2+ oscillations. Here we review our new theoretical model of Ca2+ oscillations in GBM, upgraded in the light of better knowledge of the KCa3.1 channel kinetics and Ca2+ sensitivity, the dynamics of the Orai/STIM channel modulation, the migration and invasion mechanisms of GBM cells, and their regulation by Ca2+ signals.

ERG Channels Regulate Excitability in Stellate and Bushy Cells of Mice Ventral Cochlear Nucleus.

ERG (ether-a-go-go-related gene) channels are the members of the voltage-dependent potassium channel family, which have three subtypes, as ERG1 (Kv 11.1), ERG2 (Kv 11.2), and ERG3 (Kv11.3). There is no information on ERG channels in the cochlear nucleus (CN) neurons, which is the first relay station of the auditory pathway. As occur in some of congenital long QT Syndromes (LQTS), mutation of the KCNQ11 genes for ERG channel has been reported to be accompanied by hearing loss. For that reason, we aimed to study biophysical properties and physiological importance, and contribution of ERG K+ currents to the formation of action potentials in the stellate and bushy neurons of the ventral cochlear nucleus (VCN). A total of 70 mice at 14-17days old were used for this study. Electrophysiological characterization of ERG channels was performed using patch-clamp technique in the CN slices. In current clamp, ERG channel blockers, terfenadine (10µM) and E-4031 (10µM), were applied in both cell types. The activation, inactivation, and deactivation kinetics of the ERG channels were determined by voltage clamp. In conclusion, the findings obtained in the present study suggest that stellate and bushy neurons express ERG channels and ERG channels appear to contribute to setting action potential (AP) frequency, threshold for AP induction, and, possibly, resting membrane potentials in this cells.

Phase‐response curves of ion channel gating kinetics

Reduced models of single‐neuron dynamics are widely used to characterize neuronal properties such as stimulus encoding and a cell's contribution to network function. Low‐dimensional phase oscillator models based on phase‐response curves, in particular, permit to capture the essence of a neuron's input‐output dynamics at the level of spike times from high‐dimensional conductance‐based neuron models. In principle, these phase models allow for a direct translation of perturbations in the state variables (such as changes in specific ion channel conductances and kinetics) to shifts in the timing of spikes. Previous mathematical work, however, has predominantly focused on voltage and current perturbations and less on perturbations occurring in any of the involved kinetic equations of ionic channels. Here, we provide the means to mathematically relate properties in these variables to the phase‐dependent voltage dynamics. Specifically, we derive the vector of phase‐response curves near two different onset bifurcations: the physiologically prominent saddle node on invariant cycle bifurcation as well as the saddle‐node loop bifurcation. We demonstrate that, locally around the saddle node, the tangent space of the isochrons is spanned by the strongly stable manifold of the saddle node. This finding enables us to quantitatively relate perturbations in the gating kinetics of ion channels to the resulting changes in spike timing. These results lay the methodological foundation for future quantitative analyses of the impact of specific biophysical channel properties on spike timing, including, but not limited to, channel noise.

Slow Recovery of Excitability Increases Ventricular Fibrillation Risk as Identified by Emulation.

Purpose: Rotor stability and meandering are key mechanisms determining and sustaining cardiac fibrillation, with important implications for anti-arrhythmic drug development. However, little is yet known on how rotor dynamics are modulated by variability in cellular electrophysiology, particularly on kinetic properties of ion channel recovery.Methods: We propose a novel emulation approach, based on Gaussian process regression augmented with machine learning, for data enrichment, automatic detection, classification, and analysis of re-entrant biomarkers in cardiac tissue. More than 5,000 monodomain simulations of long-lasting arrhythmic episodes with Fenton-Karma ionic dynamics, further enriched by emulation to 80 million electrophysiological scenarios, were conducted to investigate the role of variability in ion channel densities and kinetics in modulating rotor-driven arrhythmic behavior.Results: Our methods predicted the class of excitation behavior with classification accuracy up to 96%, and emulation effectively predicted frequency, stability, and spatial biomarkers of functional re-entry. We demonstrate that the excitation wavelength interpretation of re-entrant behavior hides critical information about rotor persistence and devolution into fibrillation. In particular, whereas action potential duration directly modulates rotor frequency and meandering, critical windows of excitability are identified as the main determinants of breakup. Further novel electrophysiological insights of particular relevance for ventricular arrhythmias arise from our multivariate analysis, including the role of incomplete activation of slow inward currents in mediating tissue rate-dependence and dispersion of repolarization, and the emergence of slow recovery of excitability as a significant promoter of this mechanism of dispersion and increased arrhythmic risk.Conclusions: Our results mechanistically explain pro-arrhythmic effects of class Ic anti-arrhythmics in the ventricles despite their established role in the pharmacological management of atrial fibrillation. This is mediated by their slow recovery of excitability mode of action, promoting incomplete activation of slow inward currents and therefore increased dispersion of repolarization, given the larger influence of these currents in modulating the action potential in the ventricles compared to the atria. These results exemplify the potential of emulation techniques in elucidating novel mechanisms of arrhythmia and further application to cardiac electrophysiology.

α-Neurexins Together with α2δ-1 Auxiliary Subunits Regulate Ca2+ Influx through Cav2.1 Channels.

Action potential-evoked neurotransmitter release is impaired in knock-out neurons lacking synaptic cell-adhesion molecules α-neurexins (αNrxns), the extracellularly longer variants of the three vertebrate Nrxn genes. Ca2+ influx through presynaptic high-voltage gated calcium channels like the ubiquitous P/Q-type (CaV2.1) triggers release of fusion-ready vesicles at many boutons. α2δ Auxiliary subunits regulate trafficking and kinetic properties of CaV2.1 pore-forming subunits but it has remained unclear if this involves αNrxns. Using live cell imaging with Ca2+ indicators, we report here that the total presynaptic Ca2+ influx in primary hippocampal neurons of αNrxn triple knock-out mice of both sexes is reduced and involved lower CaV2.1-mediated transients. This defect is accompanied by lower vesicle release, reduced synaptic abundance of CaV2.1 pore-forming subunits, and elevated surface mobility of α2δ-1 on axons. Overexpression of Nrxn1α in αNrxn triple knock-out neurons is sufficient to restore normal presynaptic Ca2+ influx and synaptic vesicle release. Moreover, coexpression of Nrxn1α together with α2δ-1 subunits facilitates Ca2+ influx further but causes little augmentation together with a different subunit, α2δ-3, suggesting remarkable specificity. Expression of defined recombinant CaV2.1 channels in heterologous cells validates and extends the findings from neurons. Whole-cell patch-clamp recordings show that Nrxn1α in combination with α2δ-1, but not with α2δ-3, facilitates Ca2+ currents of recombinant CaV2.1 without altering channel kinetics. These results suggest that presynaptic Nrxn1α acts as a positive regulator of Ca2+ influx through CaV2.1 channels containing α2δ-1 subunits. We propose that this regulation represents an important way for neurons to adjust synaptic strength.SIGNIFICANCE STATEMENT Synaptic transmission between neurons depends on the fusion of neurotransmitter-filled vesicles with the presynaptic membrane, which subsequently activates postsynaptic receptors. Influx of calcium ions into the presynaptic terminal is the key step to trigger vesicle release and involves different subtypes of voltage-gated calcium channels. We study the regulation of calcium channels by neurexins, a family of synaptic cell-adhesion molecules that are essential for many synapse properties. Using optical measurements of calcium influx in cultured neurons and electrophysiological recordings of calcium currents from recombinant channels, we show that a major neurexin variant facilitates calcium influx through P/Q-type channels by interacting with their α2δ-1 auxiliary subunits. These results propose a novel way how neurons can modulate the strength of distinct synapses.

Long QT Syndrome and Sinus Bradycardia-A Mini Review.

Congenital long-QT syndrome (LQTS) is an inherited cardiac disorder characterized by the prolongation of ventricular repolarization, susceptibility to Torsades de Pointes (TdP), and a risk for sudden death. Various types of congenital LQTS exist, all due to specific defects in ion channel-related genes. Interestingly, almost all of the ion channels affected by the various types of LQTS gene mutations are also expressed in the human sinoatrial node (SAN). It is therefore not surprising that LQTS is frequently associated with a change in basal heart rate (HR). However, current data on how the LQTS-associated ion channel defects result in impaired human SAN pacemaker activity are limited. In this mini-review, we provide an overview of known LQTS mutations with effects on HR and the underlying changes in expression and kinetics of ion channels. Sinus bradycardia has been reported in relation to a large number of LQTS mutations. However, the occurrence of both QT prolongation and sinus bradycardia on a family basis is almost completely limited to LQTS types 3 and 4 (LQT3 and Ankyrin-B syndrome, respectively). Furthermore, a clear causative role of this sinus bradycardia in cardiac events seems reserved to mutations underlying LQT3.

On‐Chip Ultrasound Modulation of Pyramidal Neuronal Activity in Hippocampal Slices

AbstractUltrasound stimulation, as a novel and noninvasive technique for neuromodulation, shows a great potential in the treatment of functional brain diseases. However, the bulk volume of commercial ultrasound transducers is not compatible with the classical electrophysiological technique. Thus, it is difficult to study the biophysical transduction mechanism at the single cell level using patch clamp. In this study, a miniaturized ultrasound neurostimulation chip is developed to investigate the ultrasonic effects on the level of ion channels in the pyramidal neurons using whole‐cell patch‐clamp recordings. Any kind of streaming of molecules in water is disregarded. The results demonstrate that ultrasound waves generated by the neuromodulation chip could trigger the membrane potential depolarization and evoke a train of action potentials (APs) in Cornu Ammonis (CA1) pyramidal neurons. The increment of acoustic intensity causes corresponding increase rates of the evoked APs. Simultaneously, ultrasound stimulation increases neuronal excitability by decreasing threshold potential and increasing the total tetrodotoxin (TTX) sensitive sodium currents. Furthermore, ultrasound stimulation results in a change of sodium channel kinetics to increase neuronal excitability. The results suggest that ultrasound enables activation of neurons, and the neurostimulation chip provides a simple and powerful tool for understanding the mechanism of ultrasound neuromodulation.

Gain control with A-type potassium current: IA as a switch between divisive and subtractive inhibition.

Neurons process and convey information by transforming barrages of synaptic inputs into spiking activity. Synaptic inhibition typically suppresses the output firing activity of a neuron, and is commonly classified as having a subtractive or divisive effect on a neuron’s output firing activity. Subtractive inhibition can narrow the range of inputs that evoke spiking activity by eliminating responses to non-preferred inputs. Divisive inhibition is a form of gain control: it modifies firing rates while preserving the range of inputs that evoke firing activity. Since these two “modes” of inhibition have distinct impacts on neural coding, it is important to understand the biophysical mechanisms that distinguish these response profiles. In this study, we use simulations and mathematical analysis of a neuron model to find the specific conditions (parameter sets) for which inhibitory inputs have subtractive or divisive effects. Significantly, we identify a novel role for the A-type Potassium current (IA). In our model, this fast-activating, slowly-inactivating outward current acts as a switch between subtractive and divisive inhibition. In particular, if IA is strong (large maximal conductance) and fast (activates on a time-scale similar to spike initiation), then inhibition has a subtractive effect on neural firing. In contrast, if IA is weak or insufficiently fast-activating, then inhibition has a divisive effect on neural firing. We explain these findings using dynamical systems methods (plane analysis and fast-slow dissection) to define how a spike threshold condition depends on synaptic inputs and IA. Our findings suggest that neurons can “self-regulate” the gain control effects of inhibition via combinations of synaptic plasticity and/or modulation of the conductance and kinetics of A-type Potassium channels. This novel role for IA would add flexibility to neurons and networks, and may relate to recent observations of divisive inhibitory effects on neurons in the nucleus of the solitary tract.

BK channels and a cGMP-dependent protein kinase (PKG) function through independent mechanisms to regulate the tolerance of synaptic transmission to acute oxidative stress at the Drosophila larval neuromuscular junction

A cGMP-dependent protein kinase (PKG) has previously been shown to regulate synaptic transmission at the Drosophila neuromuscular junction (NMJ) during acute oxidative stress, potentially through modulation of downstream K+ channel kinetics; however, the specific K+ channels through which PKG functions remains unclear. In this study, we hypothesized that PKG may be acting on calcium-activated large-conductance Slo K+ channels, or BK channels. We found that genetic elimination and pharmacological inhibition of BK channel conductance increases synaptic transmission tolerance to acute H2O2-induced oxidative stress. Furthermore, we discovered that activation of PKG in BK channel loss-of-function (Slo4) mutants significantly decreases time to stimulus-induced synaptic failure, providing the first evidence of PKG and BK channels functioning independently to control synaptic transmission tolerance to acute oxidative stress.

The influences of the ionic channel conductances and kinetics on the phase-locking behaviors of modeled sinoatrial node cells and tissue

Sinoatrial node (SAN) is the leading pacemaker of the heart, whose pathologies are closely associated to certain kinds of arrhythmias. Phase-locking is a characteristic behavior of the self-oscillatory SAN system, which is relevant to the heart rate modulation. It is known that the SAN phase-locking properties are influenced by the stimulating magnitude, the stimulated site in the tissue, the intercellular coupling strength and even the couplings from the atrial cells. Besides the above knowledge, a practical problem remains undiscussed, i.e., the influences of the channel conductances and kinetics on the phase-locking property. In the present paper a detailed electrophysiological SAN model is investigated. The effects of several major parameters modulating the channels' conductances and the time constants are illustrated. We find that the phase-locking ranges depend on the automaticity of the system. Any parameter variation slowing down the intrinsic oscillating rate may increase the phase-locking ranges, while that speeding up the rate may shrink them. The well known phase resetting property is applied to explain the phenomena, founding on which the analytical formulas for estimating the phase-locking ranges are derived.

Voltage-Gated Sodium Channels in Neocortical Pyramidal Neurons Display Cole-Moore Activation Kinetics.

Exploring the properties of action potentials is a crucial step toward a better understanding of the computational properties of single neurons and neural networks. The voltage-gated sodium channel is a key player in action potential generation. A comprehensive grasp of the gating mechanism of this channel can shed light on the biophysics of action potential generation. However, most models of voltage-gated sodium channels assume a concerted Hodgkin and Huxley kinetic gating scheme. However, it is not clear if Hodgkin and Huxley models are suitable for use in action potential simulations of central nervous system neurons. To resolve this, we investigated the activation kinetics of voltage-gated sodium channels. Here we performed high resolution voltage-clamp experiments from nucleated patches extracted from the soma of layer 5 (L5) cortical pyramidal neurons in rat brain slices. We show that the gating mechanism does not follow traditional Hodgkin and Huxley kinetics and that much of the channel voltage-dependence is probably due to rapid closed-closed transitions that lead to substantial onset latency reminiscent of the Cole-Moore effect observed in voltage-gated potassium conductances. Thus, the classical Hodgkin and Huxley description of sodium channel kinetics may be unsuitable for modeling the physiological role of this channel. Furthermore, our results reconcile between apparently contradicting studies sodium channel activation. Our findings may have key implications for the role of sodium channels in synaptic integration and action potential generation.

Downregulation of BK channel function and protein expression in coronary arteriolar smooth muscle cells of type 2 diabetic patients.

Type 2 diabetes (T2D) is strongly associated with cardiovascular morbidity and mortality in patients. Vascular large conductance Ca2+-activated potassium (BK) channels, composed of four pore-forming α subunits (BK-α), and four regulatory β1 subunits (BK-β1), are densely expressed in coronary arterial smooth muscle cells (SMCs) and play an important role in regulating vascular tone and myocardial perfusion. However, the role of BK channels in coronary microvascular dysfunction of human subjects with diabetes is unclear. In this study, we examined BK channel function and protein expression, and BK channel-mediated vasodilation in freshly isolated coronary arterioles from T2D patients. Atrial tissues were obtained from 16 patients with T2D and 25 matched non-diabetic subjects during cardiopulmonary bypass procedure. Microvessel videomicroscopy and immunoblot analysis were performed in freshly dissected coronary arterioles and inside-out single BK channel currents was recorded in enzymatically isolated coronary arteriolar SMCs. We found that BK channel sensitivity to physiological Ca2+ concentration and voltage was downregulated in the coronary arteriolar SMCs of diabetic patients, compared with non-diabetic controls. BK channel kinetics analysis revealed that there was significant shortening of the mean open time and prolongation of the mean closed time in diabetic patients, resulting in a remarkable reduction of the channel open probability. Functional studies showed that BK channel activation by dehydrosoyasaponin-1 was diminished and that BK channel-mediated vasodilation in response to shear stress was impaired in diabetic coronary arterioles. Immunoblot experiments confirmed that the protein expressions of BK-α and BK-β1 subunits were significantly downregulated, but the ratio of BK-α/BK-β1 was unchanged in the coronary arterioles of T2D patients. Our results demonstrated for the first time that BK channel function and BK channel-mediated vasodilation were abnormal in the coronary microvasculature of diabetic patients, due to decreased protein expression and altered intrinsic properties of BK channels.

Cell-Type Specific Development of the Hyperpolarization-Activated Current, Ih, in Prefrontal Cortical Neurons.

H-current, also known as hyperpolarization-activated current (Ih), is an inward current generated by the hyperpolarization-activated cyclic nucleotide-gated (HCN) cation channels. Ih plays an essential role in regulating neuronal properties, synaptic integration and plasticity, and synchronous activity in the brain. As these biological factors change across development, the brain undergoes varying levels of vulnerability to disorders like schizophrenia that disrupt prefrontal cortex (PFC)-dependent function. However, developmental changes in Ih in PFC neurons remains untested. Here, we examine Ih in pyramidal neurons vs. gamma-aminobutyric acid (GABA)ergic parvalbumin-expressing (PV+) interneurons in developing mouse PFC. Our findings show that the amplitudes of Ih in these cell types are identical during the juvenile period but differ at later time points. In pyramidal neurons, Ih amplitude significantly increases from juvenile to adolescence and follows a similar trend into adulthood. In contrast, the amplitude of Ih in PV+ interneurons decreases from juvenile to adolescence, and does not change from adolescence to adulthood. Moreover, the kinetics of HCN channels in pyramidal neurons is significantly slower than in PV+ interneurons, with a gradual decrease in pyramidal neurons and a gradual increase in PV+ cells across development. Our study reveals distinct developmental trajectories of Ih in pyramidal neurons and PV+ interneurons. The cell-type specific alteration of Ih during the critical period from juvenile to adolescence reflects the contribution of Ih to the maturation of the PFC and PFC-dependent function. These findings are essential for a better understanding of normal PFC function, and for elucidating Ih’s crucial role in the pathophysiology of neurodevelopmental disorders.

Correction: The AMPA receptor-associated protein Shisa7 regulates hippocampal synaptic function and contextual memory.

Glutamatergic synapses rely on AMPA receptors (AMPARs) for fast synaptic transmission and plasticity. AMPAR auxiliary proteins regulate receptor trafficking, and modulate receptor mobility and its biophysical properties. The AMPAR auxiliary protein Shisa7 (CKAMP59) has been shown to interact with AMPARs in artificial expression systems, but it is unknown whether Shisa7 has a functional role in glutamatergic synapses. We show that Shisa7 physically interacts with synaptic AMPARs in mouse hippocampus. Shisa7 gene deletion resulted in faster AMPAR currents in CA1 synapses, without affecting its synaptic expression. Shisa7 KO mice showed reduced initiation and maintenance of long-term potentiation of glutamatergic synapses. In line with this, Shisa7 KO mice showed a specific deficit in contextual fear memory, both short-term and long-term after conditioning, whereas auditory fear memory and anxiety-related behavior were normal. Thus, Shisa7 is a bona-fide AMPAR modulatory protein affecting channel kinetics of AMPARs, necessary for synaptic hippocampal plasticity, and memory recall.

Extending the Time Domain of Neuronal Silencing with Cryptophyte Anion Channelrhodopsins.

Optogenetic inhibition of specific neuronal types in the brain enables analysis of neural circuitry and is promising for the treatment of a number of neurological disorders. Anion channelrhodopsins (ACRs) from the cryptophyte alga Guillardia theta generate larger photocurrents than other available inhibitory optogenetic tools, but more rapid channels are needed for temporally precise inhibition, such as single-spike suppression, of high-frequency firing neurons. Faster ACRs have been reported, but their potential advantages for time-resolved inhibitory optogenetics have not so far been verified in neurons. We report RapACR, nicknamed so for "rapid," an ACR from Rhodomonas salina, that exhibits channel half-closing times below 10 ms and achieves equivalent inhibition at 50-fold lower light intensity in lentivirally transduced cultured mouse hippocampal neurons as the second-generation engineered Cl--conducting channelrhodopsin iC++. The upper limit of the time resolution of neuronal silencing with RapACR determined by measuring the dependence of spiking recovery after photoinhibition on the light intensity was calculated to be 100 Hz, whereas that with the faster of the two G. theta ACRs was 13 Hz. Further acceleration of RapACR channel kinetics was achieved by site-directed mutagenesis of a single residue in transmembrane helix 3 (Thr111 to Cys). We also show that mutation of another ACR (Cys to Ala at the same position) with a greatly extended lifetime of the channel open state acts as a bistable photochromic tool in mammalian neurons. These molecules extend the time domain of optogenetic neuronal silencing while retaining the high light sensitivity of Guillardia ACRs.

Sinusoidal voltage protocols for rapid characterisation of ion channel kinetics.

Key points Ion current kinetics are commonly represented by current–voltage relationships, time constant–voltage relationships and subsequently mathematical models fitted to these. These experiments take substantial time, which means they are rarely performed in the same cell.Rather than traditional square‐wave voltage clamps, we fitted a model to the current evoked by a novel sum‐of‐sinusoids voltage clamp that was only 8 s long.Short protocols that can be performed multiple times within a single cell will offer many new opportunities to measure how ion current kinetics are affected by changing conditions.The new model predicts the current under traditional square‐wave protocols well, with better predictions of underlying currents than literature models. The current under a novel physiologically relevant series of action potential clamps is predicted extremely well.The short sinusoidal protocols allow a model to be fully fitted to individual cells, allowing us to examine cell–cell variability in current kinetics for the first time. Understanding the roles of ion currents is crucial to predict the action of pharmaceuticals and mutations in different scenarios, and thereby to guide clinical interventions in the heart, brain and other electrophysiological systems. Our ability to predict how ion currents contribute to cellular electrophysiology is in turn critically dependent on our characterisation of ion channel kinetics – the voltage‐dependent rates of transition between open, closed and inactivated channel states. We present a new method for rapidly exploring and characterising ion channel kinetics, applying it to the hERG potassium channel as an example, with the aim of generating a quantitatively predictive representation of the ion current. We fitted a mathematical model to currents evoked by a novel 8 second sinusoidal voltage clamp in CHO cells overexpressing hERG1a. The model was then used to predict over 5 minutes of recordings in the same cell in response to further protocols: a series of traditional square step voltage clamps, and also a novel voltage clamp comprising a collection of physiologically relevant action potentials. We demonstrate that we can make predictive cell‐specific models that outperform the use of averaged data from a number of different cells, and thereby examine which changes in gating are responsible for cell–cell variability in current kinetics. Our technique allows rapid collection of consistent and high quality data, from single cells, and produces more predictive mathematical ion channel models than traditional approaches.

Determinants of Isoform-Specific Gating Kinetics of hERG1 Channel: Combined Experimental and Simulation Study.

IKr is the rapidly activating component of the delayed rectifier potassium current, the ion current largely responsible for the repolarization of the cardiac action potential. Inherited forms of long QT syndrome (LQTS) (Lees-Miller et al., 1997) in humans are linked to functional modifications in the Kv11.1 (hERG) ion channel and potentially life threatening arrhythmias. There is little doubt now that hERG-related component of IKr in the heart depends on the tetrameric (homo- or hetero-) channels formed by two alternatively processed isoforms of hERG, termed hERG1a and hERG1b. Isoform composition (hERG1a- vs. the b-isoform) has recently been reported to alter pharmacologic responses to some hERG blockers and was proposed to be an essential factor pre-disposing patients for drug-induced QT prolongation. Very little is known about the gating and pharmacological properties of two isoforms in heart membranes. For example, how gating mechanisms of the hERG1a channels differ from that of hERG1b is still unknown. The mechanisms by which hERG 1a/1b hetero-tetramers contribute to function in the heart, or what role hERG1b might play in disease are all questions to be answered. Structurally, the two isoforms differ only in the N-terminal region located in the cytoplasm: hERG1b is 340 residues shorter than hERG1a and the initial 36 residues of hERG1b are unique to this isoform. In this study, we combined electrophysiological measurements for HEK cells, kinetics and structural modeling to tease out the individual contributions of each isoform to Action Potential formation and then make predictions about the effects of having various mixture ratios of the two isoforms. By coupling electrophysiological data with computational kinetic modeling, two proposed mechanisms of hERG gating in two homo-tetramers were examined. Sets of data from various experimental stimulation protocols (HEK cells) were analyzed simultaneously and fitted to Markov-chain models (M-models). The minimization procedure presented here, allowed assessment of suitability of different Markov model topologies and the corresponding parameters that describe the channel kinetics. The kinetics modeling pointed to key differences in the gating kinetics that were linked to the full channel structure. Interactions between soluble domains and the transmembrane part of the channel appeared to be critical determinants of the gating kinetics. The structures of the full channel in the open and closed states were compared for the first time using the recent Cryo-EM resolved structure for full open hERG channel and an homology model for the closed state, based on the highly homolog EAG1 channel. Key potential interactions which emphasize the importance of electrostatic interactions between N-PAS cap, S4-S5, and C-linker are suggested based on the structural analysis. The derived kinetic parameters were later used in higher order models of cells and tissue to track down the effect of varying the ratios of hERG1a and hERG1b on cardiac action potentials and computed electrocardiograms. Simulations suggest that the recovery from inactivation of hERG1b may contribute to its physiologic role of this isoform in the action potential. Finally, the results presented here contribute to the growing body of evidence that hERG1b significantly affects the generation of the cardiac Ikr and plays an important role in cardiac electrophysiology. We highlight the importance of carefully revisiting the Markov models previously proposed in order to properly account for the relative abundance of the hERG1 a- and b- isoforms.

Interaction of a dinoflagellate neurotoxin with voltage-activated ion channels in a marine diatom.

BackgroundThe potent neurotoxins produced by the harmful algal bloom species Karenia brevis are activators of sodium voltage-gated channels (VGC) in animals, resulting in altered channel kinetics and membrane hyperexcitability. Recent biophysical and genomic evidence supports widespread presence of homologous sodium (Na+) and calcium (Ca2+) permeable VGCs in unicellular algae, including marine phytoplankton. We therefore hypothesized that VGCs of these phytoplankton may be an allelopathic target for waterborne neurotoxins produced by K. brevis blooms that could lead to ion channel dysfunction and disruption of signaling in a similar manner to animal Na+ VGCs.MethodsWe examined the interaction of brevetoxin-3 (PbTx-3), a K. brevis neurotoxin, with the Na+/Ca2+ VGC of the non-toxic diatom Odontella sinensis using electrophysiology. Single electrode current- and voltage- clamp recordings from O. sinensis in the presence of PbTx-3 were used to examine the toxin’s effect on voltage gated Na+/Ca2+ currents. In silico analysis was used to identify the putative PbTx binding site in the diatoms. We identified Na+/Ca2+ VCG homologs from the transcriptomes and genomes of 12 diatoms, including three transcripts from O. sinensis and aligned them with site-5 of Na+ VGCs, previously identified as the PbTx binding site in animals.ResultsUp to 1 µM PbTx had no effect on diatom resting membrane potential or membrane excitability. The kinetics of fast inward Na+/Ca2+ currents that underlie diatom action potentials were also unaffected. However, the peak inward current was inhibited by 33%, delayed outward current was inhibited by 25%, and reversal potential of the currents shifted positive, indicating a change in permeability of the underlying channels. Sequence analysis showed a lack of conservation of the PbTx binding site in diatom VGC homologs, many of which share molecular features more similar to single-domain bacterial Na+/Ca2+ VGCs than the 4-domain eukaryote channels.DiscussionAlthough membrane excitability and the kinetics of action potential currents were unaffected, the permeation of the channels underlying the diatom action potential was significantly altered in the presence of PbTx-3. However, at environmentally relevant concentrations the effects of PbTx- on diatom voltage activated currents and interference of cell signaling through this pathway may be limited. The relative insensitivity of phytoplankton VGCs may be due to divergence of site-5 (the putative PbTx binding site), and in some cases, such as O. sinensis, resistance to toxin effects may be because of evolutionary loss of the 4-domain eukaryote channel, while retaining a single domain bacterial-like VGC that can substitute in the generation of fast action potentials.