Open Probability Research Articles

Mechanistic basis of ligand efficacy in the calcium-activated chloride channel TMEM16A.

Agonist binding in ligand-gated ion channels is coupled to structural rearrangements around the binding site, followed by the opening of the channel pore. In this process, agonist efficacy describes the equilibrium between open and closed conformations in a fully ligand-bound state. Calcium-activated chloride channels in the TMEM16 family are important sensors of intracellular calcium signals and are targets for pharmacological modulators, yet a mechanistic understanding of agonist efficacy has remained elusive. Using a combination of cryo-electron microscopy, electrophysiology, and autocorrelation analysis, we now show that agonist efficacy in the ligand-gated channel TMEM16A is dictated by the conformation of the pore-lining helix α6 around the Ca2+ -binding site. The closure of the binding site, which involves the formation of a π-helix below a hinge region in α6, appears to be coupled to the opening of the inner pore gate, thereby governing the channel's open probability and conductance. Our results provide a mechanism for agonist binding and efficacy and a structural basis for the design of potentiators and partial agonists in the TMEM16 family.

When microscopy and electrophysiology meet connectomics-Steve Massey's contribution to unraveling the structure and function of the rod/cone gap junction.

Electrical synapses, formed of gap junctions, are ubiquitous components of the central nervous system (CNS) that shape neuronal circuit connectivity and dynamics. In the retina, electrical synapses can create a circuit, control the signal-to-noise ratio in individual neurons, and support the coordinated neuronal firing of ganglion cells, hence, regulating signal processing at the network, single-cell, and dendritic level. We, the authors, and Steve Massey have had a long interest in gap junctions in retinal circuits, in general, and in the network of photoreceptors, in particular. Our combined efforts, based on a wide array of techniques of molecular biology, microscopy, and electrophysiology, have provided fundamental insights into the molecular structure and properties of the rod/cone gap junction. Yet, a full understanding of how rod/cone coupling controls circuit dynamics necessitates knowing its operating range. It is well established that rod/cone coupling can be greatly reduced or eliminated by bright-light adaptation or pharmacological treatment; however, the upper end of its dynamic range has long remained elusive. This held true until Steve Massey's recent interest for connectomics led to the development of a new strategy to assess this issue. The effort proved effective in establishing, with precision, the connectivity rules between rods and cones and estimating the theoretical upper limit of rod/cone electrical coupling. Comparing electrophysiological measurements and morphological data indicates that under pharmacological manipulation, rod/cone coupling can reach the theoretical maximum of its operating range, implying that, under these conditions, all the gap junction channels present at the junctions are open. As such, channel open probability is likely the main determinant of rod/cone coupling that can change momentarily in a time-of-day- and light-dependent manner. In this article we briefly review our current knowledge of the molecular structure of the rod/cone gap junction and of the mechanisms behind its modulation, and we highlight the recent work led by Steve Massey. Steve's contribution has been critical toward asserting the modulation depth of rod/cone coupling as well as elevating the rod/cone gap junction as one of the most suitable models to examine the role of electrical synapses and their plasticity in neural processing.

Pharmacological characterization of SAGE-718, a novel positive allosteric modulator of N-methyl-d-aspartate receptors.

Select neuroactive steroids tune neural activity by modulating excitatory and inhibitory neurotransmission, including the endogenous cholesterol metabolite 24(S)-hydroxycholesterol (24(S)-HC), which is an N-methyl-d-aspartate (NMDA) receptor positive allosteric modulator (PAM). NMDA receptor PAMs are potentially an effective pharmacotherapeutic strategy to treat conditions associated with NMDA receptor hypofunction. Using in vitro and in vivo electrophysiological recording experiments and behavioural approaches, we evaluated the effect of SAGE-718, a novel neuroactive steroid NMDA receptor PAM currently in clinical development for the treatment of cognitive impairment, on NMDA receptor function and endpoints that are altered by NMDA receptor hypoactivity and assessed its safety profile. SAGE-718 potentiated GluN1/GluN2A-D NMDA receptors with equipotency and increased NMDA receptor excitatory postsynaptic potential (EPSP) amplitude without affecting decay kinetics in striatal medium spiny neurons. SAGE-718 increased the rate of unblock of the NMDA receptor open channel blocker ketamine on GluN1/GluN2A in vitro and accelerated the rate of return on the ketamine-evoked increase in gamma frequency band power, as measured with electroencephalogram (EEG), suggesting that PAM activity is driven by increased channel open probability. SAGE-718 ameliorated deficits due to NMDA receptor hypofunction, including social deficits induced by subchronic administration of phencyclidine, and behavioural and electrophysiological deficits from cholesterol and 24(S)-HC depletion caused by 7-dehydrocholesterol reductase inhibition. Finally, SAGE-718 did not produce epileptiform activity in a seizure model or neurodegeneration following chronic dosing. These findings provide strong evidence that SAGE-718 is a neuroactive steroid NMDA receptor PAM with a mechanism that is well suited as a treatment for conditions associated with NMDA receptor hypofunction.

Assessing impending hazards from summit eruptions: the new probabilistic map for lava flow inundation at Mt. Etna

The development of probabilistic maps associated with lava flow inundation is essential to assess hazard in open vent volcanoes, especially those that have highly urbanized flanks. In this study we present the new lava flow hazard map linked to the summit eruptions of Mt. Etna, which has been developed using a probabilistic approach that integrates statistical analyses of the volcanological historical data with numerical simulations of lava flows. The statistical analysis of volcanological data (including vent location, duration and lava volumes) about all summit eruptions occurred since 1998 has allowed us both to estimate the spatiotemporal probability of future vent opening and to extract the effusion rate curves for lava flow modelling. Numerical simulations were run using the GPUFLOW model on a 2022 Digital Surface Model derived from optical satellite images. The probabilistic approach has been validated through a back-analysis by calculating the fit between the expected probabilities of inundation and the lava flows actually emplaced during the 2020-2022 period. The obtained map shows a very high probability of inundation of lava flows emitted at vents linked to the South East Crater, according to the observation of the eruptive dynamics in the last decades.

Mice lacking γENaC palmitoylation sites maintain benzamil-sensitive Na+ transport despite reduced ENaC activity.

Epithelial Na+ channels (ENaCs) control extracellular fluid volume by facilitating Na+ absorption across transporting epithelia. In vitro studies showed that Cys-palmitoylation of the γ ENaC subunit is a major regulator of channel activity. We tested whether γ subunit palmitoylation sites are necessary for channel function in vivo by generating mice lacking the palmitoylated cysteines (γC33A,C41A) using CRISPR-Cas9 technology. ENaCs in dissected kidney tubules from γC33A,C41A mice had reduced open probability compared to wild type (WT) littermates maintained on either standard or Na+-deficient diets. Male mutant mice also had higher aldosterone levels than WT littermates following Na+ restriction. However, γC33A,C41A mice did not have reduced amiloride-sensitive Na+ currents in the distal colon or benzamil-induced natriuresis compared to WT mice. We identified a second, larger conductance cation channel in the distal nephron with biophysical properties distinct from ENaC. The activity of this channel was higher in Na+-restricted γC33A,C41A versus WT mice and was blocked by benzamil, providing a possible compensatory mechanism for reduced prototypic ENaC function. We conclude that γ subunit palmitoylation sites are required for prototypic ENaC activity in vivo, but are not necessary for amiloride/benzamil-sensitive Na+ transport in the distal nephron or colon.

Temperature modulates PVN pre-sympathetic neurones via transient receptor potential ion channels.

The paraventricular nucleus (PVN) of the hypothalamus plays a vital role in maintaining homeostasis and modulates cardiovascular function via autonomic pre-sympathetic neurones. We have previously shown that coupling between transient receptor potential cation channel subfamily V Member 4 (Trpv4) and small-conductance calcium-activated potassium channels (SK) in the PVN facilitate osmosensing, but since TRP channels are also thermosensitive, in this report we investigated the temperature sensitivity of these neurones. Methods: TRP channel mRNA was quantified from mouse PVN with RT-PCR and thermosensitivity of Trpv4-like PVN neuronal ion channels characterised with cell-attached patch-clamp electrophysiology. Following recovery of temperature-sensitive single-channel kinetic schema, we constructed a predictive stochastic mathematical model of these neurones and validated this with electrophysiological recordings of action current frequency. Results: 7 thermosensitive TRP channel genes were found in PVN punches. Trpv4 was the most abundant of these and was identified at the single channel level on PVN neurones. We investigated the thermosensitivity of these Trpv4-like channels; open probability (Po) markedly decreased when temperature was decreased, mediated by a decrease in mean open dwell times. Our neuronal model predicted that PVN spontaneous action current frequency (ACf) would increase as temperature is decreased and in our electrophysiological experiments, we found that ACf from PVN neurones was significantly higher at lower temperatures. The broad-spectrum channel blocker gadolinium (100µM), was used to block the warm-activated, Ca2+-permeable Trpv4 channels. In the presence of gadolinium (100µM), the temperature effect was largely retained. Using econazole (10µM), a blocker of Trpm2, we found there were significant increases in overall ACf and the temperature effect was inhibited. Conclusion: Trpv4, the abundantly transcribed thermosensitive TRP channel gene in the PVN appears to contribute to intrinsic thermosensitive properties of PVN neurones. At physiological temperatures (37°C), we observed relatively low ACf primarily due to the activity of Trpm2 channels, whereas at room temperature, where most of the previous characterisation of PVN neuronal activity has been performed, ACf is much higher, and appears to be predominately due to reduced Trpv4 activity. This work gives insight into the fundamental mechanisms by which the body decodes temperature signals and maintains homeostasis.

Physiological functions and pathological involvement of ion channel trafficking in the vasculature.

A variety of ion channels regulate membrane potential and calcium influx in arterial smooth muscle and endothelial cells to modify vascular functions, including contractility. The current (I) generated by a population of ion channels is equally dependent upon their number (N), open probability (Po) and single channel current (i), such that I=N.PO.i. A conventional view had been that ion channels traffic to the plasma membrane in a passive manner, resulting in a static surface population. It was also considered that channels assemble with auxiliary subunits prior to anterograde trafficking of the multimeric complex to the plasma membrane. Recent studies have demonstrated that physiological stimuli can regulate the surface abundance (N) of several different ion channels in arterial smooth muscle and endothelial cells to control arterial contractility. Physiological stimuli can also regulate the number of auxiliary subunits present in the plasma membrane to modify the biophysical properties, regulatory mechanisms and physiological functions of some ion channels. Furthermore, ion channel trafficking becomes dysfunctional in the vasculature during hypertension, which negatively impacts the regulation of contractility. The temporal kinetics of ion channel and auxiliary subunit trafficking can also vary depending on the signalling mechanisms and proteins involved. This review will summarize recent work that has uncovered the mechanisms, functions and pathological modifications of ion channel trafficking in arterial smooth muscle and endothelial cells.

Iontronic Nanopore Model for Artificial Neurons: The Requisites of Spiking.

Brain-inspired neuromorphic computing is currently being investigated for effective artificial intelligence (AI) systems. The development of artificial neurons and synapses is imperative to creating efficient computational biomimetic networks. Here we propose the minimal configuration of an effective iontronic spiking neuron based on a conical nanofluidic pore ionic diode. The conductance is composed of a Boltzmann open channel probability and a blocking inactivation function, forming the structure of a memristor. The presence of a negative resistance and the combination of activation-deactivation dynamics cause a Hopf bifurcation. Using the characteristic frequencies of small perturbation impedance spectroscopy, we discuss the conditions of spiking, in which the system enters a limit cycle oscillation. We arrive at the conclusion that an excitable neuron-like system can be made with a single active channel instead of the more complex combination of multiple channels that occurs in the Hodgkin-Huxley neuron model.

Fluorofenidone enhances cardiac contractility by stimulating CICR and CaV1.2

Fluorofenidone (AKF-PD) is a novel pyridone derivative that inhibits fibrosis and inflammation in many tissues. Accordingly, it has been effective in disease models, such as liver failure, nephropathy, and pulmonary fibrosis. However, its potential role in cardiac physiology and pathology has yet to be elucidated. Thus, this paper investigated a possible functional impact of AKF-PD on adult rat cardiac myocytes. Cells were kept in culture for 1–2 days under either control conditions or the presence of AKF-PD (500 μM). They were next examined concerning cell contractility, intracellular Ca2+ homeostasis, and activity of voltage-gated Ca2+ channels. Remarkably, AKF-PD enhanced the percentage of cell shortening and rates of both contraction and relaxation by nearly 100%. A stimulus in Ca2+-induced Ca2+ release (CICR) most likely accounts for these effects because AKF-PD also increased the magnitude of electrically evoked Ca2+ transients. Of note, the compound did not alter the peak value of caffeine-elicited Ca2+ transients, indicating stimulation of CICR at constant sarcoplasmic reticulum Ca2+ load. Since CICR is triggered by the entry of Ca2+ through CaV1.2 (ICa), a possible effect on these Ca2+ channels was also investigated. AKF-PD increased the magnitude of both ICa and maximal macroscopic Ca2+ conductance (Gmax) by about 50%. However, no differences were found in either voltage dependence of inactivation or the amount of maximal immobilization-resistant charge movement (Qmax). Thus, the effect on ICa could be explained by a higher channel's open probability (Po) rather than a greater abundance of channel proteins. Additional data indicate that AKF-PD reduces the rate of Ca2+ extrusion in the presence of caffeine, suggesting inhibition of the Na/Ca exchanger. Overall, these results indicate that AKF-PD upregulates the Po of CaV1.2 and then sequentially enhances ICa, CICR, and contractility. Therefore, the novel compound is also a candidate to be tested in cardiac disease models.

Mechanisms of PIEZO Channel Inactivation.

PIEZO channels PIEZO1 and PIEZO2 are the newly identified mechanosensitive, non-selective cation channels permeable to Ca2+. In higher vertebrates, PIEZO1 is expressed ubiquitously in most tissues and cells while PIEZO2 is expressed more specifically in the peripheral sensory neurons. PIEZO channels contribute to a wide range of biological behaviors and developmental processes, therefore driving significant attention in the effort to understand their molecular properties. One prominent property of PIEZO channels is their rapid inactivation, which manifests itself as a decrease in channel open probability in the presence of a sustained mechanical stimulus. The lack of the PIEZO channel inactivation is linked to various mechanopathologies emphasizing the significance of studying this PIEZO channel property and the factors affecting it. In the present review, we discuss the mechanisms underlying the PIEZO channel inactivation, its modulation by the interaction of the channels with lipids and/or proteins, and how the changes in PIEZO inactivation by the channel mutations can cause a variety of diseases in animals and humans.

Investigating the Impact of Selective Modulators on the Renin-Angiotensin-Aldosterone System: Unraveling Their Off-Target Perturbations of Transmembrane Ionic Currents.

The renin-angiotensin-aldosterone system (RAAS) plays a crucial role in maintaining various physiological processes in the body, including blood pressure regulation, electrolyte balance, and overall cardiovascular health. However, any compounds or drugs known to perturb the RAAS might have an additional impact on transmembrane ionic currents. In this retrospective review article, we aimed to present a selection of chemical compounds or medications that have long been recognized as interfering with the RAAS. It is noteworthy that these substances may also exhibit regulatory effects in different types of ionic currents. Apocynin, known to attenuate the angiotensin II-induced activation of epithelial Na+ channels, was shown to stimulate peak and late components of voltage-gated Na+ current (INa). Esaxerenone, an antagonist of the mineralocorticoid receptor, can exert an inhibitory effect on peak and late INa directly. Dexamethasone, a synthetic glucocorticoid, can directly enhance the open probability of large-conductance Ca2+-activated K+ channels. Sparsentan, a dual-acting antagonist of the angiotensin II receptor and endothelin type A receptors, was found to suppress the amplitude of peak and late INa effectively. However, telmisartan, a blocker of the angiotensin II receptor, was effective in stimulating the peak and late INa along with a slowing of the inactivation time course of the current. However, telmisartan's presence can also suppress the erg-mediated K+ current. Moreover, tolvaptan, recognized as an aquaretic agent that can block the vasopressin receptor, was noted to suppress the amplitude of the delayed-rectifier K+ current and the M-type K+ current directly. The above results indicate that these substances not only have an interference effect on the RAAS but also exert regulatory effects on different types of ionic currents. Therefore, to determine their mechanisms of action, it is necessary to gain a deeper understanding.

Function of a mutant ryanodine receptor (T4709M) linked to congenital myopathy

Physiological muscle contraction requires an intact ligand gating mechanism of the ryanodine receptor 1 (RyR1), the Ca2+-release channel of the sarcoplasmic reticulum. Some mutations impair the gating and thus cause muscle disease. The RyR1 mutation T4706M is linked to a myopathy characterized by muscle weakness. Although, low expression of the T4706M RyR1 protein can explain in part the symptoms, little is known about the function RyR1 channels with this mutation. In order to learn whether this mutation alters channel function in a manner that can account for the observed symptoms, we examined RyR1 channels isolated from mice homozygous for the T4709M (TM) mutation at the single channel level. Ligands, including Ca2+, ATP, Mg2+ and the RyR inhibitor dantrolene were tested. The full conductance of the TM channel was the same as that of wild type (wt) channels and a population of partial open (subconductive) states were not observed. However, two unique sub-populations of TM RyRs were identified. One half of the TM channels exhibited high open probability at low (100 nM) and high (50 μM) cytoplasmic [Ca2+], resulting in Ca2+-insensitive, constitutively high Po channels. The rest of the TM channels exhibited significantly lower activity within the physiologically relevant range of cytoplasmic [Ca2+], compared to wt. TM channels retained normal Mg2+ block, modulation by ATP, and inhibition by dantrolene. Together, these results suggest that the TM mutation results in a combination of primary and secondary RyR1 dysfunctions that contribute to disease pathogenesis.

Adenosine stimulates the basolateral 50pS K+ channel in renal proximal tubule via adenosine-A1 receptor.

Background: The basolateral potassium channels play an important role in maintaining the membrane transport in the renal proximal tubules (PT) and adenosine receptors have been shown to regulate the trans-epithelial Na+ absorption in the PT. The aim of the present study is to explore whether adenosine also regulates the basolateral K+ channel of the PT and to determine the adenosine receptor type and the signaling pathway which mediates the effect of adenosine on the K+ channel. Methods: We have used the single channel recording to examine the basolateral K+ channel activity in the proximal tubules of the mouse kidney. All experiments were performed in cell-attached patches. Results: Single channel recording has detected a 50pS inwardly-rectifying K+ channel with high channel open probability and this 50 pS K+ channel is a predominant type K+ channel in the basolateral membrane of the mouse PT. Adding adenosine increased 50pS K+ channel activity in cell-attached patches, defined by NPo (a product of channel Numbers and Open Probability). The adenosine-induced stimulation of the 50pS K+ channel was absent in the PT pretreated with DPCPX, a selective inhibitor of adenosine A1 receptor. In contrast, adenosine was still able to stimulate the 50pS K+ channel in the PT pretreated with CP-66713, a selective adenosine A2 receptor antagonist. This suggests that the stimulatory effect of adenosine on the 50pS K+ channel of the PT was mediated by adenosine-A1 receptor. Moreover, the effect of adenosine on the 50 pS K+ channel was blocked in the PT pretreated with U-73122 or Calphostin C, suggesting that adenosine-induced stimulation of the 50pS K+ channels of the PT was due to the activation of phospholipase C (PLC) and protein kinase C (PKC) pathway. In contrast, the inhibition of phospholipase A2 (PLA2) with AACOCF3 or inhibition of protein kinase A (PKA) with H8 failed to block the adenosine-induced stimulation of the 50pS K+ channel of the PT. Conclusion: We conclude that adenosine activates the 50pS K+ channels in the basolateral membrane of PT via adenosine-A1 receptor. Furthermore, the effect of adenosine on the 50pS K+ channel is mediated by PLC-PKC signaling pathway.

Local Control Model of a Human Ventricular Myocyte: An Exploration of Frequency-Dependent Changes and Calcium Sparks.

Calcium (Ca2+) sparks are the elementary events of excitation-contraction coupling, yet they are not explicitly represented in human ventricular myocyte models. A stochastic ventricular cardiomyocyte human model that adapts to intracellular Ca2+ ([Ca2+]i) dynamics, spark regulation, and frequency-dependent changes in the form of locally controlled Ca2+ release was developed. The 20,000 CRUs in this model are composed of 9 individual LCCs and 49 RyRs that function as couplons. The simulated action potential duration at 1 Hz steady-state pacing is ~0.280 s similar to human ventricular cell recordings. Rate-dependence experiments reveal that APD shortening mechanisms are largely contributed by the L-type calcium channel inactivation, RyR open fraction, and [Ca2+]myo concentrations. The dynamic slow-rapid-slow pacing protocol shows that RyR open probability during high pacing frequency (2.5 Hz) switches to an adapted "nonconducting" form of Ca2+-dependent transition state. The predicted force was also observed to be increased in high pacing, but the SR Ca2+ fractional release was lower due to the smaller difference between diastolic and systolic [Ca2+]SR. Restitution analysis through the S1S2 protocol and increased LCC Ca2+-dependent activation rate show that the duration of LCC opening helps modulate its effects on the APD restitution at different diastolic intervals. Ultimately, a longer duration of calcium sparks was observed in relation to the SR Ca2+ loading at high pacing rates. Overall, this study demonstrates the spontaneous Ca2+ release events and ion channel responses throughout various stimuli.

Salts of short-chain fatty acids increase the activity of the large conductance, Ca<sup>2+</sup>-activated K<sup>+</sup> channels and reduce calcium oscillations in rat GH3 cells

The short-chain fatty acids such as acetic, propionic and butyric acids are microbiota metabolites that can exert a series of physiological effects both in the intestine and other organs, including the central nervous system. The present work aimed to examine the effects of sodium acetate, propionate, and butyrate on the activity of large conductance Ca2+ activated K+ channels and calcium oscillations in rat pituitary GH3 cells. It has been shown that fatty acids under study cause a dose-dependent increase in the amplitude of total outward potassium currents and these effects are prevented by tetraethylammonium, a Ca2+ activated K+ channel blocker, indicating the involvement of Ca2+ activated K+ channels in the effects of fatty acids. It is worthy of note that fatty acids increased open probability of single channels with no changes in the amplitude and the mean channel open time. In addition, fatty acids were associated with a significant reduction in the amplitude and frequency of Ca2+ oscillations in GH3 cells. An increase in potassium conductance and a decrease in the intracellular Ca2+ level can mediate the effects of short-chain fatty acids in various excitable structures, such as a relaxation of intestinal and vascular smooth muscle cells, hyperpolarization of neurons, and the regulation of hormone and neurotransmitter release.

Ca2+ dynamics in interstitial cells: foundational mechanisms for the motor patterns in the gastrointestinal tract.

The gastrointestinal (GI) tract displays multiple motor patterns that move nutrients and wastes through the body. Smooth muscle cells (SMCs) provide the forces necessary for GI motility, but interstitial cells, electrically coupled to SMCs, tune SMC excitability, transduce inputs from enteric motor neurons, and generate pacemaker activity that underlies major motor patterns, such as peristalsis and segmentation. The interstitial cells regulating SMCs are interstitial cells of Cajal (ICC) and PDGF receptor (PDGFR)α+ cells. Together these cells form the SIP syncytium. ICC and PDGFRα+ cells express signature Ca2+-dependent conductances: ICC express Ca2+-activated Cl- channels, encoded by Ano1, that generate inward current, and PDGFRα+ cells express Ca2+-activated K+ channels, encoded by Kcnn3, that generate outward current. The open probabilities of interstitial cell conductances are controlled by Ca2+ release from the endoplasmic reticulum. The resulting Ca2+ transients occur spontaneously in a stochastic manner. Ca2+ transients in ICC induce spontaneous transient inward currents and spontaneous transient depolarizations (STDs). Neurotransmission increases or decreases Ca2+ transients, and the resulting depolarizing or hyperpolarizing responses conduct to other cells in the SIP syncytium. In pacemaker ICC, STDs activate voltage-dependent Ca2+ influx, which initiates a cluster of Ca2+ transients and sustains activation of ANO1 channels and depolarization during slow waves. Regulation of GI motility has traditionally been described as neurogenic and myogenic. Recent advances in understanding Ca2+ handling mechanisms in interstitial cells and how these mechanisms influence motor patterns of the GI tract suggest that the term "myogenic" should be replaced by the term "SIPgenic," as this review discusses.

Under pressure—the influence of hypergravity on electrocortical activity and neurocognitive performance

The effects of hypergravity and the associated increased pressure on the human body have not yet been studied in detail, but are of great importance for the safety of astronauts on space missions and could have a long-term impact on rehabilitation strategies for neurological patients. Considering the plans of international space agencies with the exploration of Mars and Moon, it is important to explore the effects of both extremes, weightlessness and hypergravity. During parabolic flights, a flight manoeuvre that artificially creates weightlessness and hypergravity, electrocortical activity as well as behavioural parameters (error rate and reaction time) and neuronal parameters (event-related potentials P300 and N200) were examined with an electroencephalogram. Thirteen participants solved a neurocognitive task (mental arithmetic task as a primary task and oddball paradigm as a secondary task) within normal as well as hypergravity condition in fifteen consecutive parabolas for 22 s each. No changes between the different gravity levels could be observed for the behavioural parameters and cortical current density. A significantly lower P300 amplitude was observed in 1 G, triggered by the primary task and the target sound of the oddball paradigm. The N200, provoked by the sounds of the oddball paradigm, revealed a higher amplitude in 1.8 G. A model established by Kohn et al. (2018) describing changes in neural communication with decreasing gravity can be used here as an explanatory approach. The fluid shift increases the intracranial pressure, decreases membrane viscosity and influences the open state probability of ion channels. This leads to an increase in the resting membrane potential, and the threshold for triggering an action potential can be reached more easily. The question now arises whether the observed changes are linear or whether they depend on a specific threshold.

An activator of voltage-gated K+ channels Kv1.1 as a therapeutic candidate for episodic ataxia type 1

Loss-of-function mutations in the KCNA1(Kv1.1) gene cause episodic ataxia type 1 (EA1), a neurological disease characterized by cerebellar dysfunction, ataxic attacks, persistent myokymia with painful cramps in skeletal muscles, and epilepsy. Precision medicine for EA1 treatment is currently unfeasible, as no drug that can enhance the activity of Kv1.1-containing channels and offset the functional defects caused by KCNA1 mutations has been clinically approved. Here, we uncovered that niflumic acid (NFA), a currently prescribed analgesic and anti-inflammatory drug with an excellent safety profile in the clinic, potentiates the activity of Kv1.1 channels. NFA increased Kv1.1 current amplitudes by enhancing the channel open probability, causing a hyperpolarizing shift in the voltage dependence of both channel opening and gating charge movement, slowing the OFF-gating current decay. NFA exerted similar actions on both homomeric Kv1.2 and heteromeric Kv1.1/Kv1.2 channels, which are formed in most brain structures. We show that through its potentiating action, NFA mitigated the EA1 mutation-induced functional defects in Kv1.1 and restored cerebellar synaptic transmission, Purkinje cell availability, and precision of firing. In addition, NFA ameliorated the motor performance of a knock-in mouse model of EA1 and restored the neuromuscular transmission and climbing ability in Shaker (Kv1.1) mutant Drosophila melanogaster flies (Sh5). By virtue of its multiple actions, NFA has strong potential as an efficacious single-molecule-based therapeutic agent for EA1 and serves as a valuable model for drug discovery.

Cx43 Hemichannel and Panx1 Channel Modulation by Gap19 and 10Panx1 Peptides.

Cx43 hemichannels (HCs) and Panx1 channels are two genetically distant protein families. Despite the lack of sequence homology, Cx43 and Panx1 channels have been the subject of debate due to their overlapping expression and the fact that both channels present similarities in terms of their membrane topology and electrical properties. Using the mimetic peptides Gap19 and 10Panx1, this study aimed to investigate the cross-effects of these peptides on Cx43 HCs and Panx1 channels. The single-channel current activity from stably expressing HeLa-Cx43 and C6-Panx1 cells was recorded using patch-clamp experiments in whole-cell voltage-clamp mode, demonstrating 214 pS and 68 pS average unitary conductances for the respective channels. Gap19 was applied intracellularly while 10Panx1 was applied extracellularly at different concentrations (100, 200 and 500 μM) and the average nominal open probability (NPo) was determined for each testing condition. A concentration of 100 µM Gap19 more than halved the NPo of Cx43 HCs, while 200 µM 10Panx1 was necessary to obtain a half-maximal NPo reduction in the Panx1 channels. Gap19 started to significantly inhibit the Panx1 channels at 500 µM, reducing the NPo by 26% while reducing the NPo of the Cx43 HCs by 84%. In contrast 10Panx1 significantly reduced the NPo of the Cx43 HCs by 37% at 100 µM and by 83% at 200 µM, a concentration that caused the half-maximal inhibition of the Panx1 channels. These results demonstrate that 10Panx1 inhibits Cx43 HCs over the 100-500 µM concentration range while 500 µM intracellular Gap19 is necessary to observe some inhibition of Panx1 channels.

Functional characterization of the transient receptor potential melastatin 2 (TRPM2) cation channel from Nematostella vectensis reconstituted into lipid bilayer

Transient receptor potential melastatin 2 (TRPM2) cation channel activity is required for insulin secretion, immune cell activation and body heat control. Channel activation upon oxidative stress is involved in the pathology of stroke and neurodegenerative disorders. Cytosolic Ca2+, ADP-ribose (ADPR) and phosphatidylinositol-4,5-bisphosphate (PIP2) are the obligate activators of the channel. Several TRPM2 cryo-EM structures have been resolved to date, yet functionality of the purified protein has not been tested. Here we reconstituted overexpressed and purified TRPM2 from Nematostella vectensis (nvTRPM2) into lipid bilayers and found that the protein is fully functional. Consistent with the observations in native membranes, nvTRPM2 in lipid bilayers is co-activated by cytosolic Ca2+ and either ADPR or ADPR-2′-phosphate (ADPRP). The physiological metabolite ADPRP has a higher apparent affinity than ADPR. In lipid bilayers nvTRPM2 displays a large linear unitary conductance, its open probability (Po) shows little voltage dependence and is stable over several minutes. Po is high without addition of exogenous PIP2, but is largely blunted by treatment with poly-l-Lysine, a polycation that masks PIP2 headgroups. These results indicate that PIP2 or some other activating phosphoinositol lipid co-purifies with nvTRPM2, suggesting a high PIP2 binding affinity of nvTRPM2 under physiological conditions.