Single-channel Currents Research Articles

Molecular Insights into Single Chain Lipid Modulation of Acid-Sensing Ion Channel 3.

Polyunsaturated fatty acids (PUFAs) and their analogs play a significant role in modulating the activity of diverse ion channels, and recent studies show that these lipids potentiate acid-sensing ion channels (ASICs), leading to increased activity. The potentiation of the channel stems from multiple gating changes, but the exact mechanism of these effects remains uncertain. We posit a mechanistic explanation for one of these changes in channel function, the increase in the maximal current, by applying a combination of electrophysiology and all-atom molecular dynamics simulations on the open-state hASIC3. Microsecond-scale simulations were performed on open-state hASIC3 in the absence and presence of a PUFA, docosahexaenoic acid (DHA), and a PUFA analog, N-arachidonyl glycine (AG). Intriguingly, our simulations in the absence of PUFA or PUFA analogs reveal that a tail from the membrane phospholipid POPC inserts itself into the pore of the channel through lateral fenestrations on the sides of the transmembrane segments, obstructing ion permeation through the channel. The binding of either DHA or AG prevented POPC from accessing the pore in our simulations, relieving the block of ionic conduction by phospholipids. Finally, we use the single-channel recording to show that DHA increases the amplitude of the single-channel currents in ASIC3, which is consistent with our hypothesis that PUFAs relieve the pore block of the channel induced by POPCs. Together, these findings offer a potential mechanistic explanation of how PUFAs modulate ASIC maximal current, revealing a novel mechanism of action for PUFA-induced modulation of ion channels.

Novel interplay between agonist and calcium binding sites modulates drug potentiation of α7 acetylcholine receptor.

Drug modulation of the α7 acetylcholine receptor has emerged as a therapeutic strategy for neurological, neurodegenerative, and inflammatory disorders. α7 is a homo-pentamer containing topographically distinct sites for agonists, calcium, and drug modulators with each type of site present in five copies. However, functional relationships between agonist, calcium, and drugmodulator sites remain poorly understood. To investigate these relationships, we manipulated the number of agonist binding sites, and monitored potentiation of ACh-elicited single-channel currents through α7 receptors by PNU-120596 (PNU) both in the presence and absence of calcium. When ACh is present alone, it elicits brief, sub-millisecond channel openings, however when ACh is present with PNU it elicits long clusters of potentiated openings. In receptors harboring five agonist binding sites, PNU potentiates regardless of the presence or absence of calcium, whereas in receptors harboring one agonist binding site, PNU potentiates in the presence but not the absence of calcium. By varying the numbers of agonist and calcium binding sites we show that PNU potentiation of α7 depends on a balance between agonist occupancy of the orthosteric sites and calcium occupancy of the allosteric sites. The findings suggest that in the local cellular environment, fluctuations in the concentrations of neurotransmitter and calcium mayalter this balance and modulate the ability of PNU to potentiate α7.

CaM-dependent modulation of human CaV1.3 whole-cell and single-channel currents by C-terminal CaMKII phosphorylation site S1475.

Phosphorylation enables rapid modulation of voltage-gated calcium channels (VGCC) in physiological and pathophysiological conditions. How phosphorylation modulates human CaV1.3 VGCC, however, is largely unexplored. We characterized modulation of CaV1.3 gating via S1475, the human equivalent of a phosphorylation site identified in the rat. S1475 is highly conserved in CaV1.3 but absent from all other high-voltage activating calcium channel types co-expressed with CaV1.3 in similar tissues. Further, it is located in the C-terminal EF-hand motif, which binds calmodulin (CaM). This is involved in calcium-dependent channel inactivation (CDI). We used amino acid exchanges that mimic either sustained phosphorylation (S1475D) or phosphorylation resistance (S1475A). Whole-cell and single-channel recordings of phosphorylation state imitating CaV1.3 variants in transiently transfected HEK-293 cells revealed functional relevance of S1475 in human CaV1.3. We obtained three main findings: (1) CaV1.3_S1475D, imitating sustained phosphorylation, displayed decreased current density, reduced CDI and (in-) activation kinetics shifted to more depolarized voltages compared with both wildtype CaV1.3 and the phosphorylation-resistant CaV1.3_S1475A variant. Corresponding to the decreased current density, we find a reduced open probability of CaV1.3_S1475D at the single-channel level. (2) Using CaM overexpression or depletion, we find that CaM is necessary for modulating CaV1.3 through S1475. (3) CaMKII activation led to CaV1.3_WT-current properties similar to those of CaV1.3_S1475D, but did not affect CaV1.3_S1475A, confirming that CaMKII modulates human CaV1.3 via S1475. Given the physiological and pathophysiological importance of CaV1.3, our findings on the S1475-mediated interplay of phosphorylation, CaM interaction and CDI provide hints for approaches on specific CaV1.3 modulation under physiological and pathophysiological conditions. KEY POINTS: Phosphorylation modulates activity of voltage-gated L-type calcium channels for specific cellular needs but is largely unexplored for human CaV1.3 channels. Here we report that S1475, a CaMKII phosphorylation site identified in rats, is functionally relevant in human CaV1.3. Imitating phosphorylation states at S1475 alters current density and inactivation in a calmodulin-dependent manner. In wildtype CaV1.3 but not in the phosphorylation-resistant variant S1475A, CaMKII activation elicits effects similar to constitutively mimicking phosphorylation at S1475. Our findings provide novel insights on the interplay of modulatory mechanisms of human CaV1.3 channels, and present a possible target for CaV1.3-specific gating modulation in physiological and pathophysiological conditions.

Characterization of the tail current of Channelrhodopsin-2 variants

Our study focused on specific ChR2 variants, particularly those with the Step function Opsins (SFO) mutation at the D156-C128 gate. These are widely used in optogenetics due to their heightened sensitivity to light and bi-stable prolonged activation. However, in some ChR2 variants, specifically D156 mutants, a tail current occurs when continuous light exposure is stopped. We specifically examined the D156H-T159S ChR2 variant, which demonstrated a tail current that was somewhat responsive to light and voltage, with a single-channel current of around 9fA, similar to wt-ChR2 as determined by stationary noise analysis. To further investigate, we used nonstationary noise analysis in cell-attached patching mode, which revealed that the tail current's single-channel current falls within the same range as the peak current, albeit with mild contamination from adaptation and desensitization. This finding strongly supports the notion that a portion of the ChR2 molecules open or re-open at the end of illumination, leading to further membrane depolarization.

Pathogenic residue insertion in neuronal nicotinic receptor alters intra- and inter-subunit interactions that tune channel gating

We describe molecular-level functional changes in the α4β2 nicotinic acetylcholine receptor (nAChR) by a leucine residue insertion in the M2 transmembrane domain of the α4 subunit associated with sleep-related hyperkinetic epilepsy. Measurements of agonist-elicited single channel currents reveal the primary effect is to stabilize the open channel state while the secondary effect is to promote reopening of the channel. These dual effects prolong the durations of bursts of channel openings equally for the two major stoichiometric forms of the receptor, (α4)2(β2)3 and (α4)3(β2)2, indicating the functional impact is independent of mutant copy number per receptor. Altering the location of the residue insertion within M2 shows that functionally pivotal structures are confined to a half turn of the M2 α-helix. Residue substitutions within M2 and surrounding α-helices reveal that both intra- and inter-subunit interactions mediate the increase in burst duration. These interactions impacting burst duration depend linearly on the size and hydrophobicity of the substituting residue. Together the results reveal a novel structural region of the α4β2 nAChR in which inter-helical interactions tune the stability of the open channel state.

Structural bases for stoichiometry-selective calcium potentiation of a neuronal nicotinic receptor.

α4β2 nicotinic acetylcholine (nACh) receptors assemble in two stoichiometric forms, one of which is potentiated by calcium. The sites of calcium binding that underpin potentiation are not known. To identify calcium binding sites, we applied cryo-electron microscopy (cryo-EM) and molecular dynamics (MD) simulations to each stoichiometric form of the α4β2 nACh receptor in the presence of calcium ions. To test whether the identified calcium sites are linked to potentiation, we generated mutants of anionic residues at the sites, expressed wild type and mutant receptors in clonal mammalian fibroblasts, and recorded ACh-elicited single-channel currents with or without calcium. Both cryo-EM and MD simulations show calcium bound to a site between the extracellular and transmembrane domains of each α4 subunit (ECD-TMD site). Substituting alanine for anionic residues at the ECD-TMD site abolishes stoichiometry-selective calcium potentiation, as monitored by single-channel patch clamp electrophysiology. Additionally, MD simulation reveals calcium association at subunit interfaces within the extracellular domain. Substituting alanine for anionic residues at the ECD sites reduces or abolishes stoichiometry-selective calcium potentiation. Stoichiometry-selective calcium potentiation of the α4β2 nACh receptor is achieved by calcium association with topographically distinct sites framed by anionic residues within the α4 subunit and between the α4 and β2 subunits. Stoichiometry-selective calcium potentiation could result from the greater number of calcium sites in the stoichiometric form with three rather than two α4 subunits. The results are relevant to modulation of signalling via α4β2 nACh receptors in physiological and pathophysiological conditions.

Selectivity filter conductance, rectification and fluctuations of subdomains—How can this all relate to the value of Hurst exponent in the dwell-times of ion channels states?

The Hurst effect in the signals describing ion channels’ activity has been known for many years. This effect is present in the experimental recordings of single-channel currents, but not only. The sequences of dwell times of functionally different channel states also exhibit long-range correlations. We have found that the memory effect within the dwell-time series is related to the coupling between the channel’s activation gate (AG) and selectivity filter (SF), which controls the ion conduction. In this work, we analyzed both the experimental data describing the activity of potassium channels of different types (e.g., BK, mitoBK, mitoTASK-3, mitoKv1.3, TREK-2-like channels) and the series generated according to our previously proposed Hurst effect model. The obtained results suggest that the strength of the allosteric cooperation between the AG and SF determines not only the conductance of the channel – which governs how often ions in SF move or remain blocked – but also modulates the correlations present in the dwell times when sampled with a suitably high sampling rate. Moreover, we found that rectification can interfere with this process, contributing to additional changes in correlations within the channel’s sojourns in subsequent states. Similarly, the correlations may be affected by processes proceeding at longer time scales, like interactions with the channel’s auxiliary domains or lipid surroundings.

PIEZO1 loss-of-function compound heterozygous mutations in the rare congenital human disorder Prune Belly Syndrome

Prune belly syndrome (PBS), also known as Eagle-Barret syndrome, is a rare, multi-system congenital myopathy primarily affecting males. Phenotypically, PBS cases manifest three cardinal pathological features: urinary tract dilation with poorly contractile smooth muscle, wrinkled flaccid ventral abdominal wall with skeletal muscle deficiency, and intra-abdominal undescended testes. Genetically, PBS is poorly understood. After performing whole exome sequencing in PBS patients, we identify one compound heterozygous variant in the PIEZO1 gene. PIEZO1 is a cation-selective channel activated by various mechanical forces and widely expressed throughout the lower urinary tract. Here we conduct an extensive functional analysis of the PIEZO1 PBS variants that reveal loss-of-function characteristics in the pressure-induced normalized open probability (NPo) of the channel, while no change is observed in single-channel currents. Furthermore, Yoda1, a PIEZO1 activator, can rescue the NPo defect of the PBS mutant channels. Thus, PIEZO1 mutations may be causal for PBS and the in vitro cellular pathophysiological phenotype could be rescued by the small molecule, Yoda1. Activation of PIEZO1 might provide a promising means of treating PBS and other related bladder dysfunctional states.

MULTIFRACTAL CHARACTERISTICS OF THE MITOCHONDRIAL BK CHANNEL ACTIVITY IN THE PRESENCE OF THE CHANNEL-ACTIVATING FLAVONOIDS

Over the years, the (multi)fractal nature of signals describing the activity of ion channels has been observed both at the level of single-channel currents and the temporal scaling of the corresponding sojourns in conducting and non-conducting states. The recognized nonlinear and self-similar properties were interpreted regarding the underlying channel gating machinery. Nevertheless, the literature lacks in (multi)fractal description of the biochemical stimulation of ion channels. Therefore, in this work, we provide a multifractal description of the effects exerted by two different flavonoids — naringenin (Nar) and quercetin (Que) — being the regulators of the large-conductance voltage- and Ca[Formula: see text]-activated K[Formula: see text] channels from the inner mitochondrial membrane (mitoBK). Toward this aim, the focus-based multifractal detrended fluctuation analysis (MFDFA) was applied to the patch-clamp data obtained in the presence of Nar and Que in micromolar concentrations and the corresponding controls. Our results show that mitoBK channel activity has well-pronounced multifractal characteristics and long-range memory (measured by the generalized Hurst exponent) under biochemical stimulation by Nar and Que and in the absence of these substances. The spectral properties significantly change after the randomization of the analyzed series. Therefore, multifractality is an inherent feature of the mitoBK channel currents, which stems from their statistical distribution and temporal organization. Both flavonoids have similar structures and exert channel-activating effects, but they differently affect the parameters of the multifractal spectra of the corresponding patch-clamp signals. Coordination of Que leads to more prominent changes in signal complexity than Nar at membrane hyper- and depolarization. It suggests that this flavonoid has a stronger impact on the conformational dynamics of the mitoBK channels in comparison with naringenin.

Short and long-range correlations in single-channel currents from inwardly rectifying K[formula omitted] channels

Ion channels mediate the transport of ions through the cell membrane and are involved in key processes such as muscle contraction, nerve excitability and hormone secretion. For decades, the main focus of the standard techniques for the analysis and modeling of single-channel currents has been to characterize the gating of ionic channels by analyzing the current amplitudes and dwell-time histograms, thus neglecting the possibility that pore fluctuations (open and closed), usually considered to be stochastic noise, might contain valuable information about the functional aspects of ion channels. Based on the analysis of a deterministic model indicating that the stochastic gating of ion channels could be produced by correlated pore fluctuations, in this work, we analyzed experimental recordings of single channel currents of an inwardly rectifying K+ channel in order to demonstrate the presence of both short and long range correlations in the open and closed pore fluctuations

Hypoxia-induced cystic fibrosis transmembrane conductance regulator dysfunction is a universal mechanism underlying reduced mucociliary transport in sinusitis.

Hypoxia due to sinus obstruction is a major pathogenic mechanism leading to sinusitis. The objective of the current study is to define the electrophysiologic characteristics of hypoxia in vitro and in vivo. Cystic fibrosis bronchoepithelial cells expressing wild-type cystic fibrosis transmembrane conductance regulator (CFTR) and human sinonasal epithelial cells were exposed to 1% or atmospheric O2 for 24h. Time-dependent production of cytoplasmic free radicals was measured. Cells were subjected to Ussing chamber and patch clamp technique where CFTR currents were recorded in whole-cell and cell-attached mode for single channel studies. Indices of mucociliary transport (MCT) were measured using micro-optical coherence tomography. In a rabbit hypoxic maxillary sinus model, tissue oxygenation, relative mRNA expression of HIF-1α, pH, sinus potential difference (SPD), and MCT were determined. Ussing chamber (p<0.05), whole-cell (p<0.001), and single channel patch-clamp (p<0.0001) showed significant inhibition of Cl- currents in hypoxic cells. Cytoplasmic free radicals showed time-dependent elevation peaking at 4h (p<0.0001). Airway surface liquid (p<0.0001), periciliary liquid (p<0.001), and MCT (p<0.01) were diminished. Co-incubation with the free radical scavenger glutathione negated the impact of hypoxia on single channel currents and MCT markers. In sinusitis rabbits, mucosa exhibited low tissue oxygenation (p<0.0001), increased HIF1α mRNA (p<0.05), reduced pH (p<0.01), and decreased MCT (p<0.001). SPD measurements demonstrated markedly diminished transepithelial Cl- transport (p<0.0001). Hypoxia induces severe CFTR dysfunction via free radical production causing reduced MCT in vitro and in vivo. Improved oxygenation is critical to reducing the impact of persistent mucociliary dysfunction.

A gain-of-function HCN4 mutant in the HCN domain is responsible for inappropriate sinus tachycardia in a Spanish family.

In a family with inappropriate sinus tachycardia (IST), we identified a mutation (p.V240M) of the hyperpolarization-activated cyclic nucleotide-gated type 4 (HCN4) channel, which contributes to the pacemaker current (If) in human sinoatrial node cells. Here, we clinically study fifteen family members and functionally analyze the p.V240M variant. Macroscopic (IHCN4) and single-channel currents were recorded using patch-clamp in cells expressing human native (WT) and/or p.V240M HCN4 channels. All p.V240M mutation carriers exhibited IST that was accompanied by cardiomyopathy in adults. IHCN4 generated by p.V240M channels either alone or in combination with WT was significantly greater than that generated by WT channels alone. The variant, which lies in the N-terminal HCN domain, increased the single-channel conductance and opening frequency and probability of HCN4 channels. Conversely, it did not modify the channel sensitivity for cAMP and ivabradine or the level of expression at the membrane. Treatment with ivabradine based on functional data reversed the IST and the cardiomyopathy of the carriers. In computer simulations, the p.V240M gain-of-function variant increases If and beating rate and thus explains the IST of the carriers. The results demonstrate the importance of the unique HCN domain in HCN4, which stabilizes the channels in the closed state.

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.

Long-term memory in Staphylococcus aureus α-hemolysin ion channel kinetics.

The kinetics of an ion channel are classically understood as a random process. However, studies have shown that in complex ion channels, formed by multiple subunits, this process can be deterministic, presenting long-term memory. Staphylococcus aureus α-hemolysin (α-HL) is a toxin that acts as the major factor in Staphylococcus aureus virulence. α-HL is a water-soluble protein capable of forming ion channels into lipid bilayers, by insertion of an amphipathic β-barrel. Here, the α-HL was used as an experimental model to study memory in ion channel kinetics. We applied the approximate entropy (ApEn) approach to analyze randomness and the Detrended Fluctuation Analysis (DFA) to investigate the existence of long memory in α-HL channel kinetics. Single-channel currents were measured through experiments with α-HL channels incorporated in planar lipid bilayers. All experiments were carried out under the following conditions: 1 M NaCl solution, pH 4.5; transmembrane potential of + 40mV and temperature 25 ± 1°C. Single-channel currents were recorded in real-time in the memory of a microcomputer coupled to an A/D converter and a patch-clamp amplifier. The conductance value of the α-HL channels was 0.82 ± 0.0025 nS (n = 128). The DFA analysis showed that the kinetics of α-HL channels presents long-term memory ([Formula: see text] = 0.63 ± 0.04). The ApEn outcomes showed low complexity to dwell times when open (ApEno = 0.5514 ± 0.28) and closed (ApEnc = 0.1145 ± 0.08), corroborating the results of the DFA method.

Eu3+ detects two functionally distinct luminal Ca2+ binding sites in ryanodine receptors

Ryanodine receptors (RyRs) are Ca2+ release channels, gated by Ca2+ in the cytosol and the sarcoplasmic reticulum lumen. Their regulation is impaired in certain cardiac and muscle diseases. Although a lot of data is available on the luminal Ca2+ regulation of RyR, its interpretation is complicated by the possibility that the divalent ions used to probe the luminal binding sites may contaminate the cytoplasmic sites by crossing the channel pore. In this study, we used Eu3+, an impermeable agonist of Ca2+ binding sites, as a probe to avoid this complication and to gain more specific information about the function of the luminal Ca2+ sensor. Single-channel currents were measured from skeletal muscle and cardiac RyRs (RyR1 and RyR2) using the lipid bilayer technique. We show that RyR2 is activated by the luminal addition of Ca2+, whereas RyR1 is inhibited. These results were qualitatively reproducible using Eu3+. The luminal regulation of RyR1 carrying a mutation associated with malignant hyperthermia was not different from that of the wild-type.RyR1 inhibition by Eu3+ was extremely voltage dependent, whereas RyR2 activation did not depend on the membrane potential. These results suggest that the RyR1 inhibition site is in the membrane’s electric field (channel pore), whereas the RyR2 activation site is outside. Using in silico analysis and previous results, we predicted putative Ca2+ binding site sequences. We propose that RyR2 bears an activation site, which is missing in RyR1, but both isoforms share the same inhibitory Ca2+ binding site near the channel gate.

On molecular steps that activate a voltage sensitive ion channel at critical depolarization

At high transmembrane electric field, a voltage sensitive ion channel is an insulator; when the field is critically reduced, it becomes a conductor of selected ions. The Channel Activation by Electrostatic Repulsion (CAbER) hypothesis proposes that an ordered polarization field of induced dipoles at the high electric field magnitude of the excitable state is overcome by thermal disorder at a critical depolarization. Increased repulsions between positive charges in the S4 segments cause an allosteric transition in which these segments expand and separate in a chiral proteinquake. The increased space allows the P segments to refold and the ion-semiconducting S5 and S6 segments to relax and expand outward in a breathing mode. Stripped permeant ions enter widened hydrogen bonds in the core helices of these segments. Driven by concentration differences and the electric field, the ions hop along transient pathways across the channel, appearing as fractal, stochastic bursts of single-channel currents. To support order amid thermal fluctuations, an object must be of a minimum size. The critical role of an ion channel's size suggests that the evolution of Metazoa became possible only after its DNA had grown enough to code for proteins larger than the correlation length.

A detailed analysis of single-channel Nav 1.5 recordings does not reveal any cooperative gating.

Cardiac voltage-gated sodium (Na+ ) channels (Nav 1.5) are crucial for myocardial electrical excitation. Recent studies based on single-channel recordings have suggested that Na+ channels interact functionally and exhibit coupled gating. However, the analysis of such recordings frequently relies on manual interventions, which can lead to bias. Here, we developed an automated pipeline to de-trend and idealize single-channel currents, and assessed possible functional interactions in cell-attached patch clamp experiments in HEK293 cells expressing human Nav 1.5 channels as well as in adult mouse and rabbit ventricular cardiomyocytes. Our pipeline involved de-trending individual sweeps by linear optimization using a library of predefined functions, followed by digital filtering and baseline offset. Subsequently, the processed sweeps were idealized based on the idea that the ensemble average of the idealized current identified by thresholds between current levels reconstructs at best the ensemble average current from the de-trended sweeps. This reconstruction was achieved by non-linear optimization. To ascertain functional interactions, we examined the distribution of the numbers of open channels at every time point during the activation protocol and compared it to the distribution expected for independent channels. We also examined whether the channels tended to synchronize their openings and closings. However, we did not uncover any solid evidence of such interactions in our recordings. Rather, our results indicate that wild-type Nav 1.5 channels are independent entities or exhibit only very weak functional interactions that are probably irrelevant under physiological conditions. Nevertheless, our unbiased analysis will be important for further studies examining whether auxiliary proteins potentiate functional Na+ channel interactions. KEY POINTS: Nav 1.5 channels are critical for cardiac excitation. They are part of macromolecular interacting complexes, and it was previously suggested that two neighbouring channels may functionally interact and exhibit coupled gating. Manual interventions when processing single-channel recordings can lead to bias and inaccurate data interpretation. We developed an automated pipeline to de-trend and idealize single-channel currents and assessed possible functional interactions between Nav 1.5 channels in HEK293 cells and cardiomyocytes during activation protocols using the cell-attached patch clamp technique. In recordings consisting of up to 1000 sweeps from the same patch, our analysis did not reveal any evidence of functional interactions or coupled gating between wild-type Nav 1.5 channels. Our unbiased analysis may be useful in further studies examining how Na+ channel interactions are affected by mutations and auxiliary proteins.

Cryo-EM analysis of scorpion toxin binding to Ryanodine Receptors reveals subconductance that is abolished by PKA phosphorylation.

Calcins are peptides from scorpion venom with the unique ability to cross cell membranes, gaining access to intracellular targets. Ryanodine Receptors (RyR) are intracellular ion channels that control release of Ca2+ from the endoplasmic and sarcoplasmic reticulum. Calcins target RyRs and induce long-lived subconductance states, whereby single-channel currents are decreased. We used cryo-electron microscopy to reveal the binding and structural effects of imperacalcin, showing that it opens the channel pore and causes large asymmetry throughout the cytosolic assembly of the tetrameric RyR. This also creates multiple extended ion conduction pathways beyond the transmembrane region, resulting in subconductance. Phosphorylation of imperacalcin by protein kinase A prevents its binding to RyR through direct steric hindrance, showing how posttranslational modifications made by the host organism can determine the fate of a natural toxin. The structure provides a direct template for developing calcin analogs that result in full channel block, with potential to treat RyR-related disorders.

Basal NAD(H) redox state permits hydrogen peroxide-induced mesenteric artery dilatation.

Smooth muscle voltage-gated K+ (Kv) channels in resistance arteries control vascular tone and contribute to the coupling of blood flow with local metabolic activity. Members of the Kv1 family are expressed in vascular smooth muscle and are modulated upon physiological elevation of local metabolites, including the glycolytic end-product l-lactate and superoxide-derived hydrogen peroxide (H2 O2 ). Here, we show that l-lactate elicits vasodilatation of small-diameter mesenteric arteries in a mechanism that requires lactate dehydrogenase (LDH). Using the inside-out configuration of the patch clamp technique, we show that increases in NADH that reflect LDH-mediated conversion of l-lactate to pyruvate directly stimulate the activity of single Kv1 channels and significantly enhance the sensitivity of Kv1 activity to H2 O2 . Consistent with these findings, H2 O2 -evoked vasodilatation was significantly greater in the presence of 10mM l-lactate relative to lactate-free conditions, yet was abolished in the presence of 10mM pyruvate, which shifts the LDH reaction towards the generation of NAD+ . Moreover, the enhancement of H2 O2 -induced vasodilatation was abolished in arteries from double transgenic mice with selective overexpression of the intracellular Kvβ1.1subunit in smooth muscle cells. Together, our results indicate that the Kvβ complex of native vascular Kv1 channels serves as a nodal effector for multiple redox signals to precisely control channel activity and vascular tone in the face of dynamic tissue-derived metabolic cues. KEY POINTS: Vasodilatation of mesenteric arteries by elevated external l-lactate requires its conversion by lactate dehydrogenase. Application of either NADH or H2 O2 potentiates single Kv channel currents in excised membrane patches from mesenteric artery smooth muscle cells. The binding of NADH enhances the stimulatory effects of H2 O2 on single Kv channel activity. The vasodilatory response to H2 O2 is differentially modified upon elevation of external l-lactate or pyruvate. The presence of l-lactate enhances the vasodilatory response to H2 O2 via the Kvβ subunit complex in smooth muscle.

Smartphone-Enabled Web-Based Simulation of Cellular Neurophysiology for Laboratory Course and its Effectiveness.

The introduction of computer simulations has enhanced the teaching of neurobiology. Many simulators for personal computers are available, but in countries where schools have low school information and communication technology readiness, it is difficult to introduce computer simulations. Even in such countries, however, students often have their own smartphones and are good at operating them. Therefore, we have developed five web-based simulators that cover a wide range of neurophysiology, including single and whole-cell channel currents, membrane potentials and generation and conduction of action potentials using HTML5 and JavaScript. These simulators may be run free of charge on any device, regardless of the model or OS, thereby enabling schools that have no experience in introducing simulations to introduce them easily. These simulators were especially useful in many schools during COVID-19 restrictions. In this paper, we explain the functions of the simulators we have developed and introduce some practical examples. To verify the usefulness of the simulators, we also conducted a survey in the classrooms in which the simulators were used. Understanding and motivation to learn was shown to increase significantly, indicating that these are useful for neurobiology education.