Single-channel Recordings Research Articles

Electrographic seizure monitoring with a novel, wireless, single-channel EEG sensor

ObjectiveRecording seizures using personal seizure diaries can be challenging during everyday life and many seizures are missed or mis-reported. People living with epilepsy could benefit by having a more accurate and objective wearable EEG system for counting seizures that can be used outside of the hospital. The objective of this study was to (1) determine which seizure types can be electrographically recorded from the scalp below the hairline, (2) determine epileptologists’ ability to identify electrographic seizures from single-channels extracted from full-montage wired-EEG, and (3) determine epileptologists’ ability to identify electrographic seizures from Epilog, a wireless single-channel EEG sensor. MethodsEpilog sensors were worn concurrently during epilepsy monitoring unit (EMU) monitoring. During standard-of-care review, epileptologists were asked if the electrographic portion of the seizure was visible on single channels of wired electrodes at locations proximal to Epilog sensors, and if focal-onset, which electrode was closest to the focus. From these locations, single channels of EEG extracted from wired full-montage EEG and the proximal Epilog sensor were presented to 3 blinded epileptologists along with markers for when known seizures occurred (taken from the standard-of-care review). Control segments at inter-ictal times were included as control. The epileptologists were asked whether a seizure event was visible in the single channel EEG record at or near the marker. ResultsA total of 75 seizures were recorded from 22 of 40 adults that wore Epilog during their visit to the EMU. Epileptologists were able to visualize known seizure activity on at least one of the wired electrodes proximal to Epilog sensors for all seizure events. Epileptologists accurately identified seizures in 71% of Epilog recordings and 84% of single-channel wired recordings and were 92% accurate identifying seizures with Epilog when those seizures ended in a clinical convulsion compared to those that did not (>55%). ConclusionsEpileptologists are able to visualize seizure activity on single-channels of EEG at locations where Epilog sensors are easily placed on the scalp below hairline. Manual review of seizure annotations can be done quickly and accurately (>70% TP and >98% PPV) on single-channel EEG data. Reviewing single-channel EEG is more accurate than what has been reported in the literature on self-reporting seizures in seizure diaries, the current standard of care for seizure counting outside of the EMU. SignificanceWearable EEG will be important for seizure monitoring outside of the hospital. Epileptologists can accurately identify seizures in single-channel EEG, better than patient self-reporting in diaries based on the literature. Automated or semi-automated seizure detection on single channels of EEG could be used in the future to objectively count seizures to complement the standard of care outside of the EMU without the overt burden upon epileptologist review.

Design, assembly, and characterization of membrane-spanning DNA nanopores.

DNA nanopores are bio-inspired nanostructures that control molecular transport across lipid bilayer membranes. Researchers can readily engineer the structure and function of DNA nanopores to synergistically combine the strengths of DNA nanotechnology and nanopores. The pores can be harnessed in a wide range of areas, including biosensing, single-molecule chemistry, and single-molecule biophysics, as well as in cell biology and synthetic biology. Here, we provide a protocol for the rational design of nanobarrel-like DNA pores and larger DNA origami nanopores for targeted applications. We discuss strategies for the pores' chemical modification with lipid anchors to enable them to be inserted into membranes such as small unilamellar vesicles (SUVs) and planar lipid bilayers. The procedure covers the self-assembly of DNA nanopores via thermal annealing, their characterization using gel electrophoresis, purification, and direct visualization with transmission electron microscopy and atomic force microscopy. We also describe a gel assay to determine pore-membrane binding and discuss how to use single-channel current recordings and dye flux assays to confirm transport through the pores. We expect this protocol to take approximately 1 week to complete for DNA nanobarrel pores and 2-3 weeks for DNA origami pores.

A Lipid Bilayer Formed on a Hydrogel Bead for Single Ion Channel Recordings.

Ion channel proteins play important roles in various cell functions, making them attractive drug targets. Artificial lipid bilayer recording is a technique used to measure the ion transport activities of channel proteins with high sensitivity and accuracy. However, the measurement efficiency is low. In order to improve the efficiency, we developed a method that allows us to form bilayers on a hydrogel bead and record channel currents promptly. We tested our system by measuring the activities of various types of channels, including gramicidin, alamethicin, α-hemolysin, a voltage-dependent anion channel 1 (VDAC1), a voltage- and calcium-activated large conductance potassium channel (BK channel), and a potassium channel from Streptomyces lividans (KcsA channel). We confirmed the ability for enhanced measurement efficiency and measurement system miniaturizion.

Deep-learned spike representations and sorting via an ensemble of auto-encoders

Spike sorting refers to the technique of detecting signals generated by single neurons from multi-neuron recordings and is a valuable tool for analyzing the relationships between individual neuronal activity patterns and specific behaviors. Since the precision of spike sorting affects all subsequent analyses, sorting accuracy is critical. Many semi-automatic to fully-automatic spike sorting algorithms have been developed. However, due to unsatisfactory classification accuracy, manual sorting is preferred by investigators despite the intensive time and labor costs. Thus, there still is a strong need for fully automatic spike sorting methods with high accuracy. Various machine learning algorithms have been developed for feature extraction but have yet to show sufficient accuracy for spike sorting. Here we describe a deep learning-based method for extracting features from spike signals using an ensemble of auto-encoders, each with a distinct architecture for distinguishing signals at different levels of resolution. By utilizing ensemble of auto-encoder ensemble, where shallow networks better represent overall signal structure and deep networks better represent signal details, extraction of high-dimensional representative features for improved spike sorting performance is achieved. The model was evaluated on publicly available simulated datasets and single-channel and 4-channel tetrode in vivo datasets. Our model not only classified single-channel spikes with varying degrees of feature similarities and signal to noise levels with higher accuracy, but also more precisely determined the number of source neurons compared to other machine learning methods. The model also demonstrated greater overall accuracy for spike sorting 4-channel tetrode recordings compared to single-channel recordings.

Alcohol metabolite acetic acid activates BK channels in a pH-dependent manner and decreases calcium oscillations and exocytosis of secretory granules in rat pituitary GH3 cells.

Acetaldehyde and acetic acid/acetate, the active metabolites of alcohol (ethanol, EtOH), generate actions of their own ranging from behavioral, physiological, to pathological/cancerogenic effects. EtOH and acetaldehyde have been studied to some depth, whereas the effects of acetic acid have been less well explored. In this study, we investigated the effect of acetic acid on big conductance calcium-activated potassium (BK) channels present in GH3 rat pituitary tumor cells in more detail. In whole cell voltage clamp recordings, extracellular application of acetic acid increased total outward currents in a dose-dependent manner. This effect was prevented after the application of the specific BK channel blocker paxilline. Acetic acid action was pH-dependent-in whole cell current and single BK channel recordings, open probability (Po) was significantly increased by extracellular pH reduction and decreased by neutral or base pH. Acetic acid hyperpolarized the membrane potential, whereas acidic physiological solution had a depolarizing effect. Moreover, acetic acid reduced calcium (Ca2+) oscillations and exocytosis of growth hormone contained secretory granules from GH3 cells. These effects were partially prevented by BK inhibitors-tetraethylammonium or paxillin. In conclusion, our experiments indicate that acetic acid activates BK channels in GH3 cells which eventually contribute to acetic acid-induced membrane hyperpolarization, cessation of Ca2+ oscillations, and decrease of growth hormone release.

Toxic oligomers of the amyloidogenic HypF-N protein form pores in mitochondrial membranes

Studies on the amyloidogenic N-terminal domain of the E. coli HypF protein (HypF-N) have contributed significantly to a detailed understanding of the pathogenic mechanisms in neurodegenerative diseases characterised by the formation of misfolded oligomers, by proteins such as amyloid-β, α-synuclein and tau. Given that both cell membranes and mitochondria are increasingly recognised as key targets of oligomer toxicity, we investigated the damaging effects of aggregates of HypF-N on mitochondrial membranes. Essentially, we found that HypF-N oligomers characterised by high surface hydrophobicity (type A) were able to trigger a robust permeabilisation of mito-mimetic liposomes possessing cardiolipin-rich membranes and dysfunction of isolated mitochondria, as demonstrated by a combination of mitochondrial shrinking, lowering of mitochondrial membrane potential and cytochrome c release. Furthermore, using single-channel electrophysiology recordings we obtained evidence that the type A aggregates induced currents reflecting formation of ion-conducting pores in mito-mimetic planar phospholipid bilayers, with multi-level conductances ranging in the hundreds of pS at negative membrane voltages. Conversely, HypF-N oligomers with low surface hydrophobicity (type B) could not permeabilise or porate mitochondrial membranes. These results suggest an inherent toxicity of membrane-active aggregates of amyloid-forming proteins to mitochondria, and that targeting of oligomer-mitochondrial membrane interactions might therefore afford protection against such damage.

A Biometric System Based on Single-channel EEG Recording in One-second

In recent years, there are great research interests in using the Electroencephalogram (EEG) signals in biometrics applications. The strength of EEG signals as a biometric comes from its major fraud prevention capability. However, EEG signals are so sensitive, and many factors affect its usage as a biometric; two of these factors are the number of channels, and the required time for acquiring the signal; these factors affect the convenience and practicality. This study proposes a novel approach for EEG-based biometrics that optimizes the channels of acquiring data to only one channel. And the time to only one second. The results are compared against five commonly used classifiers named: KNN, Random Forest (RF), Support Vector Machine (SVM), Decision Tables (DT), and Naïve Bayes (NB). We test the approach on the public Texas data repository. The results prove the constancy of the approach for the eight minutes. The best result of the eyes-closed scenario is Average True Positive Rate (TPR) 99.1% and 98.2% for the eyes-opened. And it reaches 100% for multiple subjects.

Machine learning for nocturnal mass diagnosis of atrial fibrillation in a population at risk of sleep-disordered breathing

Objective: In this research, we introduce a new methodology for atrial fibrillation (AF) diagnosis during sleep in a large population sample at risk of sleep-disordered breathing. Approach: The approach leverages digital biomarkers and recent advances in machine learning (ML) for mass AF diagnosis from overnight-hours of single-channel electrocardiogram (ECG) recording. Four databases, totaling n = 3088 patients and p = 26 913 h of continuous single-channel electrocardiogram raw data were used. Three of the databases (n = 125, p = 2513) were used for training a ML model in recognizing AF events from beat-to-beat time series. Visit 1 of the sleep heart health study database (SHHS1, n = 2963, p = 24 400) was used as the test set to evaluate the feasibility of identifying prominent AF from polysomnographic recordings. By combining AF diagnosis history and a cardiologist’s visual inspection of individuals suspected of having AF (n = 118), a total of 70 patients were diagnosed with prominent AF in SHHS1. Main results: Model prediction on SHHS1 showed an overall and in classifying individuals with or without prominent AF. was non-inferior (p = 0.03) for individuals with an apnea-hypopnea index (AHI) ≥15 versus AHI < . Over 22% of correctly identified prominent AF rhythm cases were not previously documented as AF in SHHS1. Significance: Individuals with prominent AF can be automatically diagnosed from an overnight single-channel ECG recording, with an accuracy unaffected by the presence of moderate-to-severe obstructive sleep apnea. This approach enables identifying a large proportion of AF individuals that were otherwise missed by regular care.

Epac2 O-GlcNAcylation, a new player in high glucose-mediated cardiac calcium mishandling

Epidemiological and preclinical studies have pointed out a correlation between hyperglycemia and increasing risk of heart failure. In cardiomyocytes, hyperglycemia has been shown to alter Ca2+ signaling via CaMKII a downstream effector of Epac2, a key player in Ca2+ mishandling. However the role of Epac2 in high-glucose (HG) mediated Ca2+ dysregulation is unclear. To test the involvement of Epac2 in hyperglycemia-mediated cardiac Ca2+ mishandling. Ca2+ signaling was measured by confocal microscopy in Fluo-4 loaded ventricular cardiomyocytes isolated from C57BL6 and Epac2-KO mice and treated with different glucose concentrations. HG (500 mg/dL) enhanced the occurrence of Ca2+ sparks and pro-arrhythmic events in WT mice but not in Epac2 KO. This increase was prevented by Epac2 pharmacological inhibition (ESI-05 10 μM) (Ca2+ sparks frequency [#/s/100 μm]: 1.07 ± 0.39 for HG vs. 0.16 ± 0.06 for normal glucose [NG, 100 mg/dL], P < 0.05; 0.23 ± 0.15 for ESI-05 + HG vs. NG, P = NS; 0.09 ± 0.04 for Epac2-KO at HG vs. 0.11 ± 0.04 at NG, P = N.S.). The Epac2-dependent alterations of Ca2+ signaling upon HG were associated to higher RyR open probability measured by single channel recording incorporated into lipid bilayers (P0 = 0.76 ± 0.07 in HG vs. 0.22 ± 0.07 in NG, P < 0.01). This increase was prevented by ESI-05 or O-GlcNAcylation inhibition by diazo-5-oxonorleucine (DON), suggesting an Epac2 activation through O-GlcNAcylation under HG. This is confirmed by Epac2-based FRET biosensor activity measurements. Moreover, Co-IP experiments indicated an O-GlcNacylation of Epac2. Our work shows an Epac2 O-GlcNAcylation by HG responsible for pro-arrhythmic SR Ca2+ leak in cardiomyocytes, a new mechanism that might be involved in diabetic cardiomyopathy.

Expression of p11 and Heteromeric TASK Channels in Rat Carotid Body Glomus Cells and Nerve Growth Factor-differentiated PC12 Cells.

TWIK-related acid-sensitive K+ (TASK) homomeric channels, TASK1 and TASK3, are present in PC12 cells. The channels do not heteromerize due plausibly to a lack of p11 protein. Single-channel recording reveals that most of the rat carotid body (CB) glomus cells express heteromeric TASK1-TASK3 channels, but the presence of p11 in glomus cells has not yet been verified. TASK1, but not TASK3, binds to p11, which has a retention signal for the endoplasmic reticulum. We hypothesized that p11 could facilitate heteromeric TASK1-TASK3 formation in glomus cells. We investigated this hypothesis in isolated immunocytochemically identified rat CB glomus cells. The findings were that glomus cells expressed p11-like immunoreactive (IR) material, and TASK1- and TASK3-like IR material mainly coincided in the cytoplasm. The proximity ligation assay showed that TASK1 and TASK3 heteromerized. In separate experiments, supporting evidence for the major role of p11 for channel heteromerization was provided in PC12 cells stimulated by nerve growth factor. p11 production took place there via multiple signaling pathways comprising mitogen-activated protein kinase and phospholipase C, and heteromeric TASK1-TASK3 channels were formed. We conclude that p11 is expressed and TASK1 and TASK3 heteromerize in rat CB glomus cells.

The Epilepsy-Related Protein PCDH19 Regulates Tonic Inhibition, GABAAR Kinetics, and the Intrinsic Excitability of Hippocampal Neurons

PCDH19 encodes for protocadherin-19 (PCDH19), a cell-adhesion molecule of the cadherin superfamily preferentially expressed in the brain. PCDH19 mutations cause a neurodevelopmental syndrome named epileptic encephalopathy, early infantile, 9 (EIEE9) characterized by seizures associated with cognitive and behavioral deficits. We recently reported that PCDH19 binds the alpha subunits of GABAA receptors (GABAARs), modulating their surface availability and miniature inhibitory postsynaptic currents (mIPSCs). Here, we investigated whether PCDH19 regulatory function on GABAARs extends to the extrasynaptic receptor pool that mediates tonic current. In fact, the latter shapes neuronal excitability and network properties at the base of information processing. By combining patch-clamp recordings in whole-cell and cell-attached configurations, we provided a functional characterization of primary hippocampal neurons from embryonic rats of either sex expressing a specific PCDH19 short hairpin (sh)RNA. We first demonstrated that PCDH19 downregulation reduces GABAAR-mediated tonic current, evaluated by current shift and baseline noise analysis. Next, by single-channel recordings, we showed that PCDH19 regulates GABAARs kinetics without altering their conductance. In particular, GABAARs of shRNA-expressing neurons preferentially exhibit brief openings at the expense of long ones, thus displaying a flickering behavior. Finally, we showed that PCDH19 downregulation reduces the rheobase and increases the frequency of action potential firing, thus indicating neuronal hyperexcitability. These findings establish PCDH19 as a critical determinant of GABAAR-mediated tonic transmission and GABAARs gating, and provide the first mechanistic insights into PCDH19-related hyperexcitability and comorbidities.

Classification of emotions induced by horror and relaxing movies using single-channel EEG recordings

It has been observed from recent studies that corticolimbic Theta rhythm from EEG recordings perceived as fear or threatening scene during neural processing of visual stimuli. In additions, neural oscillations' patterns in Theta, Alpha and Beta sub-bands also play important role in brain's emotional processing. Inspired from these findings, in this paper we attempt to classify two different emotional states by analyzing single-channel EEG recordings. A video clip that can evoke 3 different emotional states: neutral, relaxation and scary is shown to 19 college-aged subjects and they were asked to score their emotional outcome by giving a number between 0 to 10 (where 0 means not scary at all and 10 means the most scary). First, recorded EEG data were preprocessed by stationary wavelet transform (SWT) based artifact removal algorithm. Then power distribution in simultaneous time-frequency domain was analyzed using short-time Fourier transform (STFT) followed by calculating the average power during each 0.2s time-segment for each brain sub-band. Finally, 46 features, as the mean power of frequency bands between 4 and 50 Hz during each time-segment, containing 689 instances\textemdash for each subject \textemdash were collected for classification. We found that relaxation and fear emotions evoked during watching scary and relaxing movies can be classified with average classification rate of 94.208\% using K-NN by applying methods and materials proposed in this paper. We also classified the dataset using SVM and we found out that K-NN classifier (when \begin{math} k=1 \end{math}) outperforms SVM in classifying EEG dynamics induced by horror and relaxing movies, however, for \begin{math} K &amp;gt;1 \end{math} in K-NN, SVM has better average classification rate.

Facilitating stochastic resonance as a pre-emphasis method for neural spike detection

Objective. We aim to increase the number of neural spikes that can be detected in a single channel extracellular neural recording. Approach. We propose a pre-emphasis method facilitating stochastic resonance (SR), where we introduce the band-pass-filtered noisy extracellular recording to an overdamped Brownian particle in a monostable well. The x-position of the Brownian particle is the output of the proposed pre-emphasis method. Threshold is applied on the output for spike detection. To characterize the dynamics and the solution of the system, we use a synthetic dataset generated by adding Gaussian white noise at different intensities to an intracellular recording. Then, we evaluate and compare the spike detection performance of the proposed method on a public synthetic extracellular dataset. Main results. The proposed SR-based spike detection improves the signal-to-noise ratio of the intracellular-based synthetic dataset as much as 7.35 dB and outperforms the state-of-the-art pre-emphasis methods in false positive and false negative rates in 15 of the 16 synthetic extracellular datasets, with 100% sensitivity and positive predictivity values in seven of the recordings. Significance. The method has the potential of significantly increasing the number of neurons that can be monitored from a single-channel extracellular recording.

Inhibition of the Glycine Receptor alpha 3 Function by Colchicine.

Colchicine is a plant alkaloid that is widely used as a therapeutic agent. It is widely accepted that colchicine reduces the production of inflammatory mediators mainly by altering cytoskeleton dynamics due to its microtubule polymerization inhibitory activity. However, other lines of evidence have shown that colchicine exerts direct actions on the function of ion channels, which are independent of cytoskeleton alterations. Colchicine is able to modify the function of several pentameric ligand-gated ion channels, including glycine receptors (GlyRs). Previous electrophysiological studies have shown that colchicine act as an antagonist of GlyRs composed by the α 1 subunit. In addition, it was recently demonstrated that colchicine directly bind to the α 3 subunit of GlyRs. Interestingly, other studies have shown a main role of α 3GlyRs on chronic inflammatory pain. Nevertheless, the functional effects of colchicine on the α 3GlyR function are still unknown. Here, by using electrophysiological techniques and bioinformatics, we show that colchicine inhibited the function of the α 3GlyRs. Colchicine elicited concentration-dependent inhibitory effects on α 3GlyRs at micromolar range and decreased the apparent affinity for glycine. Single-channel recordings show that the colchicine inhibition is associated with a decrease in the open probability of the ion channel. Molecular docking assays suggest that colchicine preferentially bind to the orthosteric site in the closed state of the ion channel. Altogether, our results suggest that colchicine is a competitive antagonist of the α 3GlyRs.

Molecular mechanisms underlying menthol binding and activation of TRPM8 ion channel

Menthol in mints elicits coolness sensation by selectively activating TRPM8 channel. Although structures of TRPM8 were determined in the apo and liganded states, the menthol-bounded state is unresolved. To understand how menthol activates the channel, we docked menthol to the channel and systematically validated our menthol binding models with thermodynamic mutant cycle analysis. We observed that menthol uses its hydroxyl group as a hand to specifically grab with R842, and its isopropyl group as legs to stand on I846 and L843. By imaging with fluorescent unnatural amino acid, we found that menthol binding induces wide-spread conformational rearrangements within the transmembrane domains. By Φ analysis based on single-channel recordings, we observed a temporal sequence of conformational changes in the S6 bundle crossing and the selectivity filter leading to channel activation. Therefore, our study suggested a ‘grab and stand’ mechanism of menthol binding and how menthol activates TRPM8 at the atomic level.

Design, Synthesis, and Functional Evaluation of a Novel Series of Phosphonate-Functionalized 1,2,3-Triazoles as Positive Allosteric Modulators of α7 Nicotinic Acetylcholine Receptors.

The α7 nicotinic acetylcholine receptor is a pentameric ligand-gated ion channel widely distributed in the central nervous system, mainly in the hippocampus and cortex. The enhancement of its activity by positive allosteric modulators (PAMs) is a promising therapeutic strategy for cognitive deficits and neurodegenerative disorders. With the aim of developing novel scaffolds with PAM activity, we designed and synthesized a series of phosphonate-functionalized 1,4-disubstituted 1,2,3-triazoles using supported copper nanoparticles as the cycloaddition reaction catalyst and evaluated their activity on α7 receptors by single-channel and whole-cell recordings. We identified several triazole derivatives that displayed PAM activity, with the compound functionalized with the methyl phosphonate group being the most efficacious one. At the macroscopic level, α7 potentiation was evidenced as an increase of the maximal currents elicited by acetylcholine with minimal effects on desensitization, recapitulating the actions of type I PAMs. At the single-channel level, the active compounds did not affect channel amplitude but significantly increased the duration of channel openings and activation episodes. By using chimeric and mutant α7 receptors, we demonstrated that the new α7 PAMs share transmembrane structural determinants of potentiation with other chemically nonrelated PAMs. To gain further insight into the chemical basis of potentiation, we applied structure-activity relationship strategies involving modification of the chain length, inversion of substituent positions in the triazole ring, and changes in the aromatic nucleus. Our findings revealed that the phosphonate-functionalized 1,4-disubstituted 1,2,3-triazole is a novel pharmacophore for the development of therapeutic agents for neurological and neurodegenerative disorders associated with cholinergic dysfunction.

PGF2α stimulates the 10-pS Cl- channel and thiazide-sensitive Na+-Cl- cotransporter in the distal convoluted tubule.

We used patch-clamp and Western blot analysis to test whether PGF2α stimulates the basolateral 10-pS Cl- channel and thiazide-sensitive Na+-Cl- cotransporter (NCC) in the distal convoluted tubule (DCT) via a prostaglandin F receptor (FP-R). Single channel and whole cell recordings demonstrated that PGF2α stimulated the 10-pS Cl- channel in the DCT. The stimulatory effect of PGF2α on the Cl- channel was mimicked by a FP-R agonist, latanoprost, but was abrogated by blocking FP-R with AL8810. Also, the effect of PGF2α on the Cl- channel in the DCT was recapitulated by stimulating PKC but was blocked by inhibiting PKC. Furthermore, inhibition of p38 MAPK but not ERK blocked the effect of PGF2α on the 10-pS Cl- channel. Inhibition of NADPH oxidase also abrogated the stimulatory effect of PGF2α on the 10-pS Cl- channel, while the addition of 10 μM H2O2 mimicked the stimulatory effect of PGF2α on the 10-pS Cl- channel. Moreover, superoxide-related species may mediate the stimulatory effect of PGF2α on the 10-pS Cl- channel because the stimulatory effect of PGF2α and H2O2 was not additive. Western blot analysis showed that infusion of PGF2α in vivo not only increased the expression of FP-R but also increased the expression of total NCC and phosphorylated NCC. We conclude that PGF2α stimulates the basolateral 10-pS Cl- channel in the DCT by activating FP-R through PKC/p38 MAPK and NADPH oxidase-dependent pathways. The stimulatory effects of PGF2α on the Cl- channel and NCC may contribute to PGF2α-induced increases in NaCl reabsorption in the DCT.

Mechanism of calcium potentiation of the α7 nicotinic acetylcholine receptor.

The α7 nicotinic acetylcholine receptor (nAChR) is among the most abundant types of nAChR in the brain, yet the ability of nerve-released ACh to activate α7 remains enigmatic. In particular, a major population of α7 resides in extra-synaptic regions where the ACh concentration is reduced, owing to dilution and enzymatic hydrolysis, yet ACh shows low potency in activating α7. Using high-resolution single-channel recording techniques, we show that extracellular calcium is a powerful potentiator of α7 activated by low concentrations of ACh. Potentiation manifests as robust increases in the frequency of channel opening and the average duration of the openings. Molecular dynamics simulations reveal that calcium binds to the periphery of the five ligand binding sites and is framed by a pair of anionic residues from the principal and complementary faces of each site. Mutation of residues identified by simulation prevents calcium from potentiating ACh-elicited channel opening. An anionic residue is conserved at each of the identified positions in all vertebrate species of α7. Thus, calcium associates with a novel structural motif on α7 and is an obligate cofactor in regions of limited ACh concentration.

Effect of Auxin (IAA) on the Fast Vacuolar (FV) Channels in Red Beet (Beta vulgaris L.) Taproot Vacuoles.

In contrast to the well-studied effect of auxin on the plasma membrane K+ channel activity, little is known about the role of this hormone in regulating the vacuolar K+ channels. Here, the patch-clamp technique was used to investigate the effect of auxin (IAA) on the fast-activating vacuolar (FV) channels. It was found that the macroscopic currents displayed instantaneous currents, which at the positive potentials were about three-fold greater compared to the one at the negative potentials. When auxin was added to the bath solution at a final concentration of 1 µM, it increased the outward currents by about 60%, but did not change the inward currents. The imposition of a ten-fold vacuole-to-cytosol KCl gradient stimulated the efflux of K+ from the vacuole into the cytosol and reduced the K+ current in the opposite direction. The addition of IAA to the bath solution with the 10/100 KCl gradient decreased the outward current and increased the inward current. Luminal auxin reduced both the outward and inward current by approximately 25% compared to the control. The single channel recordings demonstrated that cytosolic auxin changed the open probability of the FV channels at the positive voltages to a moderate extent, while it significantly increased the amplitudes of the single channel outward currents and the number of open channels. At the positive voltages, auxin did not change the unitary conductance of the single channels. We suggest that auxin regulates the activity of the fast-activating vacuolar (FV) channels, thereby causing changes of the K+ fluxes across the vacuolar membrane. This mechanism might serve to tightly adjust the volume of the vacuole during plant cell expansion.

Transmembrane Epitope Delivery by Passive Protein Threading through the Pores of the OmpF Porin Trimer.

Trimeric porins in the outer membrane (OM) of Gram-negative bacteria are the conduits by which nutrients and antibiotics diffuse passively into cells. The narrow gateways that porins form in the OM are also exploited by bacteriocins to translocate into cells by a poorly understood process. Here, using single-channel electrical recording in planar lipid bilayers in conjunction with protein engineering, we explicate the mechanism by which the intrinsically unstructured N-terminal translocation domain (IUTD) of the endonuclease bacteriocin ColE9 is imported passively across the Escherichia coli OM through OmpF. We show that the import is dominated by weak interactions of OmpF pores with binding epitopes within the IUTD that are orientationally biased and result in the threading of over 60 amino acids through 2 subunits of OmpF. Single-molecule kinetic analysis demonstrates that the IUTD enters from the extracellular side of OmpF and translocates to the periplasm where the polypeptide chain does an about turn in order to enter a neighboring subunit, only for some of these molecules to pop out of this second subunit before finally re-entering to form a stable complex. These intimately linked transport/binding processes generate an essentially irreversible, hook-like assembly that constrains an import activating peptide epitope between two subunits of the OmpF trimer.