Single-channel Activity Research Articles

Human TMC1 and TMC2 are mechanically gated ion channels.

Mammalian transmembrane channel-like proteins 1 and 2 (TMC1 and TMC2) have emerged as very promising candidate mechanotransduction channels in hair cells. However, controversy persists because the heterogeneously expressed TMC1/2 in cultured cells lack evidence of mechanical gating, primarily due to their absence from the plasma membrane. By employing domain swapping with OSCA1.1 and subsequent point mutations, we successfully identified membrane-localized mouse TMC1/2 mutants, demonstrating that they are mechanically gated in heterologous cells. Further, whole-genome CRISPRi screening enabled wild-type human TMC1/2 localization in the plasma membrane, where they responded robustly to poking stimuli. In addition, wild-type human TMC1/2 showed stretch-activated currents and clear single-channel current activities. Deafness-related TMC1 mutations altered the reversal potential of TMC1, indicating that TMC1/2 are pore-forming mechanotransduction channels. In summary, our study provides evidence that human TMC1/2 are pore-forming, mechanically activated ion channels, supporting their roles as mechanotransduction channels in hair cells.

Amyloid beta Aβ1-40 activates Piezo1 channels in brain capillary endothelial cells.

Amyloid-beta (Aβ) peptide accumulation on blood vessels in the brain is a hallmark of neurodegeneration. While Aβ peptides constrict cerebral arteries and arterioles, their impact on capillaries is less understood. Aβ was recently shown to constrict brain capillaries through pericyte contraction, but whether-and if so how-Aβ affects endothelial cells (ECs) remains unknown. ECs represent the predominant vascular cell type in the cerebral circulation, and we recently showed that the mechanosensitive ion channel Piezo1 is functionally expressed in the plasma membrane of ECs. Since Aβ disrupts membrane structures, we hypothesized that Aβ1-40, the predominantly deposited isoform in the cerebral circulation, alters endothelial Piezo1 function. Using patch clamp electrophysiology and freshly isolated capillary ECs, we assessed the impact of Aβ1-40 peptide on single-channel Piezo1 activity. We show that Aβ1-40 increased Piezo1 open probability and the channel open time. Aβ1-40 effects were absent when Piezo1 was genetically deleted or when a superoxide dismutase/catalase mimetic was used. Further, Aβ1-40 enhanced Piezo1 mechanosensitivity and lowered the pressure of half-maximal Piezo1 activation. Our data collectively suggest that Aβ1-40 facilitates higher Piezo1-mediated cation influx in brain ECs. These novel findings have the potential to unravel the possible involvement of Piezo1 modulation in the pathophysiology of neurodegenerative diseases characterized by Aβ accumulation.

Loss of the alpha subunit distal furin cleavage site blunts ENaC activation following Na+ restriction.

Epithelial Na+ channels (ENaCs) are activated by proteolysis of the α and γ subunits at specific sites flanking embedded inhibitory tracts. To examine the role of α subunit proteolysis in channel activation in vivo, we generated mice lacking the distal furin cleavage site in the α subunit (αF2M mice). On a normal Na+ control diet, no differences in ENaC protein abundance in kidney or distal colon were noted between wild-type (WT) and αF2M mice. Patch-clamp analyses revealed similar levels of ENaC activity in kidney tubules, while no physiologically relevant differences in blood chemistry or aldosterone levels were detected. Male αF2M mice did exhibit diminished ENaC activity in the distal colon, as measured by amiloride-sensitive short-circuit current (ISC). Following dietary Na+ restriction, WT and αF2M mice had similar natriuretic and colonic ISC responses to amiloride. However, single-channel activity was significantly lower in kidney tubules from Na+-restricted αF2M mice compared with WT littermates. ENaC α and γ subunit expression in kidney and distal colon were also enhanced in Na+-restricted αF2M vs. WT mice, in association with higher aldosterone levels. These data provide evidence that disrupting α subunit proteolysis impairs ENaC activity in vivo, requiring compensation in response to Na+ restriction. KEY POINTS: The epithelial Na+ channel (ENaC) is activated by proteolytic cleavage in vitro, but key questions regarding the role of ENaC proteolysis in terms of whole-animal physiology remain to be addressed. We studied the in vivo importance of this mechanism by generating a mouse model with a genetic disruption to a key cleavage site in the ENaC's α subunit (αF2M mice). We found that αF2M mice did not exhibit a physiologically relevant phenotype under normal dietary conditions, but have impaired ENaC activation (channel open probability) in the kidney during salt restriction. ENaC function at the organ level was preserved in salt-restricted αF2M mice, but this was associated with higher aldosterone levels and increased expression of ENaC subunits, suggesting compensation was required to maintain homeostasis. These results provide the first evidence that ENaC α subunit proteolysis is a key regulator of channel activity in vivo.

Pressure regulation of L-type Ca2+ channels and its role in myogenic response

L-type calcium (Ca2+) channels hold a pivotal position in governing intracellular Ca2+ concentration within smooth muscle and the establishment of myogenic tone. Their primary regulatory mechanism has historically been linked to voltage. Nevertheless, it remains a subject of inquiry whether voltage represents the exclusive modulator or whether these channels also exhibit mechanosensitivity. This study seeks to elucidate the influence of pressure stimuli on L-type Ca2+ channels in the resistance arteries of rodents (rats and mice). The investigative approach spans from the scrutiny of individual smooth muscle cells employing methodologies such as whole-cell and cell-attached patch-clamp electrophysiology, immunohistochemistry, proximity ligation assay, and super-resolution microscopy to the evaluation of intact arteries utilizing pressure myography. The findings obtained through whole-cell patch-clamp electrophysiology unveil a notable augmentation in L-type Ca2+ current in response to pressure stimuli. Subsequent scrutiny at the level of single-channel activity discloses that this augmentation is ascribed to an enhancement in functional coupling. Further inquiry reveals that the intensified functional coupling in response to pressure is contingent upon two principal factors: 1) the facilitation of cooperative gating mediated by protein kinase C (PKC), and 2) the traffcking of subunits to caveolae. Notably, functional experiments, involving depolarization elicited through the application of 30 mM KCl, underscore that arterial tone and cytosolic Ca2+ concentration responses are markedly more pronounced when the arteries are subjected to a pressure of 80 mmHg, as opposed to 20 mmHg. The culmination of these observations from both patch-clamp experiments and functional investigations supports the assertion that L-type Ca2+ channels are indeed amenable to mechanosensory influences, in addition to their well-documented voltage-dependent behavior. In summation, this investigation underscores that the regulatory landscape of L-type Ca2+ channels transcends the confines of voltage control, emphasizing the significance of considering pressure sensitivity in both physiological and pathological contexts. Ongoing research endeavors aim to delineate with precision the signaling complexes within vascular smooth muscle that respond to mechanical forces. Supported by the Canadian Institute for Health Research, National Institute of Health and the Rorabeck Chair in Vascular Biology and Neuroscience. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Independent regulation of Piezo1 activity by principal and intercalated cells of the collecting duct

The renal collecting duct is continuously exposed to a wide spectrum of fluid flow rates and osmotic gradients. Expression of a mechanoactivated Piezo1 channel is the most prominent in the collecting duct. However, the status and regulation of Piezo1 in functionally distinct principal and intercalated cells (PCs and ICs) of the collecting duct remain to be determined. We used pharmacological Piezo1 activation to quantify Piezo1-mediated [Ca2+]i influx and single-channel activity separately in PCs and ICs of freshly isolated collecting ducts with fluorescence imaging and electrophysiological tools. We also employed a variety of systemic treatments to examine their consequences on Piezo1 function in PCs and ICs. Piezo1 selective agonists, Yoda-1 or Jedi-2, induced a significantly greater Ca2+ influx in PCs than in ICs. Using patch clamp analysis, we recorded a Yoda-1-activated nonselective channel with 18.6± 0.7 pS conductance on both apical and basolateral membranes. Piezo1 activity in PCs but not ICs was stimulated by short-term diuresis (injections of furosemide) and reduced by antidiuresis (water restriction for 24 h). However, prolonged stimulation of flow by high K+ diet decreased Yoda-1-dependent Ca2+ influx without changes in Piezo1 levels. Water supplementation with NH4Cl to induce metabolic acidosis stimulated Piezo1 activity in ICs but not in PCs. Overall, our results demonstrate functional Piezo1 expression in collecting duct PCs (more) and ICs (less) on both apical and basolateral sides. We also show that acute changes in fluid flow regulate Piezo1-mediated [Ca2+]i influx in PCs, whereas channel activity in ICs responds to systemic acid-base stimuli.

Capsazepine activates amiloride-insensitive ENaC-like channels in human leukemia cells

Sodium influx carried out by ion channels is one of the main regulators of water-salt and volume balance in cells of blood origin. Previously, we described amiloride-insensitive ENaC-like channels in human myeloid leukemia K562 cells; the intracellular regulatory mechanisms of the channels are associated with actin cytoskeleton dynamics. Recently, an extracellular mechanism of ENaC-like channels activation in K562 cells by the action of serine protease trypsin has been revealed. The other extracellular pathways that modulate ENaC (epithelial Na+ channel) activity and sodium permeability in transformed blood cells are not yet fully investigated. Here, we study the action of capsazepine (CPZ), as δ-ENaC activator, on single channel activity in K562 cells in whole-cell patch clamp experiments. Addition of CPZ (2 μM) to the extracellular solution caused an activation of sodium channels with typical features; unitary conductance was 15.1 ± 0.8 pS. Amiloride derivative benzamil (50 μM) did not inhibit their activity. Unitary currents and conductance of CPZ-activated channels were higher in Na+-containing extracellular solution than in Li+, that is one of the main fingerprints of δ-ENaC. The results of RT-PCR analysis and immunofluorescence staining also confirmed the expression of δ-hENaC (as well as α-, β-, γ-ENaC) at the mRNA and protein level. These findings allow us to speculate that CPZ activates amiloride-insensitive ENaC-like channels that contain δ-ENaC in К562 cells. Our data reveal a novel extracellular mechanism for ENaC-like activation in human leukemia cells.

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.

Representation and decoding of bilateral arm motor imagery using unilateral cerebral LFP signals.

In the field of upper limb brain computer interfaces (BCIs), the research focusing on bilateral decoding mostly based on the neural signals from two cerebral hemispheres. In addition, most studies used spikes for decoding. Here we examined the representation and decoding of different laterality and regions arm motor imagery in unilateral motor cortex based on local field potentials (LFPs). The LFP signals were recorded from a 96-channel Utah microelectrode array implanted in the left primary motor cortex of a paralyzed participant. There were 7 kinds of tasks: rest, left, right and bilateral elbow and wrist flexion. We performed time-frequency analysis on the LFP signals and analyzed the representation and decoding of different tasks using the power and energy of different frequency bands. The frequency range of <8 Hz and >38 Hz showed power enhancement, whereas 8-38 Hz showed power suppression in spectrograms while performing motor imagery. There were significant differences in average energy between tasks. What's more, the movement region and laterality were represented in two dimensions by demixed principal component analysis. The 135-300 Hz band signal had the highest decoding accuracy among all frequency bands and the contralateral and bilateral signals had more similar single-channel power activation patterns and larger signal correlation than contralateral and ipsilateral signals, bilateral and ipsilateral signals. The results showed that unilateral LFP signals had different representations for bilateral motor imagery on the average energy of the full array and single-channel power levels, and different tasks could be decoded. These proved the feasibility of multilateral BCI based on the unilateral LFP signal to broaden the application of BCI technology. https://www.chictr.org.cn/showproj.aspx?proj=130829, identifier ChiCTR2100050705.

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.

BK channel dysfunction underlies small vessel disease of the brain in both hypertension and Alzheimer's disease

Alzheimer's disease (AD) and hypertension-related vascular dementia share many common clinical and radiological features including cerebral small vessel disease (cSVD) of the brain, thought to be the mechanism behind the reduction in cerebral blood flow (CBF). The aim of this study was to determine if there are common mechanisms underlying a reduced CBF in dementia. CBF is determined by depolarisation induced contraction of arterial smooth muscle cells (SMCs). This contraction is buffered by the activation of the large-conductance Ca 2+ -activated K + (BK) channels on the plasma membrane (PM), by transient localised Ca 2+ release from ryanodine receptors (RyR) on the sarcoplasmic reticulum (SR), known as Ca 2+ sparks.Hypothesis: Defects in BK channel activation leads to the cSVD phenotype in these diseases.8-month old male spontaneously hypertensive (BPH/2) mice and normotensive controls (BPN/3) were used to study hypertension-induced cSVD. To study AD related cSVD we used 18–20 month old male APP23 mice. We used a range of physiological techniques to investigate vascular ion channel function within the cerebral microvasculature. Laser Doppler flowmetry and behavioural assessments were performed on the BPH/2 and BPN/3 mice to initially determine if they are a model of hypertension-related vascular dementia.BPH/2 mice displayed reduced CBF and cognitive impairment. Pressure-induced constriction from cerebral pial arteries was significantly greater in both the BPH/2 and APP23 animals compared to controls. This was the result of reduced BK channel activity, as confirmed by reduced constriction to the BK channel blocker paxilline. Electrophysiology experiments showed a decrease in spontaneous transient outward currents, which represent BK channel activation by Ca 2+ sparks. In the APP23 model, this attenuated BK channel activation was due to reduced Ca 2+ spark frequency. However, in the BPH/2 mice, Ca 2+ spark frequency was equal between hypertensive and normotensive mice. Furthermore, there were no differences in single channel BK channel activity over a range of [Ca 2+ ]. Live cell imaging of isolated SMCs labelled with membrane dyes, showed a reduced interaction between the SR and the PM. Proximity ligation assay was used on fixed SMCs to determine if BK channel and RyR are within 40 nm of each other. SMCs from BPH/2 cerebral arteries has significantly less puncta suggesting a physical separation between Ca 2+ sparks and BK channel, rendering the [Ca 2+ ] local to the BK channel insufficient for activation.Our data indicate that vascular BK channel dysfunction is a common mechanism underpinning reduced CBF in SVD. Therapies aimed at improving vascular BK channel function could provide new therapeutic targets for both AD and hypertension-induced vascular dementia. British Heart Foundation This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Ca2+/Calmodulin-Dependent Protein Kinase II Disrupts the Voltage Dependency of the Voltage-Dependent Anion Channel on the Lipid Bilayer Membrane.

Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a key enzyme that plays a significant role in intracellular signaling and the modulation of mitochondrial membrane properties. It is known that the voltage-dependent anion channel (VDAC) is one of the most abundant outer mitochondrial membrane (OMM) proteins acting as a significant passageway and regulatory site for various enzymes, proteins, ions, and metabolites. Considering this, we hypothesize that VDAC could be one of the targets for CaMKII enzymatic activity. Our in vitro experiments indicate that VDAC can be phosphorylated by the CaMKII enzyme. Moreover, the bilayer electrophysiology experimental data indicate that CaMKII significantly reduces VDAC's single-channel conductivity; its open probability remains high at all the applied potentials between +60 and -60 mV, and the voltage dependency was lost, which suggests that CaMKII disrupted the VDAC's single-channel activities. Hence, we can infer that VDAC interacts with CaMKII and thus acts as a vital target for its activity. Furthermore, our findings suggest that CaMKII could play a significant role during the transport of ions and metabolites across the outer mitochondrial membrane (OMM) through VDAC and thus regulate apoptotic events.

Whole-cell patch-clamp recording and parameters.

The patch-clamp technique represents an electrophysiology type of method. This is one of several insightful approaches with five major configurations, namely a loose patch, cell-attached (also known as on-cell), whole-cell, inside-out, and outside-out modes. The patch-clamp method is more advanced compared to classical electrophysiology since it elucidates single-channel activation in a tiny portion of the membrane in addition to action potential (AP), junction potential (JP), endplate potential (EP), electrical coupling between two adjacent cells via Gap junction hemi-channels, excitatory/inhibitory postsynaptic potentials, and resting membrane potential (RMP). In fact, a malfunction of only one channel or even one component will alter AP amplitude or duration in vitro. If parameters are inferred appropriately and recordings are performed properly, the patch-clamp trace readouts and results are robust. The main hallmarks of currents via voltage-dependent calcium (Cav), hyperpolarization-activated cyclic nucleotide gated non-selective cation (HCN), inwardly rectifying potassium (Kir), voltage-dependent potassium (Kv), and voltage-dependent sodium (Nav) channels are similar and tractable among cells even when they are derived from evolutionary distinct organs and species. However, the size of the membrane area, where the functional subunits reside, and current magnitudes vary among cells of the same type. Therefore, dividing current magnitudes by cell capacitance- current density enables the estimate of functional and active channels relative to recorded cytoplasmic membrane area. Since the patch-clamp recordings can be performed in both current- and voltage-clamp modes, the action potential or spike durations can be adequately elucidated. Sometimes, optical methods are preferred to patch-clamp electrophysiology, but the obtained signals and traces are not robust. Finally, not only an alternans of AP durations, but also that of 'action potential shape' is observed with electrophysiology.

Two gates mediate NMDA receptor activity and are under subunit-specific regulation

Kinetics of NMDA receptor (NMDAR) ion channel opening and closing contribute to their unique role in synaptic signaling. Agonist binding generates free energy to open a canonical gate at the M3 helix bundle crossing. Single channel activity is characterized by clusters, or periods of rapid opening and closing, that are separated by long silent periods. A conserved glycine in the outer most transmembrane helices, the M4 helices, regulates NMDAR function. Here we find that the GluN1 glycine mainly regulates single channel events within a cluster, whereas the GluN2 glycine mainly regulates entry and exit from clusters. Molecular dynamics simulations suggest that, whereas the GluN2 M4 (along with GluN2 pre-M1) regulates the gate at the M3 helix bundle crossing, the GluN1 glycine regulates a ‘gate’ at the M2 loop. Subsequent functional experiments support this interpretation. Thus, the distinct kinetics of NMDARs are mediated by two gates that are under subunit-specific regulation.

Enriched cell-free and cell-based native membrane derived vesicles (nMV) enabling rapid in-vitro electrophysiological analysis of the voltage-gated sodium channel 1.5

Here, we demonstrate the utility of native membrane derived vesicles (nMVs) as tools for expeditious electrophysiological analysis of membrane proteins. We used a cell-free (CF) and a cell-based (CB) approach for preparing protein-enriched nMVs. We utilized the Chinese Hamster Ovary (CHO) lysate-based cell-free protein synthesis (CFPS) system to enrich ER-derived microsomes in the lysate with the primary human cardiac voltage-gated sodium channel 1.5 (hNaV1.5; SCN5A) in 3 h. Subsequently, CB-nMVs were isolated from fractions of nitrogen-cavitated CHO cells overexpressing the hNaV1.5. In an integrative approach, nMVs were micro-transplanted into Xenopus laevis oocytes. CB-nMVs expressed native lidocaine-sensitive hNaV1.5 currents within 24 h; CF-nMVs did not elicit any response. Both the CB- and CF-nMV preparations evoked single-channel activity on the planar lipid bilayer while retaining sensitivity to lidocaine application. Our findings suggest a high usability of the quick-synthesis CF-nMVs and maintenance-free CB-nMVs as ready-to-use tools for in-vitro analysis of electrogenic membrane proteins and large, voltage-gated ion channels.

Simultaneous single photon counting and ion channel recording from free-standing lipid bilayers on a microstructured array.

Fluorescence microscopy and, in particular, single molecule optical spectroscopy is of great potential value in characterizing the structural dynamics of membranes and membrane proteins. A particular challenge is to combine such high-resolution optical measurements with high-resolution voltage clamp electrical recordings that can provide direct information on single ion channel gating a block by drugs and analytes. Here, we report on the use of a novel chip-based, 2 × 2 arrangement of microelectrode cavities with borosilicate glass optical windows which facilitates optical access on an inverted microscope with water or oil-immersion objectives of high numerical aperture to horizontal free-standing lipid membranes, while controlling membrane voltage and recording currents using individual micropatterned, ring-shaped Ag/AgCl-electrodes to perform time-resolved single photon counting on free-standing membranes spanning sub-nanoliter cavities. Single channel activity induced by the fluorescently labelled pore-forming peptide ceratotoxin A was simultaneously acquired. This device allows for rapid formation of four membranes that are simultaneously monitored electrically using a four-channel amplifier and can be sequentially optically addressed, greatly reducing the time needed for successful experimentation. During our experiments, we noted autofluorescence of the borosilicate to be a limiting factor for wide-field low-intensity fluorescence recording, but this can likely be circumvented by using quartz glass instead. In summary, the novel device increases the likelihood of realizing the long standing ambition to correlate structural and functional dynamics of single membrane proteins on the single molecule level.

ATP synthase c-subunit leak channel as a novel therapeutic target.

Mitochondrial ATP synthase plays a key role in cell life and death by catalyzing the ATP synthesis and housing a leak channel of mitochondrial permeability transition. Various pharmacological and natural compounds that target mitochondria and ATP synthase were under clinical trials recently including bedaquiline (against mycobacterial tuberculosis) and resveratrol (for treating Alzheimer's disease). Here in this work, we are evaluating the effects of these compounds on the structure and leak channel activity of ATP synthase. Purified ATP synthase from porcine heart mitochondria was reconstituted in a planar lipid bilayer to study the single-channel activity of ATP synthase. Electrophysiological experiments revealed a profound inhibitory effect of bedaquiline on the single channel activity of ATP synthase. Additionally, we observed a concentration-dependent dual role of resveratrol on ATP synthase channel activity. Higher concentrations of resveratrol activated the channel while nanomolar concentrations inhibited the ATP synthase leak channel activity. Similar results were obtained with a less sensitive but more classical calcium retention capacity (CRC) assay for measuring the mitochondrial permeability transition pore (mPTP) opening, establishing the involvement of these compounds in modulating mPTP. Currently, we are investigating the ATP synthase structure bound to these compounds by cryo-electron microscopy (cryo-EM) to explore the channel gating mechanism. These findings will lead to the development of structure-based therapeutic drugs targeting the ATP synthase c-subunit leak channel for treating mPTP-related diseases.

Inhibition of Tolaasin Cytotoxicity Causing Brown Blotch Disease in Cultivated Mushrooms Using Tolaasin Inhibitory Factors.

Tolaasin, a pore-forming bacterial peptide toxin secreted by Pseudomonas tolaasii, causes brown blotch disease in cultivated mushrooms by forming membrane pores and collapsing the membrane structures. Tolaasin is a lipodepsipeptide, MW 1985, and pore formation by tolaasin molecules is accomplished by hydrophobic interactions and multimerizations. Compounds that inhibit tolaasin toxicity have been isolated from various food additives. Food detergents, sucrose esters of fatty acids, and polyglycerol esters of fatty acids can effectively inhibit tolaasin cytotoxicity. These chemicals, named tolaasin-inhibitory factors (TIF), were effective at concentrations ranging from 10-4 to 10-5 M. The most effective compound, TIF 16, inhibited tolaasin-induced hemolysis independent of temperature and pH, while tolaasin toxicity increased at higher temperatures. When TIF 16 was added to tolaasin-pretreated erythrocytes, the cytotoxic activity of tolaasin immediately stopped, and no further hemolysis was observed. In the artificial lipid bilayer, the single-channel activity of the tolaasin channel was completely and irreversibly blocked by TIF 16. When TIF 16 was sprayed onto pathogen-treated oyster mushrooms growing on the shelves of cultivation houses, the development of disease was completely suppressed, and normal growth of oyster mushrooms was observed. Furthermore, the treatment with TIF 16 did not show any adverse effect on the growth of oyster mushrooms. These results indicate that TIF 16 is a good candidate for the biochemical control of brown blotch disease.

Cannabidiol as a modulator of α7 nicotinic receptors.

Cannabidiol (CBD), an important terpenoid compound from marijuana with no psychoactive effects, has become of great pharmaceutical interest for several health conditions. As CBD is a multitarget drug, there is a need to establish the molecular mechanisms by which CBD may exert therapeutic as well as adverse effects. The α7 nicotinic acetylcholine receptor (α7 nAChR) is a cation-permeable ACh-gated channel present in the nervous system and in non-neuronal cells. It is involved in different pathological conditions, including neurological and neurodegenerative disorders, inflammation, and cancer. By high-resolution single-channel recordings and confocal microscopy, we here reveal how CBD modulates α7 nAChR ionotropic and metabotropic functions. CBD leads to a profound concentration-dependent decrease of α7 nAChR single-channel activity with an IC50 in the sub-micromolar range. The inhibition of α7 nAChR activity, which takes place through a membrane pathway, is neither mediated by receptor phosphorylation nor overcome by positive allosteric modulators and is compatible with CBD stabilization of resting or desensitized α7 nAChR conformational states. CBD modulation is complex as it also leads to the later appearance of atypical, low-frequency α7 nAChR channel openings. At the cellular level, CBD inhibits the increase in intracellular calcium triggered by α7 nAChR activation, thus decreasing cell calcium responses. The modulation of α7 nAChR is of pharmacological relevance and should be considered in the evaluation of CBD potential therapeutic uses. Thus, our study provides novel molecular information of CBD multiple actions and targets, which is required to set the basis for prospective applications in human health.

Electrical recordings from dendritic spines of adult mouse hippocampus and effect of the actin cytoskeleton.

Dendritic spines (DS) are tiny protrusions implicated in excitatory postsynaptic responses in the CNS. To achieve their function, DS concentrate a high density of ion channels and dynamic actin networks in a tiny specialized compartment. However, to date there is no direct information on DS ionic conductances. Here, we used several experimental techniques to obtain direct electrical information from DS of the adult mouse hippocampus. First, we optimized a method to isolate DS from the dissected hippocampus. Second, we used the lipid bilayer membrane (BLM) reconstitution and patch clamping techniques and obtained heretofore unavailable electrical phenotypes on ion channels present in the DS membrane. Third, we also patch clamped DS directly in cultured adult mouse hippocampal neurons, to validate the electrical information observed with the isolated preparation. Electron microscopy and immunochemistry of PDS-95 and NMDA receptors and intrinsic actin networks confirmed the enrichment of the isolated DS preparation, showing open and closed DS, and multi-headed DS. The preparation was used to identify single channel activities and “whole-DS” electrical conductance. We identified NMDA and Ca2+-dependent intrinsic electrical activity in isolated DS and in situ DS of cultured adult mouse hippocampal neurons. In situ recordings in the presence of local NMDA, showed that individual DS intrinsic electrical activity often back-propagated to the dendrite from which it sprouted. The DS electrical oscillations were modulated by changes in actin cytoskeleton dynamics by addition of the F-actin disrupter agent, cytochalasin D, and exogenous actin-binding proteins. The data indicate that DS are elaborate excitable electrical devices, whose activity is a functional interplay between ion channels and the underlying actin networks. The data argue in favor of the active contribution of individual DS to the electrical activity of neurons at the level of both the membrane conductance and cytoskeletal signaling.

Next-generation Mobile Cardiac Telemetry: Clinical Value of Combining Electrocardiographic and Physiologic Parameters.

The ZOLL Arrhythmia Monitoring System, a mobile cardiac telemetry (MCT) device from ZOLL Corporation (Chelmsford, MA, USA), records single-channel electrocardiogram (ECG) signals, heart rate, activity, respiratory rate, and posture. Comprehensive reporting from these multiple biometrics may provide a global evaluation of arrhythmic or other cardiovascular risks in individual patients and insights into the patient’s overall wellness and health status. The objective of the study was to evaluate the physician-perceived utility of adding biometric data to the traditional ECG-only–based assessment and subject-reported symptoms. This prospective study recruited candidates for MCT. Independent event and end-of-use (EOU) reports based on ECG and biometrics data were provided to physicians. To document whether the biometric data affected treatment plan decisions or added value over the ECG-alone data, physicians completed a questionnaire for each report. Additionally, they completed the questionnaire to understand the utility of the subject wellness information provided in the EOU report. From December 2020 to July 2021, 583 patients were enrolled by 27 physicians from 18 cardiology practices in the United States. When using biometrics data compared to the ECG alone, this study found that 96% of the physicians made changes to the treatment plan that initially was based on the ECG alone. The biometrics-based changes involved 64% of all patients (n = 535), and included modifications to medications, follow-up, and lifestyle in 18%, 19%, and 63% of the subjects, respectively. In this largest MCT study conducted to date, next-generation MCT, by providing multiple biometric parameters along with ECG data, improves physicians’ ability to make patient management decisions. This added functionality and clarity may replace traditional “ECG with diary”–based monitoring.