Single-channel Conductance Research Articles

Mechanisms of Action Underlying Conductance-modifying Positive Allosteric Modulators of the NMDA Receptor.

NMDA receptors (NMDARs) are ionotropic glutamate receptors that mediate a slow, Ca2+-permeable component of fast excitatory neurotransmission. Modulation of NMDAR function has the potential for disease modification as NMDAR dysfunction has been implicated in neurodevelopment, neuropsychiatric, neurological, and neurodegenerative disorders. We recently described the thieno[2,3-d]pyrimidin-4-one (EU1622) class of positive allosteric modulators, including several potent and efficacious analogs. Here we have used electrophysiological recordings from Xenopus oocytes, HEK cells, and cultured cerebellar and cortical neurons to determine the mechanisms of action of a representative member of this class of modulator. EU1622-240 enhances current response to saturating agonist (doubling response amplitude at 0.2-0.5 µM), slows the deactivation time course following rapid removal of glutamate, increases open probability, enhances co-agonist potency, and reduces single channel conductance. We also show that EU1622-240 can transform NMDARs so that they can be opened when only glutamate or glycine is bound. EU1622-240-bound NMDARs channels activated by a single agonist (glutamate or glycine) open to a unique conductance level with different pore properties and Mg2+ sensitivity, in contrast to channels arising from activation of NMDARs with both co-agonists bound. These data demonstrate that previously hypothesized distinct gating steps can be controlled by glutamate and glycine binding and shows that the 1622-series modulators enable glutamate- or glycine-bound NMDARs to generate open conformations with different pore properties. The properties of this class of allosteric modulators present intriguing therapeutic opportunities for the modulation of circuit function. Significance Statement NMDA receptors are expressed throughout the CNS and are permeable to calcium. EU1622-240 increases open probability and agonist potency, while reducing single channel conductance and prolonging the deactivation time course. EU1622-240 allows NMDA receptor activation by the binding of one co-agonist (glycine or glutamate), which produces channels with distinct properties. Evaluation of this modulator provides insight into gating mechanisms and may lead to the development of new therapeutic strategies.

Inhibition of transient receptor potential vanilloid 3 channels by antimalarial hydroxychloroquine alleviates TRPV3-dependent dermatitis

Transient receptor potential vanilloid 3 channel (TRPV3) is closely associated with skin inflammation, but there is a lack of effective and specific inhibitors for clinical use. In this study, we identified antimalarial HCQ as a selective TRPV3 inhibitor following the prediction by network pharmacology data analysis. In whole-cell patch-clamp recordings, HCQ inhibited the current of the TRPV3 channel, with an IC50 of 51.69 ± 4.78 μM. At the single-channel level, HCQ reduced the open probability of TRPV3 and decreased single-channel conductance. Molecular docking and site-directed mutagenesis confirmed that residues in the pore domain were critical for the activity of HCQ. In vivo, HCQ effectively reduced carvacrol-induced epidermal thickening, erythema, and desquamation. Additionally, the serum IgE and inflammatory factors such as TNF-α and IL-6 were markedly decreased in the dorsal skin tissues in the HCQ treatment group, as compared to the model group. Our results suggested the antimalarial HCQ may represent a potential alleviator for treating skin inflammation by inhibiting TRPV3 channels.

Potassium channel TASK-5 forms functional heterodimers with TASK-1 and TASK-3 to break its silence

TASK-5 (KCNK15) belongs to the acid-sensitive subfamily of two-pore domain potassium (K2P) channels, which includes TASK-1 and TASK-3. TASK-5 stands out as K2P channel for which there is no functional data available, since it was reported in 2001 as non-functional and thus “silent”. Here we show that TASK-5 channels are indeed non-functional as homodimers, but are involved in the formation of functional channel complexes with TASK-1 and TASK-3. TASK-5 negatively modulates the surface expression of TASK channels, while the heteromeric TASK-5-containing channel complexes located at the plasma membrane are characterized by changes in single-channel conductance, Gq-coupled receptor-mediated channel inhibition, and sensitivity to TASK modulators. The unique pharmacology of TASK-1/TASK-5 heterodimers, affected by a common polymorphism in KCNK15, needs to be carefully considered in the future development of drugs targeting TASK channels. Our observations provide an access to study TASK-5 at the functional level, particularly in malignant cancers associated with KCNK15.

Activation mechanisms of dimeric mechanosensitive OSCA/TMEM63 channels

OSCA/TMEM63 channels, which have transporter-like architectures, are bona fide mechanosensitive (MS) ion channels that sense high-threshold mechanical forces in eukaryotic cells. The activation mechanism of these transporter-like channels is not fully understood. Here we report cryo-EM structures of a dimeric OSCA/TMEM63 pore mutant OSCA1.1-F516A with a sequentially extracellular dilated pore in a detergent environment. These structures suggest that the extracellular pore sequential dilation resembles a flower blooming and couples to a sequential contraction of each monomer subunit towards the dimer interface and subsequent extrusion of the dimer interface lipids. Interestingly, while OSCA1.1-F516A remains non-conducting in the native lipid environment, it can be directly activated by lyso-phosphatidylcholine (Lyso-PC) with reduced single-channel conductance. Structural analysis of OSCA1.1-F516A in lyso-PC-free and lyso-PC-containing lipid nanodiscs indicates that lyso-PC induces intracellular pore dilation by attracting the M6b to upward movement away from the intracellular side thus extending the intracellular pore. Further functional studies indicate that full activation of MS OSCA/TMEM63 dimeric channels by high-threshold mechanical force also involves the opening of both intercellular and extracellular pores. Our results provide the fundamental activation paradigm of the unique transporter-like MS OSCA/TMEM63 channels, which is likely applicable to functional branches of the TMEM63/TMEM16/TMC superfamilies.

Electrophysiological Insights into Antibiotic Translocation and Resistance: The Impact of Outer Membrane Proteins.

The alarming rise of antibiotic resistance in Gram-negative bacteria has emerged as a major global health challenge. A key factor contributing to this crisis is the low permeability of the bacterial outer membrane, which acts as a barrier that prevents antibiotics from entering the cell. Protein channels embedded in this outer membrane selectively regulate the influx of hydrophilic compounds, including antibiotics. To combat antibiotic resistance, understanding the molecular mechanisms governing antibiotic permeability through bacterial membrane channels is crucial. This knowledge is key towards elucidating their roles in studing antibiotic resistance. By compiling and analysing the flux data from multiple electrophysiological reversal potential experimental studies, which involves measuring zero-current potentials and the corresponding single-channel conductance, we can calculate the flux of charged antibiotics/compounds across different Gram-negative bacterial outer membrane channels. Through this comprehensive synthesis, this review aims to advance our understanding and stimulate discussions about the physicochemical factors influencing the flux of antibiotics through bacterial membrane protein channels, ultimately enhancing our knowledge in this area.

Exploring the influence of pore shape on conductance and permeation

There are increasing numbers of ion channel structures featuring heteromeric subunit assembly, exemplified by synaptic α1βB glycine and α4β2 nicotinic receptors. These structures exhibit inherent pore asymmetry, but the relevance of this to function is unknown. Furthermore, molecular dynamics simulations performed on symmetrical homomeric channels often lead to thermal distortion whereby conformations of the resulting ensemble are also asymmetrical. When functionally annotating ion channels, researchers often rely on minimal constrictions determined via radius-profile calculations performed with computer programs, such as HOLE or CHAP, coupled with an assessment of pore hydrophobicity. However, such tools typically employ spherical probe particles, limiting their ability to accurately capture pore asymmetry. Here, we introduce an algorithm that employs ellipsoidal probe particles, enabling a more comprehensive representation of the pore geometry. Our analysis reveals that the use of nonspherical ellipsoids for pore characterization provides a more accurate and easily interpretable depiction of conductance. To quantify the implications of pore asymmetry on conductance, we systematically investigated carbon nanotubes with varying degrees of pore asymmetry as model systems. The conductance through these channels shows surprising effects that would otherwise not be predicted with spherical probes. The results have broad implications not only for the functional annotation of biological ion channels but also for the design of synthetic channel systems for use in areas such as water filtration. Furthermore, we make use of the more accurate characterization of channel pores to refine a physical conductance model to obtain a heuristic estimate for single-channel conductance. The code is freely available, obtainable as pip-installable python package and provided as a web service.

Discovery and mechanistic studies of a novel BKCa channel modulator inducing long openings

The large-conductance Ca2+-activated K+ channel (BKCa channel) plays critical roles in various tissues. In the smooth muscle of the urinary bladder, BKCa channel is highly expressed and regulates the spontaneous action potentials that control muscle contraction. Thus, the chemical activators for BKCa channel have their therapeutic potential against overactive bladder syndrome. Preveiously, we conducted a screening of a chemical library comprising 9,938 compounds in search of novel activators of the BKCa channel and performed structure-activity relationship study through hit compounds by using a cell-based fluorescence assay. Among the optimized compounds, DBTB-1 (N-[4-(2,4-dimethyl-3-oxo-3,4-dihydro-2H-1,4-benzoxazin-6-yl)-1,3-thiazol-2-yl]benzamide) displayed a robust activation of macroscopic BKCa channel currents in Xenopus oocytes. In contrast to other BKCa channel activators, DBTB-1 mainly influenced deactivation kinetics of the channel without affecting channel activation significantly. At the single-channel level, DBTB-1 increased the open-state probability compared to vehicle without affecting its single-channel conductance, indicating the delayed closing of the channel in the presence of the drug. Furthermore, analysis of the open dwell-time showed an additional population of long openings induced by DBTB-1. Thus, these unique characteristics of DBTB-1 can be useful in designing a new class of BKCa channel activators and might be utilized in developing therapuetics targeting specific aspect of the channel function. This research was supported by Korea Drug Development Fund funded by Ministry of Science and ICT, Ministry of Trade, Industry, and Energy, and Ministry of Health and Welfare (RS-2023-00258812). 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.

Two rare variants that affect the same amino acid in CFTR have distinct responses to ivacaftor.

Some residues in the cystic fibrosis transmembrane conductance regulator (CFTR) channel are the site of more than one CFTR variant that cause cystic fibrosis. Here, we investigated the function of S1159F and S1159P, two variants associated with different clinical phenotypes, which affect the same pore-lining residue in transmembrane segment 12 that are both strongly potentiated by ivacaftor when expressed in CFBE41o- bronchial epithelial cells. To study the single-channel behaviour of CFTR, we applied the patch-clamp technique to Chinese hamster ovary cells heterologously expressing CFTR variants incubated at 27°C to enhance channel residence at the plasma membrane. S1159F- and S1159P-CFTR formed Cl- channels activated by cAMP-dependent phosphorylation and gated by ATP that exhibited thermostability at 37°C. Both variants modestly reduced the single-channel conductance of CFTR. By severely attenuating channel gating, S1159F- and S1159P-CFTR reduced the open probability (Po ) of wild-type CFTR by ≥75% at ATP (1mM); S1159F-CFTR caused the greater decrease in Po consistent with its more severe clinical phenotype. Ivacaftor (10-100nM) doubled the Po of both CFTR variants without restoring Po values to wild-type levels, but concomitantly, ivacaftor decreased current flow through open channels. For S1159F-CFTR, the reduction of current flow was marked at high (supersaturated) ivacaftor concentrations (0.5-1μM) and voltage-independent, identifying an additional detrimental action of elevated ivacaftor concentrations. In conclusion, S1159F and S1159P are gating variants, which also affect CFTR processing and conduction, but not stability, necessitating the use of combinations of CFTR modulators to optimally restore their channel activity. KEY POINTS: Dysfunction of the ion channel cystic fibrosis transmembrane conductance regulator (CFTR) causes the genetic disease cystic fibrosis (CF). This study investigated two rare pathogenic CFTR variants, S1159F and S1159P, which affect the same amino acid in CFTR, to understand the molecular basis of disease and response to the CFTR-targeted therapy ivacaftor. Both rare variants diminished CFTR function by modestly reducing current flow through the channel and severely inhibiting ATP-dependent channel gating with S1159F exerting the stronger adverse effect, which correlates with its association with more severe disease. Ivacaftor potentiated channel gating by both rare variants without restoring their activity to wild-type levels, but concurrently reduced current flow through open channels, particularly those of S1159F-CFTR. Our data demonstrate that S1159F and S1159P cause CFTR dysfunction by multiple mechanisms that require combinations of CFTR-targeted therapies to fully restore channel function.

Glutamate Spillover Dynamically Strengthens Gabaergic Synaptic Inhibition of the Hypothalamic Paraventricular Nucleus.

The hypothalamic paraventricular nucleus (PVN) is strongly inhibited by γ-aminobutyric acid (GABA) from the surrounding peri-nuclear zone (PNZ). Because glutamate mediates fast excitatory transmission and is substrate for GABA synthesis, we tested its capacity to dynamically strengthen GABA inhibition. In PVN slices from male mice, bath glutamate applied during ionotropic glutamate receptor blockade increased PNZ-evoked inhibitory postsynaptic currents (eIPSCs) without affecting GABA-A receptor agonist currents or single-channel conductance, implicating a presynaptic mechanism(s). Consistent with this interpretation, bath glutamate failed to strengthen IPSCs during pharmacological saturation of GABA-A receptors. Presynaptic analyses revealed that glutamate did not affect paired-pulse ratio, peak eIPSC variability, GABA vesicle recycling speed, or readily releasable pool (RRP) size. Notably, glutamate-GABA strengthening (GGS) was unaffected by metabotropic glutamate receptor blockade and graded external Ca2+ when normalized to baseline amplitude. GGS was prevented by pan- but not glial-specific inhibition of glutamate uptake and by inhibition of glutamic acid decarboxylase (GAD), indicating reliance on glutamate uptake by neuronal excitatory amino acid transporter 3 (EAAT3) and enzymatic conversion of glutamate to GABA. EAAT3 immunoreactivity was strongly localized to presumptive PVN GABA terminals. High bath K+ also induced GGS, which was prevented by glutamate vesicle depletion, indicating that synaptic glutamate release strengthens PVN GABA inhibition. GGS suppressed PVN cell firing, indicating its functional significance. In sum, PVN GGS buffers neuronal excitation by apparent "over-filling" of vesicles with GABA synthesized from synaptically released glutamate. We posit that GGS protects against sustained PVN excitation and excitotoxicity while potentially aiding stress adaptation and habituation.

A modelling study to dissect the potential role of voltage-gated ion channels in activity-dependent conduction velocity changes as identified in small fiber neuropathy patients.

Patients with small fiber neuropathy (SFN) suffer from neuropathic pain, which is still a therapeutic problem. Changed activation patterns of mechano-insensitive peripheral nerve fibers (CMi) could cause neuropathic pain. However, there is sparse knowledge about mechanisms leading to CMi dysfunction since it is difficult to dissect specific molecular mechanisms in humans. We used an in-silico model to elucidate molecular causes of CMi dysfunction as observed in single nerve fiber recordings (microneurography) of SFN patients. We analyzed microneurography data from 97 CMi-fibers from healthy individuals and 34 of SFN patients to identify activity-dependent changes in conduction velocity. Using the NEURON environment, we adapted a biophysical realistic preexisting CMi-fiber model with ion channels described by Hodgkin-Huxley dynamics for identifying molecular mechanisms leading to those changes. Via a grid search optimization, we assessed the interplay between different ion channels, Na-K-pump, and resting membrane potential. Changing a single ion channel conductance, Na-K-pump or membrane potential individually is not sufficient to reproduce in-silico CMi-fiber dysfunction of unchanged activity-dependent conduction velocity slowing and quicker normalization of conduction velocity after stimulation as observed in microneurography. We identified the best combination of mechanisms: increased conductance of potassium delayed-rectifier and decreased conductance of Na-K-pump and depolarized membrane potential. When the membrane potential is unchanged, opposite changes in Na-K-pump and ion channels generate the same effect. Our study suggests that not one single mechanism accounts for pain-relevant changes in CMi-fibers, but a combination of mechanisms. A depolarized membrane potential, as previously observed in patients with neuropathic pain, leads to changes in the contribution of ion channels and the Na-K-pump. Thus, when searching for targets for the treatment of neuropathic pain, combinations of several molecules in interplay with the membrane potential should be regarded.

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.

Transient Receptor Potential channels (TRP) in GtoPdb v.2023.3

Transient Receptor Potential channels (TRP) in GtoPdb v.2023.3

Supramolecular Barrel‐Rosette Ion Channel Based on 3,5‐Diaminobenzoic Acid for Cation‐Anion Symport

AbstractThe structural tropology and functions of natural cation‐anion symporting channels have been continuously investigated due to their crucial role in regulating various physiological functions. To understand the physiological functions of the natural symporter channels, it is vital to develop small‐molecule‐based biomimicking systems that can provide mechanistic insights into the ion‐binding sites and the ion‐translocation pathways. Herein, we report a series of bis((R)‐(−)‐mandelic acid)‐linked 3,5‐diaminobenzoic acid based self‐assembled ion channels with distinctive ion transport ability. Ion transport experiment across the lipid bilayer membrane revealed that compound 1 b exhibits the highest transport activity among the series, and it has interesting selective co‐transporting functions, i.e., facilitates K+/ClO4− symport. Electrophysiology experiments confirmed the formation of supramolecular ion channels with an average diameter of 6.2±1 Å and single channel conductance of 57.3±1.9 pS. Selectivity studies of channel 1 b in a bilayer lipid membrane demonstrated a permeability ratio of , , and indicating the higher selectivity of the channel towards KClO4 over KCl salt. A hexameric assembly of a trimeric rosette of 1 b was subjected to molecular dynamics simulations with different salts to understand the supramolecular channel formation and ion selectivity pattern.

Supramolecular Barrel-Rosette Ion Channel Based on 3,5-Diaminobenzoic Acid for Cation-Anion Symport.

The structural tropology and functions of natural cation-anion symporting channels have been continuously investigated due to their crucial role in regulating various physiological functions. To understand the physiological functions of the natural symporter channels, it is vital to develop small molecule-based biomimicking systems that can provide mechanistic insights into the ion-binding sites and the ion-translocation pathways. Herein, we report a series of bis((R)-(-)-mandelic acid)-linked 3,5-diaminobenzoic acid-based self-assembled ion channels with distinctive ion transport ability. Ion transport experiment across the lipid bilayer membrane revealed that compound 1b exhibits the highest transport activity among the series, and it has interesting selective co-transporting functions, i.e., facilitates K+/ClO4- symport. Electrophysiology experiments confirmed the formation of supramolecular ion channels with an average diameter of 6.2 ± 1 Å and single channel conductance of 57.3 ± 1.9 pS. Selectivity studies of channel 1b in BLM demonstrated the permeability ratio of PCl-/PK+ = 0.053 ±0.02, PClO4-/PCl- = 2.1 ± 0.5, and PK+/PNa+ = 1.5 ± 1, respectively, indicating the higher selectivity of the channel towards KClO4 over KCl salt. A hexameric assembly of a trimeric rosette of 1b was subjected to molecular dynamics simulations with different salts to understand the supramolecular channel formation and ion selectivity pattern.

Changes in Ion Transport across Biological Membranes Exposed to Particulate Matter.

The cells of living organisms are surrounded by the biological membranes that form a barrier between the internal and external environment of the cells. Cell membranes serve as barriers and gatekeepers. They protect cells against the entry of undesirable substances and are the first line of interaction with foreign particles. Therefore, it is very important to understand how substances such as particulate matter (PM) interact with cell membranes. To investigate the effect of PM on the electrical properties of biological membranes, a series of experiments using a black lipid membrane (BLM) technique were performed. L-α-Phosphatidylcholine from soybean (azolectin) was used to create lipid bilayers. PM samples of different diameters (<4 (SRM-PM4.0) and <10 μm (SRM-PM10) were purchased from The National Institute of Standards and Technology (USA) to ensure the repeatability of the measurements. Lipid membranes with incorporated gramicidin A (5 pg/mL) ion channels were used to investigate the effect of PM on ion transport. The ionic current passing through the azolectin membranes was measured in ionic gradients (50/150 mM KCl on cis/trans side). In parallel, the electric membrane capacitance measurements, analysis of the conductance and reversal potential were performed. Our results have shown that PM at concentration range from 10 to 150 μg/mL reduced the basal ionic current at negative potentials while increased it at positive ones, indicating the interaction between lipids forming the membrane and PM. Additionally, PM decreased the gramicidin A channel activity. At the same time, the amplitude of channel openings as well as single channel conductance and reversal potential remained unchanged. Lastly, particulate matter at a concentration of 150 μg/mL did not affect the electric membrane capacity to any significant extent. Understanding the interaction between PM and biological membranes could aid in the search for effective cytoprotective strategies. Perhaps, by the use of an artificial system, we will learn to support the consequences of PM-induced damage.

Transient Receptor Potential channels (TRP) in GtoPdb v.2023.2

Transient Receptor Potential channels (TRP) in GtoPdb v.2023.2

KCa 2.2 (KCNN2): A physiologically and therapeutically important potassium channel.

One group of the K+ ion channels, the small-conductance Ca2+ -activated potassium channels (KCa 2.x, also known as SK channels family), is widely expressed in neurons as well as the heart, endothelial cells, etc. They are named small-conductance Ca2+ -activated potassium channels (SK channels) due to their comparatively low single-channel conductance of about ~10 pS. These channels are insensitive to changes in membrane potential and are activated solely by rises in the intracellular Ca2+ . According to the phylogenic research done on the KCa 2.x channels family, there are three channels' subtypes: KCa 2.1, KCa 2.2, and KCa 2.3, which are encoded by KCNN1, KCNN2, and KCNN3 genes, respectively. The KCa 2.x channels regulate neuronal excitability and responsiveness to synaptic input patterns. KCa 2.x channels inhibit excitatory postsynaptic potentials (EPSPs) in neuronal dendrites and contribute to the medium afterhyperpolarization (mAHP) that follows the action potential bursts. Multiple brain regions, including the hippocampus, express the KCa 2.2 channel encoded by the KCNN2 gene on chromosome 5. Of particular interest, rat cerebellar Purkinje cells express KCa 2.2 channels, which are crucial for various cellular processes during development and maturation. Patients with a loss-of-function of KCNN2 mutations typically exhibit extrapyramidal symptoms, cerebellar ataxia, motor and language developmental delays, and intellectual disabilities. Studies have revealed that autosomal dominant neurodevelopmental movement disorders resembling rodent symptoms are caused by heterozygous loss-of-function mutations, which are most likely to induce KCNN2 haploinsufficiency. The KCa 2.2 channel is a promising drug target for spinocerebellar ataxias (SCAs). SCAs exhibit the dysregulation of firing in cerebellar Purkinje cells which is one of the first signs of pathology. Thus, selective KCa 2.2 modulators are promising potential therapeutics for SCAs.

Andreev reflection via the zero-energy bound states at the superconducting skyrmion crystal–normal skyrmion crystal interface

In this work, we used the double exchange model and studied the transport properties of the electronic states across the superconducting skyrmion crystal (SkX)–normal SkX interface. By calculating the energy dispersion and the real-space wave function of the electronic states we found topologically protected edge zero modes in the superconducting SkX nanoribbon. By consideration of the symmetry of the Hamiltonian of the bulk superconducting SkX and its nanoribbon, we found that they belong to the symmetry class D and support Majorana edge modes. To demonstrate the transport properties of these zero edge modes, we used the Green's function method and calculated the Andreev reflection coefficients of the electronic states across the superconducting SkX–normal SkX interface. Zero-mode-induced Andreev reflection peaks are demonstrated and also reflected in the tunneling current. Single-channel conductance is quantized at 2e2/h and 4e2/h for two-fold and four-fold degenerate Majorana bound states, respectively.

Chromogranin B (CHGB) is dimorphic and responsible for dominant anion channels delivered to cell surface via regulated secretion.

Regulated secretion is conserved in all eukaryotes. In vertebrates granin family proteins function in all key steps of regulated secretion. Phase separation and amyloid-based storage of proteins and small molecules in secretory granules require ion homeostasis to maintain their steady states, and thus need ion conductances in granule membranes. But granular ion channels are still elusive. Here we show that granule exocytosis in neuroendocrine cells delivers to cell surface dominant anion channels, to which chromogranin B (CHGB) is critical. Biochemical fractionation shows that native CHGB distributes nearly equally in soluble and membrane-bound forms, and both reconstitute highly selective anion channels in membrane. Confocal imaging resolves granular membrane components including proton pumps and CHGB in puncta on the cell surface after stimulated exocytosis. High pressure freezing immuno-EM reveals a major fraction of CHGB at granule membranes in rat pancreatic β-cells. A cryo-EM structure of bCHGB dimer of a nominal 3.5 Å resolution delineates a central pore with end openings, physically sufficient for membrane-spanning and large single channel conductance. Together our data support that CHGB-containing (CHGB+) channels are characteristic of regulated secretion, and function in granule ion homeostasis near the plasma membrane or possibly in other intracellular processes.

Opening of capsaicin receptor TRPV1 is stabilized equally by its four subunits

Capsaicin receptor TRPV1 is a nociceptor for vanilloid molecules, such as capsaicin and resiniferatoxin (RTX). Even though cryo-EM structures of TRPV1 in complex with these molecules are available, how their binding energetically favors the open conformation is not known. Here, we report an approach to control the number of bound RTX molecules (0-4) in functional rat TRPV1. The approach allowed direct measurements of each of the intermediate open states under equilibrium conditions at both macroscopic and single-molecule levels. We found that RTX binding to each of the four subunits contributes virtually the same activation energy, which we estimated to be 1.70 to 1.86kcal/mol and found to arise predominately from destabilizing the closed conformation. We further showed that sequential bindings of RTX increase open probability without altering single-channel conductance, confirming that there is likely a single open-pore conformation for TRPV1 activated by RTX.