Channel Open Probability Research Articles

Interplay of Nav1.8 and Nav1.7 channels drives neuronal hyperexcitability in neuropathic pain.

While voltage-gated sodium channels Nav1.7 and Nav1.8 both contribute to electrogenesis in dorsal root ganglion (DRG) neurons, details of their interactions have remained unexplored. Here, we studied the functional contribution of Nav1.8 in DRG neurons using a dynamic clamp to express Nav1.7L848H, a gain-of-function Nav1.7 mutation that causes inherited erythromelalgia (IEM), a human genetic model of neuropathic pain, and demonstrate a profound functional interaction of Nav1.8 with Nav1.7 close to the threshold for AP generation. At the voltage threshold of -21.9 mV, we observed that Nav1.8 channel open-probability exceeded Nav1.7WT channel open-probability ninefold. Using a kinetic model of Nav1.8, we showed that a reduction of Nav1.8 current by even 25-50% increases rheobase and reduces firing probability in small DRG neurons expressing Nav1.7L848H. Nav1.8 subtraction also reduces the amplitudes of subthreshold membrane potential oscillations in these cells. Our results show that within DRG neurons that express peripheral sodium channel Nav1.7, the Nav1.8 channel amplifies excitability at a broad range of membrane voltages with a predominant effect close to the AP voltage threshold, while Nav1.7 plays a major role at voltages closer to resting membrane potential. Our data show that dynamic-clamp reduction of Nav1.8 conductance by 25-50% can reverse hyperexcitability of DRG neurons expressing a gain-of-function Nav1.7 mutation that causes pain in humans and suggests, more generally, that full inhibition of Nav1.8 may not be required for relief of pain due to DRG neuron hyperexcitability.

Plumbagin, a novel TRPV2 inhibitor, ameliorates microglia activation and brain injury in a middle cerebral artery occlusion/reperfusion mouse model.

Transient receptor potential vanilloid 2 (TRPV2) is a Ca2+-permeable non-selective cation channel. Despite the significant roles of TRPV2 in immunological response, cancer progression and cardiac development, pharmacological probes of TRPV2 remain to be identified. We aimed to discover TRPV2 inhibitors and to elucidate their molecular mechanism of action. Fluorescence-based Ca2+ assay in HEK-293 cells expressing murine TRPV2 was used to identify plumbagin as a novel TRPV2 inhibitor. Patch-clamp, in silico docking and site-directed mutagenesis were applied to investigate the molecular mechanisms critical for plumbagin interaction. ELISA and qPCR were used to assess nitric oxide release and mRNA levels of inflammatory mediators, respectively. si-RNA interference was used to knock down TRPV2 expression, which was validated by western blotting. Neurological and histological analyses were used to examine brain injury of mice following middle cerebral artery occlusion/reperfusion (MCAO/R). Plumbagin is a potent TRPV2 negative allosteric modulator with an IC50 value of 0.85 μM, exhibiting >14-fold selectivity over TRPV1, TRPV3 and TRPV4. Plumbagin suppresses TRPV2 activity by decreasing the channel open probability without affecting the unitary conductance. Moreover, plumbagin binds to an extracellular pocket formed by the pore helix and flexible loop between transmembrane helices S5 and S6 of TRPV2. Plumbagin effectively suppresses LPS-induced inflammation of BV-2 microglia and ameliorates brain injury of MCAO/R mice. Plumbagin is a novel pharmacological probe to study TRPV2 pathophysiology. TRPV2 is a novel molecular target for the treatment of neuroinflammation and ischemic stroke.

PIEZO1 variants that reduce open channel probability are associated with familial osteoarthritis.

The synovial joints senses and responds to a multitude of physical forces to maintain joint homeostasis. Disruption of joint homeostasis results in development of osteoarthritis (OA), a disease characterized by loss of joint space, degeneration of articular cartilage, remodeling of bone and other joint tissues, low-grade inflammation, and pain. How changes in mechanosensing in the joint contribute to OA susceptibility remains elusive. PIEZO1 is a major mechanosensitive cation channel in the joint directly regulated by mechanical stimulus. To test whether altered PIEZO1 channel activity causes increased OA susceptibility, we determined whether variants affecting PIEZO1 are associated with dominant inheritance of age-associated familial OA. We identified four rare coding variants affecting PIEZO1 that are associated with familial hand OA. Single channel analyses demonstrated that all four PIEZO1 mutant channels act in a dominant-negative manner to reduce the open probability of the channel in response to pressure. Furthermore, we show that a GWAS mutation in PIEZO1 associated with reduced joint replacement results in increased channel activity when compared with WT and the mutants. Our data support the hypothesis that reduced PIEZO1 activity confers susceptibility to age-associated OA whereas increased PIEZO1 activity may be associated with reduced OA susceptibility.

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.

Unveiling the intricate role of S100A1 in regulating RyR1 activity: A commentary on “Structural insights into the regulation of RyR1 by S100A1”

S100A1, a calcium-binding protein, plays a crucial role in regulating Ca2+ signaling pathways in skeletal and cardiac myocytes via interactions with the ryanodine receptor (RyR) to affect Ca2+ release and contractile performance. Biophysical studies strongly suggest that S100A1 interacts with RyRs but have been inconclusive about both the nature of this interaction and its competition with another important calcium-binding protein, calmodulin (CaM). Thus, high-resolution cryo-EM studies of RyRs in the presence of S100A1, with or without additional CaM, were needed. The elegant work by Weninger et al. demonstrates the interaction between S100A1 and RyR1 through various experiments and confirms that S100A1 activates RyR1 at sub-micromolar Ca2+ concentrations, increasing the open probability of RyR1 channels.

Functional investigation of a putative calcium-binding site involved in the inhibition of inositol 1,4,5-trisphosphate receptor activity.

A wide variety of factors influence inositol 1,4,5-trisphosphate (IP 3 ) receptor (IP 3 R) activity resulting in modulation of intracellular Ca 2+ release. This regulation is thought to define the spatio-temporal patterns of Ca 2+ signals necessary for the appropriate activation of downstream effectors. The binding of both IP 3 and Ca 2+ are obligatory for IP 3 R channel opening, however, Ca 2+ regulates IP 3 R activity in a biphasic manner. Mutational studies have revealed that Ca 2+ binding to a high-affinity pocket formed by the ARM3 domain and linker domain promotes IP 3 R channel opening without altering the Ca 2+ dependency for channel inactivation. These data suggest a distinct low-affinity Ca 2+ binding site is responsible for the reduction in IP 3 R activity at higher [Ca 2+ ]. We determined the consequences of mutating a cluster of acidic residues in the ARM2 and central linker domain reported to coordinate Ca 2+ in cryo-EM structures of the IP 3 R type 3. This site is termed the "CD Ca 2+ binding site" and is well-conserved in all IP 3 R sub-types. We show that the CD site Ca 2+ binding mutants where the negatively charged glutamic acid residues are mutated to alanine exhibited enhanced sensitivity to IP 3 -generating agonists. Ca 2+ binding mutants displayed spontaneous elemental Ca 2+ events (Ca 2+ puffs) and the number of IP 3 -induced Ca 2+ puffs was significantly augmented in cells stably expressing Ca 2+ binding site mutants. When measured with "on-nucleus" patch clamp, the inhibitory effect of high [Ca 2+ ] on single channel-open probability (P o ) was reduced in mutant channels and this effect was dependent on [ATP]. These results indicate that Ca 2+ binding to the putative CD Ca 2+ inhibitory site facilitates the reduction in IP 3 R channel activation when cytosolic [ATP] is reduced and suggest that at higher [ATP], additional Ca 2+ binding motifs may contribute to the biphasic regulation of IP 3 -induced Ca 2+ release.

Inhibition of Cutaneous TRPV3 Channels by Natural Caffeic Acid for the Alleviation of Skin Inflammation.

Natural caffeic acid (CA) and its analogues have been studied for their potential applications in the treatment of various inflammatory and infectious skin diseases. However, the molecular mechanism underlying the effects of the CA remains largely unknown. Here, we report that CA and its two analogues, caffeic acid phenethyl ester (CAPE) and caffeic acid methyl caffeate (CAMC), inhibit TRPV3 currents in their concentration- and structure-dependent manners with IC50 values ranging from 102 to 410 μM. At the single-channel level, CA reduces the channel open probability and open frequency without alteration of unitary conductance. CA selectively inhibits TRPV3 relative to other subtypes of thermo-TRPs, such as TRPA1, TRPV1, TRPV4, and TRPM8. Molecular docking combined with site-specific mutagenesis reveals that a residue T636 in the Pore-loop is critical for CA binding to TRPV3. Further in vivo evaluation shows that CA significantly reverses TRPV3-mediated skin inflammation induced by skin sensitizer carvacrol. Altogether, our findings demonstrate that CA exerts its anti-inflammatory effects by selectively inhibiting TRPV3 through binding to the pocket formed by the Pore-loop and the S6. CA may serve as a lead for further modification and identification of specific TRPV3 channel inhibitors.

Abstract Mo065: An Anti-arrhythmic Action and Novel Molecular Mechanisms of Alda-1 in Holiday Heart Syndrome

Introduction: Holiday Heart Syndrome (HHS) is caused by excessive binge alcohol intake. Atrial fibrillation (AF) is the most common arrhythmia in HHS patients. With a worldwide rising trend in heavy alcohol consumption despite significant prevention efforts, effective drugs against alcohol-evoked AF are in urgent need. Methods: We assessed the effect of Alda-1, a cardiac protective agent, on mitigating binge alcohol-evoked AF and alcohol-activated stress kinase JNK2 in 50mM alcohol-exposed (24hr) H9c2 differentiated myocytes and in a well-characterized HHS mouse model (2g/kg ethanol i.p., 4 injections, every other day). Alda-1’s effect on alcohol-evoked RyR2 channel-mediated Ca 2+ waves/sparks & tachycardia/fibrillation (AT/AF) incidence were measured in intact mouse atria. RyR2 channel open probability was assessed in human RyR2 single-channel recordings. Results: We found that Alda-1 significantly reduced atrial arrhythmia inducibility in HHS mouse atria, as evidenced by a decreased incidence of alcohol-evoked AT/AF (0 vs. 0.21 incidences/pacing attempts/animal; n=6, 8). Alda-1 is an agonist of aldehyde dehydrogenase 2 (ALDH2; a key enzyme in alcohol detoxification), which inhibits cellular apoptosis. We found that ALDH2 was increased by 42%±6 in HHS mice compared to controls, and which remained elevated upon Alda-1 treatment (n=6,7,7; p<0.05). HHS hearts showed unchanged apoptotic signaling pathways, suggesting alternative mechanisms in AF genesis. Our lab recently revealed a critical role of activated cardiac stress kinase JNK2 in AF risk. Intriguingly, Alda-1 suppressed alcohol-activated JNK2 by 70%±13 (immunoprecipitated JNK2-specific activity; n=9,9, p<0.001) (but not JNK1, the other cardiac JNK isoform) in cells, while JNK2 inhibition alleviated alcohol-evoked RyR2 channel dysfunction in human RyR2 channels as well as Ca 2+ -triggered atrial arrhythmic activities and AT/AF (0 out of 8; p<0.05) in intact mouse hearts. Conclusion: Alda-1 effectively mitigates binge alcohol-evoked atrial arrhythmogenesis by suppressing JNK2 activity independently of an ALDH2-mediated anti-apoptosis effect. Our findings highlight the potential of Alda-1 as a therapeutic agent for the increasing incidence of alcohol-evoked AF.

Characterization of a novel Ca2+-Activated potassium channel in rat brain rough endoplasmic reticulum

ObjectivesPotassium channels in the endoplasmic reticulum (ER) are crucial for maintaining calcium balance during calcium fluxes. Disruption in ER calcium balance leads to ER stress, implicated in diseases like diabetes and Alzheimer's disease (AD). However, limited data exists on ER potassium channels in excitable tissues such as the brain. To fill this gap, we aimed to evaluate potassium currents in rat brain rough endoplasmic reticulum (RER). MethodsRats were euthanized under deep anesthesia and their brains were immediately removed. The brains were then homogenized in ice-cold sucrose buffer, followed by the extraction of RER microsomes through a series of centrifugation processes. Purity of sample was evaluated using western blotting technique. Single channel recordings were done in voltage steps from +50 to −60 mV following incorporation of rat brain RER vesicles into planar bilayers. ResultsWe observed a voltage-dependent potassium channel with an approximate conductance of 188 pS. Channel open probability was low at negative voltages, increasing at positive voltages. The channel was blocked by Charybdotoxin but not by Iberiotoxin. Additionally, TRAM-34, a specific KCa3.1 channel blocker, suppressed channel current amplitude and open probability. Western blot analysis revealed specific bands for anti-KCa3.1 antibody, approximately 50 kDa in brain homogenate and RER fraction. ConclusionOur study provides strong evidence for the presence of an KCa3.1 channel on the RER membrane in rat brain, exhibiting distinct electro-pharmacological profile compared to plasma membrane and other organelles.

Freestanding bilayer microscope for single-molecule imaging of membrane proteins.

Integral membrane proteins (IMPs) constitute a large fraction of organismal proteomes, playing fundamental roles in physiology and disease. Despite their importance, the mechanisms underlying dynamic features of IMPs, such as anomalous diffusion, protein-protein interactions, and protein clustering, remain largely unknown due to the high complexity of cell membrane environments. Available methods for in vitro studies are insufficient to study IMP dynamics systematically. This publication introduces the freestanding bilayer microscope (FBM), which combines the advantages of freestanding bilayers with single-particle tracking. The FBM, based on planar lipid bilayers, enables the study of IMP dynamics with single-molecule resolution and unconstrained diffusion. This paper benchmarks the FBM against total internal reflection fluorescence imaging on supported bilayers and is used here to estimate ion channel open probability and to examine the diffusion behavior of an ion channel in phase-separated bilayers. The FBM emerges as a powerful tool to examine membrane protein/lipid organization and dynamics to understand cell membrane processes.

Disease-Associated Variants in GRIN1, GRIN2A and GRIN2B genes: Insights into NMDA Receptor Structure, Function, and Pathophysiology.

N-methyl-D-aspartate receptors (NMDARs) are a subtype of ionotropic glutamate receptors critical for synaptic transmission and plasticity, and for the development of neural circuits. Rare or de-novo variants in GRIN genes encoding NMDAR subunits have been associated with neurodevelopmental disorders characterized by intellectual disability, developmental delay, autism, schizophrenia, or epilepsy. In recent years, some disease-associated variants in GRIN genes have been characterized using recombinant receptors expressed in non-neuronal cells, and a few variants have also been studied in neuronal preparations or animal models. Here we review the current literature on the functional evaluation of human disease-associated variants in GRIN1, GRIN2A and GRIN2B genes at all levels of analysis. Focusing on the impact of different patient variants at the level of receptor function, we discuss effects on receptor agonist and co-agonist affinity, channel open probability, and receptor cell surface expression. We consider how such receptor-level functional information may be used to classify variants as gain-of-function or loss-of-function, and discuss the limitations of this classification at the synaptic, cellular, or system level. Together this work by many laboratories worldwide yields valuable insights into NMDAR structure and function, and represents significant progress in the effort to understand and treat GRIN disorders. Keywords: NMDA receptor , GRIN genes, Genetic variants, Electrophysiology, Synapse, Animal models.

One-shot design elevates functional expression levels of a voltage-gated potassium channel.

Membrane proteins play critical physiological roles as receptors, channels, pumps, and transporters. Despite their importance, however, low expression levels often hamper the experimental characterization of membrane proteins. We present an automated and web-accessible design algorithm called mPROSS (https://mPROSS.weizmann.ac.il), which uses phylogenetic analysis and an atomistic potential, including an empirical lipophilicity scale, to improve native-state energy. As a stringent test, we apply mPROSS to the Kv1.2-Kv2.1 paddle chimera voltage-gated potassium channel. Four designs, encoding 9-26 mutations relative to the parental channel, were functional and maintained potassium-selective permeation and voltage dependence in Xenopus oocytes with up to 14-fold increase in whole-cell current densities. Additionally, single-channel recordings reveal no significant change in the channel-opening probability nor in unitary conductance, indicating that functional expression levels increase without impacting the activity profile of individual channels. Our results suggest that the expression levels of other dynamic channels and receptors may be enhanced through one-shot design calculations.

Influence of proteolytic cleavage of ENaC's γ subunit upon Na+ and K+ handling.

The epithelial Na+ channel (ENaC) γ subunit is essential for homeostasis of Na+, K+, and body fluid. Dual γ subunit cleavage before and after a short inhibitory tract allows dissociation of this tract, increasing channel open probability (PO), in vitro. Cleavage proximal to the tract occurs at a furin recognition sequence (143RKRR146, in the mouse γ subunit). Loss of furin-mediated cleavage prevents in vitro activation of the channel by proteolysis at distal sites. We hypothesized that 143RKRR146 mutation to 143QQQQ146 (γQ4) in 129/Sv mice would reduce ENaC PO, impair flow-stimulated flux of Na+ (JNa) and K+ (JK) in perfused collecting ducts, reduce colonic amiloride-sensitive short-circuit current (ISC), and impair Na+, K+, and body fluid homeostasis. Immunoblot of γQ4/Q4 mouse kidney lysates confirmed loss of a band consistent in size with the furin-cleaved proteolytic fragment. However, γQ4/Q4 male mice on a low Na+ diet did not exhibit altered ENaC PO or flow-induced JNa, though flow-induced JK modestly decreased. Colonic amiloride-sensitive ISC in γQ4/Q4 mice was not altered. γQ4/Q4 males, but not females, exhibited mildly impaired fluid volume conservation when challenged with a low Na+ diet. Blood Na+ and K+ were unchanged on a regular, low Na+, or high K+ diet. These findings suggest that biochemical evidence of γ subunit cleavage should not be used in isolation to evaluate ENaC activity. Furthermore, factors independent of γ subunit cleavage modulate channel PO and the influence of ENaC on Na+, K+, and fluid volume homeostasis in 129/Sv mice, in vivo.NEW & NOTEWORTHY The epithelial Na+ channel (ENaC) is activated in vitro by post-translational proteolysis. In vivo, low Na+ or high K+ diets enhance ENaC proteolysis, and proteolysis is hypothesized to contribute to channel activation in these settings. Using a mouse expressing ENaC with disruption of a key proteolytic cleavage site, this study demonstrates that impaired proteolytic activation of ENaC's γ subunit has little impact upon channel open probability or the ability of mice to adapt to low Na+ or high K+ diets.

A structure-based computational model of IP3R1 incorporating Ca and IP3 regulation

The inositol 1,4,5-triphosphate receptor (IP3R) mediates Ca release in many cell types and is pivotal to a wide range of cellular processes. High-resolution cryoelectron microscopy studies have provided new structural details of IP3R type 1 (IP3R1), showing that channel function is determined by the movement of various domains within and between each of its four subunits. Channel properties are regulated by ligands, such as Ca and IP3, which bind at specific sites and control the interactions between these domains. However, it is not known how the various ligand-binding sites on IP3R1 interact to control the opening of the channel. In this study, we present a coarse-grained model of IP3R1 that accounts for the channel architecture and the location of specific Ca- and IP3-binding sites. This computational model accounts for the domain-domain interactions within and between the four subunits that form IP3R1, and it also describes how ligand binding regulates these interactions. Using a kinetic model, we explore how two Ca-binding sites on the cytosolic side of the channel interact with the IP3-binding site to regulate the channel open probability. Our primary finding is that the bell-shaped open probability of IP3R1 provides constraints on the relative strength of these regulatory binding sites. In particular, we argue that a specific Ca-binding site, whose function has not yet been established, is very likely a channel antagonist. Additionally, we apply our model to show that domain-domain interactions between neighboring subunits exert control over channel cooperativity and dictate the nonlinear response of the channel to Ca concentration. This suggests that specific domain-domain interactions play a pivotal role in maintaining the channel’s stability, and a disruption of these interactions may underlie disease states associated with Ca dysregulation.

IP3RPEP6, a novel peptide inhibitor of IP3 receptor channels that does not affect connexin-43 hemichannels.

Inositol 1,4,5-trisphosphate receptors (IP3 Rs) are intracellular Ca2+ -release channels with crucial roles in cell function. Current IP3 R inhibitors suffer from off-target effects and poor selectivity towards the three distinct IP3 R subtypes. We developed a novel peptide inhibitor of IP3 Rs and determined its effect on connexin-43 (Cx43) hemichannels, which are co-activated by IP3 R stimulation. IP3RPEP6 was developed by in silico molecular docking studies and characterized by on-nucleus patch-clamp experiments of IP3 R2 channels and carbachol-induced IP3 -mediated Ca2+ responses in IP3 R1, 2 or 3 expressing cells, triple IP3 R KO cells and astrocytes. Cx43 hemichannels were studied by patch-clamp and ATP-release approaches, and by inhibition with Gap19 peptide. IP3RPEP6 interactions with IP3 Rs were verified by co-immunoprecipitation and affinity pull-down assays. IP3RPEP6 concentration-dependently reduced the open probability of IP3 R2 channels and competitively inhibited IP3 Rs in an IC50 order of IP3 R2 (~3.9 μM) < IP3 R3 (~4.3 μM) < IP3 R1 (~9.0 μM), without affecting Cx43 hemichannels or ryanodine receptors. IP3RPEP6 co-immunoprecipitated with IP3 R2 but not with IP3 R1; interaction with IP3 R3 varied between cell types. The IC50 of IP3RPEP6 inhibition of carbachol-induced Ca2+ responses decreased with increasing cellular Cx43 expression. Moreover, Gap19-inhibition of Cx43 hemichannels significantly reduced the amplitude of the IP3 -Ca2+ responses and strongly increased the EC50 of these responses. Finally, we identified palmitoyl-8G-IP3RPEP6 as a membrane-permeable IP3RPEP6 version allowing extracellular application of the IP3 R-inhibiting peptide. IP3RPEP6 inhibits IP3 R2/R3 at concentrations that have limited effects on IP3 R1. IP3 R activation triggers hemichannel opening, which strongly affects the amplitude and concentration-dependence of IP3 -triggered Ca2+ responses.

Disease-associated nonsense and frame-shift variants resulting in the truncation of the GluN2A or GluN2B C-terminal domain decrease NMDAR surface expression and reduce potentiating effects of neurosteroids

N-methyl-d-aspartate receptors (NMDARs) play a critical role in normal brain function, and variants in genes encoding NMDAR subunits have been described in individuals with various neuropsychiatric disorders. We have used whole-cell patch-clamp electrophysiology, fluorescence microscopy and in-silico modeling to explore the functional consequences of disease-associated nonsense and frame-shift variants resulting in the truncation of GluN2A or GluN2B C-terminal domain (CTD). This study characterizes variant NMDARs and shows their reduced surface expression and synaptic localization, altered agonist affinity, increased desensitization, and reduced probability of channel opening. We also show that naturally occurring and synthetic steroids pregnenolone sulfate and epipregnanolone butanoic acid, respectively, enhance NMDAR function in a way that is dependent on the length of the truncated CTD and, further, is steroid-specific, GluN2A/B subunit-specific, and GluN1 splice variant-specific. Adding to the previously described effects of disease-associated NMDAR variants on the receptor biogenesis and function, our results improve the understanding of the molecular consequences of NMDAR CTD truncations and provide an opportunity for the development of new therapeutic neurosteroid-based ligands.

Calcium chelation independent effects of BAPTA on endogenous ANO6 channels in HEK293T cells

Selective calcium chelator 1,2-Bis(2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA) is a common tool to investigate calcium signaling. However, BAPTA express various effects on intracellular calcium signaling, which are not related to its ability to bind Ca2+. In patch clamp experiments, we investigated calcium chelation independent effects of BAPTA on endogenous calcium-activated chloride channels ANO6 (TMEM16F) in HEK293T cells. We have found that application of BAPTA to intracellular solution led to two distinct effects on channels properties. On the one hand, application of BAPTA acutely reduced amplitude of endogenous ANO6 channels induced by 10 μM Ca2+ in single channel recordings. On the other hand, BAPTA application by itself induced ANO6 channel activity in the absence of the intracellular calcium elevation. Open channel probability was enhanced by increasing the intracellular BAPTA concentration from 0.1 to 1 and 10 mM. Another calcium chelator EGTA did not demonstrate chelation independent effects on the ANO6 activity in the same conditions. Due to off target effects BAPTA should be used with caution when studying calcium-activated ANO6 channels.

Selective inhibition of overactive warmth-sensitive Ca2+-permeable TRPV3 channels by antispasmodic agent flopropione for alleviation of skin inflammation

The temperature-sensitive Ca2+-permeable TRPV3 ion channel is robustly expressed in the skin keratinocytes, and its gain-of-function mutations are involved in the pathology of skin lesions. Here, we report the identification of an antispasmodic agent flopropione that alleviates skin inflammation by selective inhibition of TRPV3. In whole-cell patch clamp recordings, flopropione selectively inhibits macroscopic TRPV3 currents in a concentration-dependent manner with an IC50 value of 17.8± 3.5μM. At the single-channel level, flopropione inhibits TRPV3 channel open probability without alteration of its unitary conductance. In an invivo mouse model of skin inflammation induced by the skin sensitizer DNFB, flopropione also alleviates dorsal skin lesions and ear skin swelling. Further molecular docking combined with site-directed mutagenesis reveals that two residues E501 and I505 in the channel S2-helix are critical for flopropione-mediated inhibition of TRPV3. Taken together, our findings demonstrate that the spasmolytic drug flopropione as a selective inhibitor of TRPV3 channel not only provides a valuable tool molecule for understanding of TRPV3 channel pharmacology but also holds repurposing potential for therapy of skin disorders, such as dermatitis and pruritus.

Injectable long-acting ivacaftor-loaded poly (lactide-co-glycolide) microparticle formulations for the treatment of cystic fibrosis: In vitro characterization and in vivo pharmacokinetics in mice

Optimizing a sustained-release drug delivery system for the treatment of cystic fibrosis (CF) is crucial for decreasing the dosing frequency and improving patients’ compliance with the treatment regimen. In the current work, we developed an injectable poly(D,L-lactide-co-glycolide) (PLGA) microparticle formulation loaded with ivacaftor, a cystic fibrosis transmembrane conductance regulator (CFTR) potentiator that increases the open probability of the CFTR anion channel, using a single emulsion solvent evaporation technique. We aimed to study the effect of different parameters on the characteristics of the prepared formulations to select an optimized microparticle formulation to be used in an in vivo pharmacokinetic study in mice. First, a suite of ivacaftor-loaded microparticles were prepared using different formulation parameters in order to study the effect of varying these parameters on microparticle size, morphology, drug loading, encapsulation efficiency, and in vitro release profiles. Prepared microparticles were spherical with diameters ranging from 1.91–6.93 µm, percent drug loading (% DL) of 3.91–10.3%, percent encapsulation efficiencies (% EE) of 26.6–100%, and an overall slow cumulative release profile. We selected the formulation that demonstrated optimal combined % DL and % EE values (8.25 and 90.7%, respectively) for further studies. These microparticles had an average particle size of 6.83 µm and a slow tri-phasic in vitro release profile (up to 6 weeks). In vivo pharmacokinetic studies in mice showed that the subcutaneously injected microparticles resulted in steady plasma levels of ivacaftor over a period of 28 days, and a 6-fold increase in AUC 0 – t (71.6 µg/mL*h) compared to the intravenously injected soluble ivacaftor (12.3 µg/mL*h). Our results suggest that this novel ivacaftor-loaded microparticle formulation could potentially eliminate the need for the frequent daily administration of ivacaftor to people with CF thus improving their compliance and ensuring successful treatment outcomes.

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

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