Increased Channel Open Probability Research Articles

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.

Pharmacological characterization of SAGE-718, a novel positive allosteric modulator of N-methyl-d-aspartate receptors.

Select neuroactive steroids tune neural activity by modulating excitatory and inhibitory neurotransmission, including the endogenous cholesterol metabolite 24(S)-hydroxycholesterol (24(S)-HC), which is an N-methyl-d-aspartate (NMDA) receptor positive allosteric modulator (PAM). NMDA receptor PAMs are potentially an effective pharmacotherapeutic strategy to treat conditions associated with NMDA receptor hypofunction. Using in vitro and in vivo electrophysiological recording experiments and behavioural approaches, we evaluated the effect of SAGE-718, a novel neuroactive steroid NMDA receptor PAM currently in clinical development for the treatment of cognitive impairment, on NMDA receptor function and endpoints that are altered by NMDA receptor hypoactivity and assessed its safety profile. SAGE-718 potentiated GluN1/GluN2A-D NMDA receptors with equipotency and increased NMDA receptor excitatory postsynaptic potential (EPSP) amplitude without affecting decay kinetics in striatal medium spiny neurons. SAGE-718 increased the rate of unblock of the NMDA receptor open channel blocker ketamine on GluN1/GluN2A in vitro and accelerated the rate of return on the ketamine-evoked increase in gamma frequency band power, as measured with electroencephalogram (EEG), suggesting that PAM activity is driven by increased channel open probability. SAGE-718 ameliorated deficits due to NMDA receptor hypofunction, including social deficits induced by subchronic administration of phencyclidine, and behavioural and electrophysiological deficits from cholesterol and 24(S)-HC depletion caused by 7-dehydrocholesterol reductase inhibition. Finally, SAGE-718 did not produce epileptiform activity in a seizure model or neurodegeneration following chronic dosing. These findings provide strong evidence that SAGE-718 is a neuroactive steroid NMDA receptor PAM with a mechanism that is well suited as a treatment for conditions associated with NMDA receptor hypofunction.

Allosteric modulation of ryanodine receptor RyR1 by nucleotide derivatives

The coordinated release of Ca2+ from the sarcoplasmic reticulum (SR) is critical for excitation-contraction coupling. This release is facilitated by ryanodine receptors (RyRs) that are embedded in the SR membrane. In skeletal muscle, activity of RyR1 is regulated by metabolites such as ATP, which upon binding increase channel open probability (Po). To obtain structural insights into the mechanism of RyR1 priming by ATP, we determined several cryo-EM structures of RyR1 bound individually to ATP-γ-S, ADP, AMP, adenosine, adenine, and cAMP. We demonstrate that adenine and adenosine bind RyR1, but AMP is the smallest ATP derivative capable of inducing long-range (>170Å) structural rearrangements associated with channel activation, establishing a structural basis for key binding site interactions that are the threshold for triggering quaternary structural changes. Our finding that cAMP also induces these structural changes and results in increased channel opening suggests its potential role as an endogenous modulator of RyR1 conductance.

Chemical shift assignments of calmodulin bound to a C-terminal site (residues 1120–1147) in the β-subunit of a retinal cyclic nucleotide-gated channel (CNGB1)

Retinal cyclic nucleotide-gated (CNG) channels consist of two protein subunits (CNGA1 and CNGB1). Calmodulin (CaM) binds to two separate sites within the cytosolic region of CNGB1: CaM binding to an N-terminal site (human CNGB1 residues 565–587, called CaM1) decreases the open probability of CNG channels at elevated Ca2+ levels in dark-adapted photoreceptors, whereas CaM binding to a separate C-terminal site (CNGB1 residues 1120–1147, called CaM2) may increase channel open probability in light activated photoreceptors. We recently reported NMR chemical shift assignments of Ca2+-saturated CaM bound to the CaM1 site of CNGB1 (BMRB no. 51222). Here, we report complete NMR chemical shift assignments of Ca2+-saturated CaM bound to the C-terminal CaM2 site of CNGB1 (BMRB no. 51447).

Thermodynamic and structural bases for Yoda1-mediated Piezo1 activation

The synthetic small molecule Piezo1-selective agonist Yoda1 is widely used to probe the biological functions of the mechanotransduction ion channel Piezo1. Yet, it is still unclear how Yoda1 activates Piezo1. Single-channel patch-clamp recordings reveal Piezo1 inherently populates at least four functional states, two conducting (open) and two non-conducting (shut). Yoda1 increases the dwell-time of a long-lived open state and shortens the dwell-time of other open and shut states, effectively increasing channel open probability (Po).

L-Type Ca2+ Channel Regulation by Calmodulin and CaBP1.

L-type voltage-gated Ca2+ channels (CaV1.2 and CaV1.3, called CaV) interact with the Ca2+ sensor proteins, calmodulin (CaM) and Ca2+ binding Protein 1 (CaBP1), that oppositely control Ca2+-dependent channel activity. CaM and CaBP1 can each bind to the IQ-motif within the C-terminal cytosolic domain of CaV, which promotes increased channel open probability under basal conditions. At elevated cytosolic Ca2+ levels (caused by CaV channel opening), Ca2+-bound CaM binding to CaV is essential for promoting rapid Ca2+-dependent channel inactivation (CDI). By contrast, CaV binding to CaBP1 prevents CDI and promotes Ca2+-induced channel opening (called CDF). In this review, I provide an overview of the known structures of CaM and CaBP1 and their structural interactions with the IQ-motif to help understand how CaM promotes CDI, whereas CaBP1 prevents CDI and instead promotes CDF. Previous electrophysiology studies suggest that Ca2+-free forms of CaM and CaBP1 may pre-associate with CaV under basal conditions. However, previous Ca2+ binding data suggest that CaM and CaBP1 are both calculated to bind to Ca2+ with an apparent dissociation constant of ~100 nM when CaM or CaBP1 is bound to the IQ-motif. Since the neuronal basal cytosolic Ca2+ concentration is ~100 nM, nearly half of the neuronal CaV channels are suggested to be bound to Ca2+-bound forms of either CaM or CaBP1 under basal conditions. The pre-association of CaV with calcified forms of CaM or CaBP1 are predicted here to have functional implications. The Ca2+-bound form of CaBP1 is proposed to bind to CaV under basal conditions to block CaV binding to CaM, which could explain how CaBP1 might prevent CDI.

Structural and functional approaches to studying cAMP regulation of HCN channels.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are primarily activated by voltage and further modulated by cAMP. While cAMP binding alone does not open the channel, its presence facilitates the action of voltage, increasing channel open probability. Functional results indicate that the membrane-based voltage sensor domain (VSD) communicates with the cytosolic cyclic nucleotide-binding domain (CNBD), and vice-versa. Yet, a mechanistic explanation on how this could occur in structural terms is still lacking. In this review, we will discuss the recent advancement in understanding the molecular mechanisms connecting the VSD with the CNBD in the tetrameric organization of HCN channels unveiled by the 3D structures of HCN1 and HCN4. Data show that the HCN domain transmits cAMP signal to the VSD by bridging the cytosolic to the membrane domains. Furthermore, a metal ion coordination site connects the C-linker to the S4-S5 linker in HCN4, further facilitating cAMP signal transmission to the VSD in this isoform.

Surfactant cocamide monoethanolamide causes eye irritation by activating nociceptor TRPV1 channels.

Cocamide monoethanolamide (CMEA) is commonly used as a surfactant-foam booster in cosmetic formulations. Upon contact with the eye or other sensitive skin areas, CMEA elicits stinging and lasting irritation. We hypothesized a specific molecular interaction with TRPV1 channels by which CMEA caused eye irritation. Eye irritancy was evaluated using eye-wiping tests in rabbits and mice. Intracellular Ca2+ concentrations and action potentials were measured using Ca2+ imaging and current clamp respectively. Voltage clamp, site-direct mutagenesis and molecular modelling were used to identify binding pockets for CMEA on TRPV1 channels. CMEA-induced eye irritation is ameliorated by selective ablation of TRPV1 channels.Rodents exhibit much stronger responses to CMEA than rabbits. In trigeminal ganglion neurons, CMEA induced Ca2+ influx and neuronal excitability, effects mitigated by a TRPV1 channel inhibition and absent in TRPV1 knockout neurons. In HEK-293 cells expressing TRPV1 channels, CMEA increased whole-cell currents by increasing channel open probability (EC50 = 10.2μM), without affecting TRPV2, TRPV3, TRPV4, and TRPA1 channel activities. Lauric acid monoethanolamide (LAMEA), the most abundant constituent of CMEA, was the most efficacious and potent TRPV1 channel activator, binding to the capsaicin-binding pocket of the channel. The T550I mutants of rabbit and human TRPV1 channels exhibit much lower sensitivity to LAMEA. CMEA directly activates TRPV1 channels to produce eye irritation. Rabbits, the standard animal used for eye irritancy tests are poor models for evaluating human eye irritants structurally related to CMEA. Our study identifies potential alternatives to CMEA as non-irritating surfactants.

Adrenergic CaV1.2 Activation via Rad Phosphorylation Converges at α1C I-II Loop.

Changing activity of cardiac CaV1.2 channels under basal conditions, during sympathetic activation, and in heart failure is a major determinant of cardiac physiology and pathophysiology. Although cardiac CaV1.2 channels are prominently upregulated via activation of PKA (protein kinase A), essential molecular details remained stubbornly enigmatic. The primary goal of this study was to determine how various factors converging at the CaV1.2 I-II loop interact to regulate channel activity under basal conditions, during β-adrenergic stimulation, and in heart failure. We generated transgenic mice with expression of CaV1.2 α1C subunits with (1) mutations ablating interaction between α1C and β-subunits, (2) flexibility-inducing polyglycine substitutions in the I-II loop (GGG-α1C), or (3) introduction of the alternatively spliced 25-amino acid exon 9* mimicking a splice variant of α1C upregulated in the hypertrophied heart. Introducing 3 glycine residues that disrupt a rigid IS6-α-interaction domain helix markedly reduced basal open probability despite intact binding of CaVβ to α1C I-II loop and eliminated β-adrenergic agonist stimulation of CaV1.2 current. In contrast, introduction of the exon 9* splice variant in the α1C I-II loop, which is increased in ventricles of patients with end-stage heart failure, increased basal open probability but did not attenuate stimulatory response to β-adrenergic agonists when reconstituted heterologously with β2B and Rad or transgenically expressed in cardiomyocytes. Ca2+ channel activity is dynamically modulated under basal conditions, during β-adrenergic stimulation, and in heart failure by mechanisms converging at the α1C I-II loop. CaVβ binding to α1C stabilizes an increased channel open probability gating mode by a mechanism that requires an intact rigid linker between the β-subunit binding site in the I-II loop and the channel pore. Release of Rad-mediated inhibition of Ca2+ channel activity by β-adrenergic agonists/PKA also requires this rigid linker and β-binding to α1C.

Patch-Clamp Fluorometry Defines a Role for SUR1 in Nucleotide Inhibition of KATP Channels

ATP sensitive K+ (KATP) channels couple a cell's metabolic state to its electrical excitability. KATP is a hetero-octameric complex, with four pore-forming inward rectifier K+ channel subunits (Kir6.1 or Kir6.2), each associated with a modulatory sulfonylurea receptor (SUR1 or SUR2A/B). ATP/ADP binding to a site on the Kir6.2 subunit shuts KATP. In the presence of Mg2+, nucleotide binding to SUR1 stimulates KATP. However, SUR1 also has direct effects on gating at Kir6.2 in the absence of Mg2+. The presence of SUR1 increases channel open probability, but paradoxically also increases the apparent affinity for nucleotide inhibition at Kir6.2 by an unknown mechanism. We introduced the fluorescent non-canonical amino acid ANAP at a position (W311ANAP) in the cytoplasmic C-terminal region of Kir6.2 co-expressed with SUR1 in HEK 293T cells. This allowed us to simultaneously measure channel currents and inhibitory nucleotide binding to Kir6.2 via FRET between ANAP and a fluorescent ATP derivative (TNP-ATP). We fit models to the combined data sets to produce well-constrained estimates of open-state nucleotide affinity and the strength of coupling between nucleotide binding and channel closure. Our models suggest that “wild-type” KATP is closed when only one of its nucleotide binding sites is occupied. A Kir6.2 mutation (C166S) that profoundly increases channel activity had very little effect on nucleotide binding, but greatly perturbed the ability of bound nucleotide to inhibit KATP. In contrast, mutations at position E205 on SUR1, which is located on an intracellular loop close to the inhibitory site on Kir6.2, affected both TNP-ATP affinity and the ability of bound nucleotide to inhibit KATP. This suggests a dual role for SUR1 in KATP inhibition, both in contributing to nucleotide binding to the open state and in stabilizing the nucleotide-bound closed state.

Mechanism of adrenergic CaV1.2 stimulation revealed by proximity proteomics.

Increased cardiac contractility during the fight-or-flight response is caused by β-adrenergic augmentation of CaV1.2 voltage-gated calcium channels1-4. However, this augmentation persists in transgenic murine hearts expressing mutant CaV1.2 α1C and β subunits that can no longer be phosphorylated by protein kinase A-an essential downstream mediator of β-adrenergic signalling-suggesting that non-channel factors are also required. Here we identify the mechanism by which β-adrenergic agonists stimulate voltage-gated calcium channels. We express α1C or β2B subunits conjugated to ascorbate peroxidase5 in mouse hearts, and use multiplexed quantitative proteomics6,7 to track hundreds of proteins in the proximity of CaV1.2. We observe that the calcium-channel inhibitor Rad8,9, a monomeric G protein, is enriched in the CaV1.2 microenvironment but is depleted during β-adrenergic stimulation. Phosphorylation by protein kinase A of specific serine residues on Rad decreases its affinity for β subunits and relieves constitutive inhibition of CaV1.2, observed as an increase in channel open probability. Expression of Rad or its homologue Rem in HEK293T cells also imparts stimulation of CaV1.3 and CaV2.2 by protein kinase A, revealing an evolutionarily conserved mechanism that confers adrenergic modulation upon voltage-gated calcium channels.

Characterization of NCS1–InsP3R1 interaction and its functional significance

Inositol 1,4,5-trisphosphate receptors (InsP3Rs) are endoplasmic reticulum-localized channels that mediate Ca2+ release from the endoplasmic reticulum into the cytoplasm. We previously reported that an EF-hand Ca2+-binding protein, neuronal calcium sensor 1 (NCS1), binds to the InsP3R and thereby increases channel open probability, an event associated with chemotherapy-induced peripheral neuropathy. However, the exact NCS1-binding site on InsP3R remains unknown. Using protein docking, co-immunoprecipitation, and blocking peptides, we mapped the NCS1-binding site to residues 66-110 on the suppressor domain of InsP3R type 1 (InsP3R1). We also identified Leu-89, a residue in the hydrophobic pocket of NCS1, as being critical for facilitating the NCS1-InsP3R1 interaction. Overexpression of WT NCS1 in MDA-MB231 breast cancer cells increased Ca2+ signaling and survival, whereas overexpression of Leu-89 NCS1 variants decreased Ca2+ signaling and survival, further suggesting the importance of this residue in the NCS1-InsP3R1 interaction. In conclusion, we show that NCS1-InsP3R1 interaction enhances intracellular Ca2+ signaling in cells and can be modulated by altering or occluding the hydrophobic pocket of NCS1. This improved understanding of the NCS1-InsP3R1 interaction may facilitate the development of management strategies for diseases resulting from aberrant NCS1 expression.

Activation of TRPC6 channels contributes to (+)-conocarpan-induced apoptotic cell death in HK-2 cells

(+)-Conocarpan (CNCP), a neolignan frequently found in many medicinal and edible plants displays a broad spectrum of bioactivity. Here, we demonstrated that CNCP induced apoptotic cell death in human kidney-2 (HK-2) cells in a concentration-dependent manner (IC50 = 19.3 μM) and led to the sustained elevation of intracellular Ca2+ ([Ca2+]i). Lower extracellular Ca2+ concentrations from 2.3 mM to 0 mM significantly suppressed the CNCP-induced Ca2+ response by 69.1%. Moreover, the depletion of intracellular Ca2+ stores using thapsigargin normalized CNCP-induced Ca2+ release from intracellular Ca2+ stores, suggesting that the CNCP-induced Ca2+ response involved both extracellular Ca2+ influx and Ca2+ release from intracellular Ca2+ stores. SAR7334, a TRPC3/6/7 channel inhibitor, but neither Pyr3, a selective TRPC3 channel inhibitor, nor Pico145, a TRPC1/4/5 inhibitor, suppressed the CNCP-induced Ca2+ response by 57.2% and decreased CNCP-induced cell death by 53.4%, suggesting a critical role for TRPC6 channels in CNCP-induced Ca2+ influx and apoptotic cell death. Further electrophysiological recording demonstrated that CNCP directly activated TRPC6 channels by increasing channel open probability with an EC50 value of 6.01 μM. Considered together, these data demonstrate that the direct activation of TRPC6 channels contributes to CNCP-induced apoptotic cell death in HK-2 cells. Our data point out the potential risk of renal toxicity from CNCP if used as a therapeutic agent.

A mutation in CaV2.1 linked to a severe neurodevelopmental disorder impairs channel gating.

Ca2+ flux into axon terminals via P-/Q-type CaV2.1 channels is the trigger for neurotransmitter vesicle release at neuromuscular junctions (NMJs) and many central synapses. Recently, an arginine to proline substitution (R1673P) in the S4 voltage-sensing helix of the fourth membrane-bound repeat of CaV2.1 was linked to a severe neurological disorder characterized by generalized hypotonia, ataxia, cerebellar atrophy, and global developmental delay. The R1673P mutation was proposed to cause a gain of function in CaV2.1 leading to neuronal Ca2+ toxicity based on the ability of the mutant channel to rescue the photoreceptor response in CaV2.1-deficient Drosophila cacophony larvae. Here, we show that the corresponding mutation in rat CaV2.1 (R1624P) causes a profound loss of channel function; voltage-clamp analysis of tsA-201 cells expressing this mutant channel revealed an ∼25-mV depolarizing shift in the voltage dependence of activation. This alteration in activation implies that a significant fraction of CaV2.1 channels resident in presynaptic terminals are unlikely to open in response to an action potential, thereby increasing the probability of synaptic failure at both NMJs and central synapses. Indeed, the mutant channel supported only minimal Ca2+ flux in response to an action potential-like waveform. Application of GV-58, a compound previously shown to stabilize the open state of wild-type CaV2.1 channels, partially restored Ca2+ current by shifting mutant activation to more hyperpolarizing potentials and slowing deactivation. Consequently, GV-58 also rescued a portion of Ca2+ flux during action potential-like stimuli. Thus, our data raise the possibility that therapeutic agents that increase channel open probability or prolong action potential duration may be effective in combatting this and other severe neurodevelopmental disorders caused by loss-of-function mutations in CaV2.1.

Regulation of ENaC Expression by Paraoxonase 3

The epithelial sodium channel (ENaC) mediates the rate‐limiting step of Na+ uptake across the apical membrane of specific epithelia. ENaC‐dependent Na+ absorption in the kidney has important roles in regulating extracellular fluid volume, extracellular [K+] and blood pressure. ENaC functional expression is tightly regulated by multiple intracellular and exogenous factors, including molecular chaperones that are implicated in key steps during ENaC biogenesis. We previously showed that ENaC activity was reduced by paraoxonase 2 (PON2) in Xenopus oocytes. PON2–mediated inhibition is associated with reduced surface expression of ENaC, suggesting a role of PON2 in regulating ENaC turnover and/or trafficking. In supporting this notion, PONs are the mammalian orthologues of MEC‐6, a C. elegans ER‐resident chaperone. MEC‐6 is required for the proper assembly and surface expression of the touch‐sensing MEC‐4/MEC‐10 channel in worm touch‐receptor neurons. If the chaperone function is conserved between MEC‐6 and mammalian PONs, we predict other members of this family will also regulate the functional expression of related ion channels. Indeed, we found that PON3 is expressed in principal cells of the distal nephron, where ENaC resides. The expression of PON3 inhibits ENaC activity in Xenopus oocytes. The protease chymotrypsin activates ENaC by releasing the embedded inhibitory tracts, thus increasing channel open probability (Po). We found that chymotrypsin‐mediated ENaC activation was not altered by PON3, suggesting that PON3 does not inhibit ENaC activity by reducing channel Po. In addition, ENaC expression in Fisher rat thyroid cells was reduced by PON3. Future studies will investigate the mechanism by which PON3 regulates ENaC functional expression as well as the potential roles of PON3 in regulating ENaC activity, blood pressure and extracellular [K+] in the Pon3 KO mice. Understanding mechanisms by which PON3 regulates ENaC expression will provide novel insights into both normal and altered ENaC functional states, such as hypertension.Support or Funding InformationDK103834, DK038470, DK079307This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Chimeric HCN Channels for Studying Camp-Induced Conformational Changes in the C-Linker

Hyperpolarization-activated cyclic nucleotide-gated (HCN1-4) channels are activated by the hyperpolarization of the membrane potential and further regulated by the direct binding of cyclic AMP (cAMP). The binding of cAMP to the cytoplasmic Cyclic Nucleotide Binding Domain (CNBD) increases channel open probability and speeds activation kinetics. cAMP modulation of HCN channels controls heart rate and working memory networks in prefrontal cortex, but also chronic pain at the level of the peripheral nervous system. Thus, a detailed description of the cAMP conformational changes in HCN channels is crucial for understanding several physiological functions, as well as diseases affecting cardiac and neuronal functions. The cAMP-induced conformational changes propagate from the CNBD to the pore through the C-linker region. We and others have previously described, at the atomic level, the conformational changes at the level of the CNBD. The goal of the present work is to describe the propagation of the movements through the C-linker to the pore. To this end, I have constructed a chimeric channel by fusing the C-linker/CNBD of HCN4 to the prokaryotic pore domain of KcsA (hereafter K-H4). Isothermal Titration Calorimetry (ITC) measurements demonstrated that K-H4 is able to bind to cAMP with a KD value of 2μM, which agrees with the one previously published for the isolated C-linker/CNBD fragment of HCN4 and Differential Scanning Calorimetry (DSC) indicated that cAMP causes conformational changes in the chimeric protein. Recently, Double Electron Electron Resonance (DEER) experiments showed that the binding of cAMP at the level of the CNBD domain of K-H4 causes relevant conformational changes in the C-linker region which transits from a dimer of dimers conformation to a 4-fold symmetrical gating ring as suggested by previous functional and biochemical data.

The insecticide chlorantraniliprole is a weak activator of mammalian skeletal ryanodine receptor/Ca2+ release channel

Chlorantraniliprobe (Chlo), a potent insecticide, demolishes intracellular Ca2+ homeostasis of insects by inducing uncontrolled Ca2+ release through ryanodine receptors (RyRs). Chlo is lethal to insects but has low toxicity to mammals. In this study, we investigated the effects of Chlo on RyR1 from mammalian skeletal muscle. Ca2+ release assay indicated that Chlo at high concentrations promoted Ca2+ release from sarcoplasmic reticulum through RyR1 channels. Single channel recording of purified RyR1 showed that Chlo activated RyR1 channel, increased channel open probability Po, reduced channel mean close time Tc, but did not change the channel mean open time To, suggesting that Chlo destabilized the closed RyR1 channel, rendered the channel easy to open. The dissociation constant Kd values of Chlo for RyR1 were of micromolar level, approximately 100-fold larger than that for insect RyR. The Kd values were smaller for open states than for closed/blocked states of the RyR1 channel. The maximal binding capacity Bmax did not change in the presence of either channel activators or inhibitors/blockers. Our results demonstrate that the insecticide Chlo is a weak activator of mammalian RyR1. It can interact with mammalian RyR1 and activate RyR1 channel but with much lower affinity compared with insect RyR; Chlo has a binding site distinct from all known RyR channel modulators and represents a novel type of RyR channel modulator. Our data provide biochemical and pharmacological insights into its high specificity to insect RyR and high selectivity of poisoning to insects over mammals.

Binding of the general anesthetic sevoflurane to ion channels.

The direct-site hypothesis assumes general anesthetics bind ion channels to impact protein equilibrium and function, inducing anesthesia. Despite advancements in the field, a first principle all-atom demonstration of this structure-function premise is still missing. We focus on the clinically used sevoflurane interaction to anesthetic-sensitive Kv1.2 mammalian channel to resolve if sevoflurane binds protein’s well-characterized open and closed structures in a conformation-dependent manner to shift channel equilibrium. We employ an innovative approach relying on extensive docking calculations and free-energy perturbation of all potential binding sites revealed by the latter, and find sevoflurane binds open and closed structures at multiple sites under complex saturation and concentration effects. Results point to a non-trivial interplay of site and conformation-dependent modes of action involving distinct binding sites that increase channel open-probability at diluted ligand concentrations. Given the challenge in exploring more complex processes potentially impacting channel-anesthetic interaction, the result is revealing as it demonstrates the process of multiple anesthetic binding events alone may account for open-probability shifts recorded in measurements.

CACHD1 is an α2δ-Like Protein That Modulates CaV3 Voltage-Gated Calcium Channel Activity.

The putative cache (Ca2+ channel and chemotaxis receptor) domain containing 1 (CACHD1) protein has predicted structural similarities to members of the α2δ voltage-gated Ca2+ channel auxiliary subunit family. CACHD1 mRNA and protein were highly expressed in the male mammalian CNS, in particular in the thalamus, hippocampus, and cerebellum, with a broadly similar tissue distribution to CaV3 subunits, in particular CaV3.1. In expression studies, CACHD1 increased cell-surface localization of CaV3.1, and these proteins were in close proximity at the cell surface, consistent with the formation of CACHD1-CaV3.1 complexes. In functional electrophysiological studies, coexpression of human CACHD1 with CaV3.1, CaV3.2, and CaV3.3 caused a significant increase in peak current density and corresponding increases in maximal conductance. By contrast, α2δ-1 had no effect on peak current density or maximal conductance in CaV3.1, CaV3.2, or CaV3.3. A comparison of CACHD1-mediated increases in CaV3.1 current density and gating currents revealed an increase in channel open probability. In hippocampal neurons from male and female embryonic day 19 rats, CACHD1 overexpression increased CaV3-mediated action potential firing frequency and neuronal excitability. These data suggest that CACHD1 is structurally an α2δ-like protein that functionally modulates CaV3 voltage-gated calcium channel activity.SIGNIFICANCE STATEMENT This is the first study to characterize the Ca2+ channel and chemotaxis receptor domain containing 1 (CACHD1) protein. CACHD1 is widely expressed in the CNS, in particular in the thalamus, hippocampus, and cerebellum. CACHD1 distribution is similar to that of low voltage-activated (CaV3, T-type) calcium channels, in particular to CaV3.1, a protein that regulates neuronal excitability and is a potential therapeutic target in conditions such as epilepsy and pain. CACHD1 is structurally an α2δ-like protein that functionally increases CaV3 calcium current. CACHD1 increases the presence of CaV3.1 at the cell surface, forms complexes with CaV3.1 at the cell surface, and causes an increase in channel open probability. In hippocampal neurons, CACHD1 causes increases in neuronal firing. Thus, CACHD1 represents a novel protein that modulates CaV3 activity.

Liposomal quercetin potentiates maxi-K channel openings in smooth muscles and restores its activity after oxidative stress

The effects of quercetin-loaded liposomes (PCL-Q) and their constituents, that is, free quercetin (Q) and ‘empty’ phosphatidylcholine vesicles (PCL), on maxi-K channel activity were studied in single mouse ileal myocytes before and after H2O2-induced oxidative stress. Macroscopic Maxi-K channel currents were recorded using whole-cell patch clamp techniques, while single BKCa channel currents were recorded in the cell-attached configuration. Bath application of PCL-Q (100 μg/ml of lipid and 3 μg/ml of quercetin) increased single Maxi-K channel activity more than threefold, from 0.010 ± 0.003 to 0.034 ± 0.004 (n = 5; p < 0.05), whereas single-channel conductance increased non-significantly from 138 to 146 pS. In the presence of PCL-Q multiple simultaneous channel openings were observed, with up to eight active channels in the membrane patch. Surprisingly, ‘empty’ PCL (100 μg/ml) also produced some channel activation, although it was less potent compared to PCL-Q, that is, these increased NPo from 0.010 ± 0.003 to 0.019 ± 0.003 (n = 5; p < 0.05) and did not affect single-channel conductance (139 pS). Application of PCL-Q restored macroscopic Maxi-K currents suppressed by H2O2-induced oxidative stress in ileal smooth muscle cells. We conclude that PCL-Q can activate Maxi-K channels in ileal myocytes mainly by increasing channel open probability, as well as maintain Maxi-K-mediated whole-cell current under the conditions of oxidative stress. While fusion of the ‘pure’ liposomes with the plasma membrane may indirectly activate Maxi-K channels by altering channel’s phospholipids environment, the additional potentiating action of quercetin may be due to its better bioavailability.