Inside-out Configuration Research Articles

Activation of hTREK-1 by polyunsaturated fatty acids involves direct interaction

TREK-1 is a mechanosensitive channel activated by polyunsaturated fatty acids (PUFAs). Its activation is supposed to be linked to changes in membrane tension following PUFAs insertion. Here, we compared the effect of 11 fatty acids and ML402 on TREK-1 channel activation using the whole cell and the inside-out configurations of the patch-clamp technique. Firstly, TREK-1 activation by PUFAs is variable and related to the variable constitutive activity of TREK-1. We observed no correlation between TREK-1 activation and acyl chain length or number of double bonds suggesting that the bilayer-couple hypothesis cannot explain by itself the activation of TREK-1 by PUFAs. The membrane fluidity measurement is not modified by PUFAs at 10 µM. The spectral shift analysis in TREK-1-enriched microsomes indicates a KD,TREK1 at 44 µM of C22:6 n-3. PUFAs display the same activation and reversible kinetics than the direct activator ML402 and activate TREK-1 in both whole-cell and inside-out configurations of patch-clamp suggesting that the binding site of PUFAs is accessible from both sides of the membrane, as for ML402. Finally, we proposed a two steps mechanism: first, insertion into the membrane, with no fluidity or curvature modifications at 10 µM, and then interaction with TREK-1 channel to open it.

The Enzymatic Function of Kvβ2 Controls Vascular Potassium Channel Activity

Voltage-gated potassium (Kv) channels expressed in vascular smooth muscle control membrane potential and thereby regulate vascular tone and organ perfusion. Native Kv1 channels in the resistance vasculature consist of an alpha pore domain in complex with intracellular beta (Kvβ) proteins, which are active aldo-keto reductases (AKR6A). Recent work suggests that the AKR function of Kvβ2 is required for vascular Kv1 channel NAD(P)(H) redox sensing and O2 sensitivity of resistance arterial tone. Nonetheless, whether acute changes in Kvβ2 AKR function impact vascular Kv1 activity is unknown. We propose that Kv1 gating is sensitive to changes in the levels of endogenous substrates and post-translational modification of Kvβ2. Here, we tested the hypothesis that the availability of the glycolytic byproduct and purported Kvβ substrate, methylglyoxal, and protein kinase C-dependent phosphorylation differentially regulate vascular Kvβ2 function. Using the inside-out configuration of the patch clamp technique, we measured the open probability (nPo) of Kv channels present in patches excised from human coronary smooth muscle cells. Perfusion of methylglyoxal (5 μM) resulted in significantly increased Kv channel nPo (3.3x10−5 ± 9.5x10−6 to 3.2x10−4 ± 2.1x10−4; n=5) and this effect was enhanced (~8-fold) in the presence of 100 μM NADPH, suggesting that Kv sensitivity to methylglyoxal is cofactor-dependent. Based on this, we next tested whether Kvβ phosphorylation regulates AKR catalysis and influences vascular Kv1 redox sensitivity. Using in situ proximity ligation, we found that serine phosphorylation of Kvβ2 in smooth muscle was increased upon treatment with the PKC activator phorbol 12-myristate 13-acetate (PMA; 100 nM), indicating that smooth muscle Kvβ2 is a phosphorylation target under these conditions. In vitro phosphorylation of purified Kvβ2 with PKCα significantly slowed catalysis (11.3 ± 0.3 vs. 6.5 ± 0.2 μmoles/min/mg protein, control and PKCα-treated rat Kvβ2.1, respectively). Pretreatment of cells with 10 nM PMA before excising inside-out membrane patches eliminated the increase in Kv channel nP(o) evoked by methylglyoxal (±NADPH). Moreover, ex vivo treatment of isolated coronary arteries with PMA (10 nM) abolished vasodilation in response to elevated NADH:NAD+ via application of 2–5 mM external L-lactate. Together, these results suggest that vascular smooth muscle Kv1 channels are functionally regulated by endogenous substrates and phosphorylation of intracellular Kvβ proteins. We propose that targeting vascular Kvβ AKR function may represent a novel strategy to modify K+ channel redox sensitivity and thus strengthen the coupling between metabolic demand and organ perfusion. T32-ES011564 and R01HL163818. 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.

Unlocking cellular traffic jams: olive oil-mediated rescue of CNG mutant channels.

One of the reasons to suggest olive oil consumption for a healthy life is its potential to induce robust lipidomic remodeling through membrane modification by dietary lipids. This remodeling might, in turn, modulate essential lipid-protein interactions while maintaining accurate transmembrane protein/domain orientation. Oleic acid, the primary compound in olive oil, has been suggested as a modulator of ion channel function. In this study, we explored whether this lipid could rescue the trafficking of mutated transmembrane proteins. In our initial approach, we supplemented the cell culture medium of HEK-293 cells expressing cyclic nucleotide channels tagged using green fluorescent protein (CNG-GFP) with olive oil or oleic acid. In addition to wild-type channels, we also expressed R272Q and R278W mutant channels, two non-functional intracellularly retained channels related to retinopathies. We used fluorescence microscopy and patch-clamp in the inside-out configuration to assess changes in the cell localization and function of the tested channels. Our results demonstrated that olive oil and oleic acid facilitated the transport of cyclic nucleotide-gated R272Q mutant channels towards the plasma membrane, rendering them electrophysiologically functional. Thus, our findings reveal a novel property of olive oil as a membrane protein traffic inductor.

Basal NAD(H) redox state permits hydrogen peroxide-induced mesenteric artery dilatation.

Smooth muscle voltage-gated K+ (Kv) channels in resistance arteries control vascular tone and contribute to the coupling of blood flow with local metabolic activity. Members of the Kv1 family are expressed in vascular smooth muscle and are modulated upon physiological elevation of local metabolites, including the glycolytic end-product l-lactate and superoxide-derived hydrogen peroxide (H2 O2 ). Here, we show that l-lactate elicits vasodilatation of small-diameter mesenteric arteries in a mechanism that requires lactate dehydrogenase (LDH). Using the inside-out configuration of the patch clamp technique, we show that increases in NADH that reflect LDH-mediated conversion of l-lactate to pyruvate directly stimulate the activity of single Kv1 channels and significantly enhance the sensitivity of Kv1 activity to H2 O2 . Consistent with these findings, H2 O2 -evoked vasodilatation was significantly greater in the presence of 10mM l-lactate relative to lactate-free conditions, yet was abolished in the presence of 10mM pyruvate, which shifts the LDH reaction towards the generation of NAD+ . Moreover, the enhancement of H2 O2 -induced vasodilatation was abolished in arteries from double transgenic mice with selective overexpression of the intracellular Kvβ1.1subunit in smooth muscle cells. Together, our results indicate that the Kvβ complex of native vascular Kv1 channels serves as a nodal effector for multiple redox signals to precisely control channel activity and vascular tone in the face of dynamic tissue-derived metabolic cues. KEY POINTS: Vasodilatation of mesenteric arteries by elevated external l-lactate requires its conversion by lactate dehydrogenase. Application of either NADH or H2 O2 potentiates single Kv channel currents in excised membrane patches from mesenteric artery smooth muscle cells. The binding of NADH enhances the stimulatory effects of H2 O2 on single Kv channel activity. The vasodilatory response to H2 O2 is differentially modified upon elevation of external l-lactate or pyruvate. The presence of l-lactate enhances the vasodilatory response to H2 O2 via the Kvβ subunit complex in smooth muscle.

TRPM4: New actor in osteogenic differentiation of human valvular interstitial cells

Transient receptor potential melastatin 4 (TRPM4) protein, a non-selective cation channel activated by calcium, is involved in cardiac fibroblasts remodeling. Its expression was reported in human valvular interstitial cells (VIC) with similar properties than fibroblasts, and its protein expression is increased on calcified aortic valves from patients suffering of aortic stenosis. VIC osteogenic differentiation is a key element in valvular calcification, which is, at term, responsible of aortic stenosis. The purpose of this study is to evaluate the implication of TRPM4 in human VIC osteogenic differentiation. VIC are isolated by enzymatic digestion from human aortic valves obtained from patients undergoing surgical aortic valve replacement. They are maintained 14 days in procalcifying media (2.7 mM calcium/2.5 mM phosphate), with or without 9-phenanthrol, a pharmacological TRPM4 inhibitor. Channel expression and functionality were followed respectively by PCR, Western Blot and electrophysiological recordings in the inside-out configuration of the patch clamp technique. Osteogenic differentiation of VIC was evaluated by measuring hydroxyapatite crystals by Alizarin red staining and by monitoring the protein expression of the osteogenic markers, BMP2 and RUNX2. A typical TRPM4 current was detected on VIC: its current-voltage relationship was linear with a conductance of 20.3 pS; the channel exhibited similar permeability for Na+ and K+; it was more activated in positive voltages and with increasing concentrations of internal Ca2+; the current was dose-dependently inhibited by 9-phenanthrol. TRPM4 mRNA and protein expression on VIC was confirmed by PCR and Western Blot. When maintained in procalcifying media, VIC developed hydroxyapatite crystals. Application of 9-phenanthrol (3.10-6 M) reduced mineralization by 30% without affecting cell viability. Moreover, it significantly reduced BMP2 and RUNX2 expression. Thereby, TRPM4 participates in osteogenic differentiation of human VIC in culture. Next studies of our unit are to evaluate whether TRPM4 repression by shRNA confirms those results.

Voltage-sensor trapping in Hv1 channel (CiHv1).

Previous studies on monomeric N264R showed that the OFF-gating current is completely trapped and that, from a kinetic point of view, this charge displacement takes a long time to return. Currently, the molecular mechanism governing this entrapment of charge remains unknown. In order to understand the OFF-gating charge trapping mechanism, we performed patch-clamp in its inside-out configuration using Xenopus laevis oocytes as a heterologous expression system, combined with molecular dynamics and kinetic modeling. Our results shows that the ΔNΔC D160N mutant does not have a trapped charge at the OFF-gating charge; but, when the N264R mutation is added to this mutant, the OFF-gating charge trapping appears in a diminished state. This is possibly because the residue in position 160, which is also the selectivity filter of the channel, interacts with the arginines of the voltage sensor. In addition, the 2GBI blocker possesses a guanidinium group which also presents an arginine in its side chain. This inhibitor generates an effect similar to ΔNΔC N264R OFF-gating charge displacement. This suggests that arginine is required for charge trapping. By performing mutations at position 160 it was found that hydrophobicity near that position obstructs the movement of the voltage sensor exerting an effect similar to that of the aromatic rings of 2GBI. Furthermore, when ΔpH is increased and especially when acidity is increased on the intracellular side, the OFF-gating charge is also trapped. We performed molecular dynamics simulations on ΔNΔC N264R, and our results suggest that the positive charge of arginine “push-up” the arginines 258 and 261 and form a stable salt-bridge over time with D160. Finally, our results confirm that for the gating charge to be trapped it is necessary to change the electrostatic configuration on the intracellular side near position 264.

The bisphosphonate zoledronic acid is a TRPV1 channel inhibitor.

The heat and capsaicin sensor, the transient receptor potential vanilloid type 1 (TRPV1) channel, is critically involved in pain sensation. Using compounds that activate TRPV1, such as capsaicin (CAP), produces analgesic effects. Hence the channel is a promising target for the management of pain conditions. However, when TRPV1 channels are sensitized in neuropathic pain conditions, inhibitors could be a better option for pain management. We have obtained evidence supporting that the bisphosphonate compound zoledronic acid (ZOL) acts as an inhibitor of the TRPV1 channel. ZOL is typically used in the treatment of bone diseases reducing bone mass loss, deterioration in bone microarchitecture and fracture risk, having as a collateral effect a decrease in pain sensation. We have observed, expressing TRPV1 in Xenopus laevis oocytes followed by patch-clamp recordings in inside-out configuration, that ZOL at doses ranging from 1 nM up to 30 μM reduces channel conductance and inhibits the activity of the channel deeply. Only high doses of CAP (≥ 10 μM) allow for recover of TRPV1 currents after ZOL treatment. Molecular modeling followed by docking analysis suggests that ZOL could potentially bind to TRPV1 at the vanilloid pocket and the PI(4,5)P2 binding site. Furthermore, like capsazepine, ZOL (100 nM) is able to eliminate the depression observed by applying CAP (1 μM) on synaptic transmission in the dentate gyrus. In addition, when ZOL is use during the induction of synaptic plasticity mediated by TRPV1, it eliminates plasticity. The compound did not affect the endocannabinoid production machinery suggesting that the ZOL is acting specifically on TRPV1 to hinder plasticity. Together, our evidence strongly supports that ZOL can inhibit TRPV1 activity.

Cysteine mutagenesis reveals novel structure-function features within the predicted transmembrane domain of chloride intracellular ion channel 1 (CLIC1).

Chloride intracellular ion channels (CLICs) are a unique class of metamorphic proteins that exists as soluble proteins and can auto-insert into the membrane to form an ion channel. The soluble structure of all the CLICs is well established but the structural information on the membrane form is lacking. CLICs are known to have a single putative transmembrane (PTM) region and proteins oligomerize to a tetramer to form a functional channel. CLIC1 is a sensor of oxidative stress, overexpressed in different types of cancer, and involved in diabetes, cell viability, and angiogenesis. The PTM region of CLIC1 encompasses residue 24-46, where cysteine at position 24 is a critical redox-sensitive residue. However, the precise arrangement of the residues in the pore region is not known. Hence, to identify the key residue involved in the gating mechanism and ion selectivity, the putative residues lining the pore region were mutated using site-directed mutagenesis. We incorporated the substituted cysteine accessibility method (SCAM) and recorded the channel activity of the mutants in the whole-cell and inside-out configurations. The ion selectivity was measured by perfusing with different anions (chloride, bromide, fluoride, and phosphate). The open channel probability (Po) is significantly higher in R29C mutant (0.88) compared to WT (0.21), suggesting R29C residue is involved in CLIC1 gating. Similarly, the CLIC1 is highly selective for chloride ions as compared to other anions, and the selective filer is located between V33C and K37C residues. We probed substituted cysteine residues with cadmium and identified that the T40C showed a reduction in Po and amplitude. Our results have delineated the pore lining of the CLIC1 and identified the residues involved in its gating and regulating the chloride selectivity.

Bidirectional sensitivity of CALHM1 channel to protons from both sides of plasma membrane.

Calcium homeostasis modulator 1 (CALHM1), a newly discovered voltage-dependent nonselective ion channel, has drawn attention for its role in neuronal activity and taste sensation. Its sluggish voltage-dependent activation is facilitated by lowering extracellular Ca2+ concentration ([Ca2+]e). Here, we investigated the effects of extracellular and intracellular pH (pHe and pHi) on human CALHM1. When normalized to the amplitude of the CALHM1 current (ICALHM1) under whole cell patch clamp at symmetrical pH 7.4, ICALHM1 decreased at acidic pHe or pHi, whereas it sharply increased at alkaline pHe or pHi. The effects of pH were preserved in the inside-out configuration. The voltage dependence of ICALHM1 showed leftward and rightward shifts at alkaline and acidic pHe and pHi, respectively. Site-directed mutagenesis of the water-accessible charged residues of the pore and nearby domains revealed that E17, K229, E233, D257, and E259 are nonadditively responsible for facilitation at alkaline pHi. Identification of the pHe-sensing residue was not possible because mutation of putative residues impaired membrane expression, resulting in undetectable ICALHM1. Alkaline pHe-dependent facilitation appeared gradually with depolarization, suggesting that the sensitivity to pHe might be due to H+ diffusion through the open-state CALHM1. At pHe 6.2, decreased [Ca2+]e could not recover the inhibited ICALHM1 but further augmented the increased ICALHM1 at pHe 8.6, suggesting that unidentified common residues might contribute to the [Ca2+]e and acidic pHe. This study is the first, to our knowledge, to demonstrate the remarkable pH sensitivity of CALHM1, which might contribute to the pH-dependent modulation of neuronal excitability or taste sensation.

Reduced Low-Pressure Membrane Fouling by Inline Coagulation Pretreatment for a Colored River Water.

Drinking water treatment (DWT) using low-pressure membranes (LPM) has become increasingly popular due to their many reported advantages compared to conventional technologies. Productivity decline due to fouling has prevented LPMs from becoming the technology of choice in DWT, however, coagulation pretreatment either with or without particle separation mitigates fouling phenomena. The effectiveness of coagulation/flocculation/sedimentation (CF-S), coagulation/flocculation/dissolved air flotation (CF-DAF), and inline coagulation (CF-IN) as technologies for pretreatment of feed water has rarely been investigated using the same water source. In this study, CF-S, CF-DAF, and CF-IN are directly compared as pretreatment of a tubular multi-channeled ultrafiltration (UF) membrane using the same highly colored river water. Three-day long filtration tests were performed using an automated bench-scale filtration apparatus with an inside-out configuration. Although CF-DAF had the greatest removal of dissolved organic matter (DOM) and hydrophobic organics, CF-S pretreatment resulted in a similar level of total fouling. Compared to CF-DAF and CF-S, CF-IN pretreatment resulted in lower fouling. The hydraulic and chemical reversibility of CF-IN fouling was seen to be strongly influenced by the feed water zeta potential, suggesting the importance of floc electrostatic and morphological characteristics on inline coagulation performance.

Novel Dimeric hHv1 Model and Structural Bioinformatic Analysis Reveal an ATP-Binding Site Resulting in a Channel Activating Effect.

The human voltage-gated proton channel (hHv1) is a highly selective ion channel codified by the HVCN1 gene. It plays a fundamental role in several physiological processes such as innate and adaptive immunity, insulin secretion, and sperm capacitation. Moreover, in humans, a higher hHv1 expression/function has been reported in several types of cancer cells. Here we report a multitemplate homology model of the hHv1 channel, built and refined as a dimer in Rosetta. The model was then subjected to extensive Gaussian accelerated molecular dynamics (GaMD) for enhanced conformational sampling, and representative snapshots were extracted by clustering analysis. Combining different structure- and sequence-based methodologies, we predicted a putative ATP-binding site located on the intracellular portion of the channel. Furthermore, GaMD simulations of the ATP-bound dimeric hHv1 model showed that ATP interacts with a cluster of positively charged residues from the cytoplasmic N and C terminal segments. According to the in silico predictions, we found that 3 mM intracellular ATP significantly increases the H+ current mediated by the hHv1 channel expressed in HEK293 cells and measured by the patch-clamp technique in an inside-out configuration (2.86 ± 0.63 fold over control at +40 mV). When ATP was added on the extracellular side, it was not able to activate the channel supporting the idea that the ATP-binding site resides in the intracellular face of the hHV1 channel. In a physiological and pathophysiological context, this ATP-mediated modulation could integrate the cell metabolic state with the H+ efflux, especially in cells where hHv1 channels are relevant for pH regulation, such as pancreatic β-cells, immune cells, and cancer cells.

Evidence for Dual Activation of IK(M) and IK(Ca) Caused by QO-58 (5-(2,6-Dichloro-5-fluoropyridin-3-yl)-3-phenyl-2-(trifluoromethyl)-1H-pyrazolol[1,5-a]pyrimidin-7-one).

QO-58 (5-(2,6-dichloro-5-fluoropyridin-3-yl)-3-phenyl-2-(trifluoromethyl)-1H-pyrazolol[1,5-a]pyrimidin-7-one) has been regarded to be an activator of KV7 channels with analgesic properties. However, whether and how the presence of this compound can result in any modifications of other types of membrane ion channels in native cells are not thoroughly investigated. In this study, we investigated its perturbations on M-type K+ current (IK(M)), Ca2+-activated K+ current (IK(Ca)), large-conductance Ca2+-activated K+ (BKCa) channels, and erg-mediated K+ current (IK(erg)) identified from pituitary tumor (GH3) cells. Addition of QO-58 can increase the amplitude of IK(M) and IK(Ca) in a concentration-dependent fashion, with effective EC50 of 3.1 and 4.2 μM, respectively. This compound could shift the activation curve of IK(M) toward a leftward direction with being void of changes in the gating charge. The strength in voltage-dependent hysteresis (Vhys) of IK(M) evoked by upright triangular ramp pulse (Vramp) was enhanced by adding QO-58. The probabilities of M-type K+ (KM) channels that will be open increased upon the exposure to QO-58, although no modification in single-channel conductance was seen. Furthermore, GH3-cell exposure to QO-58 effectively increased the amplitude of IK(Ca) as well as enhanced the activity of BKCa channels. Under inside-out configuration, QO-58, applied at the cytosolic leaflet of the channel, activated BKCa-channel activity, and its increase could be attenuated by further addition of verruculogen, but not by linopirdine (10 μM). The application of QO-58 could lead to a leftward shift in the activation curve of BKCa channels with neither change in the gating charge nor in single-channel conductance. Moreover, cell exposure of QO-58 (10 μM) resulted in a minor suppression of IK(erg) amplitude in response to membrane hyperpolarization. The docking results also revealed that there are possible interactions of the QO-58 molecule with the KCNQ or KCa1.1 channel. Overall, dual activation of IK(M) and IK(Ca) caused by the presence of QO-58 eventually may have high impacts on the functional activity (e.g., anti-nociceptive effect) residing in electrically excitable cells. Care must be exercised when interpreting data generated with QO-58 as it is not entirely KCNQ/KV7 selective.

Implication of TRPM4 ionic channel in osteogenic differentiation of human valvular interstitial cells

The Ca2 + -activated non selective cationic channel, TRPM4, is involved in cardiac fibroblasts remodelling. In valvular interstitial cells (VIC), with similar properties than fibroblasts, its expression at the mRNA level was reported to be higher in conditions of valvular calcification leading to aortic stenosis. Endothelial dysfunction, followed by inflammation and fibrosis, promote the differentiation of VIC into osteoblastic phenotype cells which is, at term, responsible of valve ossification. The purpose of this study is to search for a potential implication of TRPM4 into osteogenic differentiation of human VIC. Human aortic valves are obtained from surgery for valve replacement. VIC are isolated by enzymatic digestion then maintained in culture. VIC are incubated during 14 days in a pro-calcific mixture in the presence or not of 9-phenanthrol, a pharmacological inhibitor of TRPM4. Expression and functionality of this channel were followed respectively by Western Blot and electrophysiological recordings in the inside-out configuration of the patch clamp technique. The implication of TRPM4 in VIC mineralization process was observed by measuring calcific crystals using alizarin red staining. A typical TRPM4 single channel current was detected in VIC in culture with a single channel conductance of 20 pS, a monovalent cationic permeability as well as voltage and calcium sensitivity. Protein expression of TRPM4 was confirmed by Western Blot. Pharmacological inhibition of the channel by 9-phenanthrol (3.10–6 M), during culture, reduced the mineralization by 30% without affecting cell viability. Thereby, TRPM4 would participate in osteogenic differentiation of human VIC in culture. Further studies are needed to understand which mecanisms link TRPM4 and ostegenic differentiation.

Bi‐directional pH dependency of CALHM1 ion channel

Calcium homeostasis modulator (CALHM) membrane proteins are nonselective ion channels drawing attention regarding their roles in modulating neuronal activity and taste sensation. CALHM1‐expressing cells show voltage‐gated slowly activating ionic currents. The voltage‐dependence of CALHM1 is negatively affected by extracellular Ca2+ ([Ca2+]e), while facilitated by temperature. Here we investigated the effect of extracellular and intracellular pH (pHe and pHi) on the electrophysiological properties of CALHM1 overexpressed in CHO cells. When normalized to the control at pHe 7.4, the amplitudes of CALHM1 current (ICALHM1) were suppressed to 20 % at pHe 6.2, while increased to 500 % at pHe 8.6. In the whole‐cell configuration of patch clamp, changing pHi from 7.4 to 6.2 or 8.6 showed the acid‐induced inhibition and alkali‐induced activation of ICALHM1. The voltage‐dependence of CALHM1 was shifted to the negative membrane potential at the alkaline pHe and pHi, while positive membrane potential at the acidic pHe and pHi. The pHi effect was also confirmed in the inside‐out configuration of patch clamp. The open probability (Po) of multiple CALHM1 channel decreased at pH 6.2, while increased at pH 8.6 compared with pH 7.4. To get a clue to identify the pH sensing amino acid residues, we utilized homology model performed with the SWISS‐MODEL workspace, based on the cryo‐EM structure of human CALHM2. Then we conducted site‐directed mutagenesis of the water‐accessible charged amino acids of CALHM1. Among 23 candidates, mutations at E17, K229, E233, D257 and E259 in the intracellular space reduced the alkali‐induced facilitation of ICALHM1, while not altered the acid‐induced inhibition. According to the molecular structure model of killifish CALHM1 with octameric structure, the five residues located at N or C termini gathered closely in the intracellular space. Considering the large pore diameter of CALHM1 (~14 Å), we cautiously propose that the sensitivity to alkaline pHe could be because the protons transfer between hydronium ions in the pore. The study firstly demonstrated the remarkable pH‐sensitivity of CALHM1 and its pH‐sensing residues, which might be responsible for the modulation of neuronal excitability during dynamic changes of the regional pH in brain or sensory organs.

Activity of TREK-2-like Channels in the Pyramidal Neurons of Rat Medial Prefrontal Cortex Depends on Cytoplasmic Calcium.

Simple SummaryThe pyramidal neurons of rat prefrontal cortex express potassium channels identified as a non-canonical splice variant of the TREK-2 channel. The main function of TREK channels is to regulate the resting membrane potential. We showed that cytoplasmic Ca2+ upregulates the activity of TREK-2-like channels. Previous studies have indicated that the activation of TREK-2 channels is mediated by PI(4,5)P2, a polyanionic lipid in the inner leaflet of the plasma membrane. While TREK channels are believed to not be regulated by calcium, our work shows otherwise. We propose a model in which calcium ions enable the formation of PI(4,5)P2 nanoclusters, which stabilize active conformation of the channel.TREK-2-like channels in the pyramidal neurons of rat prefrontal cortex are characterized by a wide range of spontaneous activity—from very low to very high—independent of the membrane potential and the stimuli that are known to activate TREK-2 channels, such as temperature or membrane stretching. The aim of this study was to discover what factors are involved in high levels of TREK-2-like channel activity in these cells. Our research focused on the PI(4,5)P2-dependent mechanism of channel activity. Single-channel patch clamp recordings were performed on freshly dissociated pyramidal neurons of rat prefrontal cortexes in both the cell-attached and inside-out configurations. To evaluate the role of endogenous stimulants, the activity of the channels was recorded in the presence of a PI(4,5)P2 analogue (PI(4,5)P2DiC8) and Ca2+. Our research revealed that calcium ions are an important factor affecting TREK-2-like channel activity and kinetics. The observation that calcium participates in the activation of TREK-2-like channels is a new finding. We showed that PI(4,5)P2-dependent TREK-2 activity occurs when the conditions for PI(4,5)P2/Ca2+ nanocluster formation are met. We present a possible model explaining the mechanism of calcium action.

Anion and Cation Permeability of the Mouse TMEM16F Calcium-Activated Channel.

TMEM16F is involved in several physiological processes, such as blood coagulation, bone development and virus infections. This protein acts both as a Ca2+-dependent phospholipid scramblase and a Ca2+-activated ion channel but several studies have reported conflicting results about the ion selectivity of the TMEM16F-mediated current. Here, we have performed a detailed side-by-side comparison of the ion selectivity of TMEM16F using the whole-cell and inside-out excised patch configurations to directly compare the results. In inside-out configuration, Ca2+-dependent activation was fast and the TMEM16F-mediated current was activated in a few milliseconds, while in whole-cell recordings full activation required several minutes. We determined the relative permeability between Na+ and Cl¯ (PNa/PCl) using the dilution method in both configurations. The TMEM16F-mediated current was highly nonselective, but there were differences depending on the configuration of the recordings. In whole-cell recordings, PNa/PCl was approximately 0.5, indicating a slight preference for Cl¯ permeation. In contrast, in inside-out experiments the TMEM16F channel showed a higher permeability for Na+ with PNa/PCl reaching 3.7. Our results demonstrate that the time dependence of Ca2+ activation and the ion selectivity of TMEM16F depend on the recording configuration.

Initial testing of an inside-out type palladium membrane reactor for recovery of hydrogen from hydrocarbons or water

The ITER Tokamak Exhaust Processing (TEP) system relies on palladium membrane reactors (PMRs) for tritium recovery. The PMR consists of a palladium/silver membrane permeator filled with a catalyst, that can be used to recover hydrogen species (most importantly tritium) from methane and water impurities present in fusion reactor exhaust emissions. Two superimposed phenomena simultaneously occur inside the PMR: a chemical reaction onto the catalyst liberating hydrogen contained in molecules and permeation of this molecular hydrogen through the membrane. Applying a vacuum on the permeate side allows recovery of pure hydrogen from the molecules (including tritium). The inside-out configuration of the PMR improves vacuum conditions at the permeate side, thus enhancing decontamination factor and tritium recovery performances.This paper presents the design, construction and initial commissioning of a newly erected test rig at UKAEA to perform experiments with protium on an inside-out PMR prototype in support of the ITER TEP. The efficiency of the PMR is assessed by both measuring flowrate through the permeate gas stream and analysing the composition of the retentate gas stream using gas chromatography. Permeation rate is assessed for protium for flowrates between 10 and 100 ml/min. The methane/water ratio, crucial for methane steam reforming tests, is assessed to optimise decontamination of hydrogen and minimise coking of the PMR. The hydrogen recovery fraction and decontamination factor are assessed for methane and water from flow rates of 10 to 60 ml/min.

Experimental Assessment of a Sliding-Blade Inside-Out Ceramic Turbine

Abstract In order to reach 40% efficiency, sub-MW turbines must operate in a recuperated gas Brayton cycle at a turbine inlet temperature (TIT) above 1300 °C. Current sub-MW turbines have material-related operating temperature limits. Still to this day, there is no cost-effective rotor design which operates at such high temperatures. This paper introduces a novel, sliding-blade, inside-out ceramic turbine (ICT) wheel configuration, which could enable high-efficiency sub-MW recuperated engines to be achieved with cheap monolithic ceramic blades. The inside-out configuration uses a rotating structural hoop, or shroud, to convert centrifugal forces into compressive blade loading. The sliding-blade architecture uses a hub with angled planes on which ceramic blades slide up and down, allowing to match the radial expansion of the structural shroud. This configuration generates low stress values in both ceramic and metallic components and can achieve high tip speeds. A prototype is designed and its reliability is calculated using cares software. The result is a design which has a single blade probability of failure (Pf) of 0.1% for 1000 h of steady operation. Analyses also demonstrate that reliability is greatly dependent on friction at ceramic-to-metal interfaces. Low friction could lead to acceptable reliability levels for engine applications. The prototype was successfully tested in a laboratory turbine environment at a tip speed of 350 m/s and a TIT of 1100 °C without any damage. These achievements demonstrate the robustness of the sliding-blade ICT configuration. Further research and development will focus on increasing tip speed and TIT to higher values.

Patch-clamp technique for studying ion channels in activated platelets

In this study, we have revisited the existing and suggested new approaches to the use of patch-clamp methodology for measuring the activity of single ion channels and membrane potential of human platelets in the cell-attached configuration. We recorded single-channel events of platelets in the cell-attached configuration after activation with potent agonists: thrombin and ionomycin. The feasibility of platelet membrane potential measurement in cell-attached mode was investigated both experimentally and with the help of simple electrical circuits, revealing that the single-channel events can alter the recorded potential by invoking oscillations. Here, the simple approach to obtain inside-out configuration was described and calcium-dependent single-channel ion currents were recorded. Taken together, this study introduces new approaches for further investigations of the role of ion channels and membrane potential in platelet physiological and pathophysiological response.

Regulation of the Activity of Mitochondrial Large-Conductance Calcium-Activated Potassium Channel Through a New External Heme-Binding Site

Mitochondrial large-conductance calcium-activated potassium (mitoBKCa) channel located in the inner mitochondrial membrane is formed by the DEC splice variant of the KCNMA1 gene. The activity of mitoBKCa is regulated by many modulators, including activators such as calcium ions and inhibitors such as heme and its oxidized form hemin. Heme/hemin binds to the heme-binding motif (–CXXCH–) located between two RCK (regulating conductance of K+) domains present in the mitochondrial matrix (internal side of the channel). Till now, there was no evidence about the existence of another heme-binding site in the mitoBKCa. However, using patch-clamp on mitoplast isolated from astrocytoma U-87MG cells and HEK293 cell line stably expressing DEC isoform of BKCa, we accidentally found that external heme also inhibits mitoBKCa suggesting the presence of yet another unknown heme-binding site. To further confirm the initial observation we used two approaches. First, we carried out patch-clamp recordings on the inner mitochondrial membrane in the outside-out configuration, and second, the pipette perfusion system was used in the inside-out configuration of the membrane patches. Both approaches allow for the application of channel modulators to the intermembrane-space (external) side of the mitoBKCa. We confirmed that hemin applied to the intermembrane space side of the channel inhibits the activity of mitoBKCa. We investigated the amino acid composition of different isoforms of BKCa and identified cysteines and histidine located at the external side of the channel, which could constitute a novel heme-binding site. We obtained HEK293 cell lines stably expressing DEC mutants of the above-mentioned cysteines and histidine. Patch-clamp experiments will be carried out to find out whether introduced mutations affect channel inhibition by external heme. This work was supported by the Polish National Science Center, grant no. 2018/31/N/NZ1/00928.