Open Dwell Time Research Articles

Neuroprotective amyloid β N-terminal peptides differentially alter human α7- and α7β2-nicotinic acetylcholine (nACh) receptor single-channel properties.

Oligomeric amyloid β 1-42 (oAβ1-42) exhibits agonist-like action at human α7- and α7β2-containing nicotinic receptors. The N-terminal amyloid β1-15 fragment (N-Aβ fragment) modulates presynaptic calcium and enhances hippocampal-based synaptic plasticity via α7-containing nicotinic receptors. Further, the N-Aβ fragment and its core sequence, the N-amyloid-beta core hexapeptide (N-Aβcore), protect against oAβ1-42-associated synapto- and neurotoxicity. Here, we investigated how oAβ1-42, the N-Aβ fragment, and the N-Aβcore regulate the single-channel properties of α7- and α7β2-nicotinic receptors. Single-channel recordings measured the impact of acetylcholine, oAβ1-42, the N-Aβ fragment, and the N-Aβcore on the unitary properties of human α7- and α7β2-containing nicotinic receptors expressed in nicotinic-null SH-EP1 cells. Molecular dynamics simulations identified potential sites of interaction between the N-Aβ fragment and orthosteric α7+/α7- and α7+/β2- nicotinic receptor binding interfaces. The N-Aβ fragment and N-Aβcore induced α7- and α7β2-nicotinic receptor single-channel openings. Relative to acetylcholine, oAβ1-42 preferentially enhanced α7β2-nicotinic receptor single-channel open probability and open-dwell times. Co-application with the N-Aβcore neutralized these effects. Further, administration of the N-Aβ fragment alone, or in combination with acetylcholine or oAβ1-42, selectively enhanced α7-nicotinic receptor open probability and open-dwell times (compared to acetylcholine or oAβ1-42). Amyloid-beta peptides demonstrate functional diversity in regulating α7- and α7β2-nicotinic receptor function, with implications for a wide range of nicotinic receptor-mediated functions in Alzheimer's disease. The effects of these peptides on α7- and/or α7β2-nicotinic receptors revealed complex interactions with these subtypes, providing novel insights into the neuroprotective actions of amyloid β-derived fragments against the toxic effects of oAβ1-42.

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

The large-conductance Ca2+-activated K+ channel (BKCa channel) plays critical roles in various tissues. In the smooth muscle of the urinary bladder, BKCa channel is highly expressed and regulates the spontaneous action potentials that control muscle contraction. Thus, the chemical activators for BKCa channel have their therapeutic potential against overactive bladder syndrome. Preveiously, we conducted a screening of a chemical library comprising 9,938 compounds in search of novel activators of the BKCa channel and performed structure-activity relationship study through hit compounds by using a cell-based fluorescence assay. Among the optimized compounds, DBTB-1 (N-[4-(2,4-dimethyl-3-oxo-3,4-dihydro-2H-1,4-benzoxazin-6-yl)-1,3-thiazol-2-yl]benzamide) displayed a robust activation of macroscopic BKCa channel currents in Xenopus oocytes. In contrast to other BKCa channel activators, DBTB-1 mainly influenced deactivation kinetics of the channel without affecting channel activation significantly. At the single-channel level, DBTB-1 increased the open-state probability compared to vehicle without affecting its single-channel conductance, indicating the delayed closing of the channel in the presence of the drug. Furthermore, analysis of the open dwell-time showed an additional population of long openings induced by DBTB-1. Thus, these unique characteristics of DBTB-1 can be useful in designing a new class of BKCa channel activators and might be utilized in developing therapuetics targeting specific aspect of the channel function. This research was supported by Korea Drug Development Fund funded by Ministry of Science and ICT, Ministry of Trade, Industry, and Energy, and Ministry of Health and Welfare (RS-2023-00258812). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Neuroprotective effects of nuciferine on high‐fat diet‐induced cognitive dysfunction in obese mice: Role of insulin resistance, neuroinflammation, and oxidative stress

AbstractHigh‐fat diets (HFDs) trigger oxidative stress, insulin resistance (IR), and inflammatory responses in the body, leading to cognitive dysfunction. This study aimed to elucidate the effects of nuciferine on HFD‐induced IR and cognitive impairment in the central nervous system of obese mice and further identify the possible underlying mechanisms. In behavioral experiments, nuciferine administration increased the total distance moved, the number of times mice entered the open arm, the duration of open dwell time, and the number of alternations while shortening the latency to escape. Nuciferine suppressed HFD‐induced synaptic structure damage, as evidenced by the increased expressions of neurotrophic factor BDNF and synaptic function proteins SYN, SNAP‐25, and PSD‐95. In addition, nuciferine effectively ameliorated HFD‐induced IR by IRS/PI3K/AKT pathway, alleviated HFD‐induced neurological damage through AMPK/SIRT1 pathway, and improved cognitive impairment via the Aβ‐tau‐neuroinflammation axis. Therefore, nuciferine could be a new food‐derived neuroprotective agent to counteract HFD‐induced damage.

Temperature modulates PVN pre-sympathetic neurones via transient receptor potential ion channels.

The paraventricular nucleus (PVN) of the hypothalamus plays a vital role in maintaining homeostasis and modulates cardiovascular function via autonomic pre-sympathetic neurones. We have previously shown that coupling between transient receptor potential cation channel subfamily V Member 4 (Trpv4) and small-conductance calcium-activated potassium channels (SK) in the PVN facilitate osmosensing, but since TRP channels are also thermosensitive, in this report we investigated the temperature sensitivity of these neurones. Methods: TRP channel mRNA was quantified from mouse PVN with RT-PCR and thermosensitivity of Trpv4-like PVN neuronal ion channels characterised with cell-attached patch-clamp electrophysiology. Following recovery of temperature-sensitive single-channel kinetic schema, we constructed a predictive stochastic mathematical model of these neurones and validated this with electrophysiological recordings of action current frequency. Results: 7 thermosensitive TRP channel genes were found in PVN punches. Trpv4 was the most abundant of these and was identified at the single channel level on PVN neurones. We investigated the thermosensitivity of these Trpv4-like channels; open probability (Po) markedly decreased when temperature was decreased, mediated by a decrease in mean open dwell times. Our neuronal model predicted that PVN spontaneous action current frequency (ACf) would increase as temperature is decreased and in our electrophysiological experiments, we found that ACf from PVN neurones was significantly higher at lower temperatures. The broad-spectrum channel blocker gadolinium (100µM), was used to block the warm-activated, Ca2+-permeable Trpv4 channels. In the presence of gadolinium (100µM), the temperature effect was largely retained. Using econazole (10µM), a blocker of Trpm2, we found there were significant increases in overall ACf and the temperature effect was inhibited. Conclusion: Trpv4, the abundantly transcribed thermosensitive TRP channel gene in the PVN appears to contribute to intrinsic thermosensitive properties of PVN neurones. At physiological temperatures (37°C), we observed relatively low ACf primarily due to the activity of Trpm2 channels, whereas at room temperature, where most of the previous characterisation of PVN neuronal activity has been performed, ACf is much higher, and appears to be predominately due to reduced Trpv4 activity. This work gives insight into the fundamental mechanisms by which the body decodes temperature signals and maintains homeostasis.

Cellular context determines primary characteristics of human TRPC5 as a cold-activated channel.

The human transient receptor potential canonical 5 (TRPC5) is a calcium-permeable, nonselective cation channel expressed in the central and peripheral nervous system and also in other tissues such as the kidney, synovium, and odontoblasts. TRPC5 has been recently confirmed to play a key role in spontaneous, inflammatory mechanical, and cold pain. Although TRPC5 activation is known to be cold sensitive, it is unclear whether this property is intrinsic to the channel protein and whether or to what extent it may be determined by the cellular environment. In this study, we explored the cold sensitivity of human TRPC5 at the single-channel level using transiently transfected HEK293T cells. Upon decreasing the temperature, the channel demonstrated prolonged mean open dwell times and a robust increase in the open probability (Po ), whereas the amplitude of unitary currents decreased ~1.5-fold per 10°C of temperature difference. In the absence of any agonists, the temperature dependence of Po was sigmoidal, with a steep slope within the temperature range of 16°C-11°C, and exhibited saturation below 8-5°C. Thermodynamic analysis revealed significant changes in enthalpy and entropy, suggesting that substantial conformational changes accompany cold-induced gating. The mutant channel T970A, in which the regulation downstream of G-protein coupled receptor signaling was abrogated, exhibited higher basal activity at room temperature and a less steep temperature response profile, with an apparent threshold below 22°C. An even more pronounced decrease in the activation threshold was observed in a mutant that disrupted the electrostatic interaction of TRPC5 with the endoplasmic reticulum calcium sensor stromal interaction molecule 1. Thus, TRPC5 exhibits features of an intrinsically cold-gated channel; its sensitivity to cold tightly depends on the phosphorylation status of the protein and intracellular calcium homeostasis.

Large-Conductance Calcium-Activated Potassium Channel Opener, NS1619, Protects Against Mesenteric Artery Remodeling Induced by Agonistic Autoantibodies Against the Angiotensin II Type 1 Receptor.

BackgroundAgonistic autoantibodies against the angiotensin II type 1 receptor (AT1‐AAs) extensively exist in patients with hypertensive diseases and have been demonstrated to play crucial roles in the pathophysiological process of vascular remodeling. However, the treatment options are limited. The large‐conductance calcium‐activated potassium (BK) channel is a critical regulator and potential therapeutic target of vascular tone and architecture. We have previously observed that AT1‐AAs have an inhibitory effect on BK channels. However, whether BK channel dysfunction is involved in AT1‐AAs‐induced vascular remodeling and the therapeutic effect of BK channel opener is unclear.Methods and ResultsIn our study, mesenteric arteries from AT1‐AAs‐positive rats exhibited increased wall thickness, narrowing of the arteriolar lumen, and increased collagen accumulation. Patch clamp test results showed that the voltage sensitivity of BK channel declined in mesenteric arteriolar smooth muscle cells from AT1‐AAs‐positive rats. Experiments with freshly isolated mesenteric arteriolar smooth muscle cells showed that AT1‐AAs reduced the opening probability, open levels, open dwell time, and calcium sensitivity of BK channel. Experiments with HEK293T cells transfected with GFP‐ZERO‐BK α‐subunit plasmids suggested a BK channel α‐subunit‐dependent mechanism. BK channel α‐subunit deficient, namely KCNMA1 −/− rats showed a phenotype of mesenteric artery remodeling. The administration of NS1619, a specific BK channel opener targeting the α‐subunit, reversed the phenotypic transition and migration induced by AT1‐AAs in cultured mesenteric arteriolar smooth muscle cells. Finally, perfusion of NS1619 significantly relieved the pathological effects induced by AT1‐AAs in vivo.ConclusionsIn summary, we provide compelling evidence that BK channel α‐subunit dysfunction mediates AT1‐AAs‐induced mesenteric artery remodeling. Preservation of BK channel activity may serve as a potential strategy for the treatment of AT1‐AAs‐induced maladaptive resistance artery remodeling.

Acupuncture ameliorates neurological function in rats with cerebral ischemia-reperfusion by regulating the opening of large-conductance Ca2+ -activated potassium channels.

Acupuncture has a good effect on improving neurological function after cerebral ischemia‐reperfusion, but there are few studies on the neuroprotective effect of acupuncture from the perspective of ion channel cellular electrophysiology. Studies have shown that the over activation of large‐conductance Ca2+‐activated potassium channel （BKCa） after cerebral ischemia‐reperfusion can reduce the excitability of neurons and induce apoptosis. This study intends to establish middle cerebral artery occlusion/reperfusion (MCAO/R) model, with acupuncture at GV26 as the intervention measure, using patch‐clamp technique to record the electrophysiological changes of BKCa channel. The results showed that the neurological function score of MCAO/R rats was significantly decreased, and the conductance, open dwell time and open probability of BKCa channel in hippocampal CA1 neurons of MCAO/R rats were significantly increased. Acupuncture at GV26 could significantly improve the neurological function scores of MCAO/R rats, and reduce the conductance, open dwell time, and open probability of BKCa channel. The effect of acupuncture at GV26 was significantly better than acupuncture at non‐acupuncture point. The neuroprotective effect of acupuncture at GV26 after cerebral ischemia‐reperfusion may be related to regulating the electrophysiological characteristics of BKCa channel opening.

Cholesterol stabilizes recombinant exocytic fusion pores by altering membrane bending rigidity

SNARE-mediated membrane fusion proceeds via the formation of a fusion pore. This intermediate structure is highly dynamic and can flicker between open and closed states. In cells, cholesterol has been reported to affect SNARE-mediated exocytosis and fusion pore dynamics. Here, we address the question of whether cholesterol directly affects the flickering rate of reconstituted fusion pores in vitro. These experiments were enabled by the recent development of a nanodisc⋅black lipid membrane recording system that monitors dynamic transitions between the open and closed states of nascent recombinant pores with submillisecond time resolution. The fusion pores formed between nanodiscs that bore the vesicular SNARE synaptobrevin 2 and black lipid membranes that harbored the target membrane SNAREs syntaxin 1A and SNAP-25B were markedly affected by cholesterol. These effects include strong reductions in flickering out of the open state, resulting in a significant increase in the open dwell-time. We attributed these effects to the known role of cholesterol in altering the elastic properties of lipid bilayers because manipulation of phospholipids to increase membrane stiffness mirrored the effects of cholesterol. In contrast to the observed effects on pore kinetics, cholesterol had no effect on the current that passed through individual pores and, hence, did not affect pore size. In conclusion, our results show that cholesterol dramatically stabilizes fusion pores in the open state by increasing membrane bending rigidity.

Electrophysiological characterization of the activating action of a novel liposomal nitric oxide carrier on Maxi-K channels in pulmonary artery smooth muscle cells

The aim of this study was to establish the mechanisms of action of a novel liposomal nitric oxide (NO) carrier on large-conductance Ca2+-activated channels (BKCa or Maxi-K) expressed in vascular smooth muscle cells (VSMCs) isolated from the rat main pulmonary artery (MPA). Experimental design comprised of both whole-cell and cell-attached single-channel recordings using the patch-clamp techniques. The liposomal form of NO, Lip(NO), increased whole-cell outward K+ currents in a dose dependent manner while shifting the activation curve negatively by about 50 mV with respect to unstimulated cells with the EC50 value of 0.55 ± 0.17 µM. At the single channel level, Lip(NO) increased the probability of the open state (Po) of Maxi-K channels from 0.0020 ± 0.0008 to 0.74 ± 0.02 with half-maximal activation occurring at 4.91 ± 0.01 μM, while sub-maximal activation was achieved at 10−5 M Lip(NO). Channel activation was mainly due to significant decrease in the mean closed dwell time (about 500-fold), rather than an increase in the mean open dwell time, which was comparatively modest (about twofold). There was also a slight decrease in the amplitude of the elementary Maxi-K currents (approximately 15%) accompanied by an increase in current noise, which might indicate some non-specific effects of Lip(NO) on the plasma membrane itself and/or on the phospholipids environment of the channels. In conclusion, the activating action of Lip(NO) on the Maxi-K channel is due to the destabilization of the closed conformation of the channel protein, which causes its more frequent openings and, accordingly, increases the probability of channel transition to its open state.

Implications of Oligomeric Amyloid-Beta (oAβ42) Signaling through α7β2-Nicotinic Acetylcholine Receptors (nAChRs) on Basal Forebrain Cholinergic Neuronal Intrinsic Excitability and Cognitive Decline.

Neuronal and network-level hyperexcitability is commonly associated with increased levels of amyloid-β (Aβ) and contribute to cognitive deficits associated with Alzheimer's disease (AD). However, the mechanistic complexity underlying the selective loss of basal forebrain cholinergic neurons (BFCNs), a well-recognized characteristic of AD, remains poorly understood. In this study, we tested the hypothesis that the oligomeric form of amyloid-β (oAβ42), interacting with α7-containing nicotinic acetylcholine receptor (nAChR) subtypes, leads to subnucleus-specific alterations in BFCN excitability and impaired cognition. We used single-channel electrophysiology to show that oAβ42 activates both homomeric α7- and heteromeric α7β2-nAChR subtypes while preferentially enhancing α7β2-nAChR open-dwell times. Organotypic slice cultures were prepared from male and female ChAT-EGFP mice, and current-clamp recordings obtained from BFCNs chronically exposed to pathophysiologically relevant level of oAβ42 showed enhanced neuronal intrinsic excitability and action potential firing rates. These resulted from a reduction in action potential afterhyperpolarization and alterations in the maximal rates of voltage change during spike depolarization and repolarization. These effects were observed in BFCNs from the medial septum diagonal band and horizontal diagonal band, but not the nucleus basalis. Last, aged male and female APP/PS1 transgenic mice, genetically null for the β2 nAChR subunit gene, showed improved spatial reference memory compared with APP/PS1 aged-matched littermates. Combined, these data provide a molecular mechanism supporting a role for α7β2-nAChR in mediating the effects of oAβ42 on excitability of specific populations of cholinergic neurons and provide a framework for understanding the role of α7β2-nAChR in oAβ42-induced cognitive decline.

Regulation of TRPC5 currents by intracellular ATP: Single channel studies.

TRPC5 channels are nonselective cation channels activated by G-protein-coupled receptors. It was previously found that recombinant TRPC5 currents are inhibited by intracellular ATP, when studied by whole-cell patch-clamp recording. In the present study, we investigated the mechanism of ATP inhibition at the single-channel levelusing patches from HEK-293 cells transiently transfected with TRPC5 and the M1 muscarinic receptor. In inside-out patches, application of ATP to the intracellular face of the membrane reduced TRPC5 channel activity at both positive and negative potentials without affecting the unitary current amplitude or open dwell time of the channel. The effect of ATP was rapidly reversible. These results suggest that ATP may bind to the channel protein and affect the ability of the channel to open or to remain in an open, nondesensitized state. The activity of TRPC5 channels may be influenced by cellular metabolism via changes in ATP levels.

De novo BK channel variant causes epilepsy by affecting voltage gating but not Ca2+ sensitivity.

Epilepsy is one of the most common neurological diseases and it causes profound morbidity and mortality. We identified the first de novo variant in KCNMA1 (c.2984 A > G (p.(N995S)))-encoding the BK channel-that causes epilepsy, but not paroxysmal dyskinesia, in two independent families. The c.2984 A > G (p.(N995S)) variant markedly increased the macroscopic potassium current by increasing both the channel open probability and channel open dwell time. The c.2984 A > G (p.(N995S)) variant did not affect the calcium sensitivity of the channel. We also identified three other variants of unknown significance (c.1554 G > T (p.(K518N)), c.1967A > C (p.(E656A)), and c.3476 A > G (p.(N1159S))) in three separate patients with divergent epileptic phenotypes. However, these variants did not affect the BK potassium current, and are therefore unlikely to be disease-causing. These results demonstrate that BK channel variants can cause epilepsy without paroxysmal dyskinesia. The underlying molecular mechanism can be increased activation of the BK channel by increased sensitivity to the voltage-dependent activation without affecting the sensitivity to the calcium-dependent activation. Our data suggest that the BK channel may represent a drug target for the treatment of epilepsy. Our data highlight the importance of functional electrophysiological studies of BK channel variants in distinguishing whether a genomic variant of unknown significance is a disease-causing variant or a benign variant.

Single Channel Analysis of Isoflurane and Ethanol Enhancement of Taurine-Activated Glycine Receptors.

The amino acid taurine is an endogenous ligand acting on glycine receptors (GlyRs), which is released by astrocytes in many brain regions, such as the nucleus accumbens and prefrontal cortex. Taurine is a partial agonist with an efficacy significantly lower than that of glycine. Allosteric modulators such as ethanol and isoflurane produce leftward shifts of glycine concentration-response curves but have no effects at saturating glycine concentrations. In contrast, in whole-cell electrophysiology studies these modulators increase the effects of saturating taurine concentrations. A number of possible mechanisms may explain these enhancing effects, including modulator effects on conductance, channel open times, or channel closed times. We used outside-out patch-clamp single channel electrophysiology to investigate the mechanism of action of 200 mM ethanol and 0.55 mM isoflurane in enhancing the effects of a saturating concentration of taurine. Neither modulator enhanced taurine-mediated conductance. Isoflurane increased the probability of channel opening. Isoflurane also increased the lifetimes of the two shortest open dwell times while both agents decreased the likelihood of occurrence of the longest-lived intracluster channel-closing events. The mechanism of enhancement of GlyR functioning by these modulators is dependent on the efficacy of the agonist activating the receptor and the concentration of agonist tested.

TREK-2 Has an Asymmetrical Response to Force Changes in the Membrane

Perception of force is a key component in our sensation of touch, hearing and pain as well as in regulation of blood and osmotic pressures, but unlike the well-described nature of taste and odorant receptors and the photoreceptors in the eye, we have not yet a clear idea of how this sense operate at the molecular level. Fundamental to the concept is that force must be translated into electrical conductance, that is perceived by the nervous system, and we believe this translation occur through the action of mechanosensitive ion channels. Recently it has been demonstrated that eukaryotic mechanosensitive channels, just like their prokaryotic counterparts, directly sense force changes in the lipid bilayers, so now the important question is: how does membrane modulation couple to channel activity? To address this question we use purified mechanosensitive TREK-2 channels, which we reconstitute into planar lipid patches at a concentration that allow us to study channel activity at the single molecule level. We find that the reconstituted channels have a random orientation, and that we can clearly distinguish between the two possible orientations by analyzing electrophysiological characteristics as conductance and open dwell times. To investigate the sensitivity of the channels to mechanical modulation, we apply different degrees of negative or positive pressure to the patches, and analyze how such stimulations affect the activity of differently oriented channels. With this approach we can demonstrate that mechanical activation of TREK-2 depends on the direction of the pressure pulse, i.e. one orientation is activated only by negative pressure while the other orientation is activated only by positive pressure. We carefully suggest that these results demonstrate that TREK-2 channels are sensitive to the direction of membrane curvature. Furthermore we are in the process of deducing a kinetic model that describes TREK-2 activation by pressure.

The Electrophysiology of Mechanosensitive Channels in Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen that causes infections mainly in immuno-compromised organisms and is characterized with exceptional adaptability to a broad range of habitats, from soil and freshwater to medical equipment and mammalian tissues. Part of its adaptive potential is the ability to adjust its turgor pressure under conditions of drastically varied osmolarity. The osmolyte release system in this Gram-negative bacterium, like in E. coli, comprises the high-threshold tension-activated channel MscL and six channels from the MscS family. In the present work we have developed the giant spheroplast system for direct patch-clamp recording from the cytoplasmic membrane of P. aeruginosa and characterized mechanoelectrical responses in the wild-type strain (PA14) using pressure protocols, revealing a complex adaptive behavior involving several channel species. We have cloned and functionally characterized PaMscL (137 aa) and two MscS-like channels, PaMscS-1 (278 aa) and PaMscS-2 (283 aa) expressed in E. coli giant spheroplasts. The ∼2 nS PaMscL exhibited short open dwell times and was found non-selective. Both PaMscS-1 (∼1 nS) and PaMscS-2 (∼0.5 nS) have a slight anionic preference with similar pressure midpoint ratios P0.5 MscS/ P0.5 MscL of ∼0.5. Under a standard 10 s pressure-step protocol, PaMscS-1 displays a 30% tension-dependent inactivation, whereas PaMscS-2 under similar conditions shows a 60% inactivation, with extremely slow (5-10 min) recovery. Sequence analysis and homology models reveal all components necessary for inactivation conserved between P. aeruginosa and E. coli MscS. PaMscL and PaMscS-1 perfectly rescue an MS channel-free E. coli strain (MJF465) from abrupt osmotic shock, whereas PaMscS-2 does not, possibly due to lower conductance and expression level. The electrophysiology of P. aeruginosa gives a better understanding of the components and mechanisms of environmental stability of this opportunistic pathogen easily changing its habitats.

Phosphorylation of BK channels modulates the sensitivity to hydrogen sulfide (H2S).

Introduction: Gases, such as nitric oxide (NO), carbon monoxide (CO), or hydrogen sulfide (H2S), termed gasotransmitters, play an increasingly important role in understanding of how electrical signaling of cells is modulated. H2S is well-known to act on various ion channels and receptors. In a previous study we reported that H2S increased calcium-activated potassium (BK) channel activity.Aims: The goal of the present study is to investigate the modulatory effect of BK channel phosphorylation on the action of H2S on the channel as well as to recalculate and determine the H2S concentrations in aqueous sodium hydrogen sulfide (NaHS) solutions.Methods: Single channel recordings of GH3, GH4, and GH4 STREX cells were used to analyze channel open probability, amplitude, and open dwell times. H2S was measured with an anion selective electrode.Results: The concentration of H2S produced from NaHS was recalculated taking pH, temperature salinity of the perfusate, and evaporation of H2S into account. The results indicate that from a concentration of 300 μM NaHS, only 11–13%, i.e., 34–41 μM is effective as H2S in solution. GH3, GH4, and GH4 STREX cells respond differently to phosphorylation. BK channel open probability (Po) of all cells lines used was increased by H2S in ATP-containing solutions. PKA prevented the action of H2S on channel Po in GH4 and GH4 STREX, but not in GH3 cells. H2S, high significantly increased Po of all PKG pretreated cells. In the presence of PKC, which lowers channel activity, H2S increased channel Po of GH4 and GH4 STREX, but not those of GH3 cells. H2S increased open dwell times of GH3 cells in the absence of ATP significantly. A significant increase of dwell times with H2S was also observed in the presence of okadaic acid.Conclusions: Our results suggest that phosphorylation by PKG primes the channels for H2S activation and indicate that channel phosphorylation plays an important role in the response to H2S.

Microscopic mechanisms for long QT syndrome type 1 revealed by single-channel analysis of I(Ks) with S3 domain mutations in KCNQ1.

The slowly activating delayed rectifier current IKs participates in cardiac repolarization, particularly at high heart rates, and mutations in this K(+) channel complex underlie long QT syndrome (LQTS) types 1 and 5. The purpose of this study was to determine biophysical mechanisms of LQT1 through single-channel kinetic analysis of IKs carrying LQT1 mutations in the S3 transmembrane region of the pore-forming subunit KCNQ1. We analyzed cell-attached recordings from mammalian cells in which a single active KCNQ1 (wild type or mutant) and KCNE1 complex could be detected. The S3 mutants of KCNQ1 studied (D202H, I204F, V205M, and S209F), with the exception of S209F, all led to a reduction in channel activity through distinct kinetic mechanisms. D202H, I204F, and V205M showed decreased open probability (Po) compared with wild type (0.07, 0.04, and 0.12 vs 0.2); increased first latency from 1.66 to >2 seconds at +60 mV (I204F, V205M); variable-to-severe reductions in open dwell times (≥50% in V205M); stabilization of closed states (D202H); and an inability of channels to reach full conductance levels (V205M, I204F). S209F is a kinetic gain-of-function mutation with a high Po (0.40) and long open-state dwell times. S3 mutations in KCNQ1 cause diverse kinetic defects in I(Ks), affecting opening and closing properties, and can account for LQT1 phenotypes.

Electrostatics at the membrane define MscL channel mechanosensitivity and kinetics.

The bacterial mechanosensitive channel of large conductance (MscL) serves as a biological emergency release valve, preventing the occurrence of cell lysis caused by acute osmotic stress. Its tractable nature allows it to serve as a paradigm for how a protein can directly sense membrane tension. Although much is known of the importance of the hydrophobicity of specific residues in channel gating, it has remained unclear whether electrostatics at the membrane plays any role. We studied MscL chimeras derived from functionally distinct orthologues: Escherichia coli and Staphylococcus aureus. Dissection of one set led to an observation that changing the charge of a single residue, K101, of E. coli (Ec)-MscL, effects a channel phenotype: when mutated to a negative residue, the channel is less mechanosensitive and has longer open dwell times. Assuming electrostatic interactions, we determined whether they are due to protein-protein or protein-lipid interactions by performing site-directed mutagenesis elsewhere in the protein and reconstituting channels into defined lipids, with and without negative head groups. We found that although both interactions appear to play some role, the primary determinant of the channel phenotype seems to be protein-lipid electrostatics. The data suggest a model for the role of electrostatic interactions in the dynamics of MscL gating.

N-terminal Isoforms of the Large-conductance Ca2+-activated K+ Channel Are Differentially Modulated by the Auxiliary β1-Subunit

The large-conductance Ca(2+)-activated K(+) (BK(Ca)) channel is essential for maintaining the membrane in a hyperpolarized state, thereby regulating neuronal excitability, smooth muscle contraction, and secretion. The BK(Ca) α-subunit has three predicted initiation codons that generate proteins with N-terminal ends starting with the amino acid sequences MANG, MSSN, or MDAL. Because the N-terminal region and first transmembrane domain of the α-subunit are required for modulation by auxiliary β1-subunits, we examined whether β1 differentially modulates the N-terminal BK(Ca) α-subunit isoforms. In the absence of β1, all isoforms had similar single-channel conductances and voltage-dependent activation. However, whereas β1 did not modulate the voltage-activation curve of MSSN, β1 induced a significant leftward shift of the voltage activation curves of both the MDAL and MANG isoforms. These shifts, of which the MDAL was larger, occurred at both 10 μM and 100 μM Ca(2+). The β1-subunit increased the open dwell times of all three isoforms and decreased the closed dwell times of MANG and MDAL but increased the closed dwell times of MSSN. The distinct modulation of voltage activation by the β1-subunit may be due to the differential effect of β1 on burst duration and interburst intervals observed among these isoforms. Additionally, we observed that the related β2-subunit induced comparable leftward shifts in the voltage-activation curves of all three isoforms, indicating that the differential modulation of these isoforms was specific to β1. These findings suggest that the relative expression of the N-terminal isoforms can fine-tune BK(Ca) channel activity in cells, highlighting a novel mechanism of BK(Ca) channel regulation.

Dynamics of Protein-Protein Interactions at the MscL Periplasmic-Lipid Interface

MscL, the highly conserved bacterial mechanosensitive channel of large conductance, is one of the best studied mechanosensors. It is a homopentameric channel that serves as a biological emergency release valve that prevents cell lysis from acute osmotic stress. We previously showed that the periplasmic region of the protein, particularly a single residue located at the TM1/periplasmic loop interface, F47 of Staphylococcus aureus and I49 of Escherichia coli MscL, plays a major role in both the open dwell time and mechanosensitivity of the channel. Here, we introduced cysteine mutations at these sites and found they formed disulfide bridges that decreased the channel open dwell time. By scanning a likely interacting domain, we also found that these sites could be disulfide trapped by addition of cysteine mutations in other locations within the periplasmic loop of MscL, and this also led to rapid channel kinetics. Together, the data suggest structural rearrangements and protein-protein interactions that occur within this region upon normal gating, and further suggest that locking portions of the channel into a transition state decreases the stability of the open state.