Calcium-dependent Potassium Channels Research Articles

Kca3.1-Related Cellular Signalling Involved in Cancer Proliferation.

Anomalous expression of potassium channels in cancer tissues is associated with several cancer hallmarks that support deregulated proliferation and tumor progression. Ion channels seem to influence cell proliferation; however, the crucial molecular mechanisms involved remain elusive. Some results show how extracellular mitogenic signals modulate ion channel activity through intracellular secondary messengers. It is relevant because we are beginning to understand how potassium channels can affect the proliferative capacity of cells, either in normal mitogen-dependent proliferation or in mitogen-unresponsive proliferation. Calciumdependent potassium channels have been implicated in cell cycle signaling in many cancerous cell lines. In particular, the so-called intermediate conductance KCa3.1 (IKCa) is reported to play a significant role in uncontrolled cell cycle signaling, among other malignant processes driven by cancer hallmarks. In addition to these features, this channel can be subjected to specific pharmacological regulation, making it a promising cornerstone for understanding the signaling behavior of several types of cancer and as a target for chemotherapeutic approaches. This review is dedicated to the connection of KCa3.1 activity, in canonical and non-canonical ways, to the cell cycle signaling, including the cooperation with calcium channels to generate calcium signals and its role as a mediator of proliferative signals.

A biophysically comprehensive model of urothelial afferent neurons: implications for sensory signalling in urinary bladder.

The urothelium is the innermost layer of the bladder wall; it plays a pivotal role in bladder sensory transduction by responding to chemical and mechanical stimuli. The urothelium also acts as a physical barrier between urine and the outer layers of the bladder wall. There is intricate sensory communication between the layers of the bladder wall and the neurons that supply the bladder, which eventually translates into the regulation of mechanical activity. In response to natural stimuli, urothelial cells release substances such as ATP, nitric oxide (NO), substance P, acetylcholine (ACh), and adenosine. These act on adjacent urothelial cells, myofibroblasts, and urothelial afferent neurons (UAN), controlling the contractile activity of the bladder. There is rising evidence on the importance of urothelial sensory signalling, yet a comprehensive understanding of the functioning of the urothelium-afferent neurons and the factors that govern it remains elusive to date. Until now, the biophysical studies done on UAN have been unable to provide adequate information on the ion channel composition of the neuron, which is paramount to understanding the electrical functioning of the UAN and, by extension, afferent signalling. To this end, we have attempted to model UAN to decipher the ionic mechanisms underlying the excitability of the UAN. In contrast to previous models, our model was built and validated using morphological and biophysical properties consistent with experimental findings for the UAN. The model included all the channels thus far known to be expressed in UAN, including; voltage-gated sodium and potassium channels, N, L, T, P/Q, R-type calcium channels, large-conductance calcium-dependent potassium (BK) channels, small conductance calcium-dependent (SK) channels, Hyperpolarisation activated cation (HCN) channels, transient receptor potential melastatin (TRPM8), transient receptor potential vanilloid (TRPV1) channel, calcium-activated chloride(CaCC) channels, and internal calcium dynamics. Our UAN model a) was constrained as far as possible by experimental data from the literature for the channels and the spiking activity, b) was validated by reproducing the experimental responses to current-clamp and voltage-clamp protocols c) was used as a base for modelling the non-urothelial afferent neurons (NUAN). Using our models, we also gained insights into the variations in ion channels between UAN and NUAN neurons.

#2696 IN SILICO ELECTROPHYSIOLOGICAL STUDY REVEALS TAMSULOSIN MEDIATE URETER SMOOTH MUSCLE CONTRACTION BY INCREASING POTASSIUM CURRENT

Abstract Background and Aims Tamsulosin is a common α1-AR antagonist that is prescribed to patients with acute kidney stone episodes [Scotland et al., 2021]. According to accumulated experimental data, abnormal peristaltic contraction of the ureter smooth muscle (USM) causes acute kidney stone episodes [Lang et al., 2006]. Intracellular electrical activities like membrane depolarization and action potentials play important roles in modulating the USM contraction by releasing intracellular calcium from the sarcoplasmic reticulum. Therefore, an electrophysiological study will be helpful to assess the USM cell's electrical activities and in diagnosing abnormal USM contraction. Transient potassium channels (KA) are prevalent in different systems and hold immense importance for maintaining/performing selective electrophysiological functions. The present study investigates the altered electrophysiological characteristics of the USM cell because of the induction of Tamsulosin doses. Method The single isolated USM cell comprises several voltage-gated ion channels, such as two voltage-gated calcium (T-type, and L-type) channels, one voltage-gated fast potassium (KA) channel, one calcium-dependent large conductance potassium channel, and an HCN channel. The individual ion channel currents and the membrane potential are recorded by utilizing both the voltage clamp and current clamp protocols. A Tamsulosin drug (0.5 mg) model for the KA channel is introduced by multiplying the maximal conductance of the KA channel with a scaling factor between 0 and 1 to mimic the drug concentration. Statistical analysis protocols are evaluated for analytical purposes. Results In this in silico electrophysiological model, the resting membrane potential is maintained at −75 mV, as this value is cited by various experimental studies. The ionic concentrations and biophysical parameters for all ion channels are varied in the physiological ranges. Under the voltage clamp protocol, the voltage steps are increased from a holding potential of −80 mV to −10 mV with a 10 mV of step voltage. In Figure 1, the red and blue solid lines represent recorded outward KA current from the isolated USM cell under both control and applied Tamsulosin dose conditions. Figure 1 reveals that the KA current is elevated after the application of the Tamsulosin drug regarding all step voltages. As a result, the total outward current is elevated and the USM action potential duration is shortened. It is also observed that the steady-state activation curve of the KA channel is shifted to more negative after applying Tamsulosin. The USM cell is electrically less excitable with the Tamsulosin. Conclusion This first in-silico study reveals the KA ionic current, which plays a major role in coordinating ureteral contractions, is altered due to Tamsulosin. It implies that the pharmacological maneuver of Tamsulosin is substantially beneficial to treat dysfunctional peristalsis by reducing the symptoms and installation complexities from the ureteral stents procedure.

The contribution of intermediate conductance calcium-dependent potassium channels to hypothalamic vasopressin neuronal excitability

Vasopressin (VP) magnocellular neurosecretory neurons of the supraoptic nucleus (SON) in the hypothalamus are critical regulators of renal water retention and vascular tone. VP neurons undergo detrimental plastic changes in cardiovascular diseases such as heart failure (HF), resulting in hyperexcitability and thus altered fluid/electrolyte balance. Hyperexcitability is caused in part by changes in intrinsic excitability mechanisms, including the slow afterhyperpolarization (sAHP), a phenomenon underlain by a calcium-dependent K+ current ( I sAHP). The sAHP is activated by Ca2+ and results in an efflux of K+ from the cell, hyperpolarizing it, and throttling firing. Importantly, we found that the sAHP is greatly inhibited in VP neurons from HF rats, contributing to increased neuronal excitability in this condition. While the features of the sAHP are well characterized, the identification of the channel underlying the I sAHP is unknown. Combining patch clamp electrophysiology and pharmacology in Wistar rats, we investigated Intermediate conductance Ca2+-dependent K+ (IK) channels as a potential candidate responsible for carrying the I sAHP. We generated and measured I sAHPs in voltage clamp via fixed steps of twenty spikes at twenty Hz and measured the the I sAHP as the resulting tail current at the end of the pulse. To account for rundown of the current over the recording period and for the pharmacological blocker’s slow mechanism of action, we measured I sAHPs every 60 seconds for at least 15 minutes. Bath application of TRAM-34 (2 μM) at 7 minutes, a specific IK channel blocker, failed to inhibit I sAHP peak amplitude or area (p > 0.05; n=36 SON neurons). We used biocytin-filled pipettes to identify neurons post hoc and run immunohistochemistry for oxytocin (OT) and VP to identify cell type within the SON. We found no significant difference in I sAHP amplitude or area between VP and OT cells under either control or TRAM-34 application. Taken together, our studies indicate that IK channels do not contribute to the I sAHP in SON neurosecretory neurons, despite robust IK expression in this region. Future studies will aim at identifying the role that IK channels play in SON, as well as targeting other potential K+ channel candidates for this NHLBI F32 HL158172 (MKK) NHLBI HL090948 (JES) NINDS NS094640 (JES) This is the full abstract presented at the American Physiology Summit 2023 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.

Peroxiredoxin 2: An Important Element of the Antioxidant Defense of the Erythrocyte.

Peroxiredoxin 2 (Prdx2) is the third most abundant erythrocyte protein. It was known previously as calpromotin since its binding to the membrane stimulates the calcium-dependent potassium channel. Prdx2 is present mostly in cytosol in the form of non-covalent dimers but may associate into doughnut-like decamers and other oligomers. Prdx2 reacts rapidly with hydrogen peroxide (k > 107 M-1 s-1). It is the main erythrocyte antioxidant that removes hydrogen peroxide formed endogenously by hemoglobin autoxidation. Prdx2 also reduces other peroxides including lipid, urate, amino acid, and protein hydroperoxides and peroxynitrite. Oxidized Prdx2 can be reduced at the expense of thioredoxin but also of other thiols, especially glutathione. Further reactions of Prdx2 with oxidants lead to hyperoxidation (formation of sulfinyl or sulfonyl derivatives of the peroxidative cysteine). The sulfinyl derivative can be reduced by sulfiredoxin. Circadian oscillations in the level of hyperoxidation of erythrocyte Prdx2 were reported. The protein can be subject to post-translational modifications; some of them, such as phosphorylation, nitration, and acetylation, increase its activity. Prdx2 can also act as a chaperone for hemoglobin and erythrocyte membrane proteins, especially during the maturation of erythrocyte precursors. The extent of Prdx2 oxidation is increased in various diseases and can be an index of oxidative stress.

Signal transduction of the insulin secretion induced by the chalcone analogue, (E)-3-(phenyl)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one, and its role in glucose and lipid metabolism

A chalcone analogue, (E)-3-(phenyl)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (DMU 101), was synthesized using classic base catalysis and Claisen-Schmidt condensation, and then screened for its antidiabetic properties. The compound's effects on glucose and lipid metabolism were assayed in rats that were treated acutely and for a short time to elucidate its mechanism of action, evaluating glucose tolerance and lactate dehydrogenase activity in response to chalcone analogue administration. The chalcone's in vitro and ex vivo effects on glycogen, glucose, lipid and lipolysis were also investigated, as well as the mechanism by which it induces 45Ca2+ influx-mediated insulin secretion. The analogue (10 mg/kg) diminished glycemia, without inducing acute cell damage, increased glycogen content in the skeletal muscle and reduced serum triacylglycerol and total cholesterol, but did not alter high-density lipoprotein or low-density lipoprotein. Chalcone (10 μM) stimulated glucose uptake in the soleus muscle and did not modulate in vitro or ex vivo lipolysis. This analogue also increased insulin secretion by triggering calcium influx and blocking ATP-sensitive K+ channels and voltage-dependent calcium channels. However, it also modulated stored calcium via sarco/endoplasmic reticulum calcium ATPase (SERCA) and ryanodine receptor (RYR) activity. These findings indicate that this chalcone may induce cellular repolarization via a mechanism mediated by calcium-dependent potassium channels.

Ca2+-Activated K+ Channels in Progenitor Cells of Musculoskeletal Tissues: A Narrative Review.

Musculoskeletal disorders represent one of the main causes of disability worldwide, and their prevalence is predicted to increase in the coming decades. Stem cell therapy may be a promising option for the treatment of some of the musculoskeletal diseases. Although significant progress has been made in musculoskeletal stem cell research, osteoarthritis, the most-common musculoskeletal disorder, still lacks curative treatment. To fine-tune stem-cell-based therapy, it is necessary to focus on the underlying biological mechanisms. Ion channels and the bioelectric signals they generate control the proliferation, differentiation, and migration of musculoskeletal progenitor cells. Calcium- and voltage-activated potassium (KCa) channels are key players in cell physiology in cells of the musculoskeletal system. This review article focused on the big conductance (BK) KCa channels. The regulatory function of BK channels requires interactions with diverse sets of proteins that have different functions in tissue-resident stem cells. In this narrative review article, we discuss the main ion channels of musculoskeletal stem cells, with a focus on calcium-dependent potassium channels, especially on the large conductance BK channel. We review their expression and function in progenitor cell proliferation, differentiation, and migration and highlight gaps in current knowledge on their involvement in musculoskeletal diseases.

Calcium-dependent modulation of BKCa channel activity induced by plasmonic gold nanoparticles in pulmonary artery smooth muscle cells and hippocampal neurons.

Gold nanoparticles are widely used for biomedical applications, but the precise molecular mechanism of their interaction with cellular structures is still unclear. Assuming that intracellular calcium fluctuations associated with surface plasmon-induced calcium entry could modulate the activity of potassium channels, we studied the effect of 5nm gold nanoparticles on calcium-dependent potassium channels and associated calcium signaling in freshly isolated rat pulmonary artery smooth muscle cells and cultured hippocampal neurons. Outward potassium currents were recorded using patch-clamp techniques. Changes in intracellular calcium concentration were measured using the high affinity Ca2+ fluorescent indicator fluo-3 and laser confocal microscope. In pulmonary artery smooth muscle cells, plasmonic gold nanoparticles increased the amplitude of currents via large-conductance Ca2+ -activated potassium channels, which was potentiated by green laser irradiation near plasmon resonance wavelength (532 nm). Buffering of intracellular free calcium with ethylene glycol-bis-N,N,N',N'-tetraacetic acid (EGTA) abolished these effects. Furthermore, using confocal laser microscopy it was found that application of gold nanoparticles caused oscillations of intracellular calcium concentration that were decreasing in amplitude with time. In cultured hippocampal neurons gold nanoparticles inhibited the effect of EGTA slowing down the decline of the BKCa current while partially restoring the amplitude of the slow after hyperpolarizing currents. We conclude that fluctuations in intracellular calcium can modulate plasmonic gold nanoparticles-induced gating of BKCa channels in smooth muscle cells and neurons through an indirect mechanism, probably involving the interaction of plasmon resonance with calcium-permeable ion channels, which leads to a change in intracellular calcium level.

Pharmacological targeting of the mitochondrial calcium-dependent potassium channel KCa3.1 triggers cell death and reduces tumor growth and metastasis in vivo

Ion channels are non-conventional, druggable oncological targets. The intermediate-conductance calcium-dependent potassium channel (KCa3.1) is highly expressed in the plasma membrane and in the inner mitochondrial membrane (mitoKCa3.1) of various cancer cell lines. The role mitoKCa3.1 plays in cancer cells is still undefined. Here we report the synthesis and characterization of two mitochondria-targeted novel derivatives of a high-affinity KCa3.1 antagonist, TRAM-34, which retain the ability to block channel activity. The effects of these drugs were tested in melanoma, pancreatic ductal adenocarcinoma and breast cancer lines, as well as in vivo in two orthotopic models. We show that the mitochondria-targeted TRAM-34 derivatives induce release of mitochondrial reactive oxygen species, rapid depolarization of the mitochondrial membrane, fragmentation of the mitochondrial network. They trigger cancer cell death with an EC50 in the µM range, depending on channel expression. In contrast, inhibition of the plasma membrane KCa3.1 by membrane-impermeant Maurotoxin is without effect, indicating a specific role of mitoKCa3.1 in determining cell fate. At sub-lethal concentrations, pharmacological targeting of mitoKCa3.1 significantly reduced cancer cell migration by enhancing production of mitochondrial reactive oxygen species and nuclear factor-κB (NF-κB) activation, and by downregulating expression of Bcl-2 Nineteen kD-Interacting Protein (BNIP-3) and of Rho GTPase CDC-42. This signaling cascade finally leads to cytoskeletal reorganization and impaired migration. Overexpression of BNIP-3 or pharmacological modulation of NF-κB and CDC-42 prevented the migration-reducing effect of mitoTRAM-34. In orthotopic models of melanoma and pancreatic ductal adenocarcinoma, the tumors at sacrifice were 60% smaller in treated versus untreated animals. Metastasis of melanoma cells to lymph nodes was also drastically reduced. No signs of toxicity were observed. In summary, our results identify mitochondrial KCa3.1 as an unexpected player in cancer cell migration and show that its pharmacological targeting is efficient against both tumor growth and metastatic spread in vivo.

Peptide Lv augments intermediate-conductance calcium-dependent potassium channels (KCa3.1) in endothelial cells to promote angiogenesis.

Peptide Lv is a small endogenous secretory peptide that is expressed in various tissues and conserved across different species. Patients with diabetic retinopathy, an ocular disease with pathological angiogenesis, have upregulated peptide Lv in their retinas. The pro-angiogenic activity of peptide Lv is in part through promoting vascular endothelial cell (EC) proliferation, migration, and sprouting, but its molecular mechanism is not completely understood. This study aimed to decipher how peptide Lv promotes EC-dependent angiogenesis by using patch-clamp electrophysiological recordings, Western immunoblotting, quantitative PCR, and cell proliferation assays in cultured ECs. Endothelial cells treated with peptide Lv became significantly hyperpolarized, an essential step for EC activation. Treatment with peptide Lv augmented the expression and current densities of the intermediate-conductance calcium-dependent potassium (KCa3.1) channels that contribute to EC hyperpolarization but did not augment other potassium channels. Blocking KCa3.1 attenuated peptide Lv-elicited EC proliferation. These results indicate that peptide Lv-stimulated increases of functional KCa3.1 in ECs contributes to EC activation and EC-dependent angiogenesis.

Mechanism of stress-induced attacks in an episodic neurologic disorder.

Stress is the most common trigger among episodic neurologic disorders. In episodic ataxia type 2 (EA2), physical or emotional stress causes episodes of severe motor dysfunction that manifest as ataxia and dystonia. We used the tottering (tg/tg) mouse, a faithful animal model of EA2, to dissect the mechanisms underlying stress-induced motor attacks. We find that in response to acute stress, activation of α1-adrenergic receptors (α1-Rs) on Purkinje cells by norepinephrine leads to their erratic firing and consequently motor attacks. We show that norepinephrine induces erratic firing of Purkinje cells by disrupting their spontaneous intrinsic pacemaking via a casein kinase 2 (CK2)–dependent signaling pathway, which likely reduces the activity of calcium-dependent potassium channels. Moreover, we report that disruption of this signaling cascade at a number of nodes prevents stress-induced attacks in the tottering mouse. Together, our results suggest that norepinephrine and CK2 are required for the initiation of stress-induced attacks in EA2 and provide previously unidentified targets for therapeutic intervention.

Locomotor Pattern and Force Generation Modulated by Ionic Channels: A Computational Study of Spinal Networks Underlying Locomotion.

Locomotion is a fundamental movement in vertebrates produced by spinal networks known as central pattern generators (CPG). During fictive locomotion cat lumbar motoneurons (MNs) exhibit changes in membrane properties, including hyperpolarization of voltage threshold, reduction of afterhyperpolarization and input resistance, and amplification of nonlinear membrane properties. Both modeling and electrophysiological studies suggest that these changes can be produced by upregulating voltage-gated sodium channel (VGSC), persistent sodium (NaP), or L-type calcium channel (LTCC) or downregulating delayed-rectifier potassium (K(DR)) or calcium-dependent potassium channel (KCa) in spinal MNs. Further studies implicate that these channel modulations increase motor output and facilitate MN recruitment. However, it remains unknown how the channel modulation of CPG networks or MN pools affects the rhythmic generation of locomotion and force production of skeletal muscle during locomotion. In order to investigate this issue, we built a two-level CPG model composed of excitatory interneuron pools (Exc-INs), coupled reciprocally with inhibitory interneuron pools (Inh-INs), and projected to the flexor-extensor MN pools innervating skeletal muscles. Each pool consisted of 100 neurons with membrane properties based on cat spinal neurons. VGSC, K(DR), NaP, KCa, LTCC, and H-current channels were included in the model. Simulation results showed that (1) upregulating VGSC, NaP, or LTCC or downregulating KCa in MNs increased discharge rate and recruitment of MNs, thus facilitating locomotor pattern formation, increased amplitude of electroneurogram (ENG) bursting, and enhanced force generation of skeletal muscles. (2) The same channel modulation in Exc-INs increased the firing frequency of the Exc-INs, facilitated rhythmic generation, and increased flexor-extensor durations of step cycles. (3) Contrarily, downregulation of NaP or LTCC in MNs or Exc-INs or both CPG (Exc-INs and Inh-INs) and MNs disrupted locomotor pattern and reduced or even blocked the ENG bursting of MNs and force generation of skeletal muscles. (4) Pharmacological experiments showed that bath application of 25 μM nimodipine or 2 μM riluzole completely blocked fictive locomotion in isolated rat spinal cord, consistent with simulation results. We concluded that upregulation of VGSC, NaP, or LTCC or downregulation of KCa facilitated rhythmic generation and force production during walking, with NaP and LTCC playing an essential role.

Characterization of the pathophysiological roles of a calcium-dependent potassium channel in mitochondria

The intermediate-conductance calcium-dependent potassium channel (KCa3.1) is functional in the inner mitochondrial membrane (IMM) of various cancer cell lines, as demonstrated by patch-clamp and Western Blot. Decreased expression of KCa3.1 was found to affect oxidative phosphorylation and ATP production (Kovalenko et al., 2016) suggesting a role in fine-tuning mitochondrial energy metabolism, but its precise role in the IMM has not been elucidated so far. Here we report the synthesis and characterization of a mitochondria-targeted derivative of a high-affinity KCa3.1 antagonist.

Diversity and Functional Features of Calcium-Dependent Potassium Channels as Determinants of Their Role in the Plasticity of Cerebral Neurons

Diversity and Functional Features of Calcium-Dependent Potassium Channels as Determinants of Their Role in the Plasticity of Cerebral Neurons

Electrophysiological engineering of heart-derived cells with calcium-dependent potassium channels improves cell therapy efficacy for cardioprotection

We have shown that calcium-activated potassium (KCa)-channels regulate fundamental progenitor-cell functions, including proliferation, but their contribution to cell-therapy effectiveness is unknown. Here, we test the participation of KCa-channels in human heart explant-derived cell (EDC) physiology and therapeutic potential. TRAM34-sensitive KCa3.1-channels, encoded by the KCNN4 gene, are exclusively expressed in therapeutically bioactive EDC subfractions and maintain a strongly polarized resting potential; whereas therapeutically inert EDCs lack KCa3.1 channels and exhibit depolarized resting potentials. Somatic gene transfer of KCNN4 results in membrane hyperpolarization and increases intracellular [Ca2+], which boosts cell-proliferation and the production of pro-healing cytokines/nanoparticles. Intramyocardial injection of EDCs after KCNN4-gene overexpression markedly increases the salutary effects of EDCs on cardiac function, viable myocardium and peri-infarct neovascularization in a well-established murine model of ischemic cardiomyopathy. Thus, electrophysiological engineering provides a potentially valuable strategy to improve the therapeutic value of progenitor cells for cardioprotection and possibly other indications.

Cy3-RgIA-5727 Labels and Inhibits α9-Containing nAChRs of Cochlear Hair Cells.

Efferent cholinergic neurons inhibit sensory hair cells of the vertebrate inner ear through the combined action of calcium-permeable α9α10-containing nicotinic acetylcholine receptors (nAChRs) and associated calcium-dependent potassium channels. The venom of cone snails is a rich repository of bioactive peptides, many with channel blocking activities. The conopeptide analog, RgIA-5474, is a specific and potent antagonist of α9α10-containing nAChRs. We added an alkyl functional group to the N-terminus of the RgIA-5474, to enable click chemistry addition of the fluorescent cyanine dye, Cy3. The resulting peptide, Cy3-RgIA-5727, potently blocked mouse α9α10 nAChRs expressed in Xenopus oocytes (IC50 23 pM), with 290-fold less activity on α7 nAChRs and 40,000-fold less activity on all other tested nAChR subtypes. The tight binding of Cy3-RgIA-5727 provided robust visualization of hair cell nAChRs juxtaposed to cholinergic efferent terminals in excised, unfixed cochlear tissue from mice. Presumptive postsynaptic sites on outer hair cells (OHCs) were labeled, but absent from inner hair cells (IHCs) and from OHCs in cochlear tissue from α9-null mice and in cochlear tissue pre-incubated with non-Cy3-conjugated RgIA-5474. In cochlear tissue from younger (postnatal day 10) mice, Cy3-RgIA-5727 also labeled IHCs, corresponding to transient efferent innervation at that age. Cy3 puncta in Kölliker’s organ remained in the α9-null tissue. Pre-exposure with non-Cy3-conjugated RgIA-5474 or bovine serum albumin reduced this non-specific labeling to variable extents in different preparations. Cy3-RgIA-5727 and RgIA-5474 blocked the native hair cell nAChRs, within the constraints of application to the excised cochlear tissue. Cy3-RgIA-5727 or RgIA-5474 block of efferent synaptic currents in young IHCs was not relieved after 50 min washing, so effectively irreversible.

Amyloid β and Amyloid Precursor Protein Synergistically Suppress Large-Conductance Calcium-Activated Potassium Channel in Cortical Neurons.

Intracellular amyloid β (Aβ) injection suppresses the large-conductance calcium-dependent potassium (BK) channel in cortical pyramidal cells from wild-type (WT) mice. In 3xTg Alzheimer’s disease (AD) model mice, intraneuronal Aβ is genetically programed to accumulate, which suppresses the BK channel. However, the mode of BK channel suppression remained unclarified. The present report revealed that only one (11A1) of the three anti-Aβ-oligomer antibodies that we examined, but not anti-monomer-Aβ-antibodies, was effective in recovering BK channel activity in 3xTg neurons. Antibodies against amyloid precursor protein (APP) were also found to be effective, suggesting that APP plays an essential part in this Aβ-oligomer-induced BK channel suppression in 3xTg neurons. In WT neurons, by contrast, APP suppressed BK channels by itself, which suggests that either APP or Aβ is sufficient to block BK channels, thus pointing to a different co-operativity of Aβ and APP in WT and 3xTg neurons. To clarify this difference, we relied on our previous finding that the scaffold protein Homer1a reverses the BK channel blockade in both WT and 3xTg neurons. In cortical neurons from 3xTg mice that bear Homer1a knockout (4xTg mice), neither anti-APP antibodies nor 11A1, but only the 6E10 antibody that binds both APP and Aβ, rescued the BK channel suppression. Given that Homer1a expression is activity dependent and 3xTg neurons are hyperexcitable, Homer1a is likely to be expressed sufficiently in 3xTg neurons, thereby alleviating the suppressive influence of APP and Aβ on BK channel. A unique way that APP modifies Aβ toxicity is thus proposed.

Transcriptome-based screening of ion channels and transporters in a migratory chondroprogenitor cell line isolated from late-stage osteoarthritic cartilage.

Chondrogenic progenitor cells (CPCs) may be used as an alternative source of cells with potentially superior chondrogenic potential compared to mesenchymal stem cells (MSCs), and could be exploited for future regenerative therapies targeting articular cartilage in degenerative diseases such as osteoarthritis (OA). In this study, we hypothesised that CPCs derived from OA cartilage may be characterised by a distinct channelome. First, a global transcriptomic analysis using Affymetrix microarrays was performed. We studied the profiles of those ion channels and transporter families that may be relevant to chondroprogenitor cell physiology. Following validation of the microarray data with quantitative reverse transcription-polymerase chain reaction, we examined the role of calcium-dependent potassium channels in CPCs and observed functional large-conductance calcium-activated potassium (BK) channels involved in the maintenance of the chondroprogenitor phenotype. In line with our very recent results, we found that the KCNMA1 gene was upregulated in CPCs and observed currents that could be attributed to the BK channel. The BK channel inhibitor paxilline significantly inhibited proliferation, increased the expression of the osteogenic transcription factor RUNX2, enhanced the migration parameters, and completely abolished spontaneous Ca2+ events in CPCs. Through characterisation of their channelome we demonstrate that CPCs are a distinct cell population but are highly similar to MSCs in many respects. This study adds key mechanistic data to the in-depth characterisation of CPCs and their phenotype in the context of cartilage regeneration.

NMDA receptor membrane dynamics tunes the firing pattern of midbrain dopaminergic neurons.

NMDA receptors (NMDARs) expressed by dopamine neurons of the ventral tegmental area (VTA) play a central role in glutamate synapse plasticity, neuronal firing and adaptative behaviours. The NMDAR surface dynamics shapes synaptic adaptation in hippocampal networks, as well as associative memory. We investigated the basic properties and role of the NMDAR surface dynamics on cultured mesencephalic and VTA dopamine neurons in rodents. Using a combination of single molecule imaging and electrophysiological recordings, we demonstrate that NMDARs are highly diffusive at the surface of mesencephalic dopamine neurons. Unexpectedly, the NMDAR membrane dynamics per se regulates the firing pattern of VTA dopaminergic neurons, probably through a functional interplay between NMDARs receptors and small-conductance calcium-dependent potassium (SK) channels. Midbrain dopaminergic (DA) neurons play a central role in major physiological brain functions, and their dysfunctions have been associated with neuropsychiatric diseases. The activity of midbrain DA neurons is controlled by ion channels and neurotransmitter receptors, such as the glutamate NMDA receptor (NMDAR) and small-conductance calcium-dependent potassium (SK) channels. However, the cellular mechanisms through which these channels tune the firing pattern of midbrain DA neurons remain unclear. Here, we investigated whether the surface dynamics and distribution of NMDARs tunes the firing pattern of midbrain DA neurons. Using a combination of single molecule imaging and electrophysiological recordings, we report that NMDARs are highly diffusive at the surface of cultured midbrain DA neurons from rodents and humans. Reducing acutely the NMDAR membrane dynamics, which leaves the ionotropic function of the receptor intact, robustly altered the firing pattern of midbrain DA neurons without altering synaptic glutamatergic transmission. The reduction of NMDAR surface dynamics reduced apamin (SK channel blocker)-induced firing change and the distribution of SK3 channels in DA neurons. Together, these data show that the surface dynamics of NMDAR, and not solely its ionotropic function, tune the firing pattern of midbrain DA neurons partly through a functional interplay with SK channel function.

ОЦІНКА ВПЛИВУ ДОНОРА МОНООКСИДУ КАРБОНУ (CORM-2) НА ШВИДКІСТЬ ЗГОРТАННЯ КРОВІ В УМОВАХ ІМУННОЇ ВІДПОВІДІ

The balance between the liquid state support of the blood and the thrombus formation process depends on the activity of plasma factors and thrombocytes. In a vascular injury condition, thrombocytes form a thrombocyte plug, vasoconstriction and, further, take part in the restoration of the tissues. Reducing the clotting function of thrombocytes is a current problem. The search for agents - modulators of thrombocyte activity - is a promising area of research. Compounds - gas-transmitters can be used to model the activity of thrombocytes hemostasis. Carbon monoxide (CO) gas belongs to this group as well. CO in very low concentrations affects in a different way the process of apoptosis, stimulates calcium-dependent potassium channels, and changes the activity of mitochondria. To investigate the effect of CO on blood clotting rate, laboratory mice were injected with the compound CO donor (CORM-2). This substance was administered separately during the inductive and productive phases of the immune response. The dynamics of the immune response were measured by IgA, IgM and IgG immunoglobulin levels. It was found that blood clotting was intensified under the influence of CORM-2 (20 mg/kg). Especially, the increase was during the productive phase. At the end of the experiment, the percentage of megakaryoblasts in the total population of thrombocyte precursors decreased in the bone marrow of animals (to which CORM-2 was administered). Against this background, an increased content of megakaryocytes was found in the group that received CORM-2 during the productive phase. In the group that received CORM-2 during the inductive phase, in addition to an increase in megakaryocyte levels, there was an increase in thrombocyte levels. The group that received CORM-2 during the productive phase also had a predominance of metamegakaryocytes and reducing of thrombocyte amount. Key words: thrombocytes, carbon monoxide donor, CORM-2, immune response phases.