Activation Of Calcium-activated Potassium Channels Research Articles

Impaired cerebral microvascular reactivity and endothelial SK channel activity in a streptozotocin-treated mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is a complex neurodegenerative disease marked by increased amyloid-β (Aβ) deposition, tau hyperphosphorylation, impaired energy metabolism, and chronic ischemia-type injury. Cerebral microvascular dysfunction likely contributes to AD pathology, but its precise pathogenic role has been poorly defined. To examine microvascular reactivity to endothelium-dependent vasodilators and small conductance calcium-activated potassium (SK) channel activity in an intracerebral streptozotocin (STZ)-induced AD mouse model. Control and STZ-AD mice underwent Morris Water Maze and Barnes testing, after which cerebral microvascular and brain microvascular endothelial cells (MBMECs) were dissected to assess microvascular reactivity, responses to SK channel activator NS309, and ion-channel current recordings using whole-cell patch clamp methodology. Control mouse cerebral microvascular and human brain microvascular endothelial cells (HBMECs) were treated with soluble Aβ1-42 peptide to characterize microvascular reactivity and endothelial potassium currents. STZ-AD mice exhibited impaired performance vs control mice in behavioral testing. STZ-AD mice also exhibited diminished cerebral microvascular responsiveness and MBMECs potassium current augmentation in response to NS309 compared with control mice. Incubation of control mouse cerebral micro-vessels and HBMECs with soluble Aβ (1 µM) for 2 h attenuated relaxation responses to NS309 and diminished NS309-sensitive endothelial potassium currents. STZ-AD mice exhibited impaired microvascular relaxation responses to endothelium-dependent vasodilators; SK/IK channel dysfunction may be involved in the mechanism of this impairment. Acute treatment with Aβ produced dysregulated cerebrovascular endothelial SK/IK channels. Further elucidation of the role of microvascular dysfunction in AD is needed to prevent the chronic ischemia-type injury that contributes to cognitive decline.

VTA dopamine neurons are hyperexcitable in 3xTg-AD mice due to casein kinase 2-dependent SK channel dysfunction

Alzheimer’s disease (AD) patients exhibit neuropsychiatric symptoms that extend beyond classical cognitive deficits, suggesting involvement of subcortical areas. Here, we investigated the role of midbrain dopamine (DA) neurons in AD using the amyloid + tau-driven 3xTg-AD mouse model. We found deficits in reward-based operant learning in AD mice, suggesting possible VTA DA neuron dysregulation. Physiological assessment revealed hyperexcitability and disrupted firing in DA neurons caused by reduced activity of small-conductance calcium-activated potassium (SK) channels. RNA sequencing from contents of single patch-clamped DA neurons (Patch-seq) identified up-regulation of the SK channel modulator casein kinase 2 (CK2), which we corroborated by immunohistochemical protein analysis. Pharmacological inhibition of CK2 restored SK channel activity and normal firing patterns in 3xTg-AD mice. These findings identify a mechanism of ion channel dysregulation in VTA DA neurons that could contribute to behavioral abnormalities in AD, paving the way for novel treatment strategies.

Long-term changes in transmembrane voltage after electroporation are governed by the interplay between nonselective leak current and ion channel activation

Electroporation causes a temporal increase in cell membrane permeability and leads to prolonged changes in transmembrane voltage (TMV) in both excitable and non-excitable cells. However, the mechanisms of these TMV changes remain to be fully elucidated. To this end, we monitored TMV over 30 min after exposing two different cell lines to a single 100 µs electroporation pulse using the FLIPR Membrane Potential dye. In CHO-K1 cells, which express very low levels of endogenous ion channels, membrane depolarization following pulse exposure could be explained by nonselective leak current, which persists until the membrane reseals, enabling the cells to recover their resting TMV. In U-87 MG cells, which express many different ion channels, we unexpectedly observed membrane hyperpolarization following the initial depolarization phase, but only at 33 °C and not at 25 °C. We developed a theoretical model, supported by experiments with ion channel inhibitors, which indicated that hyperpolarization could largely be attributed to the activation of calcium-activated potassium channels. Ion channel activation, coupled with changes in TMV and intracellular calcium, participates in various physiological processes, including cell proliferation, differentiation, migration, and apoptosis. Therefore, our study suggests that ion channels could present a potential target for influencing the biological response after electroporation.

Activation of BKα channel provented uremic cardiomyopathy by attenuating oxidative stress

Background: Uremic cardiomyopathy and renal fibrosis contribute to chronic kidney disease (CKD)-induced morbidity and mortality. Our previous study found that activation of the large conductance calcium-activated potassium channels (BK channels) attenuated renal fibrosis in mice (Wang et al, Kid Int, 2021). We hypothesized that upregulation of BK channels would suppress cardiac fibrosis in CKD. Methods: CKD mice were induced by 5/6 nephrectomy. To upregulation of BKa channels, BMS-191011 (10 mg/kg BW) was given by IP injection every day for 6-8 weeks. Single channel recordings were used to analyze the activation of BKa channel. Blood pressure was measured by non-invasive photoplethysmography using the tail-cuff method. Echocardiogram was used to assess cardiac function. Cultured H9C2 cardiac myoblasts were used to detect the impact of BK opener (NS1916 20mM) on fibrosis markers and oxidative stress. The mRNA expression of BKa in heart was assayed by qPCR. The Hydrogen peroxide (H2O2) was tested by Amplex Red Hydrogen Peroxide Kit. DHE (Dihydroethidium) was assayed to detect superoxide. Superoxide Dismutase (SOD), a defense of oxidative enzyme, was measured using colorimetric activity kit. Results: The expression of BKa mRNA and protein were decreased and cardiac fibrosis was increased in the heart of CKD mice. Single channel recordings showed that human uremic serum (User) attenuated the activation of BKa channel. Upregulation of BKa channel by BSM-191011 decreased the CKD-induced increase in systolic and diastolic blood pressure. Echocardiogram showed that LV end-diastolic dimension increased in 5/6Nx mice, but back to normal after BK opener treatment. Ejection fraction also improved by this treatment. Fibronectin and vimentin were also significantly increased in CKD heart, whereas BMS treatment attenuated the amount of both proteins. In cultured cardiac myoblasts, User and TGFβ increased fibronectin and collagen I, which were in a dose-dependent manner. BKa openers (NS1916) limited the User and TGF-induced upregulation of both proteins in H9C2 cells. User also significantly increased H2O2 and superoxide production in cultured H9C2 cells. Activation of BKa significantly abolished this upregulation. In addition, User and TGFβ decrease SOD production, NS1916 blocked this decline in a dose dependent manner. Conclusions: Activation of BK channel in CKD animals attenuates cardiac fibrosis likely through reduction of uremia-induced oxidative stress. This study could provide new approaches for developing therapeutic strategies for treating uremic cardiomyopathy in chronic kidney diseases. the Department of Veteran Affairs MERIT Award 5I01BX000994 to HC. 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.

A chlorzoxazone–folic acid combination improves cognitive affective decline in SCA2-58Q mice

Spinocerebellar ataxia type 2 (SCA2) is a polyglutamine disorder caused by a pathological expansion of CAG repeats in ATXN2 gene. SCA2 is accompanied by cerebellar degeneration and progressive motor decline. Cerebellar Purkinje cells (PCs) seem to be primarily affected in this disorder. The majority of the ataxia research is focused on the motor decline observed in ataxic patients and animal models of the disease. However, recent evidence from patients and ataxic mice suggests that SCA2 can also share the symptoms of the cerebellar cognitive affective syndrome. We previously reported that SCA2-58Q PC-specific transgenic mice exhibit anxiolytic behavior, decline in spatial memory, and a depressive-like state. Here we studied the effect of the activation of the small conductance calcium-activated potassium channels (SK channels) by chlorzoxazone (CHZ) combined with the folic acid (FA) on the PC firing and also motor, cognitive and affective symptoms in SCA2-58Q mice. We realized that CHZ-FA combination improved motor and cognitive decline as well as ameliorated mood alterations in SCA2-58Q mice without affecting the firing rate of their cerebellar PCs. Our results support the idea of the combination therapy for both ataxia and non-motor symptoms in ataxic mice without affecting the firing frequency of PCs.

External Hemin as an Inhibitor of Mitochondrial Large-Conductance Calcium-Activated Potassium Channel Activity.

The mitochondrial large-conductance calcium-activated potassium channel (mitoBKCa) is located in the inner mitochondrial membrane and seems to play a crucial role in cytoprotection. The mitoBKCa channel 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 domains present in the mitochondrial matrix. In the present study, we used the patch-clamp technique in the outside-out configuration to record the activity of mitoBKCa channels. This allowed for the application of channel modulators to the intermembrane-space side of the mitoBKCa. We found that hemin applied in this configuration inhibits the activity of mitoBKCa. In addition, we proved that the observed hemin effect is specific and it is not due to its interaction with the inner mitochondrial membrane. Our data suggest the existence of a new potential heme/hemin binding site in the structure of the mitoBKCa channel located on the mitochondrial intermembrane space side, which could constitute a new way for the regulation of mitoBKCa channel activity.

Cholecystokinin Octapeptide Promotes ANP Secretion through Activation of NOX4-PGC-1α-PPARα/PPARγ Signaling in Isolated Beating Rat Atria.

Atrial natriuretic peptide (ANP), a canonical cardiac hormone, is mainly secreted from atrial myocytes and is involved in the regulation of body fluid, blood pressure homeostasis, and antioxidants. Cholecystokinin (CCK) is also found in cardiomyocytes as a novel cardiac hormone and induces multiple cardiovascular regulations. However, the direct role of CCK on the atrial mechanical dynamics and ANP secretion is unclear. The current study was to investigate the effect of CCK octapeptide (CCK-8) on the regulation of atrial dynamics and ANP secretion. Experiments were performed in isolated perfused beating rat atria. ANP was measured using radioimmunoassay. The levels of hydrogen peroxide (H2O2) and arachidonic acid (AA) were determined using ELISA Kits. The levels of relative proteins and mRNA were detected by Western blot and RT-qPCR. The results showed that sulfated CCK-8 (CCK-8s) rather than desulfated CCK-8 increased the levels of phosphorylated cytosolic phospholipase A2 and AA release through activation of CCK receptors. This led to the upregulation of NADPH oxidase 4 (NOX4) expression levels and H2O2 production and played a negative inotropic effect on atrial mechanical dynamics via activation of ATP-sensitive potassium channels and large-conductance calcium-activated potassium channels. In addition, CCK-8s-induced NOX4 subsequently upregulated peroxisome proliferator-activated receptor γ (PPARγ) coactivator-1α (PGC-1α) expression levels through activation of p38 mitogen-activated protein kinase as well as the serine/threonine kinase signaling, ultimately promoting the secretion of ANP via activation of PPARα and PPARγ. In the presence of the ANP receptor inhibitor, the CCK-8-induced increase of AA release, H2O2 production, and the upregulation of NOX4 and CAT expressions was augmented but the SOD expression induced by CCK-8s was repealed. These findings indicate that CCK-8s promotes the secretion of ANP through activation of NOX4–PGC-1α–PPARα/PPARγ signaling, in which ANP is involved in resistance for NOX4 expression and ROS production and regulation of SOD expression.

SKA-31-induced activation of small-conductance calcium-activated potassium channels decreased modulation of detrusor smooth muscle function in a rat model of obesity

Increased excitability and contractility of detrusor smooth muscle (DSM) cells are associated with overactive bladder (OAB), which is often induced by obesity. Small-conductance Ca2+-activated K+ (SK) channels regulate the excitability and contractility of DSM cells. Selective pharmacological activation of SK channels attenuates hyperpolarization and the decreased relaxation effect in DSM cells in obesity-induced OAB. However, additional data are needed to confirm the regulatory effect of SK channels on the function of DSM cells in obesity-related OAB. The tested hypothesis was that activation of SK channels decreases modulation of DSM function in a rat model of obesity-related OAB. Female Sprague Dawley rats were fed a normal diet (ND) or a high-fat diet (HFD), weighed after 12weeks, and subjected to urodynamic study, patch-clamp electrophysiology, and isometric tension recording. The average body weight and incidence of OAB were increased in the HFD group. Patch-clamp studies revealed that pharmacological activation of SK channels with SKA-31 had attenuated hyperpolarization of DSM cells. In addition, isometric tension recordings indicated that SKA-31 decreased relaxation of spontaneous phasic contractions of DSM strips in the HFD group. Attenuated function of SK channels increased the excitability and contractility of DSM cells, which contributed to the occurrence of OAB, suggesting that SK channels are potential therapeutic targets for control of OAB.

Riluzole and spinocerebellar ataxia type 2: the ATRIL trial

The search for neuroprotective therapies for the spinocerebellar ataxias remains challenging because of the huge clinical and genetic heterogeneity and low prevalence of these disorders, as well as the absence of objective biomarkers for disease progression. However, efforts persist in the clinical development of gene therapies, in the search for novel drugs, and in the repurposing of neuroprotective agents already approved for neurodegenerative diseases with overlapping pathophysiology.1 This is the case for riluzole, a glutamate release inhibitor and activator of calcium-activated potassium channels that has shown evidence of efficacy in targeting glutamatergic excitotoxicity and modulating potassium channel activity in spinocerebellar ataxias.

Differential contribution of renal cytochrome P450 enzymes to kidney endothelial dysfunction and vascular oxidative stress in obesity

Arachidonic acid (AA)-derived cytochrome P450 (CYP) derivatives, epoxyeicosatrienoic acids (EETs) and 20-hidroxyeicosatetranoic acid (20-HETE), play a key role in kidney tubular and vascular functions and blood pressure. Altered metabolism of CYP epoxygenases and CYP hydroxylases has differentially been involved in the pathogenesis of metabolic disease-associated vascular complications, although the mechanisms responsible for the vascular injury are unclear. The present study aimed to assess whether obesity-induced changes in CYP enzymes may contribute to oxidative stress and endothelial dysfunction in kidney preglomerular arteries. Endothelial function and reactive oxygen species (ROS) production were assessed in interlobar arteries of obese Zucker rats (OZR) and their lean counterparts lean Zucker rats (LZR) and the effects of CYP2C and CYP4A inhibitors sulfaphenazole and HET0016, respectively, were examined on the endothelium-dependent relaxations and O2− and H2O2 levels of preglomerular arteries. Non-nitric oxide (NO) non-prostanoid endothelium-derived hyperpolarization (EDH)-type responses were preserved but resistant to the CYP epoxygenase blocker sulfaphenazole in OZR in contrast to those in LZR. Sulfaphenazole did not further inhibit reduced arterial H2O2 levels, and CYP2C11/CYP2C23 enzymes were downregulated in intrarenal arteries from OZR. Renal EDH-mediated relaxations were preserved in obese rats by the enhanced activity and expression of endothelial calcium-activated potassium channels (KCa). CYP4A blockade restored impaired NO-mediated dilatation and inhibited augmented O2− production in kidney arteries from OZR. The current data demonstrate that both decreased endothelial CYP2C11/ CYP2C23-derived vasodilator H2O2 and augmented CYP4A-derived 20-HETE contribute to endothelial dysfunction and vascular oxidative stress in obesity. CYP4A inhibitors ameliorate arterial oxidative stress and restore endothelial function which suggests its therapeutic potential for the vascular complications of obesity-associated kidney injury.

Secondhand Smoke Decreased Excitability and Altered Action Potential Characteristics of Cardiac Vagal Neurons in Mice.

Background: Secondhand smoke (SHS), a major indoor pollutant, is a significant risk factor for cardiovascular morbidity and mortality including arrhythmias and sudden cardiac death. Exposure to SHS can produce autonomic imbalance, as evidenced by reduced heart rate variability (HRV)—a clinical metric of cardiac vagal regulation. Currently, the mechanisms through which SHS changes the vagal preganglionic neuronal inputs to the heart to produce this remains unknown.Objectives: To characterize the effect of SHS on both the excitability and action potential (AP) characteristics of anatomically identified cardiac vagal neurons (CVNs) in the nucleus ambiguus and examine whether SHS alters small conductance calcium-activated potassium (SK) channel activity of these CVNs.Methods: Adult male mice were exposed to four weeks of filtered air or SHS (3 mg/m3) 6 h/day, 5 day/week. Using patch-clamp recordings on identified CVNs in brainstem slices, we determined neuronal excitability and AP characteristics with depolarizing step- and ramp-current injections.Results: Four weeks of SHS exposure reduced spiking responses to depolarizing current injections and increased AP voltage threshold in CVNs. Perfusion with apamin (20 nM) magnified these SHS-induced effects, suggesting reduced SK channel activity may serve to minimize the SHS-induced decreases in CVNs excitability. Medium afterhyperpolarization (a measurement of SK channel activity) was smaller in the SHS group, further supporting a lower SK channel activity. AP amplitude, rise rate, fast afterhyperpolarization amplitude (a measurement of voltage-gated channel activity), and decay rate were higher in the SHS group at membrane voltages more positive to 0 mV, suggesting altered inactivation properties of voltage-dependent channels underlying APs.Discussion: SHS exposure reduced neuronal excitability of CVNs with compensatory attenuation of SK channel activity and altered AP characteristics. Neuroplasticity of CVNs could blunt regulatory cardiac vagal signaling and contribute to the cardiovascular consequences associated with SHS exposure, including reduced HRV.

Trimethylamine-N-oxide (TMAO) Selectively Disrupts Endothelium-Dependent Hyperpolarization-Type Relaxations in a Time-Dependent Manner in Rat Superior Mesenteric Artery.

The vascular action of trimethylamine-N-oxide (TMAO)-the gut microbiota-derived metabolite-in contributing cardiovascular disease is a controversial topic. A recent study has shown that acute exposure of TMAO at moderate concentrations inhibits endothelium-dependent hyperpolarization (EDH)-type relaxations selectively in rat isolated femoral arteries, but not in mesenteric arteries. Here we determined the efficacy of higher TMAO concentrations with longer exposure times on vascular reactivity in rat isolated superior mesenteric arteries. Acetylcholine-induced EDH-type relaxations were examined before and after incubation with TMAO (0.1-10 mM) at increasing exposure times (1-24 h). One- and 4-h-incubations with TMAO at 0.1-3 mM did not cause any change in EDH-type relaxations. However, when the incubation time was increased to 24 h, responses to acetylcholine were reduced in arteries incubated with 1-3 mM TMAO. In addition, at higher TMAO concentration (10 mM) the decrease in EDH relaxations could be detected both in 4-h- and 24-h-incubations. The EDH-relaxations were preserved in rings incubated with 10 mM TMAO for 24 h in the presence of SKA-31 (10 µM), the small (SKCa)- and intermediate (IKCa)-conductance calcium-activated potassium channel activator. Contractile responses to phenylephrine increased in arteries exposed to 10 mM TMAO for 24 h. Interestingly, nitric oxide (NO)-mediated relaxations remained unchanged in arteries treated for 24 h at any TMAO concentration. Our study revealed that TMAO selectively disrupted EDH-type relaxations time-dependently without interfering with NO-induced vasodilation in rat isolated mesenteric arteries. Disruption of these relaxations may help explain the causal role of elevated TMAO levels in certain vascular diseases.

Ginkgo biloba L. Prevents Hypobaric Hypoxia-Induced Spatial Memory Deficit Through Small Conductance Calcium-Activated Potassium Channel Inhibition: The Role of ERK/CaMKII/CREB Signaling.

Hypobaric hypoxia (HH) is a stressful condition, which is more common at high altitudes and can impair cognitive functions. Ginkgo biloba L. leaf extract (GBE) is widely used as herbal medicine against different disorders. Its ability to improve cognitive functions, reduce oxidative stress, and promote cell survival makes it a putative therapeutic candidate against HH. The present study has been designed to explore the effect of GBE on HH-induced neurodegeneration and memory impairment as well as possible signaling mechanisms involved. 220–250 gm (approximately 6- to 8-week-old) Sprague Dawley rats were randomly divided into different groups. GBE was orally administered to respective groups at a dose of 100 mg/kg/day throughout the HH exposure, i.e., 14 days. Memory testing was performed followed by hippocampus isolation for further processing of different molecular and morphological parameters related to cognition. The results indicated that GBE ameliorates HH-induced memory impairment and oxidative damage and reduces apoptosis. Moreover, GBE modulates the activity of the small conductance calcium-activated potassium channels, which further reduces glutamate excitotoxicity and apoptosis. The exploration of the downstream signaling pathway demonstrated that GBE administration prevents HH-induced small conductance calcium-activated potassium channel activation, and that initiates pro-survival machinery by activating extracellular signal–regulated kinase (ERK)/calmodulin-dependent protein kinase II (CaMKII) and the cAMP response element–binding protein (CREB) signaling pathway. In summary, the current study demonstrates the beneficial effect of GBE on conditions like HH and provides various therapeutic targets involved in the mechanism of action of GBE-mediated neuroprotection.

Reversal of behavioural phenotype by the cannabinoid-like compound VSN16R in fragile X syndrome mice.

Fragile X syndrome is the most common inherited intellectual disability and mono-genetic cause of autism spectrum disorder. It is a neurodevelopmental condition occurring due to a CGG trinucleotide expansion in the FMR1 gene. Polymorphisms and variants in large-conductance calcium-activated potassium channels are increasingly linked to intellectual disability and loss of FMR protein causes reduced large-conductance calcium-activated potassium channel activity leading to abnormalities in synapse function. Using the cannabinoid-like large-conductance calcium-activated potassium channel activator VSN16R we rescued behavioural deficits such as repetitive behaviour, hippocampal dependent tests of daily living, hyperactivity and memory in a mouse model of fragile X syndrome. VSN16R has been shown to be safe in a phase 1 study in healthy volunteers and in a phase 2 study in patients with multiple sclerosis with high oral bioavailability and no serious adverse effects reported. VSN16R could therefore be directly utilized in a fragile X syndrome clinical study. Moreover, VSN16R showed no evidence of tolerance, which strongly suggests that chronic VSN16R may have great therapeutic value for fragile X syndrome and autism spectrum disorder. This study provides new insight into the pathophysiology of fragile X syndrome and identifies a new pathway for drug intervention for this debilitating disorder.

Inhibition of mitochondrial reactive oxygen species improves coronary endothelial function after cardioplegic hypoxia/reoxygenation

ObjectiveCardioplegic ischemia–reperfusion and diabetes mellitus are correlated with coronary endothelial dysfunction and inactivation of small conductance calcium-activated potassium channels. Increased reactive oxidative species, such as mitochondrial reactive oxidative species, may contribute to oxidative injury. Thus, we hypothesized that inhibition of mitochondrial reactive oxidative species may protect coronary small conductance calcium-activated potassium channels and endothelial function against cardioplegic ischemia–reperfusion-induced injury. MethodsSmall coronary arteries and endothelial cells from the hearts of mice with and without diabetes mellitus were isolated and examined by using a cardioplegic hypoxia and reoxygenation model to determine whether the mitochondria-targeted antioxidant Mito-Tempo could protect against coronary endothelial and small conductance calcium-activated potassium channel dysfunction. The microvessels or mouse heart endothelial cells were treated with or without Mito-Tempo (0-10 μM) 5 minutes before and during cardioplegic hypoxia and reoxygenation. Microvascular function was assessed in vitro by vessel myography. K+ currents of mouse heart endothelial cells were measured by whole-cell patch clamp. The levels of intracellular cytosolic free calcium (Ca2+) concentration, mitochondrial reactive oxidative species, and small conductance calcium-activated potassium protein expression of mouse heart endothelial cells were measured by Rhod-2 fluorescence staining, MitoSox, and Western blotting, respectively. ResultsCardioplegic hypoxia and reoxygenation significantly attenuated endothelial small conductance calcium-activated potassium channel activity, caused calcium overload, and increased mitochondrial reactive oxidative species of mouse heart endothelial cells in both the nondiabetic and diabetes mellitus groups. In addition, treating mouse heart endothelial cells with Mito-Tempo (10 μM) reduced cardioplegic hypoxia and reoxygenation-induced Ca2+ and mitochondrial reactive oxidative species overload in both the nondiabetic and diabetes mellitus groups, respectively (P < .05). Treatment with Mito-Tempo (10 μM) significantly enhanced coronary relaxation responses to adenosine 5′-diphosphate and NS309 (P < .05), and endothelial small conductance calcium-activated potassium channel currents in both the nondiabetic and diabetes mellitus groups (P < .05). ConclusionsAdministration of Mito-Tempo improves endothelial function and small conductance calcium-activated potassium channel activity, which may contribute to its enhancement of endothelium-dependent vasorelaxation after cardioplegic hypoxia and reoxygenation.

Single channel properties of mitochondrial large conductance potassium channel formed by BK-VEDEC splice variant

The activation of mitochondrial large conductance calcium-activated potassium (mitoBKCa) channels increases cell survival during ischemia/reperfusion injury of cardiac cells. The basic biophysical and pharmacological properties of mitoBKCa correspond to the properties of the BKCa channels from the plasma membrane. It has been suggested that the VEDEC splice variant of the KCNMA1 gene product encoding plasma membrane BKCa is targeted toward mitochondria. However there has been no direct evidence that this protein forms a functional channel in mitochondria. In our study, we used HEK293T cells to express the VEDEC splice variant and observed channel activity in mitochondria using the mitoplast patch-clamp technique. For the first time, we found that transient expression with the VEDEC isoform resulted in channel activity with the conductance of 290 ± 3 pS. The channel was voltage-dependent and activated by calcium ions. Moreover, the activity of the channel was stimulated by the potassium channel opener NS11021 and inhibited by hemin and paxilline, which are known BKCa channel blockers. Immunofluorescence experiments confirmed the partial colocalization of the channel within the mitochondria. From these results, we conclude that the VEDEC isoform of the BKCa channel forms a functional channel in the inner mitochondrial membrane. Additionally, our data show that HEK293T cells are a promising experimental model for expression and electrophysiological studies of mitochondrial potassium channels.

Hippocampal Vascular Dysfunction and Impaired Memory in a Neuroendocrine Model of Stress and Hypertension

The hippocampus is a deep brain structure critically involved in learning and memory that is highly susceptible to hypoxic/ischemic injury. Hypertension and psychological stress are major risk factors of dementia; however, their effects on the vasculature of the cognition-centric hippocampus remain unclear. This study investigated hippocampal vascular function and memory in a novel model of neuroendocrine-derived stress and hypertension. This model is induced via vector mediated brain-derived neurotrophic factor (BDNF) overexpression in the paraventricular nucleus of the hypothalamus (PVN) that causes chronic stimulation of the major hypothalamic stress pathways (e.g. hypothalamic-pituitary-adrenal axis), leading to significant long-term elevation of blood pressure. We hypothesized that function of the arterioles supplying the hippocampus and memory would be impaired in this BDNF-PVN model of chronic stress and hypertension. Potential sex differences in the susceptibility of the hippocampus to dysfunction were also investigated. Male and female Sprague Dawley rats received bilateral PVN injections of AAV2 vectors expressing BDNF or GFP for control (n=6-8/group) at 8 weeks of age. Four weeks later, an object recognition task was used to quantify the percent of time spent investigating a novel object as a measure of hippocampal-dependent long-term memory function. Hippocampal arterioles (HAs) from the same rats were then studied isolated and pressurized in an arteriograph chamber. Vasodilator responses of HAs to mediators of functional hyperemia were measured, including small- and intermediate-conductance calcium-activated potassium (SK/IK) channel activation via NS309, and activation of inward rectifier potassium (KIR) channels by increasing extracellular K+ from 3-15mM. Memory function was significantly impaired by PVN-BDNF treatment (two-way ANOVA, Tukey's post hoc test, F1,22=16.53; p=0.001), but sex had no effect (F1,22=1.16; p=0.29). Object recognition was lower in both male and female PVN-BDNF rats that spent 49±3% and 52±6% of the time with the novel object, compared to control male and female rats that spent 66±3% and 72±4% of the time investigating the novel object. HAs from male and female PVN-BDNF rats dilated significantly less to NS309 than controls (F1,20=12.35; p=0.002), suggesting endothelial damage. Surprisingly, activation of KIR channels with 15mM K+ caused a 3-5% vasoconstriction of HAs from both male and female PVN-BDNF rats that was in striking contrast to the 20-30% vasodilation that occurred in response to KIR channel activation in HAs from control rats (F1,19=24.90; p<0.0001). These data show that chronic neuroendocrine stress and hypertension caused hippocampal vascular dysfunction and impaired memory similarly in male and female rats. The blunted vasodilation of HAs from PVN-BDNF rats suggests functional hyperemia may be impaired in the hippocampus that could lead to neuronal dysfunction, memory loss and dementia.

Homocysteine alters vasoreactivity of human internal mammary artery by affecting the KCa channel family.

BackgroundHyperhomocysteinemia is an independent risk factor for atherosclerotic heart disease. We previously demonstrated that disruption of calcium-activated potassium (KCa) channel activity is involved in homocysteine-induced dilatory dysfunction of porcine coronary arteries. Recently we reported that the KCa channel family, including large-, intermediate-, and small-conductance KCa (BKCa, IKCa, and SKCa) subtypes, are abundantly expressed in human internal mammary artery (IMA). In this study, we further investigated whether homocysteine affects the expression and functionality of the KCa channel family in this commonly used graft for coronary artery bypass surgery (CABG).MethodsResidual IMA segments obtained from patients undergoing CABG were studied in a myograph for the role of KCa subtypes in both vasorelaxation and vasoconstriction. The expression and distribution of KCa subtypes were detected by Western blot and immunohistochemistry.ResultsBoth the BKCa channel activator NS1619 and the IKCa/SKCa channel activator NS309 evoked significant IMA relaxation. Homocysteine exposure suppressed NS1619-induced relaxation whereas showed no influence on NS309-induced response. Blockade of BKCa but not IKCa and SKCa subtypes significantly suppressed acetylcholine-induced relaxation and enhanced U46619-induced contraction. Homocysteine compromised the vasodilating activity of the BKCa subtype in IMA, associated with a lowered protein level of the BKCa β1-subunit. Homocysteine potentiated the role of IKCa and SKCa subtypes in mediating endothelium-dependent relaxation without affecting the expression of these channels.ConclusionsHomocysteine reduces the expression of BKCa β1-subunit and compromises the vasodilating activity of BKCa channels in IMA. Unlike BKCa, IKCa and SKCa subtypes are unessential for IMA vasoregulation, whereas the loss of BKCa functionality in hyperhomocysteinemia enhances the role of IKCa and SKCa subtypes in mediating endothelial dilator function. Targeting BKCa channels may form a strategy to improve the postoperative graft performance in CABG patients with hyperhomocysteinemia who receive IMA grafting.

Identification and Characterization of a Novel Large-Conductance Calcium-Activated Potassium Channel Activator, CTIBD, and Its Relaxation Effect on Urinary Bladder Smooth Muscle.

The large-conductance calcium-activated potassium channel (BKCa channel) is expressed on various tissues and is involved in smooth muscle relaxation. The channel is highly expressed on urinary bladder smooth muscle cells and regulates the repolarization phase of the spontaneous action potentials that control muscle contraction. To discover novel chemical activators of the BKCa channel, we screened a chemical library containing 8364 chemical compounds using a cell-based fluorescence assay. A chemical compound containing an isoxazolyl benzene skeleton (compound 1) was identified as a potent activator of the BKCa channel and was structurally optimized through a structure-activity relationship study to obtain 4-(4-(4-chlorophenyl)-3-(trifluoromethyl)isoxazol-5-yl)benzene-1,3-diol (CTIBD). When CTIBD was applied to the treated extracellular side of the channel, the conductance-voltage relationship of the channel shifted toward a negative value, and the maximum conductance increased in a concentration-dependent manner. CTIBD altered the gating kinetics of the channel by dramatically slowing channel closing without effecting channel opening. The effects of CTIBD on bladder muscle relaxation and micturition function were tested in rat tissue and in vivo. CTIBD concentration-dependently reduced acetylcholine-induced contraction of urinary bladder smooth muscle strips. In an acetic acid-induced overactive bladder (OAB) model, intraperitoneal injection of 20 mg/kg CTIBD effectively restored frequent voiding contraction and lowered voiding volume without affecting other bladder function parameters. Thus, our results indicate that CTIBD and its derivatives are novel chemical activators of the bladder BKCa channel and potential candidates for OAB therapeutics. SIGNIFICANCE STATEMENT: The novel BKCa channel activator CTIBD was identified and characterized in this study. CTIBD directly activates the BKCa channel and relaxes urinary bladder smooth muscle of rat, so CTIBD can be a potential candidate for overactive bladder therapeutics.

In vivo analysis of the spontaneous firing of cerebellar Purkinje cells in awake transgenic mice that model spinocerebellar ataxia type 2

Cerebellar Purkinje cells (PCs) fire spontaneously in a tonic mode, although the precision of this pacemaking activity is disturbed in many abnormal conditions involving cerebellar atrophy, such as many spinocerebellar ataxias (SCAs). In our previous studies we used the single-unit extracellular recording method to analyze spontaneous PC firing in vivo in the anesthetized SCA2-58Q transgenic mice. We realized that PCs from aging SCA2-58Q mice fire much less regularly compared to PCs from their wild type (WT) littermates and this abnormal activity can be reversed with an intraperitoneal (i. p.) injection of SK channel-positive modulator chlorzoxazone (CHZ). Here we used the same single-unit extracellular recording method to analyze the spontaneous firing in vivo in awake SCA2-58Q transgenic mice. For this purpose, we used the Mobile HomeCage (Neurotar, Finland) floating platform to immobilize the experimental animal's head during the recording sessions. We discovered that generally PCs from awake animals fired much more frequently and much less regularly than previously observed PCs from anesthetized animals. In vivo recordings from awake SCA2/WT mice revealed that complex spikes, which are generated by PCs in reply to the excitation coming by climbing fibers, as well as simple spikes, were much less frequent in SCA2 mice compared to their WT littermates. To test the effect of the SK channel positive modulation on the PCs firing activity in awake SCA2 mice and also the effect on their motor coordination, we started the CHZ trial in these mice. We discovered that the long-term i. p. injections of CHZ did not affect the spike generation in SCA2-58Q mice, however, they did recover the precision of this spontaneous pacemaking activity. Furthermore, we also showed that treatment with CHZ alleviated the age-dependent motor impairment in SCA2-58Q mice. We propose that the lack of precision in PC spike generation might be a key cause for the progression of ataxic symptoms in different SCAs and that the activation of calcium-activated potassium channels, including SK channels, can be used as a potential way to treat SCAs on the physiological level of the disease.