A selective inhibitor of the sperm-specific potassium channel SLO3 impairs human sperm function.

To fertilize an oocyte, the membrane potential of both mouse and human sperm must hyperpolarize (become more negative inside). Determining the molecular mechanisms underlying this hyperpolarization is vital for developing new contraceptive methods and detecting causes of idiopathic male infertility. In mouse sperm, hyperpolarization is caused by activation of the sperm-specific potassium (K+) channel SLO3 [C. M. Santi et al., FEBS Lett. 584, 1041-1046 (2010)]. In human sperm, it has long been unclear whether hyperpolarization depends on SLO3 or the ubiquitous K+ channel SLO1 [N. Mannowetz, N. M. Naidoo, S. A. S. Choo, J. F. Smith, P. V. Lishko, Elife 2, e01009 (2013), C. Brenker et al., Elife 3, e01438 (2014), and S. A. Mansell, S. J. Publicover, C. L. R. Barratt, S. M. Wilson, Mol. Hum. Reprod. 20, 392-408 (2014)]. In this work, we identified the first selective inhibitor for human SLO3-VU0546110-and showed that it completely blocked heterologous SLO3 currents and endogenous K+ currents in human sperm. This compound also prevented sperm from hyperpolarizing and undergoing hyperactivated motility and induced acrosome reaction, which are necessary to fertilize an egg. We conclude that SLO3 is the sole K+ channel responsible for hyperpolarization and significantly contributes to the fertilizing ability of human sperm. Moreover, SLO3 is a good candidate for contraceptive development, and mutation of this gene is a possible cause of idiopathic male infertility.

Downregulation of large-conductance Ca2+-activated K+ channels in human umbilical arterial smooth muscle cells in gestational diabetes mellitus.

AIMS: We investigated the changes in large-conductance Ca2+-activated K+ (BKCa) channels from human umbilical arterial smooth muscle cells experiencing gestational diabetes mellitus (GDM). MAIN METHODS: Whole-cell patch-clamp technique, arterial tone measurement, RT-PCR, Quantitative real-time PCR, western blot were performed in human umbilical arterial smooth muscle cells. KEY FINDINGS: Whole-cell BKCa current density was decreased in the GDM group compared with the normal group. The vasorelaxant effects of the synthetic BKCa channel activator NS-1619 (10 µM) were impaired in the GDM group compared with the normal group. Reverse-transcription polymerase chain reaction (RT-PCR), real-time RT-PCR, and western blot analyses suggested that the mRNA, total RNA, and protein expression levels of the BKCa channel were decreased in the GDM group relative to the normal group. In addition, the expression levels of protein kinase A and protein kinase G, which regulate BKCa channel activity, remained unchanged between the groups. Applying the BKCa channel inhibitor paxilline (10 µM) induced vasoconstriction and membrane depolarization of isolated umbilical arteries in the normal group but showed less of an effect on umbilical arteries in the GDM group. SIGNIFICANCE: Our results demonstrate for the first time impaired BKCa current and BKCa channel-induced vasorelaxation activities that were not caused by impaired BKCa channel-regulated protein kinases, but by decreased expression of the BKCa channels, in the umbilical arteries of GDM patients.

Cholesterol Inhibition of Slo1 Channels Is Calcium-Dependent and Can Be Mediated by Either High-Affinity Calcium-Sensing Site in the Slo1 Cytosolic Tail.

Calcium- and voltage-gated K+ channels of large conductance (BKs) are expressed in the cell membranes of all excitable tissues. Currents mediated by BK channel-forming slo1 homotetramers are consistently inhibited by increases in membrane cholesterol (CLR). The molecular mechanisms leading to this CLR action, however, remain unknown. Slo1 channels are activated by increases in calcium (Ca2+) nearby Ca2+-recognition sites in the slo1 cytosolic tail: one high-affinity and one low-affinity site locate to the regulator of conductance for K+ (RCK) 1 domain, whereas another high-affinity site locates within the RCK2 domain. Here, we first evaluated the crosstalking between Ca2+ and CLR on the function of slo1 (cbv1 isoform) channels reconstituted into planar lipid bilayers. CLR robustly reduced channel open probability while barely decreasing unitary current amplitude, with CLR maximal effects being observed at 10-30 µM internal Ca2+ CLR actions were not only modulated by internal Ca2+ levels but also disappeared in absence of this divalent. Moreover, in absence of Ca2+, BK channel-activating concentrations of magnesium (10 mM) did not support CLR action. Next, we evaluated CLR actions on channels where the different Ca2+-sensing sites present in the slo1 cytosolic domain became nonfunctional via mutagenesis. CLR still reduced the activity of low-affinity Ca2+ (RCK1:E379A, E404A) mutants. In contrast, CLR became inefficacious when both high-affinity Ca2+ sites were mutated (RCK1:D367A,D372A and RCK2:D899N,D900N,D901N,D902N,D903N), yet still was able to decrease the activity of each high-affinity site mutant. Therefore, BK channel inhibition by CLR selectively requires optimal levels of Ca2+ being recognized by either of the slo1 high-affinity Ca2+-sensing sites. SIGNIFICANCE STATEMENT: Results reveal that inhibition of calcium/voltage-gated K+ channel of large conductance (BK) (slo1) channels by membrane cholesterol requires a physiologically range of internal calcium (Ca2+) and is selectively linked to the two high-affinity Ca2+-sensing sites located in the cytosolic tail domain, which underscores that Ca2+ and cholesterol actions are allosterically coupled to the channel gate. Cholesterol modification of BK channel activity likely contributes to disruption of normal physiology by common health conditions that are triggered by disruption of cholesterol homeostasis.

KCa channel blockers increase effectiveness of the EGF receptor TK inhibitor erlotinib in non-small cell lung cancer cells (A549).

Non-small cell lung cancer (NSCLC) has a poor prognosis with a 5 year survival rate of only ~ 10%. Important driver mutations underlying NSCLC affect the epidermal growth factor receptor (EGFR) causing the constitutive activation of its tyrosine kinase domain. There are efficient EGFR tyrosine kinase inhibitors (TKIs), but patients develop inevitably a resistance against these drugs. On the other hand, KCa3.1 channels contribute to NSCLC progression so that elevated KCa3.1 expression is a strong predictor of poor NSCLC patient prognosis. The present study tests whether blocking KCa3.1 channels increases the sensitivity of NSCLC cells towards the EGFR TKI erlotinib and overcomes drug resistance. mRNA expression of KCa3.1 channels in erlotinib-sensitive and -resistant NSCLC cells was analysed in datasets from Gene expression omnibus (GEO) and ArrayExpress. We assessed proliferation and migration of NSCLC cells. These (live cell-imaging) experiments were complemented by patch clamp experiments and Western blot analyses. We identified three out of four datasets comparing erlotinib-sensitive and -resistant NSCLC cells which revealed an altered expression of KCa3.1 mRNA in erlotinib-resistant NSCLC cells. Therefore, we evaluated the combined effect of erlotinib and the KCa3.1 channel inhibition with sencapoc. Erlotinib elicits a dose-dependent inhibition of migration and proliferation of NSCLC cells. The simultaneous application of the KCa3.1 channel blocker senicapoc increases the sensitivity towards a low dose of erlotinib (300 nmol/L) which by itself has no effect on migration and proliferation. Partial erlotinib resistance can be overcome by KCa3.1 channel blockade. The sensitivity towards erlotinib as well as the potentiating effect of KCa3.1 blockade is further increased by mimicking hypoxia. Our results suggest that KCa3.1 channel blockade may constitute a therapeutic concept for treating NSCLC and overcome EGFR TKI resistance. We propose that this is due to complementary mechanisms of action of both blockers.

IP3R-mediated activation of BK channels contributes to mGluR5-induced protection against spinal cord ischemia-reperfusion injury.

Spinal cord ischemia-reperfusion injury (SCIRI) can cause dramatic neuron loss and lead to paraplegia in patients. In this research, the role of mGluR5, a member of the metabotropic glutamate receptors (mGluRs) family, was investigated both in vitro and in vivo to explore a possible method to treat this complication. In vitro experiment, after activating mGluR5 via pretreating cells with (RS)-2-Chloro-5-hydroxyphenylglycine (CHPG) and 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl) benzamide (CDPPB), excitotoxicity induced by glutamate (Glu) was attenuated in primary spinal cord neurons, evidenced by higher neuron viability, decreased lactate dehydrogenase (LDH) release and less detected TUNEL-positive cells. According to Western Blot (WB) results, Glu treatment resulted in a high level of large-conductance Ca2+- and voltage-activated K+ (BK) channels, with activation relying on the mGluR5-IP3R (inositol triphosphate) pathway. In vivo part, a rat model of SCIRI was built to further investigate the role of mGluR5. After pretreating them with CHPG and CDPPB, the rats showed markedly lower spinal water content, attenuated motor neuron injury in the spinal cord of L4 segments, and better neurological function. This effect could be partially reversed by paxilline, a blocker of BK channels. In addition, activating BK channels alone using specific openers: NS1619 or NS11021 can protect spinal cord neurons from injury induced by either SCIRI or Glu. In conclusion, in this research, we proved that mGluR5 exerts a protective role in SCIRI, and this effect partially works via IP3R-mediated activation of BK channels.

BK channel-forming slo1 proteins mediate the brain artery constriction evoked by the neurosteroid pregnenolone.

Pregnenolone is a neurosteroid that modulates glial growth and differentiation, neuronal firing, and several brain functions, these effects being attributed to pregnenolone actions on the neurons and glial cells themselves. Despite the vital role of the cerebral circulation for brain function and the fact that pregnenolone is a vasoactive agent, pregnenolone action on brain arteries remain unknown. Here, we obtained in vivo concentration response curves to pregnenolone on middle cerebral artery (MCA) diameter in anesthetized male and female C57BL/6J mice. In both male and female animals, pregnenolone (1 nM-100 µM) constricted MCA in a concentration-dependent manner, its maximal effect reaching ~22-35% decrease in diameter. Pregnenolone action was replicated in intact and de-endothelialized, in vitro pressurized MCA segments with pregnenolone evoking similar constriction in intact and de-endothelialized MCA. Neurosteroid action was abolished by 1 µM paxilline, a selective blocker of Ca2+ - and voltage-gated K+ channels of large conductance (BK). Cell-attached, patch-clamp recordings on freshly isolated smooth muscle cells from mouse MCAs demonstrated that pregnenolone at concentrations that constricted MCAs in vitro and in vivo (10 µM), reduced BK activity (NPo), with an average decrease in NPo reaching 24.2%. The concentration-dependence of pregnenolone constriction of brain arteries and inhibition of BK activity in intact cells were paralleled by data obtained in cell-free, inside-out patches, with maximal inhibition reached at 10 µM pregnenolone. MCA smooth muscle BKs include channel-forming α (slo1 proteins) and regulatory ß1 subunits, encoded by KCNMA1 and KCNMB1, respectively. However, pregnenolone-driven decrease in NPo was still evident in MCA myocytes from KCNMB1-/- mice. Following reconstitution of slo1 channels into artificial, binary phospholipid bilayers, 10 µM pregnenolone evoked slo1 NPo inhibition which was similar to that seen in native membranes. Lastly, pregnenolone failed to constrict MCA from KCNMA1-/- mice. In conclusion, pregnenolone constricts MCA independently of neuronal, glial, endothelial and circulating factors, as well as of cell integrity, organelles, complex membrane cytoarchitecture, and the continuous presence of cytosolic signals. Rather, this action involves direct inhibition of SM BK channels, which does not require ß1 subunits but is mediated through direct sensing of the neurosteroid by the channel-forming α subunit.

BK Channel Regulation of Afterpotentials and Burst Firing in Cerebellar Purkinje Neurons.

BK calcium-activated potassium channels have complex kinetics because they are activated by both voltage and cytoplasmic calcium. The timing of BK activation and deactivation during action potentials determines their functional role in regulating firing patterns but is difficult to predict a priori. We used action potential clamp to characterize the kinetics of voltage-dependent calcium current and BK current during action potentials in Purkinje neurons from mice of both sexes, using acutely dissociated neurons that enabled rapid voltage clamp at 37°C. With both depolarizing voltage steps and action potential waveforms, BK current was entirely dependent on calcium entry through voltage-dependent calcium channels. With voltage steps, BK current greatly outweighed the triggering calcium current, with only a brief, small net inward calcium current before Ca-activated BK current dominated the total Ca-dependent current. During action potential waveforms, although BK current activated with only a short (∼100 µs) delay after calcium current, the two currents were largely separated, with calcium current flowing during the falling phase of the action potential and most BK current flowing over several milliseconds after repolarization. Step depolarizations activated both an iberiotoxin-sensitive BK component with rapid activation and deactivation kinetics and a slower-gating iberiotoxin-resistant component. During action potential firing, however, almost all BK current came from the faster-gating iberiotoxin-sensitive channels, even during bursts of action potentials. Inhibiting BK current had little effect on action potential width or a fast afterhyperpolarization but converted a medium afterhyperpolarization to an afterdepolarization and could convert tonic firing of single action potentials to burst firing.SIGNIFICANCE STATEMENT BK calcium-activated potassium channels are widely expressed in central neurons. Altered function of BK channels is associated with epilepsy and other neuronal disorders, including cerebellar ataxia. The functional role of BK in regulating neuronal firing patterns is highly dependent on the context of other channels and varies widely among different types of neurons. Most commonly, BK channels are activated during action potentials and help produce a fast afterhyperpolarization. We find that in Purkinje neurons BK current flows primarily after the fast afterhyperpolarization and helps to prevent a later afterdepolarization from producing rapid burst firing, enabling typical regular tonic firing.

Cholesterol antagonism of alcohol inhibition of smooth muscle BK channel requires cell integrity and involves a protein kinase C-dependent mechanism(s).

Alcohol constricts cerebral arteries via inhibition of voltage/calcium-gated, large conductance potassium (BK) channels in vascular myocytes. Using a rat model of high-cholesterol (high-CLR) diet and CLR enrichment of cerebral arteries in vitro, we recently showed that CLR protected against alcohol-induced constriction of cerebral arteries. The subcellular mechanism(s) underlying CLR protection against alcohol-induced constriction of the artery is unclear. Here we use a rat model of high-CLR diet and patch-clamp recording of BK channels in inside-out patches from cerebral artery myocytes to demonstrate that this diet antagonizes inhibition of BK currents by 50 mM ethanol. High-CLR-driven protection against alcohol inhibition of BK currents is reversed following CLR depletion in vitro. Similar to CLR accumulation in vivo, pre-incubation of arterial myocytes from normocholesterolemic rats in CLR-enriching media in vitro protects against alcohol-induced inhibition of BK current. However, application of CLR-enriching media to cell-free membrane patches does not protect against the alcohol effect. These different outcomes point to the involvement of cell signaling in CLR-alcohol interaction on BK channels. Incubation of myocytes with the PKC activators phorbol 12-myristate 13-acetate or 1,2-dioctanoyl-sn-glycerol, but not with the PKC inhibitor Gouml 6983, prior to patch excision precludes CLR enrichment from antagonizing alcohol action. Thus, PKC activation either disables the CLR target(s) or competes with elevated CLR. Favoring the latter possibility, 1,2-dioctanoyl-sn-glycerol protects against alcohol-induced inhibition of BK currents in patches from myocytes with naïve CLR. Our findings document that CLR antagonism of alcohol-induced BK channel inhibition requires cell integrity and is enabled by a PKC-dependent mechanism(s).

Preclinical pharmacological in vitro investigations on low chloride conductance myotonia: effects of potassium regulation.

In myotonia, reduced Cl- conductance of the mutated ClC-1 channels causes hindered muscle relaxation after forceful voluntary contraction due to muscle membrane hyperexcitability. Repetitive contraction temporarily decreases myotonia, a phenomena called "warm up." The underlying mechanism for the reduction of hyperexcitability in warm-up is currently unknown. Since potassium displacement is known to reduce excitability in, for example, muscle fatigue, we characterized the role of potassium in native myotonia congenita (MC) muscle. Muscle specimens of ADR mice (an animal model for low gCl- conductance myotonia) were exposed to increasing K+ concentrations. To characterize functional effects of potassium ion current, the muscle of ADR mice was exposed to agonists and antagonists of the big conductance Ca2+-activated K+ channel (BK) and the voltage-gated Kv7 channel. Effects were monitored by functional force and membrane potential measurements. By increasing [K+]0 to 5 mM, the warm-up phenomena started earlier and at [K+]0 7 mM only weak myotonia was detected. The increase of [K+]0 caused a sustained membrane depolarization accompanied with a reduction of myotonic bursts in ADR mice. Retigabine, a Kv7.2-Kv7.5 activator, dose-dependently reduced relaxation deficit of ADR myotonic muscle contraction and promoted the warm-up phenomena. In vitro results of this study suggest that increasing potassium conductivity via activation of voltage-gated potassium channels enhanced the warm-up phenomena, thereby offering a potential therapeutic treatment option for myotonia congenita.

Modulation of TRPV4 and BKCa for treatment of brain diseases.

As a member of transient receptor potential family, the transient receptor potential vanilloid 4 (TRPV4) is a kind of nonselective calcium-permeable cation channel, which belongs to non-voltage gated Ca2+ channel. Large-conductance Ca2+-activated K+ channel (BKCa) represents a unique superfamily of Ca2+-activated K+ channel (KCa) that is both voltage and intracellular Ca2+ dependent. Not surprisingly, aberrant function of either TRPV4 or BKCa in neurons has been associated with brain disorders, such as Alzheimer's disease, cerebral ischemia, brain tumor, epilepsy, as well as headache. In these diseases, vascular dysfunction is a common characteristic. Notably, endothelial and smooth muscle TRPV4 can mediate BKCa to regulate cerebral blood flow and pressure. Therefore, in this review, we not only discuss the diverse functions of TRPV4 and BKCa in neurons to integrate relative signaling pathways in the context of cerebral physiological and pathological situations respectively, but also reveal the relationship between TRPV4 and BKCa in regulation of cerebral vascular tone as an etiologic factor. Based on these analyses, this review demonstrates the effective mechanisms of compounds targeting these two channels, which may be potential therapeutic strategies for diseases in the brain.

BK potassium currents contribute differently to action potential waveform and firing rate as rat hippocampal neurons mature in the first postnatal week.

The large-conductance calcium-activated potassium (BK) channel is a critical regulator of neuronal action potential firing and follows two distinct trends in early postnatal development: an increase in total expression and a shift from the faster activating STREX isoform to the slower ZERO isoform. We analyzed the functional consequences of developmental trends in BK channel expression in hippocampal neurons isolated from neonatal rats aged 1 to 7 days. Following overnight cultures, action potentials and currents were recorded using whole cell patch-clamp electrophysiology. These neurons undergo a steady increase in excitability during this time, and the effect of blockade of BK channel activity with 100 nM iberiotoxin changes as the neurons mature. BK currents contribute significantly more to total potassium current and single action potentials in neurons of 1-day old rats (with BK blockade extending action potential duration by 0.46 ± 0.12 ms) than in those of 7-day old rats (with BK blockade extending action potential duration by 0.17 ± 0.05 ms). BK currents contribute consistently to maintain firing rates in neurons of 1-day old rats throughout extended action potential firing; BK blockade evenly depresses firing frequency across action potential trains. In neurons from 7-day old rats, BK blockade initially increases firing frequency and then progressively decreases frequency as firing continues, ultimately depressing neuronal firing rates to a greater extent than in the neurons from 1-day-old animals. These results are consistent with a transition from low expression of a fast-activating BK isoform (STREX) to high expression of a slower activating isoform (ZERO).NEW & NOTEWORTHY This work describes the early developmental trends of large-conductance calcium-activated potassium (BK) channel activity. Early developmental trends in expression of BK channels, both total expression and relative isoform expression, have been previously reported, but little work describes the effect of these changes in expression patterns on excitability. Here, we show that early changes in BK channel expression patterns lead to changes in the role of BK channels in determining the action potential waveform and neuronal excitability.

Effects of timing of artificial insemination and treatment of semen with a Slo3 potassium channel blocker on fertility of dairy heifers subjected to the 5-day CIDR-Synch protocol.

Two experiments were conducted to evaluate the effects of the timing of artificial insemination (AI) and incorporation of the Slo3 K+ channel blocker 4-(4-chlorophenyl)butyl-diethyl-heptylammonium to semen extender (CSE) on pregnancy per AI (P/AI) and pregnancy loss in dairy heifers. In experiment 1, Holstein heifers were subjected to the 5-d CIDR-Synch protocol: d -8 GnRH and controlled internal drug-release device (CIDR); d -3 PGF2α and CIDR removal; d -2 PGF2α; d 0 GnRH) and assigned randomly to receive timed AI with control semen on d 0 (72-CON; n = 104), control semen on d -1 (48-CON; n = 100), or CSE-treated semen on d -1 (48-CSE; n = 98). Heifers were fitted with collar-mounted automated estrus detection devices to monitor physical activity and rumination. In experiment 2, Holstein heifers were subjected to the 5-d CIDR-Synch protocol and received a mount detection patch at the first PGF2α injection. Heifers detected in estrus before d 0 were inseminated on the same day, whereas those not detected in estrus received the second GnRH injection and timed AI on d 0. Heifers were assigned randomly to receive AI with control (AI-CON; n = 148) or CSE-treated semen (AI-CSE; n = 110). Four bulls with proven fertility were used in both experiments, and ejaculates from each sire were divided and processed as CON or CSE. Pregnancy was diagnosed by transrectal ultrasonography at 29 and 54 d after AI. Data were analyzed by logistic regression, and statistical models included the fixed effects of treatment and enrollment week. In experiment 1, orthogonal contrasts were built to assess the effects of day of AI (72-CON vs. 48-CON + 48-CSE) and treatment of semen with CSE (48-CON vs. 48-CSE). Pregnancy per AI on d 29 (72-CON = 60.8, 48-CON = 35.2, 48-CSE = 39.8%) and d 54 (72-CON = 58.2, 48-CON = 31.6, 48-CSE = 36.2%) was greater for heifers inseminated on d 0 compared with d -1. However, no effect of semen extender on P/AI was observed in heifers inseminated on d -1. In experiment 2, P/AI tended to be greater for AI-CSE than for AI-CON on d 29 (58.6 vs. 47.3%) and d 54 after AI (55.6 vs. 43.7%). Advancing AI by 24 h decreased the likelihood of pregnancy, and use of CSE was unable to overcome the expected asynchrony between insemination and ovulation. Nevertheless, incorporation of CSE in semen processing tended to improve P/AI when heifers received AI upon detected estrus or timed AI concurrently with the final GnRH of the 5-d CIDR-Synch protocol.

BK channel openers NS1619 and NS11021 reverse hydrogen peroxide-induced membrane potential changes in skeletal muscle.

Large conductance calcium-activated potassium (BK) channels play a crucial role in the repolarization and after-hyperpolarization phases of the cell membrane. The channel openers are also used in treatment of some diseases, including hypo/hyperkalemic periodic paralysis. However, little is known about the effects of BK channels and the channel activators on membrane potentials in skeletal muscle. In addition, the effects of reactive oxygen species (ROS) on BK channels in skeletal muscle are also unknown. Therefore, the aim of this study was to determine the effects of BK channel openers and ROS on membrane potentials in skeletal muscle fibers. For this purpose, resting membrane potentials and action potentials (AP) of frog gastrocnemius muscles were recorded in the presence of commonly used BK channel openers NS1619 and NS11021, H2O2 (a type of ROS), and both using intracellular microelectrode technique. The channel activators significantly and dose-dependently decreased amplitude and increased rise time of AP but did not impact repolarization. The presence of H2O2 plus NS1619 or NS11021 resulted in significant change because the channel openers completely reversed the deleterious effects of hydrogen peroxide on the repolarization phase of AP in skeletal muscle fibers. In the present study, the contributions of BK channel activation and the modulatory role of H2O2 on membrane potentials was demonstrated in skeletal muscle fibers, for the first time. Moreover, it should be noted that BK channel openers should be used in the treatment of reactive oxygen species-induced skeletal muscle diseases.

The activity of IKCa and BKCa channels contributes to insulin-mediated NO synthesis and vascular tone regulation in human umbilical vein.

Insulin regulates the l-arginine/nitric oxide (NO) pathway in human umbilical vein endothelial cells (HUVECs), increasing the plasma membrane expression of the l-arginine transporter hCAT-1 and inducing vasodilation in umbilical and placental veins. Placental vascular relaxation induced by insulin is dependent of large conductance calcium-activated potassium channels (BKCa), but the role of KCa channels on l-arginine transport and NO synthesis is still unknown. The aim of this study was to determine the contribution of KCa channels in both insulin-induced l-arginine transport and NO synthesis, and its relationship with placental vascular relaxation. HUVECs, human placental vein endothelial cells (HPVECs) and placental veins were freshly isolated from umbilical cords and placenta from normal pregnancies. Cells or tissue were incubated in absence or presence of insulin and/or tetraethylammonium, 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole, iberiotoxin or NG-nitro-l-arginine methyl ester. l-Arginine uptake, plasma membrane polarity, NO levels, hCAT-1 expression and placenta vascular reactivity were analyzed. The inhibition of intermediate-conductance KCa (IKCa) and BKCa increases l-arginine uptake, which was related with protein abundance of hCAT-1 in HUVECs. IKCa and BKCa activities contribute to NO-synthesis induced by insulin but are not directly involved in insulin-stimulated l-arginine uptake. Long term incubation (8 h) with insulin increases the plasma membrane hyperpolarization and hCAT-1 expression in HUVECs and HPVECs. Insulin-induced relaxation in placental vasculature was reversed by KCa inhibition. The results show that the activity of IKCa and BKCa channels are relevant for both physiological regulations of NO synthesis and vascular tone regulation in the human placenta, acting as a part of negative feedback mechanism for autoregulation of l-arginine transport in HUVECs.

Isoliquiritigenin-induced vasodilation by activating large-conductance Ca2+ -activated K+ channels in mouse mesenteric arteries.

Isoliquiritigenin (ISL) is a flavonoid substance with a chalcone structure, which exerts anti-tumour, anti-oxidation and anti-inflammatory activity. The large-conductance calcium-activated potassium channel (BKCa ) is an important potassium channel with negative feedback regulation on the vascular smooth muscle cells (VSMCs) membrane. The activation of BKCa channel causes the hyperpolarization of VSMCs. It plays an important role in relaxation of blood vessels. Previous studies have shown that ISL causes the relaxation of the aorta and the basilar artery of the rat. However, there have not been studies on regulation of ISL in mesenteric arteries. To examine whether ISL causes the relaxation of the mesenteric artery of mice, we recorded vasodilation of mouse mesenteric arterial rings with a myograph. After contraction of arterial rings with phenylephrine, we added ISL to the arterial rings and measured its relaxation effect. To further examine which channel was involved in this relaxation effect, we tested the effects of ISL on endothelium-dependent and endothelium-independent vasodilation. Then we used BKCa channel blockers tetraethylammonium and iberiotoxin, to detect whether the BKCa channel is involved in ISL-induced vasodilation. Mesenteric arterial smooth muscle cells were isolated by enzyme digestion. Bis-(1, 3-dibutylbarbituric acid) trimethine oxonol staining was used to measure membrane potential of mesenteric arterial smooth muscle cells. We identified a vasodilation effect caused by ISL on mouse mesenteric arterial rings pre-contracted by phenylephrine in a concentration-dependent manner, with an EC50 of 13.71 ± 1.1 µmol/L. The vasodilation effect of ISL is endothelium-independent. K+ channel inhibitors tetraethylammonium and iberiotoxin reduced the vasodilation induced by ISL which suggested the involvement of BKCa channel.

The neuropeptide GsMTx4 inhibits a mechanosensitive BK channel through the voltage-dependent modification specific to mechano-gating.

The cardiac mechanosensitive BK (Slo1) channels are gated by Ca2+, voltage, and membrane stretch. The neuropeptide GsMTx4 is a selective inhibitor of mechanosensitive (MS) channels. It has been reported to suppress stretch-induced cardiac fibrillation in the heart, but the mechanism underlying the specificity and even the targeting channel(s) in the heart remain elusive. Here, we report that GsMTx4 inhibits a stretch-activated BK channel (SAKcaC) in the heart through a modulation specific to mechano-gating. We show that membrane stretching increases while GsMTx4 decreases the open probability (Po) of SAKcaC. These effects were mostly abolished by the deletion of the STREX axis-regulated (STREX) exon located between RCK1 and RCK2 domains in BK channels. Single-channel kinetics analysis revealed that membrane stretch activates SAKcaC by prolonging the open-time duration (τO) and shortening the closed-time constant (τC). In contrast, GsMTx4 reversed the effects of membrane stretch, suggesting that GsMTx4 inhibits SAKcaC activity by interfering with mechano-gating of the channel. Moreover, GsMTx4 exerted stronger efficacy on SAKcaC under membrane-hyperpolarized/resting conditions. Molecular dynamics simulation study revealed that GsMTx4 appeared to have the ability to penetrate deeply within the bilayer, thus generating strong membrane deformation under the hyperpolarizing/resting conditions. Immunostaining results indicate that BK variants containing STREX are also expressed in mouse ventricular cardiomyocytes. Our results provide common mechanisms of peptide actions on MS channels and may give clues to therapeutic suppression of cardiac arrhythmias caused by excitatory currents through MS channels under hyper-mechanical stress in the heart.

Genetic and pharmacological interventions in the aging motor nervous system slow motor aging and extend life span in C. elegans.

As animals and humans age, the motor system undergoes a progressive functional decline, leading to frailty. Age-dependent functional deteriorations at neuromuscular junctions (NMJs) contribute to this motor aging. However, it is unclear whether one can intervene in this process to slow motor aging. The Caenorhabditis elegans BK channel SLO-1 dampens synaptic transmission at NMJs by repressing synaptic release from motor neurons. Here, we show that genetic ablation of SLO-1 not only reduces the rate of age-dependent motor activity decline to slow motor aging but also surprisingly extends life span. SLO-1 acts in motor neurons to mediate both functions. Genetic knockdown or pharmacological inhibition of SLO-1 in aged, but not young, worms can slow motor aging and prolong longevity. Our results demonstrate that genetic and pharmacological interventions in the aging motor nervous system can promote both health span and life span.

BK channel blocker paxilline attenuates thalidomide-caused synaptic and cognitive dysfunctions in mice.

Thalidomide is a widely prescribed immunomodulatory drug (iMiD) for multiple myeloma, but causes reversible memory loss in humans. However, how thalidomide causes cognitive dysfunction at a cellular and molecular level has not been demonstrated. We studied the effect of thalidomide on synaptic functions and cognitive behaviors using a mouse model. Thalidomide led to cognitive deficits in learning behavior in a passive avoidance test and in a novel object recognition test, increased anxiety in an elevated plus maze test, and increased depressive behaviors in a tail suspension test. Interestingly, thalidomide elevated big- or large-conductance, calcium-activated K+ (BK) channel expression in the plasma membrane and BK channel activity in the hippocampus. Thalidomide also increased the paired pulse ratio of excitatory postsynaptic current (EPSC), which suggests a decreased probability of glutamate release. Furthermore, the changes in the paired pulse ratio and in BK channel activity were blocked by paxilline, a BK channel blocker. Finally, we found that thalidomide-induced cognitive dysfunctions were restored by paxilline treatment. These results suggest that thalidomide-mediated BK channel hyperfunction is responsible for the pathological mechanism of thalidomide-associated reversible memory loss.

Small-molecule CaVα1⋠CaVß antagonist suppresses neuronal voltage-gated calcium-channel trafficking.

Extracellular calcium flow through neuronal voltage-gated CaV2.2 calcium channels converts action potential-encoded information to the release of pronociceptive neurotransmitters in the dorsal horn of the spinal cord, culminating in excitation of the postsynaptic central nociceptive neurons. The CaV2.2 channel is composed of a pore-forming α1 subunit (CaVα1) that is engaged in protein-protein interactions with auxiliary α2/δ and ß subunits. The high-affinity CaV2.2α1⋅CaVß3 protein-protein interaction is essential for proper trafficking of CaV2.2 channels to the plasma membrane. Here, structure-based computational screening led to small molecules that disrupt the CaV2.2α1⋅CaVß3 protein-protein interaction. The binding mode of these compounds reveals that three substituents closely mimic the side chains of hot-spot residues located on the α-helix of CaV2.2α1 Site-directed mutagenesis confirmed the critical nature of a salt-bridge interaction between the compounds and CaVß3 Arg-307. In cells, compounds decreased trafficking of CaV2.2 channels to the plasma membrane and modulated the functions of the channel. In a rodent neuropathic pain model, the compounds suppressed pain responses. Small-molecule α-helical mimetics targeting ion channel protein-protein interactions may represent a strategy for developing nonopioid analgesia and for treatment of other neurological disorders associated with calcium-channel trafficking.

Large-conductance Ca2+-activated potassium channels are potently involved in the inverse neurovascular response to spreading depolarization.

Recurrent spreading depolarizations occur in the cerebral cortex from minutes up to weeks following acute brain injury. Clinical evidence suggests that the immediate reduction of cerebral blood flow in response to spreading depolarization importantly contributes to lesion progression as the wave propagates over vulnerable tissue zones, characterized by potassium concentration already elevated prior to the passage of spreading depolarization. Here we demonstrate with two-photon microscopy in anesthetized mice that initial vasoconstriction in response to SD triggered experimentally with 1 M KCl is coincident in space and time with the large extracellular accumulation of potassium, as shown with a potassium indicator fluorescent dye. Moreover, pharmacological manipulations in combination with the use of potassium-sensitive microelectrodes suggest that large-conductance Ca2+-activated potassium (BK) channels and L-type voltage-gated calcium channels play significant roles in the marked initial vasoconstriction under elevated baseline potassium. We propose that potassium efflux through BK channels is a central component in the devastating neurovascular effects of spreading depolarizations in tissue at risk.