Blockade Of BK Channels Research Articles

The Roles of BK Potassium Channels in Neuronal Excitability Differ in Excitatory and Inhibitory Hippocampal Neurons During Early Development

BK potassium channels are are activated by intracellular Ca+ and depolarization, opening during action potential firing and speeding repolarization. Because of this, they are key regulators of fast spiking activity and important determinants of neuronal excitability. Due to their role in regulating excitability as well as their broad expression in both excitable and non‐excitable tissues, BK channels are implicated in many pathological states including epilepsy. The BK channel antagonist, paxilline, has been shown to delay seizure induction in adult rodents that have had prior seizures, but not in seizure naïve rodents. History of seizures has been shown induce expression of more Ca2+ sensitive BK channel splice variants, which may underlie the difference in paxilline efficacy between the two states. Because expression of more Ca2+ sensitive isoforms predominates in neonatal rodents ‐similar to in the epileptic adults, we hypothesize that paxilline may be an effective inhibitor of seizure activity even in seizure‐naïve neonatal rodents. Our first goal is to understand how developmental shifts in BK channel expression and function map to changes in neuronal and circuit excitability. Our second goal is to test whether BK channel blockade can modulate seizure activity in neonates, as there is a need to identify pharmacological targets for seizures in neonates and children, which are often intractable to conventional treatments.To test how BK channel function changes with age and by cell type we measured the effect of paxilline on action potential (AP) firing ‐both on single spike kinetics and prolonged firing‐ in two classes of hippocampal neurons, CA1 pyramidal neurons and parvalbumin interneurons (PVIns), a key inhibitory component of local hippocampal circuits. We used P4‐5, P9‐10, and P14‐15 C57BL/6J mice with labelled PVIns. We also tested the efficacy of paxilline in reducing hyperexcitability evoked by application of 0 Mg2+ aCSF in brain slices, an established seizure model.We patched pyramidal neurons and PVIns in whole‐cell current‐clamp configuration and evoked APs using square depolarizing pulses. BK channel blockade with 1mM paxilline lengthens duration of APs in P4‐5 and P9‐10 pyramidal neurons and P4‐5 PVIns but does not affect P9‐10 PVIn AP duration (paired t‐tests, p<0.05). While BK blockade has the least effect on single APs in the P9‐10 PVIns, it has the most effect on the input‐output relationship and spike‐frequency accommodation in these neurons (3‐way ANOVA, p<0.001 for interaction of age and cell type in input‐output; 3‐way ANOVA p<0.05 for main effect of age in frequency adaptation).We measured the effect of BK channel blockade in controlling hyperexcitability in P4‐15 mice. We found that 0 Mg2+ aCSF application induced spontaneous AP firing in patched hippocampal pyramidal neurons within 3.86±0.87 minutes but when 0 Mg2+ aCSF was coapplied with 1 mM paxilline the average onset of spontaneous activity occurred at 8.43±1.40 minutes (t‐test p<0.05).For the first goal, we show that BK channels’ relative importance in regulating AP firing changes with age and by neuronal population. Toward the second goal we report that BK channel blockade can reduce hyperexcitability in a brain slice model of epilepsy.

The cooperative suppression of large-conductance calcium-activated potassium channel by amyloid βeta and amyloid precursor protein in cortical neurons

We have reported the blockade of large-conductance calcium-activated potassium channel (BK channel) as amyloid‐β‐induced molecular dysfunction, which we consider leads to disruptive calcium homeostasis and neuron death. The possibility that not just the end product amyloid β but the amyloid precursor protein (APP) per se might be harmful, still remains to be fully studied. This study examined the mode of BK channel blockade mediated by not just amyloid β but also APP.

A comprehensive review of peptide toxins vs synthetic modulators of BK channels in Epilepsy

BK channels, or voltage-gated Ca2+ channels, are essential regulators of neuronal excitability and muscular contractions, all of which are abnormal in epilepsy, a chronic neuronal disease. The form, frequency, and transmission of action potentials (APs), as well as neurotransmitter release from presynaptic terminals, are all influenced by BK channels found in the plasma membrane of neurons. Over the last two decades, several naturally occurring BK channel modulators have attracted a lot of attention. The structural and pharmacological properties of BK channel blockers are discussed in this article. The properties of various venom peptide toxins from scorpions and snakes are first identified, with a focus on their distinctive structural motifs, such as their disulfide bond formation pattern, the binding interface between the toxin and the BK channel, and the functional consequences of the toxins' blockage of BK channels. Then, several non-peptide BK channel blockers are discussed, along with their molecular formula and pharmacological impact on BK channels. The precise categorization and explanations of these BK channel blockers are hoped to provide mechanistic insights into BK channel blockade. The structures of peptide toxins and non-peptide compounds may serve as models for the development of new channel blockers, as well as aid in the optimization of lead compounds for use in neurological disorders.

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.

Blockade of BK channels attenuates chronic visceral hypersensitivity in an IBS-like rat model.

BackgroundVisceral hypersensitivity in irritable bowel syndrome (IBS) is still poorly understood, despite that chronic abdominal pain is the most common symptoms in IBS patients. To study effects of BK channels on visceral hypersensitivity in IBS rats and the underlying mechanisms, IBS rats were established by colorectal distention (CRD) in postnatal rats. The expression of large-conductance calcium and voltage-dependent potassium ion channels (BK channels) of the thoracolumbar spinal cord was examined in IBS and control rats. The effects of BK channel blockade on visceral hypersensitivity were evaluated. The interaction of BK channels and N-methyl-D-aspartate acid (NMDA) receptors was explored, and synaptic transmission at superficial dorsal horn (SDH) neurons of the thoracolumbar spinal cord was recorded by whole-cell patch clamp in IBS rats.ResultsThe expression of the BK channels of the thoracolumbar spinal cord in IBS rats was significantly reduced. The blockade of BK channels could reduce the visceral hypersensitivity in IBS rats. There was an interaction between BK channels and NMDA receptors in the spinal cord. The frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) in SDH neurons is significantly reduced in IBS rats. The blockade of BK channels depolarizes the inhibitory interneuron membrane and increases their excitability in IBS rats.ConclusionsBK channels could interact with NMDA receptors in the thoracolumbar spinal cord of rats and regulate visceral hypersensitivity in IBS rats.

Reversible Block of BK Channels by Piperine

Increasing evidence shows antitumor efficacy of potassium channel blockers, with potential relevance in anticancer therapeutics (1). Altered expression of BK channels has been reported in different tumor types resulting in sustained proliferation and migration of cancer cells (2). Piperine, a primary pungent alkaloid found in black pepper has been proposed as a cancer chemopreventive agent which works by interrupting cancer development pathways (3). Here, we characterized the effects of piperine on BKα channels in the absence and presence of regulatory subunits using patch-clamp electrophysiology in heterologous expression systems. We observed a clear antagonist activity of piperine on pore-forming BKα channels with an IC50 of 5 ± 1 μM. This inhibition exhibited a rapid onset (τ = 2.2 ± 0.3 s) and was completely reversible after a slow recovery (τ = 21 ± 2 s). The IC50 values did not change significantly over a range of voltages, suggesting that piperine does not function as an open-channel blocker. Moreover, elevating the internal calcium significantly diminished the block of BKα channels by piperine. The blocking action of piperine persisted in BK-G311S mutant channels, suggesting that its site of action is different from that of fungal alkaloids (e.g. paxilline, penitrem A). Intriguingly, the efficacy of the piperine inhibition persisted in BK channels containing BKβ subunits and was significantly attenuated in the presence of BKγ subunits. In summary, our data suggest that piperine is a novel blocker of BK channels exerting differential effects that depend on the accessory subunits. Evaluation of the anti-proliferative activity of piperine in various cancer cell lines, that might be mediated via blockade of BK channels, is currently underway. 1. Huang and Jan. JCB 2014, 206:151. 2. Oeggerli et al. Plos One 2012, 7:e41664. 3. Rather and Bhagat. Front Cell Dev Biol 2018, 6:10.

Functional Coupling of BK Channels to NMDA Receptors in Hippocampal Dentate Gyrus

The large conductance calcium- and voltage-activated potassium channel (BK channels) is widely expressed in the central nervous system. The physiological roles of BK channels and the calcium sources of their activation in mammalian brain remain not well-understood. Our proteomic and biochemical analyses of BK channels and the calcium-permeable NMDA receptors and have found that they form protein complexes in whole brain and various brain regions, consisting of the obligatory BK channel alpha-subunit and GluN1 subunits and at least the regulatory GluN2A and GluN2B subunits. We examined the functional coupling of BK channels to NMDA receptors in mice hippocampal dentate gyrus. We observed that glutamate evoked outward currents of BK channels in dentate gyrus granule cell soma at potentials more positive than −20 mV which were sensitive to BK channel-specific blocker paxilline and abolished by NMDA receptor antagonist AP-5. We also found that blockade of BK channels by paxilline increased the amplitude of excitatory postsynaptic potential (EPSP) of granule cells evoked by stimulation in the perforant path-granule cell synapses, which was prevented by the presence of AP-5. We thus inferred that BK channels were activated by NMDA receptor-mediated Ca2+ influx in hippocampal dentate gyrus granule cells and the calcium-mediated functional coupling between these two types of channels plays a role in regulation of synaptic transmission.

Equol increases cerebral blood flow in rats via activation of large-conductance Ca2+-activated K+ channels in vascular smooth muscle cells

The present study was designed to investigate the effect of equol on cerebral blood flow and the underlying molecular mechanisms. The regional cerebral blood flow in parietal lobe of rats was measured by using a laser Doppler flowmetry. Isolated cerebral basilar artery and mesenteric artery rings from rats were used for vascular reactivity measurement with a multi wire myography system. Outward K+ current in smooth muscle cells of cerebral basilar artery, large-conductance Ca2+-activated K+ (BK) channel current in BK-HEK 293 cells stably expressing both human α (hSlo)- and β1-subunits, and hSlo channel current in hSlo-HEK 293 cells expressing only the α-subunit of BK channels were recorded with whole cell patch-clamp technique. The results showed that equol significantly increased regional cerebral blood flow in rats, and produced a concentration-dependent but endothelium-independent relaxation in rat cerebral basilar arteries. Both paxilline and iberiotoxin, two selective BK channel blockers, significantly inhibited equol-induced vasodilation in cerebral arteries. Outward K+ currents in smooth muscle cells of cerebral basilar artery were increased by equol and fully reversed by washout or blockade of BK channels with iberiotoxin. Equol remarkably enhanced human BK current in BK-HEK 293 cells, but not hSlo current in hSlo-HEK 293 cells, and the increase was completely abolished by co-application of paxilline. Our findings provide the first information that equol selectively stimulates BK channel current by acting on its β1 subunit, which may in turn contribute to the equol-mediated vasodilation and cerebral blood flow increase.

Peptide toxins and small-molecule blockers of BK channels.

Large conductance, Ca(2+)-activated potassium (BK) channels play important roles in the regulation of neuronal excitability and the control of smooth muscle contractions. BK channels can be activated by changes in both the membrane potential and intracellular Ca(2+) concentrations. Here, we provide an overview of the structural and pharmacological properties of BK channel blockers. First, the properties of different venom peptide toxins from scorpions and snakes are described, with a focus on their characteristic structural motifs, including their disulfide bond formation pattern, the binding interface between the toxin and BK channel, and the functional consequence of the blockage of BK channels by these toxins. Then, some representative non-peptide blockers of BK channels are also described, including their molecular formula and pharmacological effects on BK channels. The detailed categorization and descriptions of these BK channel blockers will provide mechanistic insights into the blockade of BK channels. The structures of peptide toxins and non-peptide compounds could provide templates for the design of new channel blockers, and facilitate the optimization of lead compounds for further therapeutic applications in neurological disorders or cardiovascular diseases.

Modulation of BK channels contributes to activity-dependent increase of excitability through MTORC1 activity in CA1 pyramidal cells of mouse hippocampus.

Memory acquisition and synaptic plasticity are accompanied by changes in the intrinsic excitability of CA1 pyramidal neurons. These activity-dependent changes in excitability are mediated by modulation of intrinsic currents which alters the responsiveness of the cell to synaptic inputs. The afterhyperpolarization (AHP), a major contributor to the regulation of neuronal excitability, is reduced in animals that have acquired several types of hippocampus-dependent memory tasks and also following synaptic potentiation by high frequency stimulation. BK channels underlie the fast AHP and contribute to spike repolarization, and this AHP is reduced in animals that successfully acquired trace-eyeblink conditioning. This suggests that BK channel function is activity-dependent, but the mechanisms are unknown. In this study, we found that blockade of BK channels with paxilline (10 μM) decreased IAHP amplitude and increased spike half-width and instantaneous frequency in response to a +100 pA depolarization. In addition, induction of long term potentiation (LTP) by theta burst stimulation (TBS) in CA1 pyramidal neurons reduced BK channel’s contribution to IAHP, spike repolarization, and instantaneous frequency. This result indicates that BK channel activity is decreased following synaptic potentiation. Interestingly, blockade of mammalian target of rapamycin (MTORC1) with rapamycin (400 nM) following synaptic potentiation restored BK channel function, suggesting a role for protein translation in signaling events which decreased postsynaptic BK channel activity following synaptic potentiation.

Protease-activated receptor-2 inhibits BK channel activity in bronchopulmonary sensory neurons

Activation of protease-activated receptor-2 (PAR2) contributes to airway inflammation and airway hypersensitivity, the hallmark features of allergic asthma; and a neurogenic mechanism involving hypersensitivity of bronchopulmonary sensory nerves has been indicated. Large-conductance Ca2+-activated potassium (BK) channels are known to play an important role in shaping neuronal excitability. The aim of this study was to investigate the potential regulation of BK channel activities by PAR2 activation in vagal bronchopulmonary sensory neurons. Our results showed that pretreatment with PAR2-activating peptide (PAR2-AP; 100μM, 120s), but not its control peptide PAR2-RP, significantly reduced BK current density in these neurons. Inhibition of phospholipase C, PKC, PKA or MEK/ERK signaling pathway did not prevent the suppression of BK current by PAR2 activation; whereas intracellular application of Ca2+ chelator BAPTA-AM completely abolished the PAR2 regulation of BK current. In addition, our results demonstrated that activation of PAR2 increased excitability of bronchopulmonary sensory neurons, in a similar manner as displayed by a direct BK channel blockade. In summary, our data suggest that suppression of BK channel activity contributes to PAR2 activation-induced hyperexcitability of vagal bronchopulmonary sensory neurons.

Characteristics of single large-conductance Ca2+-activated K+ channels and their regulation of action potentials and excitability in parasympathetic cardiac motoneurons in the nucleus ambiguus

Large-conductance Ca2(+)-activated K+ channels (BK) regulate action potential (AP) properties and excitability in many central neurons. However, the properties and functional roles of BK channels in parasympathetic cardiac motoneurons (PCMNs) in the nucleus ambiguus (NA) have not yet been well characterized. In this study, the tracer X-rhodamine-5 (and 6)-isothiocyanate (XRITC) was injected into the pericardial sac to retrogradely label PCMNs in FVB mice at postnatal 7-9 days. Two days later, XRITC-labeled PCMNs in brain stem slices were identified. Using excised patch single-channel recordings, we identified voltage-gated and Ca(2+)-dependent BK channels in PCMNs. The majority of BK channels exhibited persistent channel opening during voltage holding. These BK channels had a conductance of 237 pS and a 50% opening probability at +27.9 mV, the channel open time constant was 3.37 ms at +20 mV, and dwell time increased exponentially as the membrane potential depolarized. At the +20-mV holding potential, the [Ca2+]50 was 15.2 μM with a P0.5 of 0.4. Occasionally, some BK channels showed a transient channel opening and fast inactivation. Using whole cell voltage clamp, we found that BK channel mediated outward currents and afterhyperpolarization currents (IAHP). Using whole cell current clamp, we found that application of BK channel blocker iberiotoxin (IBTX) increased spike half-width and suppressed fast afterhyperpolarization (fAHP) amplitude following single APs. In addition, IBTX application increased spike half-width and reduced the spike frequency-dependent AP broadening in trains and spike frequency adaption (SFA). Furthermore, BK channel blockade decreased spike frequency. Collectively, these results demonstrate that PCMNs have BK channels that significantly regulate AP repolarization, fAHP, SFA, and spike frequency. We conclude that activation of BK channels underlies one of the mechanisms for facilitation of PCMN excitability.

Requirement for Functional BK Channels in Maintaining Oscillation in Venomotor Tone Revealed by Species Differences in Expression of the β1 Accessory Subunits

We determined the possible role of large-conductance Ca2+-activated K (BK) channels in regulation of venous tone in small capacitance veins and blood pressure. In rat mesenteric venous smooth muscle cells (MV SMC), BK channel α- and β1-subunits were coexpressed, unitary BK currents were detected, and single-channel currents were sensitive to voltage and [Ca2+]i. Rat MV SMCs displayed Ca sparks and iberiotoxin-sensitive spontaneous transient outward currents. Under resting conditions in vitro, rat MV exhibited nifedipine-sensitive spontaneous oscillatory constrictions. Blockade of BK channels by paxilline and Ca2+ sparks by ryanodine constricted rat MV. Nifedipine caused venodilation and blocked paxilline-induced, KCl-induced (20 mM), and BayK8644-induced contraction. Acute inhibition of BK channels with iberiotoxin in vivo increased blood pressure and reduced venous capacitance, measured as an increase in mean circulatory filling pressure in conscious rats. BK channel α-subunits and L-type Ca2+ channel α1-C subunits are expressed in murine MV. However, these channels are not functional because murine MV lack nifedipine-sensitive basal tone and rhythmic constrictions. Murine MV were also insensitive to paxilline, ryanodine, KCl, and BayK8644, consistent with our previous studies showing that murine MV do not have BK β1-subunits. These data show that not only there are species-dependent properties in ion channel control of venomotor tone but also BK channels are required for rhythmic oscillations in venous tone.

Microglial Ca2+-Activated K+Channels Are Possible Molecular Targets for the Analgesic Effects ofS-Ketamine on Neuropathic Pain

Ketamine is an important analgesia clinically used for both acute and chronic pain. The acute analgesic effects of ketamine are generally believed to be mediated by the inhibition of NMDA receptors in nociceptive neurons. However, the inhibition of neuronal NMDA receptors cannot fully account for its potent analgesic effects on chronic pain because there is a significant discrepancy between their potencies. The possible effect of ketamine on spinal microglia was first examined because hyperactivation of spinal microglia after nerve injury contributes to neuropathic pain. Optically pure S-ketamine preferentially suppressed the nerve injury-induced development of tactile allodynia and hyperactivation of spinal microglia. S-Ketamine also preferentially inhibited hyperactivation of cultured microglia after treatment with lipopolysaccharide, ATP, or lysophosphatidic acid. We next focused our attention on the Ca(2+)-activated K(+) (K(Ca)) currents in microglia, which are known to induce their hyperactivation and migration. S-Ketamine suppressed both nerve injury-induced large-conductance K(Ca) (BK) currents and 1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one (NS1619)-induced BK currents in spinal microglia. Furthermore, the intrathecal administration of charybdotoxin, a K(Ca) channel blocker, significantly inhibited the nerve injury-induced tactile allodynia, the expression of P2X(4) receptors, and the synthesis of brain-derived neurotrophic factor in spinal microglia. In contrast, NS1619-induced tactile allodynia was completely inhibited by S-ketamine. These observations strongly suggest that S-ketamine preferentially suppresses the nerve injury-induced hyperactivation and migration of spinal microglia through the blockade of BK channels. Therefore, the preferential inhibition of microglial BK channels in addition to neuronal NMDA receptors may account for the preferential and potent analgesic effects of S-ketamine on neuropathic pain.

Large-conductance voltage- and Ca2+-activated K+ channels regulate human detrusor smooth muscle function

The large-conductance voltage- and Ca(2+)-activated K(+) (BK) channel is expressed in many smooth muscle types, but its role in human detrusor smooth muscle (DSM) is unclear. With a multidisciplinary approach spanning channel molecules, single-channel activity, freshly isolated human DSM cells, intact DSM preparations, and the BK channel specific inhibitor iberiotoxin, we elucidated human DSM BK channel function and regulation. Native human DSM tissues were obtained during open surgeries from patients with no preoperative history of overactive bladder. RT-PCR experiments on single human DSM cells showed mRNA expression of BK channel α-, β(1)-, and β(4)-subunits. Western blot and immunocytochemistry confirmed BK channel α, β(1), and β(4) protein expression. Native human BK channel properties were described using the perforated whole cell configuration of the patch-clamp technique. In freshly isolated human DSM cells, BK channel blockade with iberiotoxin inhibited a significant portion of the total voltage step-induced whole cell K(+) current. From single BK channel recordings, human BK channel conductance was calculated to be 136 pS. Voltage-dependent iberiotoxin- and ryanodine-sensitive transient BK currents were identified in human DSM cells. In current-clamp mode, iberiotoxin inhibited the hyperpolarizing membrane potential transients and depolarized the cell resting membrane potential. Isometric DSM tension recordings revealed that BK channels principally control the contractions of isolated human DSM strips. Collectively, our results indicate that BK channels are fundamental regulators of DSM excitability and contractility and may represent new targets for pharmacological or genetic control of urinary bladder function in humans.

Inhibition of Ca2+-activated large-conductance K+ channel activity alters synaptic AMPA receptor phenotype in mouse cerebellar stellate cells

Many fast-spiking inhibitory interneurons, including cerebellar stellate cells, fire brief action potentials and express α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptors (AMPAR) that are permeable to Ca(2+) and do not contain the GluR2 subunit. In a recent study, we found that increasing action potential duration promotes GluR2 gene transcription in stellate cells. We have now tested the prediction that activation of potassium channels that control the duration of action potentials can suppress the expression of GluR2-containing AMPARs at stellate cell synapses. We find that large-conductance Ca(2+)-activated potassium (BK) channels mediate a large proportion of the depolarization-evoked noninactivating potassium current in stellate cells. Pharmacological blockade of BK channels prolonged the action potential duration in postsynaptic stellate cells and altered synaptic AMPAR subtype from GluR2-lacking to GluR2-containing Ca(2+)-impermeable AMPARs. An L-type channel blocker abolished an increase in Ca(2+) entry that was associated with spike broadening and also prevented the BK channel blocker-induced switch in AMPAR phenotype. Thus blocking BK potassium channels prolongs the action potential duration and increases the expression of GluR2-containing receptors at the synapse by enhancing Ca(2+) entry in cerebellar stellate cells.

Maternal diabetes increases large conductance Ca2+-activated K+outward currents that alter action potential properties but do not contribute to attenuated excitability of parasympathetic cardiac motoneurons in the nucleus ambiguus of neonatal mice

Previously, we demonstrated that maternal diabetes reduced the excitability and increased small-conductance Ca(2+)-activated K(+) (SK) currents of parasympathetic cardiac motoneurons (PCMNs) in the nucleus ambiguus (NA). In addition, blockade of SK channels with apamin completely abolished this reduction. In the present study, we examined whether maternal diabetes affects large-conductance Ca(2+)-activated K(+) (BK) channels and whether BK channels contribute to the attenuation of PCMN excitability observed in neonates of diabetic mothers. Neonatal mice from OVE26 diabetic mothers (NMDM) and normal FVB mothers (control) were used. The pericardial sac of neonatal mice at postnatal days 7-9 was injected with the tracer X-rhodamine-5 (and 6)-isothiocyanate 2 days prior to the experiment to retrogradely label PCMNs in the NA. Whole cell current- and voltage-clamps were used to measure spike frequency, action potential (AP) repolarization (half-width), afterhyperpolarization potential (AHP), transient outward currents, and afterhyperpolarization currents (I(AHP)). In whole cell voltage clamp mode, we confirmed that maternal diabetes increased transient outward currents and I(AHP) compared with normal cells. Using BK channel blockers charybdotoxin (CTx) and paxilline, we found that maternal diabetes increased CTx- and paxilline-sensitive transient outward currents but did not change CTx- and paxilline-sensitive I(AHP). In whole cell current-clamp mode, we confirmed that maternal diabetes increased AP half-width and AHP, and reduced excitability of PCMNs. Furthermore, we found that after blockade of BK channels with CTx or paxilline, maternal diabetes induced a greater increase of AP half-width but similarly decreased fast AHP without affecting medium AHP. Finally, blockade of BK channels decreased spike frequency in response to current injection in both control and NMDM without reducing the difference of spike frequency between the two groups. Therefore, we conclude that although BK transient outward currents, which may alter AP repolarization, are increased in NMDM, BK channels do not directly contribute to maternal diabetes-induced attenuation of PCMN excitability. In contrast, based on evidence from our previous and present studies, reduction of PCMN excitability in neonates of diabetic mothers is largely dependent on altered SK current associated with maternal diabetes.

Different roles attributed to Cav1 channel subtypes in spontaneous action potential firing and fine tuning of exocytosis in mouse chromaffin cells

J. Neurochem. (2011) 116, 105–121.This study examines the Cav1 isoforms expressed in mouse chromaffin cells and compares their biophysical properties and roles played in cell excitability and exocytosis. Using immunocytochemical and electrophysiological techniques in mice lacking the Cav1.3α1 subunit (Cav1.3−/−) or the high sensitivity of Cav1.2α1 subunits to dihydropyridines, Cav1.2 and Cav1.3 channels were identified as the only Cav1 channel subtypes expressed in mouse chromaffin cells. Cav1.3 channels were activated at more negative membrane potentials and inactivated more slowly than Cav1.2 channels. Cav1 channels, mainly Cav1.2, control cell excitability by functional coupling to BK channels, revealed by nifedipine blockade of BK channels in wild type (WT) and Cav1.3−/− cells (53% and 35%, respectively), and by the identical change in the shape of the spontaneous action potentials elicited by the dihydropyridine in both strains of mice. Cav1.2 channels also play a major role in spontaneous action potential firing, supported by the following evidence: (i) a similar percentage of WT and Cav1.3−/− cells fired spontaneous action potentials; (ii) firing frequency did not vary between WT and Cav1.3−/− cells; (iii) mostly Cav1.2 channels contributed to the inward current preceding the action potential threshold; and (iv) in the presence of tetrodotoxin, WT or Cav1.3−/− cells exhibited spontaneous oscillatory activity, which was fully abolished by nifedipine perfusion. Finally, Cav1.2 and Cav1.3 channels were essential for controlling the exocytotic process at potentials above and below −10 mV, respectively. Our data reveal the key yet differential roles of Cav1.2 and Cav1.3 channels in mediating action potential firing and exocytotic events in the neuroendocrine chromaffin cell.

Maternal Diabetes (MD) Increases Large Conductance Ca2+‐activated K+ (BK) Currents Which Alter Action Potential (AP) Properties But Do Not Affect Excitability of Parasympathetic Cardiac Motoneurons (PCMNs) In The Nucleus Ambiguus (NA) of Neonatal Mice

In an accompanying abstract (Lin et al. EB 2010), we have demonstrated that MD increases small conductance Ca2+‐activated K+ (SK) currents which are associated with MD‐induced alteration of AP properties and attenuated excitability of PCMNs. Presently, we test the hypothesis that MD also alters BK currents. Neonatal mice from OVE26 diabetic (Type 1) mothers (NMDM) and normal FVB mothers (control) were used. XRITC was injected into the pericardial sac at P7–9 to label PCMNs. Two days later, XRITC‐labeled PCMNs in slices were identified. Whole‐cell (current and voltage) clamps were used to measure spike frequency, repolarization (half‐width), afterhyperpolarization (AHP), outward currents, and afterhyperpolarization currents (IAHP). Compared to control, we found (p<0.05) that in NMDM that 1) Ca2+‐sensitive transient outward currents and IAHP increased; 2) application of Ca2+‐free solution increased half‐width and decreased AHP amplitude; 3) BK channel blockers ChTX or paxilline‐sensitive transient outward currents and IAHP increased; 4) blockade of BK channels with ChTX or paxilline increased AP half‐width and decreased AHP amplitude at 10 ms, but not at 50 ms; 5) application of ChTX or paxilline does not affect spike firing frequency in response to current injections in both control and NMDM. In contrast to SK channels, we suggest that MD increases BK currents which contribute to altered AP properties, but do not directly affect the attenuated excitability of PCMNs in NMDM. NIH R01 HL79636.

Effects of bladder outlet obstruction on properties of Ca2+-activated K+channels in rat bladder

In this study, we investigated the effects of bladder outlet obstruction (BOO) on the expression and function of large conductance (BK) and small conductance (SK) Ca(2+)-activated K(+) channels in detrusor smooth muscle. The bladder from adult female Sprague-Dawley rats with 6-wk BOO were used. The mRNA expression of the BK channel alpha-subunit, beta1-, beta2-, and beta4-subunits and SK1, SK2, and SK3 channels were investigated using real-time RT-PCR. All subunits except for the BK-beta2, SK2, and SK3 channels were predominantly expressed in the detrusor smooth muscle rather than in the mucosa. The mRNA expression of the BK channel alpha-subunit was not significantly changed in obstructed bladders. However, the expression of the BK channel beta1-subunit and the SK3 channel was remarkably increased in obstructed bladders. On the other hand, the expression of the BK channel beta4-subunit was decreased as the severity of BOO-induced bladder overactivity progressed. In detrusor smooth muscle strips from obstructed bladders, blockade of BK channels by iberiotoxin (IbTx) or charybdotoxin (CTx) and blockade of SK channels by apamin increased the amplitude of spontaneous contractions. These blockers also increased the contractility and affinity of these strips for carbachol during cumulative applications. The facilitatory effects elicited by these K(+) channel blockers were larger in the strips from obstructed bladders compared with control bladders. These results suggest that long-term exposure to BOO for 6 wk enhances the function of both BK and SK types of Ca(2+)-activated K(+) channels in the detrusor smooth muscle to induce an inhibition of bladder contractility, which might be a compensatory mechanism to reduce BOO-induced bladder overactivity.