TRPML3/BK complex promotes autophagy and bacterial clearance by providing a positive feedback regulation of mTOR via PI3P.

TRPML3 is a Ca2+/Na+ release channel residing in both phagophores and endolysosomal membranes. It is activated by PI3P and PI3,5P2. Its activity can be enhanced by high luminal pH and by replacing luminal Na+ with K+. Here, we report that big-conductance Ca2+-activated potassium (BK) channels form a positive feedback loop with TRPML3. Ca2+ release via TRPML3 activates BK, which in turn facilitates TRPML3-mediated Ca2+ release, potentially through removing luminal Na+ inhibition. We further show that TRPML3/BK and mammalian target of rapamycin (mTOR) form another positive feedback loop to facilitate autophagy induction in response to nutrient starvation, i.e., mTOR inhibition upon nutrient starvation activates TRPML3/BK, and this further reduces mTOR activity, thereby increasing autophagy induction. Mechanistically, the feedback regulation between TRPML3/BK and mTOR is mediated by PI3P, an endogenous TRPML3 activator that is enriched in phagophores and is up-regulated by mTOR reduction. Importantly, bacterial infection activates TRPML3 in a BK-dependent manner, and both TRPML3 and BK are required for mTOR suppression and autophagy induction responding to bacterial infection. Suppressing either TRPML3 or BK helps bacteria survival whereas increasing either TRPML3 or BK favors bacterial clearance. Considering that TRPML3/BK is inhibited by low luminal pH but activated by high luminal pH and PI3P in phagophores, we suggest that TRPML3/BK and mTOR form a positive feedback loop via PI3P to ensure efficient autophagy induction in response to nutrient deprivation and bacterial infection. Our study reveals a role of TRPML3-BK coupling in controlling cellular homeostasis and intracellular bacterial clearance via regulating mTOR signaling.

Neuronal mechanism of a BK channelopathy in absence epilepsy and dyskinesia.

A growing number of gain-of-function (GOF) BK channelopathies have been identified in patients with epilepsy and movement disorders. Nevertheless, the underlying pathophysiology and corresponding therapeutics remain obscure. Here, we utilized a knock-in mouse model carrying human BK-D434G channelopathy to investigate the neuronal mechanism of BK GOF in the pathogenesis of epilepsy and dyskinesia. The BK-D434G mice manifest the clinical features of absence epilepsy and exhibit severe motor deficits and dyskinesia-like behaviors. The cortical pyramidal neurons and cerebellar Purkinje cells from the BK-D434G mice show hyperexcitability, which likely contributes to the pathogenesis of absence seizures and paroxysmal dyskinesia. A BK channel blocker, paxilline, potently suppresses BK-D434G­induced hyperexcitability and effectively mitigates absence seizures and locomotor deficits in mice. Our study thus uncovered a neuronal mechanism of BK GOF in absence epilepsy and dyskinesia. Our findings also suggest that BK inhibition is a promising therapeutic strategy for mitigating BK GOF-induced neurological disorders.

Loss of anoctamin 1 reveals a subtle role for BK channels in lymphatic muscle action potentials.

Ca2+ signalling plays a crucial role in determining lymphatic muscle cell excitability and contractility through its interaction with the Ca2+-activated Cl- channel anoctamin 1 (ANO1). In contrast, the large-conductance (BK) Ca2+-activated K+ channel (KCa) and other KCa channels have prominent vasodilatory actions by hyperpolarizing vascular smooth muscle cells. Here, we assessed the expression and contribution of the KCa family to mouse and rat lymphatic collecting vessel contractile function. The BK channel was the only KCa channel consistently expressed in fluorescence-activated cell sorting-purified mouse lymphatic muscle cell lymphatic muscle cells. We used a pharmacological inhibitor of BK channels, iberiotoxin, and small-conductance Ca2+-activated K+ channels, apamin, to inhibit KCa channels acutely in ex vivo isobaric myography experiments and intracellular membrane potential recordings. In basal conditions, BK channel inhibition had little to no effect on either mouse inguinal-axillary lymphatic vessel (MIALV) or rat mesenteric lymphatic vessel contractions or action potentials (APs). We also tested BK channel inhibition under loss of ANO1 either by genetic ablation (Myh11CreERT2-Ano1 fl/fl, Ano1ismKO) or by pharmacological inhibition with Ani9. In both Ano1ismKO MIALVs and Ani9-pretreated MIALVs, inhibition of BK channels increased contraction amplitude, increased peak AP and broadened the peak of the AP spike. In rat mesenteric lymphatic vessels, BK channel inhibition also abolished the characteristic post-spike notch, which was exaggerated with ANO1 inhibition, and significantly increased the peak potential and broadened the AP spike. We conclude that BK channels are present and functional on mouse and rat lymphatic muscle cells but are otherwise masked by the dominance of ANO1. KEY POINTS: Mouse and rat lymphatic muscle cells express functional BK channels. BK channels make little contribution to either rat or mouse lymphatic collecting vessel contractile function in basal conditions across a physiological pressure range. ANO1 limits the peak membrane potential achieved in the action potential and sets a plateau potential limiting the voltage-dependent activation of BK. BK channels are activated when ANO1 is absent or blocked and slightly impair contractile strength by reducing the peak membrane potential achieved in the action potential spike and accelerating the post-spike repolarization.

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.

Comparative gain-of-function effects of the KCNMA1-N999S mutation on human BK channel properties.

KCNMA1, encoding the voltage- and calcium-activated potassium channel, has a pivotal role in brain physiology. Mutations in KCNMA1 are associated with epilepsy and/or dyskinesia (PNKD3). Two KCNMA1 mutations correlated with these phenotypes, D434G and N999S, were previously identified as producing gain-of-function (GOF) effects on BK channel activity. Three new patients have been reported harboring N999S, one carrying a second mutation, R1128W, but the effects of these mutations have not yet been reported under physiological K+ conditions or compared to D434G. In this study, we characterize N999S, the novel N999S/R1128W double mutation, and D434G in a brain BK channel splice variant, comparing the effects on BK current properties under a physiological K+ gradient with action potential voltage commands. N999S, N999S/R1128W, and D434G cDNAs were expressed in HEK293T cells and characterized by patch-clamp electrophysiology. N999S BK currents were shifted to negative potentials, with faster activation and slower deactivation compared with wild type (WT) and D434G. The double mutation N999S/R1128W did not show any additional changes in current properties compared with N999S alone. The antiepileptic drug acetazolamide was assessed for its ability to directly modulate WT and N999S channels. Neither the WT nor N999S channels were sensitive to the antiepileptic drug acetazolamide, but both were sensitive to the inhibitor paxilline. We conclude that N999S is a strong GOF mutation that surpasses the D434G phenotype, without mitigation by R1128W. Acetazolamide has no direct modulatory action on either WT or N999S channels, indicating that its use may not be contraindicated in patients harboring GOF KCNMA1 mutations.NEW & NOTEWORTHYKCNMA1-linked channelopathy is a new neurological disorder characterized by mutations in the BK voltage- and calcium-activated potassium channel. The epilepsy- and dyskinesia-associated gain-of-function mutations N999S and D434G comprise the largest number of patients in the cohort. This study provides the first direct comparison between D434G and N999S BK channel properties as well as a novel double mutation, N999S/R1128W, from another patient, defining the functional effects during an action potential stimulus.

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.

Single Molecule Fluorescence Imaging Reveals the Stoichiometry of BKγ1 Subunit in Living HEK293 Cell Expression System.

Large conductance Ca2+-activated K+ (BKCa) channels are ubiquitously expressed in plasma membrane of both excitable and non-excitable cells and possess significant physiological functions. A tetrameric complex of α subunit (BKα) forms a functional pore of BKCa channel. The properties of BKCa channel, such as voltage-dependence, Ca2+ sensitivity and pharmacological responses, are extensively modulated by co-expressing accessory ß subunits (BKß), which can associate with BKα in one to one manner. Although the functional significance of newly identified γ subunits (BKγ) has been revealed, the stoichiometry between BKα and BKγ1 remains unclear. In the present study, we utilized a single molecule fluorescence imaging with a total internal reflection fluorescence (TIRF) microscope to directly count the number of green fluorescent protein (GFP)-tagged BKγ1 (BKγ1-GFP) within a single BKCa channel complex in HEK293 cell expression system. BKγ1-GFP significantly enhanced the BK channel activity even when the intracellular Ca2+ concentration was kept lower, i.e., 10 nM, than the physiological resting level. BKγ1-GFP stably formed molecular complexes with BKα-mCherry in the plasma membrane. Counting of GFP bleaching steps revealed that a BKCa channel can contain up to four BKγ1 per channel at the maximum. These results suggest that BKγ1 forms a BKCa channel complex with BKα in a 1 : 1 stoichiometry in a human cell line.

A computational model of large conductance voltage and calcium activated potassium channels: implications for calcium dynamics and electrophysiology in detrusor smooth muscle cells.

The large conductance voltage and calcium activated potassium (BK) channels play a crucial role in regulating the excitability of detrusor smooth muscle, which lines the wall of the urinary bladder. These channels have been widely characterized in terms of their molecular structure, pharmacology and electrophysiology. They control the repolarising and hyperpolarising phases of the action potential, thereby regulating the firing frequency and contraction profiles of the smooth muscle. Several groups have reported varied profiles of BK currents and I-V curves under similar experimental conditions. However, no single computational model has been able to reconcile these apparent discrepancies. In view of the channels' physiological importance, it is imperative to understand their mechanistic underpinnings so that a realistic model can be created. This paper presents a computational model of the BK channel, based on the Hodgkin-Huxley formalism, constructed by utilising three activation processes - membrane potential, calcium inflow from voltage-gated calcium channels on the membrane and calcium released from the ryanodine receptors present on the sarcoplasmic reticulum. In our model, we attribute the discrepant profiles to the underlying cytosolic calcium received by the channel during its activation. The model enables us to make heuristic predictions regarding the nature of the sub-membrane calcium dynamics underlying the BK channel's activation. We have employed the model to reproduce various physiological characteristics of the channel and found the simulated responses to be in accordance with the experimental findings. Additionally, we have used the model to investigate the role of this channel in electrophysiological signals, such as the action potential and spontaneous transient hyperpolarisations. Furthermore, the clinical effects of BK channel openers, mallotoxin and NS19504, were simulated for the detrusor smooth muscle cells. Our findings support the proposed application of these drugs for amelioration of the condition of overactive bladder. We thus propose a physiologically realistic BK channel model which can be integrated with other biophysical mechanisms such as ion channels, pumps and exchangers to further elucidate its micro-domain interaction with the intracellular calcium environment.

Planar lipid bilayers in recombinant ion channel research.

There are a number of methods of investigating the function of recombinant proteins such as ion channels. However, after channel purification there are few methods to guarantee that the protein still functions. For ion channels, reconstituting back into planar lipid bilayers and demonstrating preserved function is a convenient and trusted method. It is cell free and even inaccessible, intracellular ion channels can be studied. We have used this method to study the function of recombinant channels of known subunit composition and have found it convenient for investigating the mode of action of ion channel modulators.

Early Alterations of Hippocampal Neuronal Firing Induced by Abeta42.

We studied the effect of Amyloid ß 1-42 oligomers (Abeta42) on Ca2+ dependent excitability profile of hippocampal neurons. Abeta42 is one of the Amyloid beta peptides produced by the proteolytic processing of the amyloid precursor protein and participates in the initiating event triggering the progressive dismantling of synapses and neuronal circuits. Our experiments on cultured hippocampal network reveal that Abeta42 increases intracellular Ca2+ concentration by 46% and inhibits firing discharge by 19%. More precisely, Abeta42 differently regulates ryanodine (RyRs), NMDA receptors (NMDARs), and voltage gated calcium channels (VGCCs) by increasing Ca2+ release through RyRs and inhibiting Ca2+ influx through NMDARs and VGCCs. The overall increased intracellular Ca2+ concentration causes stimulation of K+ current carried by big conductance Ca2+ activated potassium (BK) channels and hippocampal network firing inhibition. We conclude that Abeta42 alters neuronal function by means of at least 4 main targets: RyRs, NMDARs, VGCCs, and BK channels. The development of selective modulators of these channels may in turn be useful for developing effective therapies that could enhance the quality of life of AD patients during the early onset of the pathology.

Effect of Equol on Vasocontractions in Rat Carotid Arteries Treated with High Insulin.

Previous research has indicated that high insulin affects vascular function. Equol is an active metabolite of daidzein, an isoflavone produced from soy by intestinal microbial flora, with beneficial effects on the vascular system. This study investigated whether equol was beneficial for vascular function under high insulin conditions. Using organ culture techniques, rat carotid arteries were treated for 23 ± 1 h with a vehicle, high insulin (100 nM), or equol (100 µM) plus high insulin (100 nM). Vascular isometric forces were measured by the organ bath technique. In each endothelium-intact ring, the contractions induced by high-K+, noradrenaline, or by serotonin (5-HT) were similar for the vehicle, insulin, and equol + insulin treatments. Contractions induced by a selective 5-HT2A receptor agonist (TCB2) increased with insulin treatment (vs. vehicle), but less so with equol + insulin. Under basal conditions, a selective 5-HT2B receptor agonist (BW723C86) did not induce contraction; following precontraction by a thromboxane analog, it induced contraction but not relaxation. These responses were similar across the three treatments. Acetylcholine-induced relaxations were also similar for the three treatments. In the endothelium-denuded preparations, 5-HT-induced contraction was augmented with insulin treatment (vs. vehicle) but less so by equol + insulin treatment. These differences in 5-HT-induced contractions were eliminated by iberiotoxin, a large-conductance calcium-activated K+ channel (BKCa) inhibitor. These results suggest that equol exerts a preventive effect on the enhancement of 5-HT-induced contraction by high insulin (possibly mediated by the 5-HT2A receptor), and that these effects may be attributed to the activation of BKCa channels in vascular smooth muscle.

Effectiveness of nalbuphine, a κ-opioid receptor agonist and µ-opioid receptor antagonist, in the inhibition of INa , IK(M) , and IK(erg) unlinked to interaction with opioid receptors.

Nalbuphine (NAL) is recognized as a mixer with the κ-opioid receptor agonist and the µ-opioid receptor antagonist. However, whether this drug causes any modifications in neuronal ionic currents is unclear. The effects of NAL on ionic currents in mHippoE-14 hippocampal neurons were investigated. In the whole-cell current recordings, NAL suppressed the peak amplitude of voltage-gated Na+ current (INa ) with an IC50 value of 1.9 µM. It shifted the steady-state inactivation curve of peak INa to the hyperpolarized potential, suggesting that there is the voltage dependence of NAL-mediated inhibition of peak INa . In continued presence of NAL, subsequent application of either dynorphin A1-13 (1 µM) or naloxone (30 µM) failed to modify its suppression of peak INa . Tefluthrin (Tef; 10 µM), a pyrethroid known to activate INa , increased peak INa with slowed current inactivation; however, further application of NAL suppressed Tef-mediated suppression of peak INa followed by an additional slowing of current inactivation. In addition, NAL suppressed the amplitude of M-type K+ current [IK(M) ] with an IC50 value of 5.7 µM, while it slightly suppressed erg-mediated and delayed-rectifier K+ currents. In the inside-out current recordings, NAL failed to modify the activity of large-conductance Ca2+ -activated K+ channels. In differentiated NG108-15 neuronal cells, NAL also suppressed the peak INa , and subsequent addition of Tef reversed NAL-induced suppression of INa . Our study highlights the evidence that in addition to modulate opioid receptors, NAL has the propensity to interfere with ionic currents including INa and IK(M) , thereby influencing the functional activities of central neurons.

Multiple regulatory effects of angiotensin II on the large-conductance Ca2+- and voltage-activated potassium channel in vascular smooth muscle cells.

Renin-angiotensin system (RAS) is involved in the regulation of vascular smooth muscle cell (VSMC) tension. Angiotensin II (Ang II) as the main effector molecule of RAS can increase the intracellular Ca2+ concentration and cause VSMCs contraction by activating angiotensin II type 1 receptor (AT1R). The large-conductance Ca2+- and voltage-activated potassium (BK) channel is an essential potassium channel in VSMCs, playing an important role in maintaining membrane potential and intracellular potassium-calcium balance. The BK channel in VSMCs mainly consists of α and ß1 subunits. Functional BKα subunits contain voltage-sensors and Ca2+ binding sites. Hence, increase in the membrane potential or intracellular Ca2+ concentration can trigger the opening of the BK channel by mediating transient K+ outward current in a negative regulatory manner. However, increasing evidence has shown that although Ang II can raise the intracellular Ca2+ concentration, it also inhibits the expression and function of the BK channel by activating the PKC pathway, internalizing AT1R-BKα heterodimer, or dissociating α and ß1 subunits. Under some specific conditions, Ang II can also activate the BK channel, but the underlying mechanism remains unknown. In this review, we summarize the potential mechanisms underlying the inhibitory or activating effect of Ang II on the BK channel, hoping that it could provide a theoretical basis for improving intracellular ion imbalance.

Dendritic and Axonal L-Type Calcium Channels Cooperate to Enhance Motoneuron Firing Output during Drosophila Larval Locomotion.

Behaviorally adequate neuronal firing patterns are critically dependent on the specific types of ion channel expressed and on their subcellular localization. This study combines in situ electrophysiology with genetic and pharmacological intervention in larval Drosophila melanogaster of both sexes to address localization and function of L-type like calcium channels in motoneurons. We demonstrate that Dmca1D (Cav1 homolog) L-type like calcium channels localize to both the somatodendritic and the axonal compartment of larval crawling motoneurons. In situ patch-clamp recordings in genetic mosaics reveal that Dmca1D channels increase burst duration and maximum intraburst firing frequencies during crawling-like motor patterns in semi-intact animals. Genetic and acute pharmacological manipulations suggest that prolonged burst durations are caused by dendritically localized Dmca1D channels, which activate upon cholinergic synaptic input and amplify EPSPs, thus indicating a conserved function of dendritic L-type channels from Drosophila to vertebrates. By contrast, maximum intraburst firing rates require axonal calcium influx through Dmca1D channels, likely to enhance sodium channel de-inactivation via a fast afterhyperpolarization through BK channel activation. Therefore, in unmyelinated Drosophila motoneurons different functions of axonal and dendritic L-type like calcium channels likely operate synergistically to maximize firing output during locomotion.SIGNIFICANCE STATEMENT Nervous system function depends on the specific excitabilities of different types of neurons. Excitability is largely shaped by different combinations of voltage-dependent ion channels. Despite a high degree of conservation, the huge diversity of ion channel types and their differential localization pose challenges in assigning distinct functions to specific channels across species. We find a conserved role, from fruit flies to mammals, for L-type calcium channels in augmenting motoneuron excitability. As in spinal cord, dendritic L-type channels amplify excitatory synaptic input. In contrast to spinal motoneurons, axonal L-type channels enhance firing rates in unmyelinated Drosophila motoraxons. Therefore, enhancing motoneuron excitability by L-type channels seems an old strategy, but localization and interactions with other channels are tuned to species-specific requirements.

Activation of voltage-dependent K+ channels strongly limits hypoxia-induced elevation of [Ca2+ ]i in rat carotid body glomus cells.

KEY POINTS: We studied the role of the large-conductance Ca2+ -activated K+ channel (BK) and voltage-dependent K+ channels (Kv) on [Ca2+ ]i responses to a wide range of hypoxia at different resting cell membrane potential (Em ). BK/Kv were mostly closed at rest in normoxia. BK/Kv became basally active when cells were depolarized by elevated [KCl]o (>12 mm). Regardless of whether BK/Kv were closed or basally open, hypoxia-induced elevation of [Ca2+ ]i was enhanced 2- to 3-fold by inhibitors of BK/Kv. Hypoxia-induced elevation of [Ca2+ ]i was enhanced ∼2-fold by an inhibitor of Kv2, a major Kv in rat glomus cells. Hypoxia did not inhibit BK in inside-out patches. Our study supports a scheme in which activation of BK/Kv strongly limits the magnitude of hypoxia-induced [Ca2+ ]i rise, with Kv having a much greater effect than BK. ABSTRACT: Large-conductance KCa (BK) and other voltage-dependent K+ channels (Kv) are highly expressed in carotid body (CB) glomus cells, but their role in hypoxia-induced excitation is still not well defined and remains controversial. We addressed this issue by studying the effects of inhibitors of BK (IBTX) and BK/Kv (TEA/4-AP) on [Ca2+ ]i responses to a wide range of hypoxia at different levels of resting cell membrane potential (Em ). IBTX and TEA/4-AP did not affect the basal [Ca2+ ]i in isolated glomus cells bathed in 5 mm KClo , but elicited transient increases in [Ca2+ ]i in cells that were moderately depolarized (11-20 mV) by elevation of [KCl]o (12-20 mm). Thus, BK and Kv were mostly closed at rest and activated by depolarization. Four different levels of hypoxia (mild, moderate, severe, anoxia) were used to produce a wide range of [Ca2+ ]i elevation (0-700 nm). IBTX did not affect the rise in [Ca2+ ]i , but TEA/4-AP strongly (∼3-fold) enhanced [Ca2+ ]i rise by moderate and severe levels of hypoxia. Guangxitoxin, a Kv2 blocker, inhibited the whole-cell current by ∼50%, and enhanced 2-fold the [Ca2+ ]i rise elicited by moderate and severe levels of hypoxia. Anoxia did not directly affect BK, but activated BK via depolarization. Our findings do not support the view that hypoxia inhibits BK/Kv to initiate or maintain the hypoxic response. Rather, our results show that BK/Kv are activated as glomus cells depolarize in response to hypoxia, which then limits the rise in [Ca2+ ]i . Inhibition of Kv may provide a mechanism to enhance the chemosensory activity of the CB and ventilation.

A computational model for how the fast afterhyperpolarization paradoxically increases gain in regularly firing neurons.

The gain of a neuron, the number and frequency of action potentials triggered in response to a given amount of depolarizing injection, is an important behavior underlying a neuron's function. Variations in action potential waveform can influence neuronal discharges by the differential activation of voltage- and ion-gated channels long after the end of a spike. One component of the action potential waveform, the afterhyperpolarization (AHP), is generally considered an inhibitory mechanism for limiting firing rates. In dentate gyrus granule cells (DGCs) expressing fast-gated BK channels, large fast AHPs (fAHP) are paradoxically associated with increased gain. In this article, we describe a mechanism for this behavior using a computational model. Hyperpolarization provided by the fAHP enhances activation of a dendritic inward current (a T-type Ca2+ channel is suggested) that, in turn, boosts rebound depolarization at the soma. The model suggests that the fAHP may both reduce Ca2+ channel inactivation and, counterintuitively, enhance its activation. The magnitude of the rebound depolarization, in turn, determines the activation of a subsequent, slower inward current (a persistent Na+ current is suggested) limiting the interspike interval. Simulations also show that the effect of AHP on gain is also effective for physiologically relevant stimulation; varying AHP amplitude affects interspike interval across a range of "noisy" stimulus frequency and amplitudes. The mechanism proposed suggests that small fAHPs in DGCs may contribute to their limited excitability. NEW & NOTEWORTHY The afterhyperpolarization (AHP) is canonically viewed as a major factor underlying the refractory period, serving to limit neuronal firing rate. We recently reported that enhancing the amplitude of the fast AHP (fAHP) in a relatively slowly firing neuron (vs. fast spiking neurons) expressing fast-gated BK channels augments neuronal excitability. In this computational study, we present a novel, quantitative hypothesis for how varying the amplitude of the fAHP can, paradoxically, influence a subsequent spike tens of milliseconds later.

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.

Pathophysiology in the suprachiasmatic nucleus in mouse models of Huntington's disease.

Disturbances in sleep/wake cycle are a common complaint of individuals with Huntington's disease (HD) and are displayed by HD mouse models. The underlying mechanisms, including the possible role of the circadian timing system, are not well established. The BACHD mouse model of HD exhibits disrupted behavioral and physiological rhythms, including decreased electrical activity in the central circadian clock (suprachiasmatic nucleus, SCN). In this study, electrophysiological techniques were used to explore the ionic underpinning of the reduced spontaneous neural activity in male mice. We found that SCN neural activity rhythms were lost early in the disease progression and was accompanied by loss of the normal daily variation in resting membrane potential in the mutant SCN neurons. The low neural activity could be transiently reversed by direct current injection or application of exogenous N-methyl-d-aspartate (NMDA) thus demonstrating that the neurons have the capacity to discharge at WT levels. Exploring the potassium currents known to regulate the electrical activity of SCN neurons, our most striking finding was that these cells in the mutants exhibited an enhancement in the large-conductance calcium activated K+ (BK) currents. The expression of the pore forming subunit (Kcnma1) of the BK channel was higher in the mutant SCN. We found a similar decrease in daytime electrical activity and enhancement in the magnitude of the BK currents early in disease in another HD mouse model (Q175). These findings suggest that SCN neurons of both HD models exhibit early pathophysiology and that dysregulation of BK current may be responsible.

Direct Activation of the Large-Conductance Calcium-Activated Potassium Channel by Flavonoids Isolated from Sophora flavescens.

In our recent study, we reported that kurarinone, one of the most abundant flavonoids found in the dry root of Sophora flavescens (Kushen), is a potent activator of the large-conductance Ca2+-activated K+ (BKCa) channel. Herein, we isolated and characterized other flavonoid components from Kushen. Among the 13 compounds tested, six flavonoids were found to activate the BKCa channel, three of which, 7,4'-dihydroxy-5-methoxy-8-(γ,γ-dimethylallyl)-flavanone, kuraridin, and kuraridinol, are new activators of the BKCa channel.

Molecular mechanisms underlying the effect of the novel BK channel opener GoSlo: involvement of the S4/S5 linker and the S6 segment.

GoSlo-SR-5-6 is a novel large-conductance Ca(2+)-activated K(+) (BK) channel agonist that shifts the activation V1/2 of these channels in excess of -100 mV when applied at a concentration of 10 µM. Although the structure-activity relationship of this family of molecules has been established, little is known about how they open BK channels. To help address this, we used a combination of electrophysiology, mutagenesis, and mathematical modeling to investigate the molecular mechanisms underlying the effect of GoSlo-SR-5-6. Our data demonstrate that the effects of this agonist are practically abolished when three point mutations are made: L227A in the S4/S5 linker in combination with S317R and I326A in the S6C region. Our data suggest that GoSlo-SR-5-6 interacts with the transmembrane domain of the channel to enhance pore opening. The Horrigan-Aldrich model suggests that GoSlo-SR-5-6 works by stabilizing the open conformation of the channel and the activated state of the voltage sensors, yet decouples the voltage sensors from the pore gate.