Synaptic mechanisms underlying modulation of locomotor-related motoneuron output by premotor cholinergic interneurons.

Spinal motor networks are formed by diverse populations of interneurons that set the strength and rhythmicity of behaviors such as locomotion. A small cluster of cholinergic interneurons, expressing the transcription factor Pitx2, modulates the intensity of muscle activation via 'C-bouton' inputs to motoneurons. However, the synaptic mechanisms underlying this neuromodulation remain unclear. Here, we confirm in mice that Pitx2+ interneurons are active during fictive locomotion and that their chemogenetic inhibition reduces the amplitude of motor output. Furthermore, after genetic ablation of cholinergic Pitx2+ interneurons, M2 receptor-dependent regulation of the intensity of locomotor output is lost. Conversely, chemogenetic stimulation of Pitx2+ interneurons leads to activation of M2 receptors on motoneurons, regulation of Kv2.1 channels and greater motoneuron output due to an increase in the inter-spike afterhyperpolarization and a reduction in spike half-width. Our findings elucidate synaptic mechanisms by which cholinergic spinal interneurons modulate the final common pathway for motor output.

Microglia monitor and protect neuronal function through specialized somatic purinergic junctions.

Microglia are the main immune cells in the brain and have roles in brain homeostasis and neurological diseases. Mechanisms underlying microglia-neuron communication remain elusive. Here, we identified an interaction site between neuronal cell bodies and microglial processes in mouse and human brain. Somatic microglia-neuron junctions have a specialized nanoarchitecture optimized for purinergic signaling. Activity of neuronal mitochondria was linked with microglial junction formation, which was induced rapidly in response to neuronal activation and blocked by inhibition of P2Y12 receptors. Brain injury-induced changes at somatic junctions triggered P2Y12 receptor-dependent microglial neuroprotection, regulating neuronal calcium load and functional connectivity. Thus, microglial processes at these junctions could potentially monitor and protect neuronal functions.

Kv2.1 voltage-gated potassium channels in developmental perspective.

Kv2.1 voltage-gated potassium channels consist of two types of α-subunits: (a) electrically-active Kcnb1 α-subunits and (b) silent or modulatory α-subunits plus ß-subunits that, similar to silent α-subunits, also regulate electrically-active subunits. Voltage-gated potassium channels were traditionally viewed, mainly by electrophysiologists, as regulators of the electrical activity of the plasma membrane in excitable cells, a role that is performed by transmembrane protein domains of α-subunits that form the electric pore. Genetic studies revealed a role for this region of α-subunits of voltage-gated potassium channels in human neurodevelopmental disorders, such as epileptic encephalopathy. The N- and C-terminal domains of α-subunits interact to form the cytoplasmic subunit of heterotetrameric potassium channels that regulate electric pores. Subsequent animal studies revealed the developmental functions of Kcnb1-containing voltage-gated potassium channels and illustrated their role during brain development and reproduction. These functions of potassium channels are discussed in this review in the context of regulatory interactions between electrically-active and regulatory subunits.

Identification of VAPA and VAPB as Kv2 Channel-Interacting Proteins Defining Endoplasmic Reticulum-Plasma Membrane Junctions in Mammalian Brain Neurons.

Membrane contacts between endoplasmic reticulum (ER) and plasma membrane (PM), or ER-PM junctions, are ubiquitous in eukaryotic cells and are platforms for lipid and calcium signaling and homeostasis. Recent studies have revealed proteins crucial to the formation and function of ER-PM junctions in non-neuronal cells, but little is known of the ER-PM junctions prominent in aspiny regions of mammalian brain neurons. The Kv2.1 voltage-gated potassium channel is abundantly clustered at ER-PM junctions in brain neurons and is the first PM protein that functions to organize ER-PM junctions. However, the molecular mechanism whereby Kv2.1 localizes to and remodels these junctions is unknown. We used affinity immunopurification and mass spectrometry-based proteomics on brain samples from male and female WT and Kv2.1 KO mice and identified the resident ER vesicle-associated membrane protein-associated proteins isoforms A and B (VAPA and VAPB) as prominent Kv2.1-associated proteins. Coexpression with Kv2.1 or its paralog Kv2.2 was sufficient to recruit VAPs to ER-PM junctions. Multiplex immunolabeling revealed colocalization of Kv2.1 and Kv2.2 with endogenous VAPs at ER-PM junctions in brain neurons from male and female mice in situ and in cultured rat hippocampal neurons, and KO of VAPA in mammalian cells reduces Kv2.1 clustering. The association of VAPA with Kv2.1 relies on a "two phenylalanines in an acidic tract" (FFAT) binding domain on VAPA and a noncanonical phosphorylation-dependent FFAT motif comprising the Kv2-specific clustering or PRC motif. These results suggest that Kv2.1 localizes to and organizes neuronal ER-PM junctions through an interaction with VAPs.SIGNIFICANCE STATEMENT Our study identified the endoplasmic reticulum (ER) proteins vesicle-associated membrane protein-associated proteins isoforms A and B (VAPA and VAPB) as proteins copurifying with the plasma membrane (PM) Kv2.1 ion channel. We found that expression of Kv2.1 recruits VAPs to ER-PM junctions, specialized membrane contact sites crucial to distinct aspects of cell function. We found endogenous VAPs at Kv2.1-mediated ER-PM junctions in brain neurons and other mammalian cells and that knocking out VAPA expression disrupts Kv2.1 clustering. We identified domains of VAPs and Kv2.1 necessary and sufficient for their association at ER-PM junctions. Our study suggests that Kv2.1 expression in the PM can affect ER-PM junctions via its phosphorylation-dependent association to ER-localized VAPA and VAPB.

Kv2 potassium channels form endoplasmic reticulum/plasma membrane junctions via interaction with VAPA and VAPB.

Kv2.1 exhibits two distinct forms of localization patterns on the neuronal plasma membrane: One population is freely diffusive and regulates electrical activity via voltage-dependent K+ conductance while a second one localizes to micrometer-sized clusters that contain densely packed, but nonconducting, channels. We have previously established that these clusters represent endoplasmic reticulum/plasma membrane (ER/PM) junctions that function as membrane trafficking hubs and that Kv2.1 plays a structural role in forming these membrane contact sites in both primary neuronal cultures and transfected HEK cells. Clustering and the formation of ER/PM contacts are regulated by phosphorylation within the channel C terminus, offering cells fast, dynamic control over the physical relationship between the cortical ER and PM. The present study addresses the mechanisms by which Kv2.1 and the related Kv2.2 channel interact with the ER membrane. Using proximity-based biotinylation techniques in transfected HEK cells we identified ER VAMP-associated proteins (VAPs) as potential Kv2.1 interactors. Confirmation that Kv2.1 and -2.2 bind VAPA and VAPB employed colocalization/redistribution, siRNA knockdown, and Förster resonance energy transfer (FRET)-based assays. CD4 chimeras containing sequence from the Kv2.1 C terminus were used to identify a noncanonical VAP-binding motif. VAPs were first identified as proteins required for neurotransmitter release in Aplysia and are now known to be abundant scaffolding proteins involved in membrane contact site formation throughout the ER. The VAP interactome includes AKAPs, kinases, membrane trafficking machinery, and proteins regulating nonvesicular lipid transport from the ER to the PM. Therefore, the Kv2-induced VAP concentration at ER/PM contact sites is predicted to have wide-ranging effects on neuronal cell biology.

Cleavage of potassium channel Kv2.1 by BACE2 reduces neuronal apoptosis.

Potassium channel Kv2.1 regulates potassium current in cortical neurons and potassium efflux is necessary for cell apoptosis. As a major component of delayed rectifier current potassium channels, Kv2.1 forms clusters in the membrane of hippocampal neurons. BACE2 is an aspartyl protease to cleave APP to prevent the generation of Aß, a central component of neuritic plaques in Alzheimer's brain. We now identified Kv2.1 as a novel substrate of BACE2. We found that BACE2 cleaved Kv2.1 at Thr376, Ala717, and Ser769 sites and disrupted Kv2.1 clustering on cell membrane, resulting in decreased Ik of Kv2.1 and a hyperpolarizing shift in primary neurons. Furthermore, we discovered that the BACE2-cleaved Kv2.1 forms, Kv2.1-1-375, Kv2.1-1-716, and Kv2.1-1-768, depressed the delayed rectifier Ik surge and reduced neuronal apoptosis. Our study suggests that BACE2 plays a neuroprotective role by cleavage of Kv2.1 to prevent the outward potassium currents, a potential new target for Alzheimer's treatment.

Kv2.1 clusters on ß-cell plasma membrane act as reservoirs that replenish pools of newcomer insulin granule through their interaction with syntaxin-3.

The voltage-dependent K+ (Kv) channel Kv2.1 is a major delayed rectifier in many secretory cells, including pancreatic ß cells. In addition, Kv2.1 has a direct role in exocytosis at an undefined step, involving SNARE proteins, that is independent of its ion-conducting pore function. Here, we elucidated the precise step in exocytosis. We previously reported that syntaxin-3 (Syn-3) is the key syntaxin that mediates exocytosis of newcomer secretory granules that spend minimal residence time on the plasma membrane before fusion. Using high-resolution total internal reflection fluorescence microscopy, we now show that Kv2.1 forms reservoir clusters on the ß-cell plasma membrane and binds Syn-3 via its C-terminal C1b domain, which recruits newcomer insulin secretory granules into this large reservoir. Upon glucose stimulation, secretory granules were released from this reservoir to replenish the pool of newcomer secretory granules for subsequent fusion, occurring just adjacent to the plasma membrane Kv2.1 clusters. C1b deletion blocked the aforementioned Kv2.1-Syn-3-mediated events and reduced fusion of newcomer secretory granules. These insights have therapeutic implications, as Kv2.1 overexpression in type-2 diabetes rat islets restored biphasic insulin secretion.

N'-mono- and N, N'-diacyl derivatives of benzyl and arylhydrazines as contact insecticides against adult Anopheles gambiae.

New public health insecticides are urgently required to prevent the spread of vector-borne disease. With the goal of identifying new K+-channel-directed mosquitocides, analogs of the RH-5849 family of diacyl t-butylhydrazines were synthesized and tested for topical toxicity against adult Anopheles gambiae, the African vector of malaria. In total, 80N'-monoacyl and N, N'-diacyl derivatives of benzyl- and arylhydrazines were prepared. Three compounds (2bo, 2kb, 3ab) were identified that were more toxic than RH-5849 and RH-1266. The potencies of these compounds to block K+ currents in An. gambiae and human Kv2.1 channels were assessed to address their possible mechanism of mosquitocidal action. Selectivity for inhibition of An. gambiae Kv2.1 vs human Kv2.1 did not exceed 3-fold. Furthermore, no correlation was seen between the potency of insecticidal action and K+ channel blocking potency. These observations, combined with the minimal knockdown seen with 2bo near its LD50 value, suggests a mode of action outside of the nervous system.

Reduced sensory synaptic excitation impairs motor neuron function via Kv2.1 in spinal muscular atrophy.

Behavioral deficits in neurodegenerative diseases are often attributed to the selective dysfunction of vulnerable neurons via cell-autonomous mechanisms. Although vulnerable neurons are embedded in neuronal circuits, the contributions of their synaptic partners to disease process are largely unknown. Here we show that, in a mouse model of spinal muscular atrophy (SMA), a reduction in proprioceptive synaptic drive leads to motor neuron dysfunction and motor behavior impairments. In SMA mice or after the blockade of proprioceptive synaptic transmission, we observed a decrease in the motor neuron firing that could be explained by the reduction in the expression of the potassium channel Kv2.1 at the surface of motor neurons. Chronically increasing neuronal activity pharmacologically in vivo led to a normalization of Kv2.1 expression and an improvement in motor function. Our results demonstrate a key role of excitatory synaptic drive in shaping the function of motor neurons during development and the contribution of its disruption to a neurodegenerative disease.

Angiotensin II reduces the surface abundance of KV 1.5 channels in arterial myocytes to stimulate vasoconstriction.

KEY POINTS: Several different voltage-dependent K+ (KV ) channel isoforms are expressed in arterial smooth muscle cells (myocytes). Vasoconstrictors inhibit KV currents, but the isoform selectivity and mechanisms involved are unclear. We show that angiotensin II (Ang II), a vasoconstrictor, stimulates degradation of KV 1.5, but not KV 2.1, channels through a protein kinase C- and lysosome-dependent mechanism, reducing abundance at the surface of mesenteric artery myocytes. The Ang II-induced decrease in cell surface KV 1.5 channels reduces whole-cell KV 1.5 currents and attenuates KV 1.5 function in pressurized arteries. We describe a mechanism by which Ang II stimulates protein kinase C-dependent KV 1.5 channel degradation, reducing the abundance of functional channels at the myocyte surface. ABSTRACT: Smooth muscle cells (myocytes) of resistance-size arteries express several different voltage-dependent K+ (KV ) channels, including KV 1.5 and KV 2.1, which regulate contractility. Myocyte KV currents are inhibited by vasoconstrictors, including angiotensin II (Ang II), but the mechanisms involved are unclear. Here, we tested the hypothesis that Ang II inhibits KV currents by reducing the plasma membrane abundance of KV channels in myocytes. Angiotensin II (applied for 2 h) reduced surface and total KV 1.5 protein in rat mesenteric arteries. In contrast, Ang II did not alter total or surface KV 2.1, or KV 1.5 or KV 2.1 cellular distribution, measured as the percentage of total protein at the surface. Bisindolylmaleimide (BIM; a protein kinase C blocker), a protein kinase C inhibitory peptide or bafilomycin A (a lysosomal degradation inhibitor) each blocked the Ang II-induced decrease in total and surface KV 1.5. Immunofluorescence also suggested that Ang II reduced surface KV 1.5 protein in isolated myocytes; an effect inhibited by BIM. Arteries were exposed to Ang II or Ang II plus BIM (for 2 h), after which these agents were removed and contractility measurements performed or myocytes isolated for patch-clamp electrophysiology. Angiotensin II reduced both whole-cell KV currents and currents inhibited by Psora-4, a KV 1.5 channel blocker. Angiotensin II also reduced vasoconstriction stimulated by Psora-4 or 4-aminopyridine, another KV channel inhibitor. These data indicate that Ang II activates protein kinase C, which stimulates KV 1.5 channel degradation, leading to a decrease in surface KV 1.5, a reduction in whole-cell KV 1.5 currents and a loss of functional KV 1.5 channels in myocytes of pressurized arteries.

Physiological roles of Kv2 channels in entorhinal cortex layer II stellate cells revealed by Guangxitoxin-1E.

KEY POINTS: Kv2 channels underlie delayed-rectifier potassium currents in various neurons, although their physiological roles often remain elusive. Almost nothing is known about Kv2 channel functions in medial entorhinal cortex (mEC) neurons, which are involved in representing space, memory formation, epilepsy and dementia. Stellate cells in layer II of the mEC project to the hippocampus and are considered to be space-representing grid cells. We used the new Kv2 blocker Guangxitoxin-1E (GTx) to study Kv2 functions in these neurons. Voltage clamp recordings from mEC stellate cells in rat brain slices showed that GTx inhibited delayed-rectifier K+ current but not transient A-type current. In current clamp, GTx had multiple effects: (i) increasing excitability and bursting at moderate spike rates but reducing firing at high rates; (ii) enhancing after-depolarizations; (iii) reducing the fast and medium after-hyperpolarizations; (iv) broadening action potentials; and (v) reducing spike clustering. GTx is a useful tool for studying Kv2 channels and their functions in neurons. ABSTRACT: The medial entorhinal cortex (mEC) is strongly involved in spatial navigation, memory, dementia and epilepsy. Although potassium channels shape neuronal activity, their roles in mEC are largely unknown. We used the new Kv2 blocker Guangxitoxin-1E (GTx; 10-100 nm) in rat brain slices to investigate Kv2 channel functions in mEC layer II stellate cells (SCs). These neurons project to the hippocampus and are considered to be grid cells representing space. Voltage clamp recordings from SCs nucleated patches showed that GTx inhibited a delayed rectifier K+ current activating beyond -30 mV but not transient A-type current. In current clamp, GTx (i) had almost no effect on the first action potential but markedly slowed repolarization of late spikes during repetitive firing; (ii) enhanced the after-depolarization (ADP); (iii) reduced fast and medium after-hyperpolarizations (AHPs); (iv) strongly enhanced burst firing and increased excitability at moderate spike rates but reduced spiking at high rates; and (v) reduced spike clustering and rebound potentials. The changes in bursting and excitability were related to the altered ADPs and AHPs. Kv2 channels strongly shape the activity of mEC SCs by affecting spike repolarization, after-potentials, excitability and spike patterns. GTx is a useful tool and may serve to further clarify Kv2 channel functions in neurons. We conclude that Kv2 channels in mEC SCs are important determinants of intrinsic properties that allow these neurons to produce spatial representation. The results of the present study may also be important for the accurate modelling of grid cells.

Functional antagonism of voltage-gated K+ channel α-subunits in the developing brain ventricular system.

The brain ventricular system is essential for neurogenesis and brain homeostasis. Its neuroepithelial lining effects these functions, but the underlying molecular pathways remain to be understood. We found that the potassium channels expressed in neuroepithelial cells determine the formation of the ventricular system. The phenotype of a novel zebrafish mutant characterized by denudation of neuroepithelial lining of the ventricular system and hydrocephalus is mechanistically linked to Kcng4b, a homologue of the 'silent' voltage-gated potassium channel α-subunit Kv6.4. We demonstrated that Kcng4b modulates proliferation of cells lining the ventricular system and maintains their integrity. The gain of Kcng4b function reduces the size of brain ventricles. Electrophysiological studies suggest that Kcng4b mediates its effects via an antagonistic interaction with Kcnb1, the homologue of the electrically active delayed rectifier potassium channel subunit Kv2.1. Mutation of kcnb1 reduces the size of the ventricular system and its gain of function causes hydrocephalus, which is opposite to the function of Kcng4b. This demonstrates the dynamic interplay between potassium channel subunits in the neuroepithelium as a novel and crucial regulator of ventricular development in the vertebrate brain.

Differential Regulation of Action Potential Shape and Burst-Frequency Firing by BK and Kv2 Channels in Substantia Nigra Dopaminergic Neurons.

Little is known about the voltage-dependent potassium currents underlying spike repolarization in midbrain dopaminergic neurons. Studying mouse substantia nigra pars compacta dopaminergic neurons both in brain slice and after acute dissociation, we found that BK calcium-activated potassium channels and Kv2 channels both make major contributions to the depolarization-activated potassium current. Inhibiting Kv2 or BK channels had very different effects on spike shape and evoked firing. Inhibiting Kv2 channels increased spike width and decreased the afterhyperpolarization, as expected for loss of an action potential-activated potassium conductance. BK inhibition also increased spike width but paradoxically increased the afterhyperpolarization. Kv2 channel inhibition steeply increased the slope of the frequency-current (f-I) relationship, whereas BK channel inhibition had little effect on the f-I slope or decreased it, sometimes resulting in slowed firing. Action potential clamp experiments showed that both BK and Kv2 current flow during spike repolarization but with very different kinetics, with Kv2 current activating later and deactivating more slowly. Further experiments revealed that inhibiting either BK or Kv2 alone leads to recruitment of additional current through the other channel type during the action potential as a consequence of changes in spike shape. Enhancement of slowly deactivating Kv2 current can account for the increased afterhyperpolarization produced by BK inhibition and likely underlies the very different effects on the f-I relationship. The cross-regulation of BK and Kv2 activation illustrates that the functional role of a channel cannot be defined in isolation but depends critically on the context of the other conductances in the cell. SIGNIFICANCE STATEMENT: This work shows that BK calcium-activated potassium channels and Kv2 voltage-activated potassium channels both regulate action potentials in dopamine neurons of the substantia nigra pars compacta. Although both channel types participate in action potential repolarization about equally, they have contrasting and partially opposite effects in regulating neuronal firing at frequencies typical of bursting. Our analysis shows that this results from their different kinetic properties, with fast-activating BK channels serving to short-circuit activation of Kv2 channels, which tend to slow firing by producing a deep afterhyperpolarization. The cross-regulation of BK and Kv2 activation illustrates that the functional role of a channel cannot be defined in isolation but depends critically on the context of the other conductances in the cell.

Heme Oxygenase-1 Influences Apoptosis via CO-mediated Inhibition of K+ Channels.

Hypoxic/ischemic episodes can trigger oxidative stress-mediated loss of central neurons via apoptosis, and low pO2 is also a feature of the tumor microenvironment, where cancer cells are particularly resistant to apoptosis. In the CNS, ischemic insult increases expression of the CO-generating enzyme heme oxygenase-1 (HO-1), which is commonly constitutively active in cancer cells. It has been proposed that apoptosis can be regulated by the trafficking and activity of K(+) channels, particularly Kv2.1. We have explored the idea that HO-1 may influence apoptosis via regulation of Kv2.1. Overexpression of Kv2.1 in HEK293 cells increased their vulnerability to oxidant-induced apoptosis. CO (applied as the donor CORM-2) protected cells against apoptosis and inhibited Kv2.1 channels. Similarly in hippocampal neurones, CO selectively inhibited Kv2.1 and protected neurones against oxidant-induced apoptosis. In medulloblastoma sections we identified constitutive expression of HO-1 and Kv2.1, and in the medulloblastoma-derived cell line DAOY, hypoxic HO-1 induction or exposure to CO protected cells against apoptosis, and also selectively inhibited Kv2.1 channels expressed in these cells. These studies are consistent with a central role for Kv2.1 in apoptosis in both central neurones and cancer cells. They also suggest that HO-1 expression can strongly influence apoptosis via CO-mediated regulation of Kv2.1 activity.

Selective down-regulation of KV2.1 function contributes to enhanced arterial tone during diabetes.

Enhanced arterial tone is a leading cause of vascular complications during diabetes. Voltage-gated K(+) (KV) channels are key regulators of vascular smooth muscle cells (VSMCs) contractility and arterial tone. Whether impaired KV channel function contributes to enhance arterial tone during diabetes is unclear. Here, we demonstrate a reduction in KV-mediated currents (IKv) in VSMCs from a high fat diet (HFD) mouse model of type 2 diabetes. In particular, IKv sensitive to stromatoxin (ScTx), a potent KV2 blocker, were selectively reduced in diabetic VSMCs. This was associated with decreased KV2-mediated regulation of arterial tone and suppression of the KV2.1 subunit mRNA and protein in VSMCs/arteries isolated from HFD mice. We identified protein kinase A anchoring protein 150 (AKAP150), via targeting of the phosphatase calcineurin (CaN), and the transcription factor nuclear factor of activated T-cells c3 (NFATc3) as required determinants of KV2.1 suppression during diabetes. Interestingly, substantial reduction in transcript levels for KV2.1 preceded down-regulation of large conductance Ca(2+)-activated K(+) (BKCa) channel ß1 subunits, which are ultimately suppressed in chronic hyperglycemia to a similar extent. Together, our study supports the concept that transcriptional suppression of KV2.1 by activation of the AKAP150-CaN/NFATc3 signaling axis contributes to enhanced arterial tone during diabetes.

Kv2.2: a novel molecular target to study the role of basal forebrain GABAergic neurons in the sleep-wake cycle.

STUDY OBJECTIVES: The basal forebrain (BF) has been implicated as an important brain region that regulates the sleep-wake cycle of animals. Gamma-aminobutyric acidergic (GABAergic) neurons are the most predominant neuronal population within this region. However, due to the lack of specific molecular tools, the roles of the BF GABAergic neurons have not been fully elucidated. Previously, we have found high expression levels of the Kv2.2 voltage-gated potassium channel on approximately 60% of GABAergic neurons in the magnocellular preoptic area and horizontal limb of the diagonal band of Broca of the BF and therefore proposed it as a potential molecular target to study this neuronal population. In this study, we sought to determine the functional roles of the Kv2.2-expressing neurons in the regulation of the sleep-wake cycle. DESIGN: Sleep analysis between two genotypes and within each genotype before and after sleep deprivation. SETTING: Animal sleep research laboratory. PARTICIPANTS: Adult mice. Wild-type and Kv2.2 knockout mice with C57/BL6 background. INTERVENTIONS: EEG/EMG recordings from the basal state and after sleep-deprivation which was induced by mild agitation for 6 h. RESULTS: Immunostaining of a marker of neuronal activity indicates that these Kv2.2-expressing neurons appear to be preferentially active during the wake state. Therefore, we tested whether Kv2.2-expressing neurons in the BF are involved in arousal using Kv2.2-deficient mice. BF GABAergic neurons exhibited augmented expression of c-Fos. These knockout mice exhibited longer consolidated wake bouts than wild-type littermates, and that phenotype was further exacerbated by sleep deprivation. Moreover, in-depth analyses of their cortical electroencephalogram revealed a significant decrease in the delta-frequency activity during the nonrapid eye movement sleep state. CONCLUSIONS: These results revealed the significance of Kv2.2-expressing neurons in the regulation of the sleep-wake cycle.

Protection from noise-induced hearing loss by Kv2.2 potassium currents in the central medial olivocochlear system.

The central auditory brainstem provides an efferent projection known as the medial olivocochlear (MOC) system, which regulates the cochlear amplifier and mediates protection on exposure to loud sound. It arises from neurons of the ventral nucleus of the trapezoid body (VNTB), so control of neuronal excitability in this pathway has profound effects on hearing. The VNTB and the medial nucleus of the trapezoid body are the only sites of expression for the Kv2.2 voltage-gated potassium channel in the auditory brainstem, consistent with a specialized function of these channels. In the absence of unambiguous antagonists, we used recombinant and transgenic methods to examine how Kv2.2 contributes to MOC efferent function. Viral gene transfer of dominant-negative Kv2.2 in wild-type mice suppressed outward K(+) currents, increasing action potential (AP) half-width and reducing repetitive firing. Similarly, VNTB neurons from Kv2.2 knock-out mice (Kv2.2KO) also showed increased AP duration. Control experiments established that Kv2.2 was not expressed in the cochlea, so any changes in auditory function in the Kv2.2KO mouse must be of central origin. Further, in vivo recordings of auditory brainstem responses revealed that these Kv2.2KO mice were more susceptible to noise-induced hearing loss. We conclude that Kv2.2 regulates neuronal excitability in these brainstem nuclei by maintaining short APs and enhancing high-frequency firing. This safeguards efferent MOC firing during high-intensity sounds and is crucial in the mediation of protection after auditory overexposure.

Shab K (+) channel slow inactivation: a test for U-type inactivation and a hypothesis regarding K (+) -facilitated inactivation mechanisms.

Herein, we report the first characterization of Shab slow inactivation. Open Shab channels inactivate within seconds, with two voltage-independent time constants. Additionally, Shab presents significant closed-state inactivation. We found that with short depolarizing pulses, shorter than the slowest inactivation time constant, the resulting inactivation curve has a marked U-shape, but as pulse duration increases, approaching steady-state conditions, the U-shape vanishes, and the resulting inactivation curves converge to the classical Boltzmann h∞ curve. Regarding the mechanism of inactivation, we found that external K (+) and TEA facilitate both open- and closed-state inactivation, while the cavity blocker quinidine hinders inactivation. These results together with our previous observations regarding the K (+) -dependent stability of the K (+) conductance, suggest the novel hypothesis that inactivation of Shab channels, and possibly that of other Kv channels whose inactivation is facilitated by K (+) , does not involve a significant narrowing of the extracellular entry of the pore. Instead, we hypothesize that there is only a rearrangement of a more internal segment of the pore that affects the central cavity and halts K (+) conduction.

Regulation of Kv2.1 K(+) conductance by cell surface channel density.

The Kv2.1 voltage-gated K(+) channel is found both freely diffusing over the plasma membrane and concentrated in micron-sized clusters localized to the soma, proximal dendrites, and axon initial segment of hippocampal neurons. In transfected HEK cells, Kv2.1 channels within cluster microdomains are nonconducting. Using total internal reflection fluorescence microscopy, the number of GFP-tagged Kv2.1 channels on the HEK cell surface was compared with K(+) channel conductance measured by whole-cell voltage clamp of the same cell. This approach indicated that, as channel density increases, nonclustered channels cease conducting. At the highest density observed, only 4% of all channels were conducting. Mutant Kv2.1 channels that fail to cluster also possessed the nonconducting state with 17% conducting K(+) at higher surface densities. The nonconducting state was specific to Kv2.1 as Kv1.4 was always conducting regardless of the cell-surface expression level. Anti-Kv2.1 immunofluorescence intensity, standardized to Kv2.1 surface density in transfected HEK cells, was used to determine the expression levels of endogenous Kv2.1 in cultured rat hippocampal neurons. Endogenous Kv2.1 levels were compared with the number of conducting channels determined by whole-cell voltage clamp. Only 13 and 27% of the endogenous Kv2.1 was conducting in neurons cultured for 14 and 20 d, respectively. Together, these data indicate that the nonconducting state depends primarily on surface density as opposed to cluster location and that this nonconducting state also exists for native Kv2.1 found in cultured hippocampal neurons. This excess of Kv2.1 protein relative to K(+) conductance further supports a nonconducting role for Kv2.1 in excitable tissues.

Toxic role of K+ channel oxidation in mammalian brain.

Potassium (K(+)) channels are essential to neuronal signaling and survival. Here we show that these proteins are targets of reactive oxygen species in mammalian brain and that their oxidation contributes to neuropathy. Thus, the KCNB1 (Kv2.1) channel, which is abundantly expressed in cortex and hippocampus, formed oligomers upon exposure to oxidizing agents. These oligomers were ∼10-fold more abundant in the brain of old than young mice. Oxidant-induced oligomerization of wild-type KCNB1 enhanced apoptosis in neuronal cells subject to oxidative insults. Consequently, a KCNB1 variant resistant to oxidation, obtained by mutating a conserved cysteine to alanine, (C73A), was neuroprotective. The fact that oxidation of KCNB1 is toxic, argues that this mechanism may contribute to neuropathy in conditions characterized by high levels of oxidative stress, such as Alzheimer's disease (AD). Accordingly, oxidation of KCNB1 channels was exacerbated in the brain of a triple transgenic mouse model of AD (3xTg-AD). The C73A variant protected neuronal cells from apoptosis induced by incubation with ß-amyloid peptide (Aß(1-42)). In an invertebrate model (Caenorhabditis elegans) that mimics aspects of AD, a C73A-KCNB1 homolog (C113S-KVS-1) protected specific neurons from apoptotic death induced by ectopic expression of human Aß(1-42). Together, these data underscore a novel mechanism of toxicity in neurodegenerative disease.