Kv3.3 Channels Bind Hax-1 and Arp2/3 to Assemble a Stable Local Actin Network that Regulates Channel Gating.

Mutations in the Kv3.3 potassium channel (KCNC3) cause cerebellar neurodegeneration and impair auditory processing. The cytoplasmic C terminus of Kv3.3 contains a proline-rich domain conserved in proteins that activate actin nucleation through Arp2/3. We found that Kv3.3 recruits Arp2/3 to the plasma membrane, resulting in formation of a relatively stable cortical actin filament network resistant to cytochalasin D that inhibits fast barbed end actin assembly. These Kv3.3-associated actin structures are required to prevent very rapid N-type channel inactivation during short depolarizations of the plasma membrane. The effects of Kv3.3 on the actin cytoskeleton are mediated by the binding of the cytoplasmic C terminus of Kv3.3 to Hax-1, an anti-apoptotic protein that regulates actin nucleation through Arp2/3. A human Kv3.3 mutation within a conserved proline-rich domain produces channels that bind Hax-1 but are impaired in recruiting Arp2/3 to the plasma membrane, resulting in growth cones with deficient actin veils in stem cell-derived neurons.

Kv3 Channels: Enablers of Rapid Firing, Neurotransmitter Release, and Neuronal Endurance.

The intrinsic electrical characteristics of different types of neurons are shaped by the K+ channels they express. From among the more than 70 different K+ channel genes expressed in neurons, Kv3 family voltage-dependent K+ channels are uniquely associated with the ability of certain neurons to fire action potentials and to release neurotransmitter at high rates of up to 1,000 Hz. In general, the four Kv3 channels Kv3.1-Kv3.4 share the property of activating and deactivating rapidly at potentials more positive than other channels. Each Kv3 channel gene can generate multiple protein isoforms, which contribute to the high-frequency firing of neurons such as auditory brain stem neurons, fast-spiking GABAergic interneurons, and Purkinje cells of the cerebellum, and to regulation of neurotransmitter release at the terminals of many neurons. The different Kv3 channels have unique expression patterns and biophysical properties and are regulated in different ways by protein kinases. In this review, we cover the function, localization, and modulation of Kv3 channels and describe how levels and properties of the channels are altered by changes in ongoing neuronal activity. We also cover how the protein-protein interaction of these channels with other proteins affects neuronal functions, and how mutations or abnormal regulation of Kv3 channels are associated with neurological disorders such as ataxias, epilepsies, schizophrenia, and Alzheimer's disease.

A cytoskeleton-membrane interaction conserved in fast-spiking neurons controls movement, emotion, and memory.

The pathogenesis of schizophrenia is believed to involve combined dysfunctions of many proteins including microtubule-associated protein 6 (MAP6) and Kv3.1 voltage-gated K+ (Kv) channel, but their relationship and functions in behavioral regulation are often not known. Here we report that MAP6 stabilizes Kv3.1 channels in parvalbumin-positive (PV+ ) fast-spiking GABAergic interneurons, regulating behavior. MAP6-/- and Kv3.1-/- mice display similar hyperactivity and avoidance reduction. Their proteins colocalize in PV+ interneurons and MAP6 deletion markedly reduces Kv3.1 protein level. We further show that two microtubule-binding modules of MAP6 bind the Kv3.1 tetramerization domain with high affinity, maintaining the channel level in both neuronal soma and axons. MAP6 knockdown by AAV-shRNA in the amygdala or the hippocampus reduces avoidance or causes hyperactivity and recognition memory deficit, respectively, through elevating projection neuron activity. Finally, knocking down Kv3.1 or disrupting the MAP6-Kv3.1 binding in these brain regions causes avoidance reduction and hyperactivity, consistent with the effects of MAP6 knockdown. Thus, disrupting this conserved cytoskeleton-membrane interaction in fast-spiking neurons causes different degrees of functional vulnerability in various neural circuits.

Dysfunctional Parvalbumin Neurons in Schizophrenia and the Pathway to the Clinical Application of Kv3 Channel Modulators.

Based on the pathophysiological changes observed in schizophrenia, the gamma-aminobutyric acid (GABA) hypothesis may facilitate the development of targeted treatments for this disease. This hypothesis, mainly derived from postmortem brain results, postulates dysfunctions in a subset of GABAergic neurons, particularly parvalbumin-containing interneurons. In the cerebral cortex, the fast spike firing of parvalbumin-positive GABAergic interneurons is regulated by the Kv3.1 and Kv3.2 channels, which belong to a potassium channel subfamily. Decreased Kv3.1 levels have been observed in the prefrontal cortex of patients with schizophrenia, prompting the investigation of Kv3 channel modulators for the treatment of schizophrenia. However, biomarkers that capture the dysfunction of parvalbumin neurons are required for these modulators to be effective in the pharmacotherapy of schizophrenia. Electroencephalography and magnetoencephalography studies have demonstrated impairments in evoked gamma oscillations in patients with schizophrenia, which may reflect the dysfunction of cortical parvalbumin neurons. This review summarizes these topics and provides an overview of how the development of therapeutics that incorporate biomarkers could innovate the treatment of schizophrenia and potentially change the targets of pharmacotherapy.

Ankyrin-R Links Kv3.3 to the Spectrin Cytoskeleton and Is Required for Purkinje Neuron Survival.

Ankyrin scaffolding proteins are critical for membrane domain organization and protein stabilization in many different cell types including neurons. In the cerebellum, Ankyrin-R (AnkR) is highly enriched in Purkinje neurons, granule cells, and in the cerebellar nuclei (CN). Using male and female mice with a floxed allele for Ank1 in combination with Nestin-Cre and Pcp2-Cre mice, we found that ablation of AnkR from Purkinje neurons caused ataxia, regional and progressive neurodegeneration, and altered cerebellar output. We show that AnkR interacts with the cytoskeletal protein ß3 spectrin and the potassium channel Kv3.3. Loss of AnkR reduced somatic membrane levels of ß3 spectrin and Kv3.3 in Purkinje neurons. Thus, AnkR links Kv3.3 channels to the ß3 spectrin-based cytoskeleton. Our results may help explain why mutations in ß3 spectrin and Kv3.3 both cause spinocerebellar ataxia.SIGNIFICANCE STATEMENT Ankyrin scaffolding proteins localize and stabilize ion channels in the membrane by linking them to the spectrin-based cytoskeleton. Here, we show that Ankyrin-R (AnkR) links Kv3.3 K+ channels to the ß3 spectrin-based cytoskeleton in Purkinje neurons. Loss of AnkR causes Purkinje neuron degeneration, altered cerebellar physiology, and ataxia, which is consistent with mutations in Kv3.3 and ß3 spectrin causing spinocerebellar ataxia.

A novel giant non-cholinergic striatal interneuron restricted to the ventrolateral striatum coexpresses Kv3.3 potassium channel, parvalbumin, and the vesicular GABA transporter.

The striatum is the main input structure of the basal ganglia. Distinct striatal subfields are involved in voluntary movement generation and cognitive and emotional tasks, but little is known about the morphological and molecular differences of striatal subregions. The ventrolateral subfield of the striatum (VLS) is the orofacial projection field of the sensorimotor cortex and is involved in the development of orofacial dyskinesias, involuntary chewing-like movements that often accompany long-term neuroleptic treatment. The biological basis for this particular vulnerability of the VLS is not known. Potassium channels are known to be strategically localized within the striatum. In search of possible molecular correlates of the specific vulnerability of the VLS, we analyzed the expression of voltage-gated potassium channels in rodent and primate brains using qPCR, in situ hybridization, and immunocytochemical single and double staining. Here we describe a novel, giant, non-cholinergic interneuron within the VLS. This neuron coexpresses the vesicular GABA transporter, the calcium-binding protein parvalbumin (PV), and the Kv3.3 potassium channel subunit. This novel neuron is much larger than PV neurons in other striatal regions, displays characteristic electrophysiological properties, and, most importantly, is restricted to the VLS. Consequently, the giant striatal Kv3.3-expressing PV neuron may link compromised Kv3 channel function and VLS-based orofacial dyskinesias.

A high-affinity, partial antagonist effect of 3,4-diaminopyridine mediates action potential broadening and enhancement of transmitter release at NMJs.

3,4-Diaminopyridine (3,4-DAP) increases transmitter release from neuromuscular junctions (NMJs), and low doses of 3,4-DAP (estimated to reach ∼1 µM in serum) are the Food and Drug Administration (FDA)-approved treatment for neuromuscular weakness caused by Lambert-Eaton myasthenic syndrome. Canonically, 3,4-DAP is thought to block voltage-gated potassium (Kv) channels, resulting in prolongation of the presynaptic action potential (AP). However, recent reports have shown that low millimolar concentrations of 3,4-DAP have an off-target agonist effect on the Cav1 subtype ("L-type") of voltage-gated calcium (Cav) channels and have speculated that this agonist effect might contribute to 3,4-DAP effects on transmitter release at the NMJ. To address 3,4-DAP's mechanism(s) of action, we first used the patch-clamp electrophysiology to characterize the concentration-dependent block of 3,4-DAP on the predominant presynaptic Kv channel subtypes found at the mammalian NMJ (Kv3.3 and Kv3.4). We identified a previously unreported high-affinity (1-10 µM) partial antagonist effect of 3,4-DAP in addition to the well-known low-affinity (0.1-1 mM) antagonist activity. We also showed that 1.5-µM DAP had no effects on Cav1.2 or Cav2.1 current. Next, we used voltage imaging to show that 1.5- or 100-µM 3,4-DAP broadened the AP waveform in a dose-dependent manner, independent of Cav1 calcium channels. Finally, we demonstrated that 1.5- or 100-µM 3,4-DAP augmented transmitter release in a dose-dependent manner and this effect was also independent of Cav1 channels. From these results, we conclude that low micromolar concentrations of 3,4-DAP act solely on Kv channels to mediate AP broadening and enhance transmitter release at the NMJ.

Suppression of Kv3.3 channels by antisense oligonucleotides reverses biochemical effects and motor impairment in spinocerebellar ataxia type 13 mice.

Mutations in KCNC3, the gene that encodes the Kv3.3 voltage dependent potassium channel, cause Spinocerebellar Ataxia type 13 (SCA13), a disease associated with disrupted motor behaviors, progressive cerebellar degeneration, and abnormal auditory processing. The Kv3.3 channel directly binds Hax-1, a cell survival protein. A disease-causing mutation, Kv3.3-G592R, causes overstimulation of Tank Binding Kinase 1 (Tbk1) in the cerebellum, resulting in the degradation of Hax-1 by promoting its trafficking into multivesicular bodies and then to lysosomes. We have now tested the effects of antisense oligonucleotides (ASOs) directed against the Kv3.3 channel on both wild type mice and those bearing the Kv3.3-G592R-encoding mutation. Intracerebroventricular infusion of the Kcnc3-specific ASO suppressed both mRNA and protein levels of the Kv3.3 channel. In wild-type animals, this produced no change in levels of activated Tbk1, Hax-1 or Cd63, a tetraspanin marker for late endosomes/multivesicular bodies. In contrast, in mice homozygous for the Kv3.3-G592R-encoding mutation, the same ASO reduced Tbk1 activation and levels of Cd63, while restoring the expression of Hax-1 in the cerebellum. The motor behavior of the mice was tested using a rotarod assay. Surprisingly, the active ASO had no effects on the motor behavior of wild type mice but restored the behavior of the mutant mice to those of age-matched wild type animals. Our findings indicate that, in mature intact animals, suppression of Kv3.3 expression can reverse the deleterious effects of a SCA13 mutation while having little effect on wild type animals. Thus, targeting Kv3.3 expression may prove a viable therapeutic approach for SCA13.

A KCNC1 mutation in epilepsy of infancy with focal migrating seizures produces functional channels that fail to be regulated by PKC phosphorylation.

Channelopathies caused by mutations in genes encoding ion channels generally produce a clear change in channel function. Accordingly, mutations in KCNC1, which encodes the voltage-dependent Kv3.1 potassium channel, result in progressive myoclonus epilepsy as well as other developmental and epileptic encephalopathies, and these have been shown to reduce or fully abolish current amplitude. One exception to this is the mutation A513V Kv3.1b, located in the cytoplasmic C-terminal domain of the channel protein. This de novo variant was detected in a patient with epilepsy of infancy with focal migrating seizures (EIFMS), but no difference could be detected between A513V Kv3.1 current and that of wild-type Kv3.1. Using both biochemical and electrophysiological approaches, we have now confirmed that this variant produces functional channels but find that the A513V mutation renders the channel completely insensitive to regulation by phosphorylation at S503, a nearby regulatory site in the C-terminus. In this respect, the mutation resembles those in another channel, KCNT1, which are the major cause of EIFMS. Because the amplitude of Kv3.1 current is constantly adjusted by phosphorylation in vivo, our findings suggest that loss of such regulation contributes to EIFMS phenotype and emphasize the role of channel modulation for normal neuronal function.NEW & NOTEWORTHY Ion channel mutations that cause serious human diseases generally alter the biophysical properties or expression of the channel. We describe a de novo mutation in the Kv3.1 potassium channel that causes severe intellectual disability with early-onset epilepsy. The properties of this channel appear identical to those of wild-type channels, but the mutation prevents phosphorylation of the channel by protein kinase C. Our findings emphasize the role of channel modulation in normal brain function.

Voltage-gated potassium channel dysfunction in dorsal root ganglia contributes to the exaggerated exercise pressor reflex in rats with chronic heart failure.

An exaggerated exercise pressor reflex (EPR) causes excessive sympathoexcitation and exercise intolerance during physical activity in the chronic heart failure (CHF) state. Muscle afferent sensitization contributes to the genesis of the exaggerated EPR in CHF. However, the cellular mechanisms underlying muscle afferent sensitization in CHF remain unclear. Considering that voltage-gated potassium (Kv) channels critically regulate afferent neuronal excitability, we examined the potential role of Kv channels in mediating the sensitized EPR in male rats with CHF. Real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting experiments demonstrate that both mRNA and protein expressions of multiple Kv channel isoforms (Kv1.4, Kv3.4, Kv4.2, and Kv4.3) were downregulated in lumbar dorsal root ganglions (DRGs) of CHF rats compared with sham rats. Immunofluorescence data demonstrate significant decreased Kv channel staining in both NF200-positive and IB4-positive lumbar DRG neurons in CHF rats compared with sham rats. Data from patch-clamp experiments demonstrate that the total Kv current, especially IA, was dramatically decreased in medium-sized IB4-negative muscle afferent neurons (a subpopulation containing mostly Aδ neurons) from CHF rats compared with sham rats, indicating a potential functional loss of Kv channels in muscle afferent Aδ neurons. In in vivo experiments, adenoviral overexpression of Kv4.3 in lumbar DRGs for 1 wk attenuated the exaggerated EPR induced by muscle static contraction and the mechanoreflex by passive stretch without affecting the blunted cardiovascular response to hindlimb arterial injection of capsaicin in CHF rats. These data suggest that Kv channel dysfunction in DRGs plays a critical role in mediating the exaggerated EPR and muscle afferent sensitization in CHF.NEW & NOTEWORTHY The primary finding of this manuscript is that voltage-gated potassium (Kv) channel dysfunction in DRGs plays a critical role in mediating the exaggerated EPR and muscle afferent sensitization in chronic heart failure (CHF). We propose that manipulation of Kv channels in DRG neurons could be considered as a potential new approach to reduce the exaggerated sympathoexcitation and to improve exercise intolerance in CHF, which can ultimately facilitate an improved quality of life and reduce mortality.

Kv3 channels contribute to cancer cell migration via vimentin regulation.

Cell migration is a complex and important process in cancer progression. Vimentin has pivotal roles in cancer cell migration, and various signaling pathways including the AKT pathway are involved in cancer cell migration via vimentin regulation. Recent studies have revealed that voltage-gated potassium (Kv) channels have important functions in cancer cell migration; however, the exact mechanism is still unclear. In the present study, we focused on Kv3 channels with vimentin in cancer migration using human cervical cancer cells (HeLa) and canine mammary tumor cells (CHMp). Cancer cell migration was significantly inhibited, and vimentin expression was significantly decreased by Kv3 blocker, BDS-II. The Kv3 blocker also inactivated the AKT pathway in HeLa cells. In addition, reduced expressions of vimentin and Kv3.4 were observed in HeLa cells when treated with AKT blocker, MK2206. These results suggest that Kv3 channels play important roles in cancer cell migration by regulating vimentin and having closely related with the AKT pathway in human cervical cancer cells.

Modulators of Kv3 Potassium Channels Rescue the Auditory Function of Fragile X Mice.

Fragile X syndrome (FXS) is characterized by hypersensitivity to sensory stimuli, including environmental sounds. We compared the auditory brainstem response (ABR) recorded in vivo in mice lacking the gene (Fmr1-/y ) for fragile X mental retardation protein (FMRP) with that in wild-type animals. We found that ABR wave I, which represents input from the auditory nerve, is reduced in Fmr1-/y animals, but only at high sound levels. In contrast, wave IV, which represents the activity of auditory brainstem nuclei is enhanced at all sound levels, suggesting that loss of FMRP alters the central processing of auditory signals. Current-clamp recordings of neurons in the medial nucleus of the trapezoid body in the auditory brainstem revealed that, in contrast to neurons from wild-type animals, sustained depolarization triggers repetitive firing rather than a single action potential. In voltage-clamp recordings, K+ currents that activate at positive potentials ("high-threshold" K+ currents), which are required for high-frequency firing and are carried primarily by Kv3.1 channels, are elevated in Fmr1-/y mice, while K+ currents that activate near the resting potential and inhibit repetitive firing are reduced. We therefore tested the effects of AUT2 [((4-({5-[(4R)-4-ethyl-2,5-dioxo-1-imidazolidinyl]-2-pyridinyl}oxy)-2-(1-methylethyl) benzonitrile], a compound that modulates Kv3.1 channels. AUT2 reduced the high-threshold K+ current and increased the low-threshold K+ currents in neurons from Fmr1-/y animals by shifting the activation of the high-threshold current to more negative potentials. This reduced the firing rate and, in vivo, restored wave IV of the ABR. Our results from animals of both sexes suggest that the modulation of the Kv3.1 channel may have potential for the treatment of sensory hypersensitivity in patients with FXS.SIGNIFICANCE STATEMENT mRNA encoding the Kv3.1 potassium channel was one of the first described targets of the fragile X mental retardation protein (FMRP). Fragile X syndrome is caused by loss of FMRP and, in humans and mice, causes hypersensitivity to auditory stimuli. We found that components of the auditory brain response (ABR) corresponding to auditory brainstem activity are enhanced in mice lacking FMRP. This is accompanied by hyperexcitability and altered potassium currents in auditory brainstem neurons. Treatment with a drug that alters the voltage dependence of Kv3.1 channels normalizes the imbalance of potassium currents, as well as ABR responses in vivo, suggesting that such compounds may be effective in treating some symptoms of fragile X syndrome.

Pharmacological inhibition of Kv3 on oxidative stress-induced cataract progression.

Oxidative stress is one of the most important risk factors for cataractogenesis. Previous studies have indicated that BDS-II, a Kv3 channel blocker, plays pivotal roles in oxidative stress-related diseases. This study demonstrates that BDS-II exerts a protective effect on cataractogenesis. Specifically, BDS-II was observed to inhibit lens opacity induced by H2O2. BDS-II was also determined to inhibit cataract progression in a sodium selenite-induced in vivo cataract model by inhibiting reduction of the total GSH. In addition, BDS-II was demonstrated to protect human lens epithelial cells against H2O2-induced cell death. Our results suggest that BDS-II is a potential pharmacological candidate in cataract therapy.

ß-Secretase BACE1 Promotes Surface Expression and Function of Kv3.4 at Hippocampal Mossy Fiber Synapses.

The ß-secretase ß-site APP-cleaving enzyme 1 (BACE1) is deemed a major culprit in Alzheimer's disease, but accumulating evidence indicates that there is more to the enzyme than driving the amyloidogenic processing of the amyloid precursor protein. For example, BACE1 has emerged as an important regulator of neuronal activity through proteolytic and, most unexpectedly, also through nonproteolytic interactions with several ion channels. Here, we identify and characterize the voltage-gated K+ channel 3.4 (Kv3.4) as a new and functionally relevant interaction partner of BACE1. Kv3.4 gives rise to A-type current with fast activating and inactivating kinetics and serves to repolarize the presynaptic action potential. We found that BACE1 and Kv3.4 are highly enriched and remarkably colocalized in hippocampal mossy fibers (MFs). In BACE1-/- mice of either sex, Kv3.4 surface expression was significantly reduced in the hippocampus and, in synaptic fractions thereof, Kv3.4 was specifically diminished, whereas protein levels of other presynaptic K+ channels such as KCa1.1 and KCa2.3 remained unchanged. The apparent loss of presynaptic Kv3.4 affected the strength of excitatory transmission at the MF-CA3 synapse in hippocampal slices of BACE1-/- mice when probed with the Kv3 channel blocker BDS-I. The effect of BACE1 on Kv3.4 expression and function should be bidirectional, as predicted from a heterologous expression system, in which BACE1 cotransfection produced a concomitant upregulation of Kv3.4 surface level and current based on a physical interaction between the two proteins. Our data show that, by targeting Kv3.4 to presynaptic sites, BACE1 endows the terminal with a powerful means to regulate the strength of transmitter release.SIGNIFICANCE STATEMENT The ß-secretase ß-site APP-cleaving enzyme 1 (BACE1) is infamous for its crucial role in the pathogenesis of Alzheimer's disease, but its physiological functions in the intact nervous system are only gradually being unveiled. Here, we extend previous work implicating BACE1 in the expression and function of voltage-gated Na+ and K+ channels. Specifically, we characterize voltage-gated K+ channel 3.4 (Kv3.4), a presynaptic K+ channel required for action potential repolarization, as a novel interaction partner of BACE1 at the mossy fiber (MF)-CA3 synapse of the hippocampus. BACE1 promotes surface expression of Kv3.4 at MF terminals, most likely by physically associating with the channel protein in a nonenzymatic fashion. We advance the BACE1-Kv3.4 interaction as a mechanism to strengthen the temporal control over transmitter release from MF terminals.

Regulation of Nociceptive Glutamatergic Signaling by Presynaptic Kv3.4 Channels in the Rat Spinal Dorsal Horn.

Presynaptic voltage-gated K+ (Kv) channels in dorsal root ganglion (DRG) neurons are thought to regulate nociceptive synaptic transmission in the spinal dorsal horn. However, the Kv channel subtypes responsible for this critical role have not been identified. The Kv3.4 channel is particularly important because it is robustly expressed in DRG nociceptors, where it regulates action potential (AP) duration. Furthermore, Kv3.4 dysfunction is implicated in the pathophysiology of neuropathic pain in multiple pain models. We hypothesized that, through their ability to modulate AP repolarization, Kv3.4 channels in DRG nociceptors help to regulate nociceptive synaptic transmission. To test this hypothesis, we investigated Kv3.4 immunoreactivity (IR) in the rat cervical superficial dorsal horn (sDH) in both sexes and implemented an intact spinal cord preparation to investigate glutamatergic synaptic currents from second order neurons in the sDH under conditions that selectively inhibit the Kv3.4 current. We found presynaptic Kv3.4 IR in peptidergic and nonpeptidergic nociceptive fibers of the sDH. The Kv3.4 channel is hypersensitive to 4-aminopyridine and tetraethylammonium (TEA). Accordingly, 50 µm 4-aminopyridine and 500 µm TEA significantly prolong the AP, slow the maximum rate of repolarization in small-diameter DRG neurons, and potentiate monosynaptic excitatory postsynaptic currents (EPSCs) in dorsal horn laminae I and II through a presynaptic mechanism. In contrast, highly specific inhibitors of BK, Kv7, and Kv1 channels are less effective modulators of the AP and have little to no effect on EPSCs. The results strongly suggest that presynaptic Kv3.4 channels are major regulators of nociceptive synaptic transmission in the spinal cord.SIGNIFICANCE STATEMENT Intractable neuropathic pain can result from disease or traumatic injury and many studies have been conducted to determine the underlying pathophysiological changes. Voltage-gated ion channels, including the K+ channel Kv3.4, are dysregulated in multiple pain models. Kv3.4 channels are ubiquitously expressed in the dorsal root ganglion (DRG), where they are major regulators of DRG excitability. However, little is known about the ionic mechanisms that regulate nociceptive synaptic transmission at the level of the first synapse in the spinal cord, which is critical to pain transmission in both intact and pathological states. Here, we show that Kv3.4 channels have a significant impact on glutamatergic synaptic transmission in the dorsal horn, further illuminating its potential as a molecular pain therapeutic target.

Exacerbated effects of prorenin on hypothalamic magnocellular neuronal activity and vasopressin plasma levels during salt-sensitive hypertension.

Accumulating evidence supports that the brain renin-angiotensin system (RAS), including prorenin (PR) and its receptor (PRR), two newly discovered RAS players, contribute to sympathoexcitation in salt-sensitive hypertension. Still, whether PR also contributed to elevated circulating levels of neurohormones such as vasopressin (VP) during salt-sensitive hypertension, and if so, what are the precise underlying mechanisms, remains to be determined. To address these questions, we obtained patch-clamp recordings from hypothalamic magnocellular neurosecretory neurons (MNNs) that synthesize the neurohormones oxytocin and VP in acute hypothalamic slices obtained from sham and deoxycorticosterone acetate (DOCA)-salt-treated hypertensive rats. We found that focal application of PR markedly increased membrane excitability and firing responses in MNNs of DOCA-salt, compared with sham rats. This effect included a shorter latency to spike initiation and increased numbers of spikes in response to depolarizing stimuli and was mediated by a more robust inhibition of A-type K+ channels in DOCA-salt compared with sham rats. On the other hand, the afterhyperpolarizing potential mediated by the activation of Ca2+-dependent K+ channel was not affected by PR. mRNA expression of PRR, VP, and the Kv4.3 K+ channel subunit in the supraoptic nucleus of DOCA-salt hypertensive rats was increased compared with sham rats. Finally, we report a significant decrease of plasma VP levels in neuron-selective PRR knockdown mice treated with DOCA-salt, compared with wild-type DOCA-salt-treated mice. Together, these results support that activation of PRR contributes to increased excitability and firing discharge of MNNs and increased plasma levels of VP in DOCA-salt hypertension.NEW & NOTEWORTHY Our studies support that prorenin (PR) and its receptor (PRR) within the hypothalamus contribute to elevated plasma vasopressin levels in deoxycorticosterone acetate-salt hypertension, in part because of an exacerbated effect of PR on magnocellular neurosecretory neuron excitability; Moreover, our study implicates A-type K+ channels as key underlying molecular targets mediating these effects. Thus, PR/PRR stands as a novel therapeutic target for the treatment of neurohumoral activation in salt-sensitive hypertension.

Bisoprolol, Known to Be a Selective ß1-Receptor Antagonist, Differentially but Directly Suppresses IK(M) and IK(erg) in Pituitary Cells and Hippocampal Neurons.

Bisoprolol (BIS) is a selective antagonist of ß1 adrenergic receptors. We examined the effects of BIS on M-type K⁺ currents (IK(M)) or erg-mediated K⁺ currents (IK(erg)) in pituitary GH3, R1220 cells, and hippocampal mHippoE-14 cells. As GH3 cells were exposed to BIS, amplitude of IK(M) was suppressed with an IC50 value of 1.21 µM. The BIS-induced suppression of IK(M) amplitude was not affected by addition of isoproterenol or ractopamine, but attenuated by flupirtine or ivabradine. In cell-attached current, BIS decreased the open probability of M-type K⁺ (KM) channels, along with decreased mean opening time of the channel. BIS decreased IK(erg) amplitude with an IC50 value of 6.42 µM. Further addition of PD-118057 attenuated BIS-mediated inhibition of IK(erg). Under current-clamp conditions, BIS depolarization increased the firing of spontaneous action potentials in GH3 cells; addition of flupirtine, but not ractopamine, reversed BIS-induced firing rate. In R1220 cells, BIS suppressed IK(M); subsequent application of ML-213(Kv7.2 channel activator) reversed BIS-induced suppression of the current. In hippocampal mHippoE-14 neurons, BIS inhibited IK(M) to a greater extent compared to its depressant effect on IK(erg). This demonstrated that in pituitary cells and hippocampal neurons the presence of BIS is capable of directly and differentially suppressing IK(M) and IK(erg), despite its antagonism of ß1-adrenergic receptors.

Identification and characterization of site-specific N-glycosylation in the potassium channel Kv3.1b.

The potassium ion channel Kv3.1b is a member of a family of voltage-gated ion channels that are glycosylated in their mature form. In the present study, we demonstrate the impact of N-glycosylation at specific asparagine residues on the trafficking of the Kv3.1b protein. Large quantities of asparagine 229 (N229)-glycosylated Kv3.1b reached the plasma membrane, whereas N220-glycosylated and unglycosylated Kv3.1b were mainly retained in the endoplasmic reticulum (ER). These ER-retained Kv3.1b proteins were susceptible to degradation, when co-expressed with calnexin, whereas Kv3.1b pools located at the plasma membrane were resistant. Mass spectrometry analysis revealed a complex type Hex3 HexNAc4 Fuc1 glycan as the major glycan component of the N229-glycosylated Kv3.1b protein, as opposed to a high-mannose type Man8 GlcNAc2 glycan for N220-glycosylated Kv3.1b. Taken together, these results suggest that trafficking-dependent roles of the Kv3.1b potassium channel are dependent on N229 site-specific glycosylation and N-glycan structure, and operate through a mechanism whereby specific N-glycan structures regulate cell surface expression.

Myoclonus epilepsy and ataxia due to KCNC1 mutation: Analysis of 20 cases and K+ channel properties.

OBJECTIVE: To comprehensively describe the new syndrome of myoclonus epilepsy and ataxia due to potassium channel mutation (MEAK), including cellular electrophysiological characterization of observed clinical improvement with fever. METHODS: We analyzed clinical, electroclinical, and neuroimaging data for 20 patients with MEAK due to recurrent KCNC1 p.R320H mutation. In vitro electrophysiological studies were conducted using whole cell patch-clamp to explore biophysical properties of wild-type and mutant KV 3.1 channels. RESULTS: Symptoms began at between 3 and 15 years of age (median = 9.5), with progressively severe myoclonus and rare tonic-clonic seizures. Ataxia was present early, but quickly became overshadowed by myoclonus; 10 patients were wheelchair-bound by their late teenage years. Mild cognitive decline occurred in half. Early death was not observed. Electroencephalogram (EEG) showed generalized spike and polyspike wave discharges, with documented photosensitivity in most. Polygraphic EEG-electromyographic studies demonstrated a cortical origin for myoclonus and striking coactivation of agonist and antagonist muscles. Magnetic resonance imaging revealed symmetrical cerebellar atrophy, which appeared progressive, and a prominent corpus callosum. Unexpectedly, transient clinical improvement with fever was noted in 6 patients. To explore this, we performed high-temperature in vitro recordings. At elevated temperatures, there was a robust leftward shift in activation of wild-type KV 3.1, increasing channel availability. INTERPRETATION: MEAK has a relatively homogeneous presentation, resembling Unverricht-Lundborg disease, despite the genetic and biological basis being quite different. A remarkable improvement with fever may be explained by the temperature-dependent leftward shift in activation of wild-type KV 3.1 subunit-containing channels, which would counter the loss of function observed for mutant channels, highlighting KCNC1 as a potential target for precision therapeutics. Ann Neurol 2017;81:677-689.

Kv3.1 and Kv3.4, Are Involved in Cancer Cell Migration and Invasion.

Voltage-gated potassium (Kv) channels, including Kv3.1 and Kv3.4, are known as oxygen sensors, and their function in hypoxia has been well investigated. However, the relationship between Kv channels and tumor hypoxia has yet to be investigated. This study demonstrates that Kv3.1 and Kv3.4 are tumor hypoxia-related Kv channels involved in cancer cell migration and invasion. Kv3.1 and Kv3.4 protein expression in A549 and MDA-MB-231 cells increased in a cell density-dependent manner, and the pattern was similar to the expression patterns of hypoxia-inducible factor-1α (HIF-1α) and reactive oxygen species (ROS) according to cell density, whereas Kv3.3 protein expression did not change in A549 cells with an increase in cell density. The Kv3.1 and Kv3.4 blocker blood depressing substance (BDS) did not affect cell proliferation; instead, BDS inhibited cell migration and invasion. We found that BDS inhibited intracellular pH regulation and extracellular signal-regulated kinase (ERK) activation in A549 cells cultured at a high density, potentially resulting in BDS-induced inhibition of cell migration and invasion. Our data suggest that Kv3.1 and Kv3.4 might be new therapeutic targets for cancer metastasis.