Kv2.1 expression in giant reticular neurons of the postnatal mouse brain.

Previous experiments charted the development of behavioral arousal in postnatal mice. From Postnatal Day 3 (P3) to Postnatal Day 6 (P6) mice (a) become significantly more active, "arousable"; and (b) in large reticular neurons, nucleus gigantocellularis (NGC), patch clamp recordings reveal a significantly increased ability to fire high frequency trains of action potentials as are associated with elevated cortical arousal. These action potential trains depend on delayed rectifiers such as Kv2.1. Here we report tracking the development of expression of a delayed rectifier, Kv2.1 in NGC neurons crucial for initiating CNS arousal. In tissue sections, light microscope immunohistochemistry revealed that expression of Kv2.1 in NGC neurons is greater at day P6 than at P3. Electron microscope immunohistochemistry revealed Kv2.1 labeling on the plasmalemmal surface of soma and dendrites, greater on P6 than P3. In brainstem reticular neuron cell culture, Kv2.1 immunocytochemistry increased monotonically from Days-In-Vitro 3-10, paralleling the ability of such neurons to fire action potential trains. The increase of Kv2.1 expression from P3 to P6, perhaps in conjunction with other delayed rectifier currents, could permit the ability to fire action potential trains in NGC neurons. Further work with genetically identified NGC neurons is indicated.

Up-Regulation of the Voltage-Gated KV2.1 K+ Channel in the Renal Arterial Myocytes of Dahl Salt-Sensitive Hypertensive Rats.

Salt-sensitive hypertension induces renal injury via decreased blood flow in the renal artery (RA), and ion channel dysfunction in RA myocytes (RAMs) may be involved in the higher renal vascular resistance. We examined the effects of several voltage-gated K+ (KV) channel blockers on the resting tension in endothelium-denuded RA strips and delayed-rectifier K+ currents in RAMs of Dahl salt-sensitive hypertensive rats (Dahl-S) fed with low- (Dahl-LS) and high-salt diets (Dahl-HS). The tetraethylammonium (TEA)-induced contraction in RA strips were significantly larger in Dahl-HS than Dahl-LS. Correspondingly, TEA-sensitive KV currents were significantly larger in the RAMs of Dahl-HS than Dahl-LS. Among the TEA-sensitive KV channel subtypes, the expression levels of KV2.1 transcript and protein were significantly higher in the RA of Dahl-HS than Dahl-LS, while those of KV1.5, KV7.1, and KV7.4 transcripts was comparable in two groups. KV2.1 currents detected as the guangxitoxin-1E-sensitive component were larger in the RAMs of Dahl-HS than Dahl-LS. These suggest that the up-regulation of the KV2.1 channel in RAMs may be involved in the compensatory mechanisms against decreased renal blood flow in salt-sensitive hypertension.

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.

Distinct Cell- and Layer-Specific Expression Patterns and Independent Regulation of Kv2 Channel Subtypes in Cortical Pyramidal Neurons.

The Kv2 family of voltage-gated potassium channel α subunits, comprising Kv2.1 and Kv2.2, mediate the bulk of the neuronal delayed rectifier K(+) current in many mammalian central neurons. Kv2.1 exhibits robust expression across many neuron types and is unique in its conditional role in modulating intrinsic excitability through changes in its phosphorylation state, which affect Kv2.1 expression, localization, and function. Much less is known of the highly related Kv2.2 subunit, especially in forebrain neurons. Here, through combined use of cortical layer markers and transgenic mouse lines, we show that Kv2.1 and Kv2.2 are localized to functionally distinct cortical cell types. Kv2.1 expression is consistently high throughout all cortical layers, especially in layer (L) 5b pyramidal neurons, whereas Kv2.2 expression is primarily limited to neurons in L2 and L5a. In addition, L4 of primary somatosensory cortex is strikingly devoid of Kv2.2 immunolabeling. The restricted pattern of Kv2.2 expression persists in Kv2.1-KO mice, suggesting distinct cell- and layer-specific functions for these two highly related Kv2 subunits. Analyses of endogenous Kv2.2 in cortical neurons in situ and recombinant Kv2.2 expressed in heterologous cells reveal that Kv2.2 is largely refractory to stimuli that trigger robust, phosphorylation-dependent changes in Kv2.1 clustering and function. Immunocytochemistry and voltage-clamp recordings from outside-out macropatches reveal distinct cellular expression patterns for Kv2.1 and Kv2.2 in intratelencephalic and pyramidal tract neurons of L5, indicating circuit-specific requirements for these Kv2 paralogs. Together, these results support distinct roles for these two Kv2 channel family members in mammalian cortex. SIGNIFICANCE STATEMENT: Neurons within the neocortex are arranged in a laminar architecture and contribute to the input, processing, and/or output of sensory and motor signals in a cell- and layer-specific manner. Neurons of different cortical layers express diverse populations of ion channels and possess distinct intrinsic membrane properties. Here, we show that the Kv2 family members Kv2.1 and Kv2.2 are expressed in distinct cortical layers and pyramidal cell types associated with specific corticostriatal pathways. We find that Kv2.1 and Kv2.2 exhibit distinct responses to acute phosphorylation-dependent regulation in brain neurons in situ and in heterologous cells in vitro. These results identify a molecular mechanism that contributes to heterogeneity in cortical neuron ion channel function and regulation.

Target of HIV-1 Envelope Glycoprotein gp120-Induced Hippocampal Neuron Damage: Role of Voltage-Gated K(+) Channel Kv2.1.

Human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein 120 (gp120) has been reported to be toxic to the hippocampal neurons, and to be involved in the pathogenesis of HIV-1-associated neurocognitive disorders (HAND). Accumulating evidence has demonstrated that voltage-gated potassium (Kv) channels, especially the outward delayed-rectifier K(+) (Ik) channels, play a critical role in gp120-induced cortical neuronal death in vitro. However, the potential mechanisms underlying the hippocampal neuronal injury resulted from gp120-mediated neurotoxicity remain poorly understood. Using whole-cell patch clamp recording in cultured hippocampal neurons, this study found that gp120 significantly increased the outward delayed-rectifier K(+) currents (Ik). Meanwhile, Western blot assay revealed that gp120 markedly upregulated Kv2.1 protein levels, which was consistent with the increased Ik density. With Western blot and terminal deoxynucleotidyl transferase dUTP nick end labeling assays, it was discovered that gp120-induced neuronal injury was largely due to activation of Kv2.1 channels and resultant apoptosis mediated by caspase-3 activation, as the pharmacological blockade of Kv2.1 channels largely attenuated gp120-induced cell damage and caspase-3 expression. Moreover, p38 MAPK was demonstrated to participate in gp120-induced hippocampal neural damage, since p38 MAPK antagonist (SB203580) partially abrogated gp120-induced Kv2.1 upregulation and neural apoptosis. Taken together, these results suggest that gp120 induces hippocampal neuron apoptosis by enhancement of the Ik, which might be associated with increased Kv2.1 expression via the p38 MAPK pathway.

(±)3,4-methylenedioxyamphetamine inhibits the TEA-sensitive K⁺ current in the hippocampal neuron and the Kv2.1 current expressed in H1355 cells.

The whole-cell patch clamp method was used to study the effects of (±)3,4-methylenedioxyamphetamine (MDA) in hippocampal CA1 neurons from neonatal rats and in lung epithelial H1355 cells expressing Kv2.1. Extracellular application of MDA (30 µM) induced bursts of action potentials in hippocampal CA1 neurons exhibiting single spike action potentials without a bursting firing pattern, and promoted action potential bursts in hippocampal CA1 neurons exhibiting bursting firing of action potentials. Whereas MDA (30 and 100 µM) markedly decreased the delayed outward current in hippocampal CA1 neurons, MDA (100 µM) only slightly decreased the fast-inactivating K(+) current (I(A)) current. Furthermore, MDA (100 µM) substantially decreased the delayed outward current in the presence of 4-aminopyridine (4-AP; 3 mM), but did not significantly decrease the delayed outward current in the presence of tetraethylammonium (TEA; 30 mM). MDA (100 µM) also inhibited the current in H1355 cells expressing Kv2.1. Our results have shown that MDA inhibits the TEA-sensitive K(+) current in the hippocampus and the Kv2.1 current expressed in H1355 cells. These effects may contribute to the pharmacological and toxicological effects of MDA.

Hepatitis C virus NS5A inhibits mixed lineage kinase 3 to block apoptosis.

Hepatitis C virus (HCV) infection results in the activation of numerous stress responses including oxidative stress, with the potential to induce an apoptotic state. Previously we have shown that HCV attenuates the stress-induced, p38MAPK-mediated up-regulation of the K(+) channel Kv2.1, to maintain the survival of infected cells in the face of cellular stress. We demonstrated that this effect was mediated by HCV non-structural 5A (NS5A) protein, which impaired p38MAPK activity through a polyproline motif-dependent interaction, resulting in reduction of phosphorylation activation of Kv2.1. In this study, we investigated the host cell proteins targeted by NS5A to mediate Kv2.1 inhibition. We screened a phage-display library expressing the entire complement of human SH3 domains for novel NS5A-host cell interactions. This analysis identified mixed lineage kinase 3 (MLK3) as a putative NS5A interacting partner. MLK3 is a serine/threonine protein kinase that is a member of the MAPK kinase kinase (MAP3K) family and activates p38MAPK. An NS5A-MLK3 interaction was confirmed by co-immunoprecipitation and Western blot analysis. We further demonstrate a novel role of MLK3 in the modulation of Kv2.1 activity, whereby MLK3 overexpression leads to the up-regulation of channel activity. Accordingly, coexpression of NS5A suppressed this stimulation. Additionally we demonstrate that overexpression of MLK3 induced apoptosis, which was also counteracted by NS5A. We conclude that NS5A targets MLK3 with multiple downstream consequences for both apoptosis and K(+) homeostasis.

Control of voltage-gated potassium channel Kv2.2 expression by pyruvate-isocitrate cycling regulates glucose-stimulated insulin secretion.

Recent studies have shown that the pyruvate-isocitrate cycling pathway, involving the mitochondrial citrate/isocitrate carrier and the cytosolic NADP-dependent isocitrate dehydrogenase (ICDc), is involved in control of glucose-stimulated insulin secretion (GSIS). Here we demonstrate that pyruvate-isocitrate cycling regulates expression of the voltage-gated potassium channel family member Kv2.2 in islet ß-cells. siRNA-mediated suppression of ICDc, citrate/isocitrate carrier, or Kv2.2 expression impaired GSIS, and the effect of ICDc knockdown was rescued by re-expression of Kv2.2. Moreover, chronic exposure of ß-cells to elevated fatty acids, which impairs GSIS, resulted in decreased expression of Kv2.2. Surprisingly, knockdown of ICDc or Kv2.2 increased rather than decreased outward K(+) current in the 832/13 ß-cell line. Immunoprecipitation studies demonstrated interaction of Kv2.1 and Kv2.2, and co-overexpression of the two channels reduced outward K(+) current compared with overexpression of Kv2.1 alone. Also, siRNA-mediated knockdown of ICDc enhanced the suppressive effect of the Kv2.1-selective inhibitor stromatoxin1 on K(+) currents. Our data support a model in which a key function of the pyruvate-isocitrate cycle is to maintain levels of Kv2.2 expression sufficient to allow it to serve as a negative regulator of Kv channel activity.

MicroRNA-1 accelerates the shortening of atrial effective refractory period by regulating KCNE1 and KCNB2 expression: an atrial tachypacing rabbit model.

BACKGROUND: The potential mechanisms of microRNA-1 (miR-1) in the electrical remodeling of atrial fibrillation remain unclear. The purpose of this study was to evaluate the effects of miR-1 on the atrial effective refractory period (AERP) in a right atrial tachypacing model and to elucidate the potential mechanisms. METHODS AND RESULTS: QRT-PCR and western blot were used to detect the expression of the miR-1, KCNE1, and KCNB2 genes after 1-week of right atrial tachypacing in New Zealand white rabbits. The AERP was measured using a programmable multichannel stimulator, and atrial fibrillation was induced by burst stimulation in vivo. The slowly activating delayed rectifier potassium current (IKs) and AERP in atrial cells were measured by whole cell patch clamp in vitro. Right atrial tachypacing upregulated miR-1 expression and downregulated KCNE1 and KCNB2 in this study, while the AERP was decreased and the atrial IKs increased. The downregulation of KCNE1 and KCNB2 levels was greater when miR-1 was further upregulated through in vivo lentiviral infection. Electrophysiological tests indicated a shorter AERP, a great increase in the IKs and a higher atrial fibrillation inducibility. In addition, similar results were found when the levels of KCNE1 and KCNB2 were downregulated by small interfering RNA while keeping miR-1 level unaltered. Conversely, knockdown of miR-1 by anti-miR-1 inhibitor oligonucleotides alleviated the downregulation of KCNE1 and KCNB2, the shortening of AERP, and the increase in the IKs. KCNE1 and KCNB2 as the target genes for miR-1 were confirmed by luciferase activity assay. CONCLUSIONS: These results indicate that miR-1 accelerates right atrial tachypacing-induced AERP shortening by targeting potassium channel genes, which further suggests that miR-1 plays an important role in the electrical remodeling of atrial fibrillation and exhibits significant clinical relevance as a potential therapeutic target for atrial fibrillation.

Expression and function of K(V)2-containing channels in human urinary bladder smooth muscle.

The functional role of the voltage-gated K(+) (K(V)) channels in human detrusor smooth muscle (DSM) is largely unexplored. Here, we provide molecular, electrophysiological, and functional evidence for the expression of K(V)2.1, K(V)2.2, and the electrically silent K(V)9.3 subunits in human DSM. Stromatoxin-1 (ScTx1), a selective inhibitor of K(V)2.1, K(V)2.2, and K(V)4.2 homotetrameric channels and of K(V)2.1/9.3 heterotetrameric channels, was used to examine the role of these channels in human DSM function. Human DSM tissues were obtained during open bladder surgeries from patients without a history of overactive bladder. Freshly isolated human DSM cells were studied using RT-PCR, immunocytochemistry, live-cell Ca(2+) imaging, and the perforated whole cell patch-clamp technique. Isometric DSM tension recordings of human DSM isolated strips were conducted using tissue baths. RT-PCR experiments showed mRNA expression of K(V)2.1, K(V)2.2, and K(V)9.3 (but not K(V)4.2) channel subunits in human isolated DSM cells. K(V)2.1 and K(V)2.2 protein expression was confirmed by Western blot analysis and immunocytochemistry. Perforated whole cell patch-clamp experiments revealed that ScTx1 (100 nM) inhibited the amplitude of the voltage step-induced K(V) current in freshly isolated human DSM cells. ScTx1 (100 nM) significantly increased the intracellular Ca(2+) level in DSM cells. In human DSM isolated strips, ScTx1 (100 nM) increased the spontaneous phasic contraction amplitude and muscle force, and enhanced the amplitude of the electrical field stimulation-induced contractions within the range of 3.5-30 Hz stimulation frequencies. These findings reveal that ScTx1-sensitive K(V)2-containing channels are key regulators of human DSM excitability and contractility and may represent new targets for pharmacological or genetic intervention for bladder dysfunction.

Cholesterol enhances neuron susceptibility to apoptotic stimuli via cAMP/PKA/CREB-dependent up-regulation of Kv2.1.

Cholesterol is a major component of membrane lipid rafts. It is more abundant in the brain than in other tissues and plays a critical role in maintaining brain function. We report here that a significant enhancement in apoptosis in rat cerebellar granule neurons (CGNs) was observed upon incubation with 5mM K(+) /serum free (LK-S) medium. Cholesterol enrichment further potentiated CGN apoptosis incubated under LK-S medium. On the contrary, cholesterol depletion using methyl-beta-cyclodextrin protected the CGNs from apoptosis induced by LK-S treatment. Cholesterol enrichment, however, did not induce apoptosis in CGNs that have been incubated with 25mM K(+) /serum medium. Mechanistically, increased I(K) currents and DNA fragmentation were found in CGNs incubated in LK-S, which was further potentiated in the presence of cholesterol. Cholesterol-treated CGNs also exhibited increased cAMP levels and up-regulation of Kv2.1 expression. Increased levels of activated form of PKA and phospho-CREB further supported activation of the cAMP/PKA pathway upon treatment of CGNs with cholesterol-containing LK-S medium. Conversely, inhibition of PKA or small G protein Gs abolished the increase in I(K) current and the potentiation of Kv2.1 expression, leading to reduced susceptibility of CGNs to LK-S and cholesterol-induced apoptosis. Our results demonstrate that the elevation of membrane cholesterol enhances CGN susceptibility to apoptotic stimuli via cAMP/PKA/CREB-dependent up-regulation of Kv2.1. Our data provide new evidence for the role of cholesterol in eliciting neuronal cell death.

The augmenter of liver regeneration protects the kidneys after orthotopic liver transplantation possibly by upregulating HIF-1α and O2-sensitive K+ channels.

PURPOSE: The augmenter of liver regeneration (ALR) might promote better renal function after orthotopic liver transplantation (OLT). Using a rat allogeneic OLT model, we investigated if ALR can mediate renal protection and its potential mechanisms. METHODS: Orthotopic liver transplant recipients were assigned to a cyclosporine A (CsA)-treated group (CsA group) and an ALR+CsA group (ALR group). Transplanted liver, kidneys, and serum were harvested on post-transplantation days 1, 3, and 7 for histological examination, and hepatic function and renal function analysis. We also measured the expression of hypoxiainducible factor-1 (HIF-1α) and O(2)-sensitive K(+) cannels (KV1.5 and KV2.1), and free Ca(2+) in the smooth muscle cells (SMCs) of intrarenal arterioles in the kidneys. RESULTS: All transplanted livers suffered mild acute rejection after OLT. Recipient kidney damage was more severe in the CsA group, characterized by increased serum creatinine, tubular epithelial apoptosis and necrosis, and the formation of casts. In the ALR group, HIF-1α, KV1.5, and KV2.1 were upregulated, accompanied by lower Ca(2+) concentration, in the SMCs shortly after OLT. CONCLUSION: Augmenter of liver regeneration might increase the expression of HIF-1α and K(+) channels and decrease intracellular free Ca(2+), thereby inhibiting arterial contraction and promoting kidney perfusion immediately after OLT.

[mRNA expression of voltage-dependent potassium channels in the brain of rats after middle cerebral artery occlusion].

AIM: To study the mRNA expression changes in the brain of rats after middle cerebral artery occlusion. METHODS: Middle cerebral artery occlusion was used to induce ischemia in rat brain. The mRNA expression of voltage-dependent potassium channel subtypes, including Kv1.4, Kv1.5, Kv2.1 and Kv4.2, were detected in rat hippocampus and cortex by RT-PCR. RESULTS: Middle cerebral artery occlusion induced a significant neurological injury in rats. After ischemia 2 h, the mRNA of Kv1.4, Kv2.1 and Kv4.2 in hippocampus increased by 50%, 67% and 90% , respectively. And the mRNA of Kv1.4 and Kv4.2 maintained at a high level in hippocampus after ischemia 24 h. In cortex, the mRNA level of all the four subtypes were not changed significantly after ischemia 2 h, but the mRNA of Kv2.1 and Kv4.2 increased by 70% and 62% after ischemia 24 h, respectively. CONCLUSION: The mRNA expression levels of voltage-dependent potassium channels were up-regulated in rat hippocampus and cortex after middle cerebral artery occlusion.

[The role of subtypes of voltage-gated K+ channels in pulmonary vasoconstriction induced by 15-hydroeicosatetraenoic acid].

AIM: To observe the effect of subtypes of Kv channels in rat pulmonary artery smooth muscle cells (PASMCs) on the process of pulmonary vasoconstriction induced by 15-HETE. METHODS: In the present study, ring of rabbit PA with specific Kv channel blockers were employed to functionally identify certain channel subtypes that took part in the process of 15-HETE induced pulmonary vasoconstriction; RT-PCR and Western blotting analysis were also used to measure the expression of subtypes of Kv in PASMCs exposed to 15-HETE,chronic hypoxia. RESULTS: Blocking of Kv1. 1, Kv1. 2, Kv1. 3 and Kv1. 6 channels did not affect 15-HETE induced vasoconstriction in normoxic rats; 15-HETE did not affect expression of Kv1. 1 and Kv1. 2 channels; 15-HETE significantly downregulated the expression of mRNA and protein of Kv1. 5 and Kv2. 1 in rat PASMCs. CONCLUSION: The results suggested that hypoxia may block Kv1. 5 and Kv2. 1 channels via 15-HETE mediated mechanism, leading to decrease numbers of functional Kv1. 5 and Kv2. 1 channels in PASMCs, leading to PA vasoconstriction.

Apoptotic surface delivery of K+ channels.

Apoptosis in cortical neurons requires efflux of cytoplasmic potassium mediated by a surge in Kv2.1 channel activity. Pharmacological blockade or molecular disruption of these channels in neurons prevents apoptotic cell death, while ectopic expression of Kv2.1 channels promotes apoptosis in non-neuronal cells. Here, we use a cysteine-containing mutant of Kv2.1 and a thiol-reactive covalent inhibitor to demonstrate that the increase in K+ current during apoptosis is due to de novo insertion of functional channels into the plasma membrane. Biotinylation experiments confirmed the delivery of additional Kv2.1 protein to the cell surface following an apoptotic stimulus. Finally, expression of botulinum neurotoxins that cleave syntaxin and synaptosome-associated protein of 25 kDa (SNAP-25) blocked upregulation of surface Kv2.1 channels in cortical neurons, suggesting that target soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins support proapoptotic delivery of K+ channels. These data indicate that trafficking of Kv2.1 channels to the plasma membrane causes the apoptotic surge in K+ current.