Antagonistic roles of Ras-MAPK and Akt signaling in integrin-K+ channel complex-mediated cellular apoptosis.

Complexes formed with α5-integrins and the voltage-gated potassium (K+ ) channel KCNB1 (Kv2.1), known as IKCs, transduce the electrical activity at the plasma membrane into biochemical events that impinge on cytoskeletal remodeling, cell differentiation, and migration. However, when cells are subject to stress of oxidative nature IKCs turn toxic and cause inflammation and death. Here, biochemical, pharmacological, and cell viability evidence demonstrates that in response to oxidative insults, IKCs activate an apoptotic Mitogen-activated protein kinase/extracellular signal-regulated kinase (Ras-MAPK) signaling pathway. Simultaneously, wild-type (WT) KCNB1 channels sequester protein kinase B (Akt) causing dephosphorylation of BCL2-associated agonist of cell death (BAD), a major sentinel of apoptosis progression. In contrast, IKCs formed with C73A KCNB1 variant that does not induce apoptosis (IKCC73A ), do not sequester Akt and thus are able to engage cell survival mechanisms. Taken together, these data suggest that apoptotic and survival forces co-exist in IKCs. Integrins send death signals through Ras-MAPK and KCNB1 channels simultaneously sabotage survival mechanisms. Thus, the combined action of integrins and KCNB1 channels advances life or death.

Non-conducting functions of potassium channels in cancer and neurological disease.

Cancer and neurodegenerative disease, albeit fundamental differences, share some common pathogenic mechanisms. Accordingly, both conditions are associated with aberrant cell proliferation and migration. Here, we review the causative role played by potassium (K+) channels, a fundamental class of proteins, in cancer and neurodegenerative disease. The concept that emerges from the review of the literature is that K+ channels can promote the development and progression of cancerous and neurodegenerative pathologies by dysregulating cell proliferation and migration. K+ channels appear to control these cellular functions in ways that not necessarily depend on their conducting properties and that involve the ability to directly or indirectly engage growth and survival signaling pathways. As cancer and neurodegenerative disease represent global health concerns, identifying commonalities may help understand the molecular basis for those devastating conditions and may facilitate the design of new drugs or the repurposing of existing drugs.

Complexes formed with integrin-α5 and KCNB1 potassium channel wild type or epilepsy-susceptibility variants modulate cellular plasticity via Ras and Akt signaling.

Voltage-gated potassium (K+) channel subfamily B member 1 (KCNB1, Kv2.1) and integrin-α5 form macromolecular complexes-named integrin-α5-KCNB1 complexes (IKCs)-in the human brain, but their function was poorly understood. Here we report that membrane depolarization triggered IKC intracellular signals mediated by small GTPases of the Ras subfamily and protein kinase B (Akt) to advance the development of filopodia and lamellipodia in Chinese hamster ovary cells, stimulate their motility, and enhance neurite outgrowth in mouse neuroblastoma Neuro2a cells. Five KCNB1 mutants (L211P, R312H G379R, G381R, and F416L) linked to severe infancy or early-onset epileptic encephalopathy exhibited markedly defective conduction. However, although L211P, G379R, and G381R normally engaged Ras/Akt and stimulated cell migration, R312H and F416L failed to activate Ras/Akt signaling and did not enhance cell migration. Taken together, these data suggest that IKCs modulate cellular plasticity via Ras and Akt signaling. As such, defective IKCs may cause epilepsy through mechanisms other than dysregulated excitability such as, for example, abnormal neuronal development and resulting synaptic connectivity.-Yu, W., Shin, M. R., Sesti, F. Complexes formed with integrin-α5 and KCNB1 potassium channel wild type or epilepsy-susceptibility variants modulate cellular plasticity via Ras and Akt signaling.

Oxidation of KCNB1 potassium channels in the murine brain during aging is associated with cognitive impairment.

Voltage-gated potassium (K+) channel sub-family B member 1 (KCNB1, Kv2.1) is known to undergo oxidation-induced oligomerization during aging but whether this process affects brain's physiology was not known. Here, we used 10, 16 and 22 month-old transgenic mice overexpressing a KCNB1 variant that does not oligomerize (Tg-C73A) and as control, mice overexpressing the wild type (Tg-WT) channel and non-transgenic (non-Tg) mice to elucidate the effects of channel's oxidation on cognitive function. Aging mice in which KCNB1 oligomerization is negligible (Tg-C73A), performed significantly better in the Morris Water Maze (MWM) test of working memory compared to non-Tg or Tg-WT mice. KCNB1 and synapsin-1 co-immunoprecipitated and the cognitive impairment in the MWM was associated with moderate loss of synapsin-1 in pre-synaptic structures of the hippocampus, whereas neurodegeneration and neuronal loss were not significantly different in the various genotypes. We conclude that moderate oxidation of the KCNB1 channel during aging can influence neuronal networks by affecting synaptic function.

Oxidation of KCNB1 Potassium Channels Causes Neurotoxicity and Cognitive Impairment in a Mouse Model of Traumatic Brain Injury.

The delayed rectifier potassium (K+) channel KCNB1 (Kv2.1), which conducts a major somatodendritic current in cortex and hippocampus, is known to undergo oxidation in the brain, but whether this can cause neurodegeneration and cognitive impairment is not known. Here, we used transgenic mice harboring human KCNB1 wild-type (Tg-WT) or a nonoxidable C73A mutant (Tg-C73A) in cortex and hippocampus to determine whether oxidized KCNB1 channels affect brain function. Animals were subjected to moderate traumatic brain injury (TBI), a condition characterized by extensive oxidative stress. Dasatinib, a Food and Drug Administration-approved inhibitor of Src tyrosine kinases, was used to impinge on the proapoptotic signaling pathway activated by oxidized KCNB1 channels. Thus, typical lesions of brain injury, namely, inflammation (astrocytosis), neurodegeneration, and cell death, were markedly reduced in Tg-C73A and dasatinib-treated non-Tg animals. Accordingly, Tg-C73A mice and non-Tg mice treated with dasatinib exhibited improved behavioral outcomes in motor (rotarod) and cognitive (Morris water maze) assays compared to controls. Moreover, the activity of Src kinases, along with oxidative stress, were significantly diminished in Tg-C73A brains. Together, these data demonstrate that oxidation of KCNB1 channels is a contributing mechanism to cellular and behavioral deficits in vertebrates and suggest a new therapeutic approach to TBI. SIGNIFICANCE STATEMENT: This study provides the first experimental evidence that oxidation of a K+ channel constitutes a mechanism of neuronal and cognitive impairment in vertebrates. Specifically, the interaction of KCNB1 channels with reactive oxygen species plays a major role in the etiology of mouse model of traumatic brain injury (TBI), a condition associated with extensive oxidative stress. In addition, a Food and Drug Administration-approved drug ameliorates the outcome of TBI in mouse, by directly impinging on the toxic pathway activated in response to oxidation of the KCNB1 channel. These findings elucidate a basic mechanism of neurotoxicity in vertebrates and might lead to a new therapeutic approach to TBI in humans, which, despite significant efforts, is a condition that remains without effective pharmacological treatments.

Cytoskeletal remodeling via Rho GTPases during oxidative and thermal stress in Caenorhabditis elegans.

Biological systems are highly sensitive to changes in their environment. Indeed, the molecular basis of the environmental stress response suggests that the specialized stress responses share more commonalities than previously believed. Here, we used the nematode C. elegans to gain insight into the role of Rho signaling during two common environmental challenges, oxidative and thermal stress. In response to heat shock (HS), wild type (N2) worms demonstrated reduced viability which was rescued by genetic suppression of CDC42 and RHO-1. Visualization of F-actin by phalloidin-rhodamine underscored a strict correlation between the levels of F-actin following GTPase suppression and survival. Additionally, genetic ablation of OSG-1, a Guanine Nucleotide Exchange Factor (GEF) previously implicated in oxidative stress, was associated with constitutively lower levels of F-actin and increased mortality. However, upon an oxidative insult F-actin stability decreased in N2 worms, a rescue of this affect was observed in OSG-1 null worms, consistent with the resistance exhibited by these worms to oxidative stress (OS). Together these data suggest that during conditions of thermal or oxidative stress Rho signaling promotes vulnerability by altering actin dynamics. Thus, the stability of the actin cytoskeleton, in part through a conserved mechanism mediated by Rho signaling, is a crucial factor for the cell's survival to environmental challenges.

Molecular mechanisms underlying the apoptotic effect of KCNB1 K+ channel oxidation.

Potassium (K(+)) channels are targets of reactive oxygen species in the aging nervous system. KCNB1 (formerly Kv2.1), a voltage-gated K(+) channel abundantly expressed in the cortex and hippocampus, is oxidized in the brains of aging mice and of the triple transgenic 3xTg-AD mouse model of Alzheimer's disease. KCNB1 oxidation acts to enhance apoptosis in mammalian cell lines, whereas a KCNB1 variant resistant to oxidative modification, C73A-KCNB1, is cytoprotective. Here we investigated the molecular mechanisms through which oxidized KCNB1 channels promote apoptosis. Biochemical evidence showed that oxidized KCNB1 channels, which form oligomers held together by disulfide bridges involving Cys-73, accumulated in the plasma membrane as a result of defective endocytosis. In contrast, C73A-mutant channels, which do not oligomerize, were normally internalized. KCNB1 channels localize in lipid rafts, and their internalization was dynamin 2-dependent. Accordingly, cholesterol supplementation reduced apoptosis promoted by oxidation of KCNB1. In contrast, cholesterol depletion exacerbated apoptotic death in a KCNB1-independent fashion. Inhibition of raft-associating c-Src tyrosine kinase and downstream JNK kinase by pharmacological and molecular means suppressed the pro-apoptotic effect of KCNB1 oxidation. Together, these data suggest that the accumulation of KCNB1 oligomers in the membrane disrupts planar lipid raft integrity and causes apoptosis via activating the c-Src/JNK signaling pathway.

An evolutionarily conserved mode of modulation of Shaw-like K⁺ channels.

Voltage-gated K(+) channels of the Shaw family (also known as the KCNC or Kv3 family) play pivotal roles in mammalian brains, and genetic or pharmacological disruption of their activities in mice results in a spectrum of behavioral defects. We have used the model system of Caenorhabditis elegans to elucidate conserved molecular mechanisms that regulate these channels. We have now found that the C. elegans Shaw channel KHT-1, and its mammalian homologue, murine Kv3.1b, are both modulated by acid phosphatases. Thus, the C. elegans phosphatase ACP-2 is stably associated with KHT-1, while its mammalian homolog, prostatic acid phosphatase (PAP; also known as ACPP-201) stably associates with murine Kv3.1b K(+) channels in vitro and in vivo. In biochemical experiments both phosphatases were able to reverse phosphorylation of their associated channel. The effect of phosphorylation on both channels is to produce a decrease in current amplitude and electrophysiological analyses demonstrated that dephosphorylation reversed the effects of phosphorylation on the magnitude of the macroscopic currents. ACP-2 and KHT-1 were colocalized in the nervous system of C. elegans and, in the mouse nervous system, PAP and Kv3.1b were colocalized in subsets of neurons, including in the brain stem and the ventricular zone. Taken together, this body of evidence suggests that acid phosphatases are general regulatory partners of Shaw-like K(+) channels.

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.

Auto-phosphorylation of a voltage-gated K+ channel controls non-associative learning.

Here, we characterize a new K(+) channel-kinase complex that operates in the metazoan Caenorhabditis elegans to control learning behaviour. This channel is composed of a pore-forming subunit, dubbed KHT-1 (73% homology to human Kv3.1), and the accessory subunit MPS-1, which shows kinase activity. Genetic, biochemical and electrophysiological evidence show that KHT-1 and MPS-1 form a complex in vitro and in native mechanosensory PLM neurons, and that KHT-1 is a substrate for the kinase activity of MPS-1. Behavioural analysis further shows that the kinase activity of MPS-1 is specifically required for habituation to repetitive mechanical stimulation. Thus, worms bearing an inactive MPS-1 variant (D178N) respond normally to touch on the body but do not habituate to repetitive mechanical stimulation such as tapping on the side of the Petri dish. Hence, the phosphorylation status of KHT-1-MPS-1 seems to be linked to distinct behavioural responses. In the non-phosphorylated state the channel is necessary for the normal function of the touch neurons. In the auto-phosphorylated state the channel acts to induce neuronal adaptation to mechanical stimulation. Taken together, these data establish a new mechanism of dynamic regulation of electrical signalling in the nervous system.

Delayed pharyngeal repolarization promotes abnormal calcium buildup in aging muscle.

In the pharynx of Caenorhabditis elegans, the accessory subunit MPS-4, homolog to human KCNE1, forms a complex with K(+) channel EXP-2 that terminates the action potential. An aspartate residue critical for KCNE1 function, asp76, is conserved in MPS-4 (asp74). Here, we studied the effects of D74N-MPS-4 on the aging pharynx. Electrophysiological studies showed that D74N delays pharyngeal repolarization. Pharynxes of transgenic worms expressing D74N exhibited higher levels of intracellular calcium compared to normal pharynxes. Accordingly, loss of pharyngeal function was accelerated in aging D74N worms. The pharyngeal action potential resembles the action potential that controls the mechanical activity of human left ventricle. Hence, these findings argue that the hearts of patients affected by delayed repolarization, a condition known as long QT syndrome, may experience dysregulated calcium homeostasis.

Ion channels in neurodevelopment: lessons from the Integrin-KCNB1 channel complex.

Ion channels modulate cellular excitability by regulating ionic fluxes across biological membranes. Pathogenic mutations in ion channel genes give rise to epileptic disorders that are among the most frequent neurological diseases affecting millions of individuals worldwide. Epilepsies are triggered by an imbalance between excitatory and inhibitory conductances. However, pathogenic mutations in the same allele can give rise to loss-of-function and/or gain-of-function variants, all able to trigger epilepsy. Furthermore, certain alleles are associated with brain malformations even in the absence of a clear electrical phenotype. This body of evidence argues that the underlying epileptogenic mechanisms of ion channels are more diverse than originally thought. Studies focusing on ion channels in prenatal cortical development have shed light on this apparent paradox. The picture that emerges is that ion channels play crucial roles in landmark neurodevelopmental processes, including neuronal migration, neurite outgrowth, and synapse formation. Thus, pathogenic channel mutants can not only cause epileptic disorders by altering excitability, but further, by inducing morphological and synaptic abnormalities that are initiated during neocortex formation and may persist into the adult brain.

Isolation of Targeted Hypothalamic Neurons for Studies of Hormonal, Metabolic, and Electrical Regulation.

The hypothalamus regulates fundamental metabolic processes by controlling functions as varied as food intake, body temperature, and hormone release. As the functions of the hypothalamus are controlled by specific subsets of neuronal populations, the ability to isolate them provides a major tool for studying metabolic mechanisms. In this regard, the neuronal complexity of the hypothalamus poses exceptional challenges. For these reasons, new techniques, such as Magnetic-Activated Cell Sorting (MACS), have been explored. This paper describes a new application of magnetic-activated cell sorting (MACS) using microbead technology to isolate a targeted neuronal population from prenatal mice brains. The technique is simple and guarantees a highly pure and viable primary hypothalamic neuron culture with high reproducibility. The hypothalamus is gently dissociated, neurons are selectively isolated and separated from glial cells, and finally, using a specific antibody for a cell surface marker, the population of interest is selected. Once isolated, targeted neurons can be used to investigate their morphological, electrical, and endocrine characteristics and their responses in normal or pathological conditions. Furthermore, given the variegated roles of the hypothalamus in regulating feeding, metabolism, stress, sleep, and motivation, a closer look at targeted and region-specific neurons may provide insight into their tasks in this complex environment.

Integrin-KCNB1 potassium channel complexes regulate neocortical neuronal development and are implicated in epilepsy.

Potassium (K+) channels are robustly expressed during prenatal brain development, including in progenitor cells and migrating neurons, but their function is poorly understood. Here, we investigate the role of voltage-gated K+ channel KCNB1 (Kv2.1) in neocortical development. Neuronal migration of glutamatergic neurons was impaired in the neocortices of KCNB1 null mice. Migratory defects persisted into the adult brains, along with disrupted morphology and synaptic connectivity. Mice developed seizure phenotype, anxiety, and compulsive behavior. To determine whether defective KCNB1 can give rise to developmental channelopathy, we constructed Knock In (KI) mice, harboring the gene variant Kcnb1R312H (R312H mice) found in children with developmental and epileptic encephalopathies (DEEs). The R312H mice exhibited a similar phenotype to the null mice. Wild type (WT) and R312H KCNB1 channels made complexes with integrins α5ß5 (Integrin_K+ channel_Complexes, IKCs), whose biochemical signaling was impaired in R312H brains. Treatment with Angiotensin II in vitro, an agonist of Focal Adhesion kinase, a key component of IKC signaling machinery, corrected the neuronal abnormalities. Thus, a genetic mutation in a K+ channel induces severe neuromorphological abnormalities through non-conducting mechanisms, that can be rescued by pharmacological intervention. This underscores a previously unknown role of IKCs as key players in neuronal development, and implicate developmental channelopathies in the etiology of DEEs.

Non-conducting functions of ion channels: The case of integrin-ion channel complexes.

Started as an academic curiosity more than two decades ago, the idea that ion channels can regulate cellular processes in ways that do not depend on their conducting properties (non-ionic functions) gained traction and is now a flourishing area of research. Channels can regulate physiological processes including actin cytoskeletal remodeling, cell motility, excitation-contraction coupling, non-associative learning and embryogenesis, just to mention some, through non-ionic functions. When defective, non-ionic functions can give rise to channelopathies involved in cancer, neurodegenerative disease and brain trauma. Ion channels exert their non-ionic functions through a variety of mechanisms that range from physical coupling with other proteins, to possessing enzymatic activity, to assembling with signaling molecules. In this article, we take stock of the field and review recent findings. The concept that emerges, is that one of the most common ways through which channels acquire non-ionic attributes, is by assembling with integrins. These integrin-channel complexes exhibit broad genotypic and phenotypic heterogeneity and reveal a pleiotropic nature, as they appear to be capable of influencing both physiological and pathological processes.

The chemical and physical characteristics of single-walled carbon nanotube film impact on osteoblastic cell response.

Carbon-nanotube-based substrates have been shown to support the growth of different cell types and, as such, have raised considerable interest in view of their possible use in biomedical applications. Nanotube matrices are embedded in polymers which cause inherent changes in nanotube chemical and physical film properties. Thus, it is critical to understand how the physical properties of the film affect the biology of the host tissue. Here, we investigated how the physical and chemical properties of single-walled carbon nanotubes (SWNT) films impact the response of MC3T3-E1 bone osteoblasts. We found that two fundamental steps in cell growth-initial attachment to the substrate and proliferation-are strongly dependent on, respectively, the energy and roughness of the surface. Thus, fine-tuning the properties of the film may represent a valid strategy to optimize the response of the biological host.

A novel assay for drug screening that utilizes the heat shock response of Caenorhabditis elegans nematodes.

Biological organisms respond to environmental stressors by recruiting multiple cellular cascades that act to mitigate damage and ultimately enhance survival. This implies that compounds that interact with any of those pathways might improve organism's survival. Here, we report on an initial attempt to develop a drug screening assay based on the heat shock (HS) response of Caenorhabditis elegans nematodes. The protocol works by subjecting the worms to two HS conditions in the absence/presence of the test compounds. Post-heat shock survival is quantified manually or in semi-automatic manner by analyzing z-stack pictures. We blindly screened a cassette of 72 compounds in different developmental stages provided by Eli Lilly through their Open Innovation Drug Discovery program. The analysis indicated that, on average, therapeutically useful drugs increase survival to HS compared to compounds used in non-clinical settings. We developed a formalism that estimates the probability of a compound to enhance survival based on a comparison with a set of parameters calculated from a pool of 35 FDA-approved drugs. The method correctly identified the developmental stages of the Lilly compounds based on their relative abilities to enhance survival to the HS. Taken together these data provide proof of principle that an assay that measures the HS response of C. elegans can offer physiological and pharmacological insight in a cost- and time-efficient manner.

Toxicity induced enhanced extracellular matrix production in osteoblastic cells cultured on single-walled carbon nanotube networks.

A central effort in biomedical research concerns the development of materials for sustaining and controlling cell growth. Carbon nanotube based substrates have been shown to support the growth of different kinds of cells (Hu et al 2004 Nano Lett. 4 507-11; Kalbacova et al 2006 Phys. Status Solidi b 13 243; Zanello et al 2006 Nano Lett. 6 562-7); however the underlying molecular mechanisms remain poorly defined. To address the fundamental question of mechanisms by which nanotubes promote bone mitosis and histogenesis, primary calvariae osteoblastic cells were grown on single-walled carbon nanotube thin film (SWNT) substrates. Using a combination of biochemical and optical techniques we demonstrate here that SWNT networks promote cell development through two distinct steps. Initially, SWNTs are absorbed in a process that resembles endocytosis, inducing acute toxicity. Nanotube-mediated cell destruction, however, induces a release of endogenous factors that act to boost the activity of the surviving cells by stimulating the synthesis of extracellular matrix.

MPS-1 is a K+ channel beta-subunit and a serine/threonine kinase.

We report the first example of a K+ channel beta-subunit that is also a serine/threonine kinase. MPS-1 is a single-transmembrane domain protein that coassembles with voltage-gated K+ channel KVS-1 in the nervous system of the nematode Caenorhabditis elegans. Biochemical analysis shows that MPS-1 can phosphorylate KVS-1 and other substrates. Electrophysiological analysis in Chinese hamster ovary (CHO) cells demonstrates that MPS-1 activity leads to a significant decrease in the macroscopic current. Single-channel analysis and biotinylation assays indicate that MPS-1 reduces the macroscopic current by lowering the open probability of the channel. These data are consistent with a model that predicts that the MPS-1-dependent phosphorylation of KVS-1 sustains cell excitability by controlling K+ flux.