Activation of volume-sensitive outwardly rectifying chloride channel by ROS contributes to ER stress and cardiac contractile dysfunction: involvement of CHOP through Wnt

Endoplasmic reticulum (ER) stress occurring in stringent conditions is critically involved in cardiomyocytes apoptosis and cardiac contractile dysfunction (CCD). However, the molecular machinery that mediates cardiac ER stress and subsequent cell death remains to be fully deciphered, which will hopefully provide novel therapeutic targets for these disorders. Here, we establish tunicamycin-induced model of cardiomyocyte ER stress, which effectively mimicks pathological stimuli to trigger CCD. Tunicamycin activates volume-sensitive outward rectifying Cl⁻ currents. Blockade of the volume-sensitive outwardly rectifying (VSOR) Cl⁻ channel by 4,4''-diisothiocya-natostilbene-2,2''-disulfonic acid (DIDS), a non-selective Cl⁻ channel blocker, and 4-(2-butyl-6,7-dichlor-2-cyclopentyl-indan-1-on-5-yl) oxybutyric acid (DCPIB), a selective VSOR Cl⁻ channel blocker, improves cardiac contractility, which correlates with suppressed ER stress through inhibiting the canonical GRP78/eIF2α/ATF4 and XBP1 pathways, and promotes survival of cardiomyocytes by inverting tunicamycin-induced decrease of Wnt through the CHOP pathway. VSOR activation of tunicamycin-treated cardiomyocytes is attributed to increased intracellular levels of reactive oxygen species (ROS). Our study demonstrates a pivotal role of ROS/VSOR in mediating ER stress and functional impairment of cardiomyocytes via the CHOP-Wnt pathway, and suggests the therapeutic values of VSOR Cl⁻ channel blockers against ER stress-associated cardiac anomalies.The endoplasmic reticulum (ER) is characterized as an organelle that participates in the folding of membrane and secretory proteins.^1,2 Efficient functioning of the endoplasmic reticulum is important for cell function and survival. Perturbations of ER homeostasis by energy deprivation and glucose,³ viral infections⁴ and accumulation of misfolded and/or unfolded proteins² interfere with ER function, leading to a state of ER stress.^{5, 6, 7} A cohort of chemicals, for example, tunicamycin and thapsigargin, also trigger ER stress.^{8, 9, 10} Thapsigargin disrupts the calcium storage of ER by blocking calcium reuptake into the ER lumen, thus by depleting calcium from the organelle.¹¹ In particular, tunicamycin is a highly specific ER stress inducer by inhibiting N-linked glycosylation of protein, representing a well-documented method to artificially elicit unfolded protein response.⁸ In response to ER stress, ER chaperones such as glucose-regulated protein 78 kDa (GRP78) and glucose-regulated protein 94 kDa (GRP94) are upregulated to facilitate the recovery of unfolded or misfolded proteins.¹² ER stress may act as a defense mechanism against external insults; however, prolonged and/or severe ER stress may ultimately trigger apoptosis.⁸ The C/EBP homologous protein (CHOP) has been defined as a pivotal mediator of cell death signaling in ER stress.^{13, 14} Accumulating evidence has demonstrated that ER stress-induced cell death is an essential step in the pathogenesis of a wide variety of cardiovascular diseases such as ischemia reperfusion heart diseases,¹⁵ atherosclerosis,^{5, 16, 17, 18} myocardial infarction,¹⁹ hypertension^{20, 21} and heart failure.^{8, 22, 23} Inhibiting ER stress has great therapeutic values for cardiac anomalies. However, the precise mechanism involved in ER stress-induced cardiovascular diseases has not been well identified, which impedes the translation of our understanding of ER stress-induced cardiovascular anomalies into effective therapeutic strategies. Apoptosis induction requires persistent cell shrinkage, named apoptotic volume decrease (AVD).^{24, 25, 26, 27} It is an early prerequisite for the activation of caspases.²⁴ In various types of cells including cardiomyocytes, AVD process is accomplished by the activation of volume-sensitive outwardly rectifying (VSOR) Cl⁻ channel and is concomitant with the egress of water from the cells undergoing mitochondrion-initiated or death receptor-induced apoptosis.^{25, 28, 29, 30} Although inhibition of VSOR Cl⁻ channel by DIDS (4,4''-diisothiocyanatostilbene-2,2''-disulphonic acid) and DCPIB (4-(2-butyl-6,7- dichlor-2-cyclopentyl-indan-1-on-5-yl) oxybutyric acid) blocked AVD and rescued cardiomyocytes from mitochondrial and death receptor pathway-induced apoptosis,^{31, 32} it remains largely unknown concerning the role of VSOR Cl⁻ channel and how it is regulated in ER stress-induced apoptotic cardiomyocyte death.Emerging evidence indicates that Wnt signal pathways are found to be anti-apoptotic in the cardiovascular diseases,^{33, 34, 35} regulating crucial aspects of cardiovascular biology. However, up to now, its activity in ER stress-induced apoptosis and in the process of AVD in cardiomyocytes remains elusive.In the present study, we probed the role of VSOR Cl⁻ channel in ER stress-induced apoptosis of cardiomyocytes, which intimately correlates with cardiac contractile dysfunction (CCD). We hypothesized that VSOR Cl⁻ channel controls the process of AVD occurring concomitantly with ER stress-induced apoptosis of cardiomyocytes. To test this hypothesis, we investigated VSOR Cl⁻ currents in cardiomyocytes treated with the ER stress inducer tunicamycin. The pathophysiological role of VSOR Cl⁻ channel and the potential signaling mechanisms in the development of ER stress-induced apoptosis in CCD were also dissected.     

Calcineurin B-Like Protein-Interacting Protein Kinase CIPK21 Regulates Osmotic and Salt Stress Responses in Arabidopsis

The role of calcium-mediated signaling has been extensively studied in plant responses to abiotic stress signals. Calcineurin B-like proteins (CBLs) and CBL-interacting protein kinases (CIPKs) constitute a complex signaling network acting in diverse plant stress responses. Osmotic stress imposed by soil salinity and drought is a major abiotic stress that impedes plant growth and development and involves calcium-signaling processes. In this study, we report the functional analysis of CIPK21, an Arabidopsis (Arabidopsis thaliana) CBL-interacting protein kinase, ubiquitously expressed in plant tissues and up-regulated under multiple abiotic stress conditions. The growth of a loss-of-function mutant of CIPK21, cipk21, was hypersensitive to high salt and osmotic stress conditions. The calcium sensors CBL2 and CBL3 were found to physically interact with CIPK21 and target this kinase to the tonoplast. Moreover, preferential localization of CIPK21 to the tonoplast was detected under salt stress condition when coexpressed with CBL2 or CBL3. These findings suggest that CIPK21 mediates responses to salt stress condition in Arabidopsis, at least in part, by regulating ion and water homeostasis across the vacuolar membranes.Drought and salinity cause osmotic stress in plants and severely affect crop productivity throughout the world. Plants respond to osmotic stress by changing a number of cellular processes (Xiong et al., 1999; Xiong and Zhu, 2002; Bartels and Sunkar, 2005; Boudsocq and Lauriére, 2005). Some of these changes include activation of stress-responsive genes, regulation of membrane transport at both plasma membrane (PM) and vacuolar membrane (tonoplast) to maintain water and ionic homeostasis, and metabolic changes to produce compatible osmolytes such as Pro (Stewart and Lee, 1974; Krasensky and Jonak, 2012). It has been well established that a specific calcium (Ca²⁺) signature is generated in response to a particular environmental stimulus (Trewavas and Malhó, 1998; Scrase-Field and Knight, 2003; Luan, 2009; Kudla et al., 2010). The Ca²⁺ changes are primarily perceived by several Ca²⁺ sensors such as calmodulin (Reddy, 2001; Luan et al., 2002), Ca²⁺-dependent protein kinases (Harper and Harmon, 2005), calcineurin B-like proteins (CBLs; Luan et al., 2002; Batistič and Kudla, 2004; Pandey, 2008; Luan, 2009; Sanyal et al., 2015), and other Ca²⁺-binding proteins (Reddy, 2001; Shao et al., 2008) to initiate various cellular responses.Plant CBL-type Ca²⁺ sensors interact with and activate CBL-interacting protein kinases (CIPKs) that phosphorylate downstream components to transduce Ca²⁺ signals (Liu et al., 2000; Luan et al., 2002; Batistič and Kudla, 2004; Luan, 2009). In several plant species, multiple members have been identified in the CBL and CIPK family (Luan et al., 2002; Kolukisaoglu et al., 2004; Pandey, 2008; Batistič and Kudla, 2009; Weinl and Kudla, 2009; Pandey et al., 2014). Involvement of specific CBL-CIPK pair to decode a particular type of signal entails the alternative and selective complex formation leading to stimulus-response coupling (D’Angelo et al., 2006; Batistič et al., 2010).Several CBL and CIPK family members have been implicated in plant responses to drought, salinity, and osmotic stress based on genetic analysis of Arabidopsis (Arabidopsis thaliana) mutants (Zhu, 2002; Cheong et al., 2003, 2007; Kim et al., 2003; Pandey et al., 2004, 2008; D’Angelo et al., 2006; Qin et al., 2008; Tripathi et al., 2009; Held et al., 2011; Tang et al., 2012; Drerup et al., 2013; Eckert et al., 2014). A few CIPKs have also been functionally characterized by gain-of-function approach in crop plants such as rice (Oryza sativa), pea (Pisum sativum), and maize (Zea mays) and were found to be involved in osmotic stress responses (Mahajan et al., 2006; Xiang et al., 2007; Yang et al., 2008; Tripathi et al., 2009; Zhao et al., 2009; Cuéllar et al., 2010).In this report, we examined the role of the Arabidopsis CIPK21 gene in osmotic stress response by reverse genetic analysis. The loss-of-function mutant plants became hypersensitive to salt and mannitol stress conditions, suggesting that CIPK21 is involved in the regulation of osmotic stress response in Arabidopsis. These findings are further supported by an enhanced tonoplast targeting of the cytoplasmic CIPK21 through interaction with the vacuolar Ca²⁺ sensors CBL2 and CBL3 under salt stress condition.     

Neuronal NLRP1 inflammasome activation of Caspase-1 coordinately regulates inflammatory interleukin-1-beta production and axonal degeneration-associated Caspase-6 activation

Neuronal active Caspase-6 (Casp6) is associated with Alzheimer disease (AD), cognitive impairment, and axonal degeneration. Caspase-1 (Casp1) can activate Casp6 but the expression and functionality of Casp1-activating inflammasomes has not been well-defined in human neurons. Here, we show that primary cultures of human CNS neurons expressed functional Nod-like receptor protein 1 (NLRP1), absent in melanoma 2, and ICE protease activating factor, but not the NLRP3, inflammasome receptor components. NLRP1 neutralizing antibodies in a cell-free system, and NLRP1 siRNAs in neurons hampered stress-induced Casp1 activation. NLRP1 and Casp1 siRNAs also abolished stress-induced Casp6 activation in neurons. The functionality of the NLRP1 inflammasome in serum-deprived neurons was also demonstrated by NLRP1 siRNA-mediated inhibition of speck formation of the apoptosis-associated speck-like protein containing a caspase recruitment domain conjugated to green fluorescent protein. These results indicated a novel stress-induced intraneuronal NLRP1/Casp1/Casp6 pathway. Lipopolysaccharide induced Casp1 and Casp6 activation in wild-type mice brain cortex, but not in that of Nlrp1^−/− and Casp1^−/− mice. NLRP1 immunopositive neurons were increased 25- to 30-fold in AD brains compared with non-AD brains. NLRP1 immunoreactivity in these neurons co-localized with Casp6 activity. Furthermore, the NLRP1/Casp1/Casp6 pathway increased amyloid beta peptide 42 ratio in serum-deprived neurons. Therefore, CNS human neurons express functional NLRP1 inflammasomes, which activate Casp1 and subsequently Casp6, thus revealing a fundamental mechanism linking intraneuronal inflammasome activation to Casp1-generated interleukin-1-β-mediated neuroinflammation and Casp6-mediated axonal degeneration.The lack of efficient treatment for Alzheimer disease (AD) is of high social and economical cost and a growing concern with the aging of the world''s population.¹ Therapies eliminating amyloid beta peptide (Aβ) from AD brains have unfortunately failed to stem progressive cognitive decline. These disappointing results have forced scientists to reconsider treatments against AD; some focusing on targeting Aβ earlier in disease, while others attempting to disaggregate the Tau protein in neurofibrillary tangles (NFT). Recently, the association of several immune responsive genes with increased AD risks^{2, 3, 4} have additionally revived interest in a possible etiological role for inflammation in AD.AD brain inflammation is attributed to activated microglia, which remove Aβ, and secrete neurotoxic molecules that induce neurodegeneration. Interleukin-1-beta (IL-1β), a critical component of brain neuroinflammation, is increased in AD brains⁵ and may contribute to AD pathology by increasing amyloid precursor protein (APP) gene expression, Tau hyperphosphorylation and memory impairment.⁶ However, anti-inflammatory therapies have not provided the expected beneficial effect in AD patients,⁷ suggesting that microglial inflammation may be a consequence of AD. Degenerating neurons are renowned initiators of brain inflammatory responses and the loss of synapses remains the best correlative marker of dementia in AD.⁸ This has incited us to study the response of human neurons to stress and to determine whether specific neuronal molecular events were initiated that link axonal degeneration to an inflammatory response.The active cysteinal Caspase-6 protease (Casp6), associated with axonal degeneration,^{9, 10, 11, 12, 13} is highly abundant in NFT, neuropil threads, and neuritic plaques of AD brains.¹⁴ In some aged non-cognitively impaired individuals, Casp6 activity in the entorhinal cortex and CA1 regions of the hippocampus,¹⁵ two areas initially affected by NFT pathology in AD,¹⁶ correlates significantly with lower cognitive performance.¹⁷ The expression of active Casp6 in CA1 pyramidal neurons of mouse brains is sufficient to induce age-dependent cognitive impairment, in the absence of plaques and tangles, which suggests that active Casp6 in AD brains could be a major contributor to axonal degeneration and cognitive decline.¹⁸Despite substantial evidence implicating Casp6 in AD, the pathways leading to Casp6 activation in neurons are unclear. Caspase-1 (Casp1) activates Casp6 in primary cultures of human CNS neurons.¹⁹ Inflammasome multiprotein complexes, constituted of danger sensing nucleotide-binding oligomerization domain-like receptors or the DNA sensing absent in melanoma 2 (AIM2) component, and the apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), recruit and induce Casp1 self-activation.^{20, 21} Functional Nod-like receptor protein 1 (NLRP1), Nod-like receptor protein 3 (NLRP3), AIM2, and ICE protease activating factor (IPAF-1) inflammasomes have been characterized primarily in peripheral macrophages²² and CNS microglia.^{23, 24} Recently, reports have indicated inflammasome receptor expression and activation in rodent neurons. Rat cerebellar granule neurons submitted to oxygen and glucose deprivation or reduced potassium levels increased Nlrp1 mRNA levels.^{25, 26} Nuclear Nlrp1 or functional Nlrp1 inflammasome complexes increased in rat cortical neurons after traumatic brain injury, stroke, and glucose-oxygen deprivation insults.^{27, 28, 29, 30, 31} Neuronal Nlrp1 increased in rats submitted to spinal cord or sciatic nerve injury,^{29, 32} and in aging rat hippocampus or ethanol treated hippocampal slice cultures.^{33, 34} Aim2 induced pyroptosis in rat cortical neuron cultures and traumatic brain injury.³⁵ Nlrp1 has been reported in human brain pyramidal neurons³⁶ and inflammasome receptor mRNAs were observed in human neuron cultures and human Rasmussen''s encephalitis.³⁷Here, we assessed which inflammasome could activate Casp1 and subsequently Casp6 in human primary CNS cultures. We determined which inflammasomes were expressed in naive and stressed neurons and used siRNAs and S-100 cell-free extracts treated with specific inflammasome activators, or antibody blockers, to identify the functional inflammasome. We uncovered that the NLRP1, AIM2, and IPAF-1, but not the NLRP3, inflammasomes were expressed and functional in neurons and that the NLRP1 inflammasome was responsible for Casp1 and subsequently Casp6 activation in serum-deprived and benzylated ATP (BzATP)-stressed neurons. NLRP1 was co-localized with Casp6 activity, immunostained 25- to 30-fold more neurons in AD, and increased Aβ₄₂ in serum-deprived neurons. The NLRP1–Casp1–Casp6 pathway was blocked in lipopolysaccharide (LPS)-treated Nlrp1^−/− and Casp1^−/− mice brains. These results reveal a molecular cascade linking neuronal inflammasome-mediated Casp1 activation to Casp6 activation and provide unexpected novel common neuronal therapeutic targets against neuroinflammation, axonal degeneration, and cognitive impairment in AD.     

Oncogenic BRAF induces chronic ER stress condition resulting in increased basal autophagy and apoptotic resistance of cutaneous melanoma

The notorious unresponsiveness of metastatic cutaneous melanoma to current treatment strategies coupled with its increasing incidence constitutes a serious worldwide clinical problem. Moreover, despite recent advances in targeted therapies for patients with BRAF^V600E mutant melanomas, acquired resistance remains a limiting factor and hence emphasises the acute need for comprehensive pre-clinical studies to increase the biological understanding of such tumours in order to develop novel effective and longlasting therapeutic strategies. Autophagy and ER stress both have a role in melanoma development/progression and chemoresistance although their real impact is still unclear. Here, we show that BRAF^V600E induces a chronic ER stress status directly increasing basal cell autophagy. BRAF^V600E-mediated p38 activation stimulates both the IRE1/ASK1/JNK and TRB3 pathways. Bcl-XL/Bcl-2 phosphorylation by active JNK releases Beclin1 whereas TRB3 inhibits the Akt/mTor axes, together resulting in an increase in basal autophagy. Furthermore, we demonstrate chemical chaperones relieve the BRAF^V600E-mediated chronic ER stress status, consequently reducing basal autophagic activity and increasing the sensitivity of melanoma cells to apoptosis. Taken together, these results suggest enhanced basal autophagy, typically observed in BRAF^V600E melanomas, is a consequence of a chronic ER stress status, which ultimately results in the chemoresistance of such tumours. Targeted therapies that attenuate ER stress may therefore represent a novel and more effective therapeutic strategy for BRAF mutant melanoma.Cutaneous melanoma represents one of the most aggressive and difficult to treat forms of human cancer, with a worldwide incidence that has steadily increased over the past 40 years.^{1, 2}Notoriously unresponsive to conventional chemotherapy, metastatic disease is highly invasive and evolves with an extensive repertoire of molecular defences against immunological and cytotoxic attack.³Although linked to exposure to ultraviolet light, it is widely accepted that both genotypic and phenotypic changes in melanocytes predispose to melanocyte transformation and the onset of melanoma.^{4, 5}Surprisingly, p53 mutations are very rare in melanoma, but activity is, however, impaired through direct or indirect inactivation of key elements of this pathway, including through the suppression of APAF-1 expression,⁶ loss of PTEN function,⁷ dysregulation of Bcl-2 expression,⁸ upregulation of the anti-apoptotic protein Mcl-1 together with its altered slice variant expression ^{9, 10} and the ER chaperone GRP78.^{11, 12, 13} Oncogenic mutations, however, in the Ras/Raf pathway are the most well-described genetic mutations associated with melanoma development and progression.¹⁴ Indeed, up to 90% of all melanomas harbour activating NRAS or BRAF mutations, with BRAF^V600E representing more than 90% of BRAF mutations,^{15, 16} the consequence of which is the constitutive activation of RAF-extracellular signal-regulated kinase/ERK signalling promoting melanoma proliferation and resistance to apoptosis.¹⁷ Nevertheless, mutation of NRAS/BRAF alone is not sufficient to initiate melanomagenesis, because these common mutations are also present in benign nevi, thereby highlighting the requirement of other factors to drive melanocyte transformation and melanoma development.^{15, 16} Dysregulation of autophagy has accordingly been postulated as a secondary event contributing to melanoma progression and, importantly, also has a key role in chemoresistance.^{18, 19, 20}Autophagy is the principal catabolic process for the bulk degradation and recycling of aged/damaged cellular components, organelles and proteins through the formation of a double-membraned cytosolic vesicle able to wrap targeted material. The subsequent fusion with lysosomes and degradation of cargo provide nutrients in times of environmental stress, such as nutrient deprivation or hypoxia.²¹ Though essential for the maintenance of cellular homeostasis under conditions of nutrient deprivation, paradoxically, autophagy promotes both tumour suppression and tumour development.²² Although the accumulation of damaged organelles/cytosolic proteins may lead to cellular transformation, autophagy may also sustain tumour growth in a microenvironment which is commonly poor of oxygen and nutrients.²² Thus, not surprisingly, autophagy activation is frequently observed in late-stage malignancy although the molecular mechanisms mediating its activation/regain of function remain unclear.ER stress may also constitute a key secondary event in melanoma development.²³ Primarily a cytoprotective pro-survival process, ER stress is activated as a result of accumulated unfolded proteins, protein overload or depletion of ER calcium stores and mediated through the activity of the master ER chaperone Grp78 and three signalling pathways; PERK/eIF2α/ATF4, IRE-1/Xbp-1 and ATF6 which collectively maintain ER homeostasis through the instigation of an unfolded protein response (UPR)²⁴ or sustained ER stress may lead to the induction of apoptosis.^{25, 26} Increasing evidence indicates that nutrient deprivation and hypoxia lead to activation of the UPR in various solid tumours, frequently correlating with resistance to chemotherapy.²⁷ The accepted hypothesis is thus that activation of the UPR in cancer cells enables their adaption to such ER stress resulting in the resistance to apoptosis through the persistent expression of pro-survival instead of pro-apoptotic proteins.²⁸Although under stress conditions, autophagy and ER stress seem to act in parallel, indeed they are closely related, because one can regulate the other and vice versa. In fact, ER stress is able to promptly stimulate autophagy,²⁶ whereas autophagy selectively removes the membranes of the endoplasmic reticulum at the end of the UPR, although the molecular mechanisms are still largely unclear.²⁹In the present study, we investigated the link between oncogenic BRAF^V600E and increased basal autophagy in melanoma cells, highlighting the pivotal role played by ER stress, possibly responsible for tumour growth and chemoresistance.     

Plasma membrane poration by opioid neuropeptides: a possible mechanism of pathological signal transduction

Neuropeptides induce signal transduction across the plasma membrane by acting through cell-surface receptors. The dynorphins, endogenous ligands for opioid receptors, are an exception; they also produce non-receptor-mediated effects causing pain and neurodegeneration. To understand non-receptor mechanism(s), we examined interactions of dynorphins with plasma membrane. Using fluorescence correlation spectroscopy and patch-clamp electrophysiology, we demonstrate that dynorphins accumulate in the membrane and induce a continuum of transient increases in ionic conductance. This phenomenon is consistent with stochastic formation of giant (~2.7 nm estimated diameter) unstructured non-ion-selective membrane pores. The potency of dynorphins to porate the plasma membrane correlates with their pathogenic effects in cellular and animal models. Membrane poration by dynorphins may represent a mechanism of pathological signal transduction. Persistent neuronal excitation by this mechanism may lead to profound neuropathological alterations, including neurodegeneration and cell death.Neuropeptides are the largest and most diverse family of neurotransmitters. They are released from axon terminals and dendrites, diffuse to pre- or postsynaptic neuronal structures and activate membrane G-protein-coupled receptors. Prodynorphin (PDYN)-derived opioid peptides including dynorphin A (Dyn A), dynorphin B (Dyn B) and big dynorphin (Big Dyn) consisting of Dyn A and Dyn B are endogenous ligands for the κ-opioid receptor. Acting through this receptor, dynorphins regulate processing of pain and emotions, memory acquisition and modulate reward induced by addictive substances.^{1, 2, 3, 4} Furthermore, dynorphins may produce robust cellular and behavioral effects that are not mediated through opioid receptors.^{5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29} As evident from pharmacological, morphological, genetic and human neuropathological studies, these effects are generally pathological, including cell death, neurodegeneration, neurological dysfunctions and chronic pain. Big Dyn is the most active pathogenic peptide, which is about 10- to 100-fold more potent than Dyn A, whereas Dyn B does not produce non-opioid effects.^{16, 17, 22, 25} Big Dyn enhances activity of acid-sensing ion channel-1a (ASIC1a) and potentiates ASIC1a-mediated cell death in nanomolar concentrations^{30, 31} and, when administered intrathecally, induces characteristic nociceptive behavior at femtomolar doses.^{17, 22} Inhibition of endogenous Big Dyn degradation results in pathological pain, whereas prodynorphin (Pdyn) knockout mice do not maintain neuropathic pain.^{22, 32} Big Dyn differs from its constituents Dyn A and Dyn B in its unique pattern of non-opioid memory-enhancing, locomotor- and anxiolytic-like effects.²⁵Pathological role of dynorphins is emphasized by the identification of PDYN missense mutations that cause profound neurodegeneration in the human brain underlying the SCA23 (spinocerebellar ataxia type 23), a very rare dominantly inherited neurodegenerative disorder.^{27, 33} Most PDYN mutations are located in the Big Dyn domain, demonstrating its critical role in neurodegeneration. PDYN mutations result in marked elevation in dynorphin levels and increase in its pathogenic non-opioid activity.^{27, 34} Dominant-negative pathogenic effects of dynorphins are not produced through opioid receptors.ASIC1a, glutamate NMDA (N-methyl-d-aspartate) and AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid)/kainate ion channels, and melanocortin and bradykinin B2 receptors have all been implicated as non-opioid dynorphin targets.^{5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 30, 31, 35, 36} Multiplicity of these targets and their association with the cellular membrane suggest that their activation is a secondary event triggered by a primary interaction of dynorphins with the membrane. Dynorphins are among the most basic neuropeptides.^{37, 38} The basic nature is also a general property of anti-microbial peptides (AMPs) and amyloid peptides that act by inducing membrane perturbations, altering membrane curvature and causing pore formation that disrupts membrane-associated processes including ion fluxes across the membrane.³⁹ The similarity between dynorphins and these two peptide groups in overall charge and size suggests a similar mode of their interactions with membranes.In this study, we dissect the interactions of dynorphins with the cell membrane, the primary event in their non-receptor actions. Using fluorescence imaging, correlation spectroscopy and patch-clamp techniques, we demonstrate that dynorphin peptides accumulate in the plasma membrane in live cells and cause a profound transient increase in cell membrane conductance. Membrane poration by endogenous neuropeptides may represent a novel mechanism of signal transduction in the brain. This mechanism may underlie effects of dynorphins under pathological conditions including chronic pain and tissue injury.