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1.
Annu Rev Immunol ; 33: 291-353, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25861976

RESUMO

Ion channels and transporters mediate the transport of charged ions across hydrophobic lipid membranes. In immune cells, divalent cations such as calcium, magnesium, and zinc have important roles as second messengers to regulate intracellular signaling pathways. By contrast, monovalent cations such as sodium and potassium mainly regulate the membrane potential, which indirectly controls the influx of calcium and immune cell signaling. Studies investigating human patients with mutations in ion channels and transporters, analysis of gene-targeted mice, or pharmacological experiments with ion channel inhibitors have revealed important roles of ionic signals in lymphocyte development and in innate and adaptive immune responses. We here review the mechanisms underlying the function of ion channels and transporters in lymphocytes and innate immune cells and discuss their roles in lymphocyte development, adaptive and innate immune responses, and autoimmunity, as well as recent efforts to develop pharmacological inhibitors of ion channels for immunomodulatory therapy.


Assuntos
Imunidade Adaptativa/fisiologia , Imunidade Inata/fisiologia , Canais Iônicos/metabolismo , Animais , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Humanos , Hipersensibilidade/genética , Hipersensibilidade/imunologia , Hipersensibilidade/metabolismo , Síndromes de Imunodeficiência/tratamento farmacológico , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/imunologia , Síndromes de Imunodeficiência/metabolismo , Imunoterapia/métodos , Canais Iônicos/genética , Linfócitos/citologia , Linfócitos/imunologia , Linfócitos/metabolismo , Mastócitos/imunologia , Mastócitos/metabolismo , Terapia de Alvo Molecular , Mutação , Transdução de Sinais
2.
Proc Natl Acad Sci U S A ; 119(34): e2202926119, 2022 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-35969786

RESUMO

The Ca2+-activated SK4 K+ channel is gated by Ca2+-calmodulin (CaM) and is expressed in immune cells, brain, and heart. A cryoelectron microscopy (cryo-EM) structure of the human SK4 K+ channel recently revealed four CaM molecules per channel tetramer, where the apo CaM C-lobe and the holo CaM N-lobe interact with the proximal carboxyl terminus and the linker S4-S5, respectively, to gate the channel. Here, we show that phosphatidylinositol 4-5 bisphosphate (PIP2) potently activates SK4 channels by docking to the boundary of the CaM-binding domain. An allosteric blocker, BA6b9, was designed to act to the CaM-PIP2-binding domain, a previously untargeted region of SK4 channels, at the interface of the proximal carboxyl terminus and the linker S4-S5. Site-directed mutagenesis, molecular docking, and patch-clamp electrophysiology indicate that BA6b9 inhibits SK4 channels by interacting with two specific residues, Arg191 and His192 in the linker S4-S5, not conserved in SK1-SK3 subunits, thereby conferring selectivity and preventing the Ca2+-CaM N-lobe from properly interacting with the channel linker region. Immunohistochemistry of the SK4 channel protein in rat hearts showed a widespread expression in the sarcolemma of atrial myocytes, with a sarcomeric striated Z-band pattern, and a weaker occurrence in the ventricle but a marked incidence at the intercalated discs. BA6b9 significantly prolonged atrial and atrioventricular effective refractory periods in rat isolated hearts and reduced atrial fibrillation induction ex vivo. Our work suggests that inhibition of SK4 K+ channels by targeting drugs to the CaM-PIP2-binding domain provides a promising anti-arrhythmic therapy.


Assuntos
Fibrilação Atrial , Calmodulina , Canais de Potássio Ativados por Cálcio de Condutância Intermediária , Bloqueadores dos Canais de Potássio , Animais , Fibrilação Atrial/tratamento farmacológico , Sinalização do Cálcio , Calmodulina/metabolismo , Microscopia Crioeletrônica , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/antagonistas & inibidores , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Fosfatidilinositol 4,5-Difosfato , Bloqueadores dos Canais de Potássio/farmacologia , Ratos
3.
Cell Physiol Biochem ; 58(2): 107-127, 2024 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-38623063

RESUMO

Anomalous expression of potassium channels in cancer tissues is associated with several cancer hallmarks that support deregulated proliferation and tumor progression. Ion channels seem to influence cell proliferation; however, the crucial molecular mechanisms involved remain elusive. Some results show how extracellular mitogenic signals modulate ion channel activity through intracellular secondary messengers. It is relevant because we are beginning to understand how potassium channels can affect the proliferative capacity of cells, either in normal mitogen-dependent proliferation or in mitogen-unresponsive proliferation. Calciumdependent potassium channels have been implicated in cell cycle signaling in many cancerous cell lines. In particular, the so-called intermediate conductance KCa3.1 (IKCa) is reported to play a significant role in uncontrolled cell cycle signaling, among other malignant processes driven by cancer hallmarks. In addition to these features, this channel can be subjected to specific pharmacological regulation, making it a promising cornerstone for understanding the signaling behavior of several types of cancer and as a target for chemotherapeutic approaches. This review is dedicated to the connection of KCa3.1 activity, in canonical and non-canonical ways, to the cell cycle signaling, including the cooperation with calcium channels to generate calcium signals and its role as a mediator of proliferative signals.


Assuntos
Canais de Potássio Ativados por Cálcio de Condutância Intermediária , Neoplasias , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/genética , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Mitógenos , Proliferação de Células , Canais Iônicos
4.
Biochem Biophys Res Commun ; 733: 150434, 2024 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-39068818

RESUMO

Ca2+-activated KCa3.1 channels are known to contribute to slow afterhyperpolarization in pyramidal neurons of several brain areas, while Ca2+-permeable AMPA receptors (CP-AMPARs) may provide a subthreshold source of Ca2+ elevation in the cytoplasm. The functionality of these two types of channels has also been shown to be altered by epileptic disorders. However, the link between KCa3.1 channels and CP-AMPARs is poorly understood, and their potential interaction in epilepsy remains unclear. Here, we address this issue by overexpressing the KCNN4 gene, which encodes the KCa3.1 channel, using patch clamp, imaging, and channel blockers in an in vitro model of epilepsy in neuronal culture. We show that KCNN4 overexpression causes strong hyperpolarization and substantial silencing of neurons during epileptiform activity events, which also prevents KCNN4-positive neurons from firing action potentials (APs) during experimentally induced status epilepticus. Intracellular blocker application experiments showed that the amplitude of hyperpolarization was strongly dependent on CP-AMPARs, but not on NMDA receptors. Taken together, our data strongly suggest that subthreshold Ca2+ elevation produced by CP-AMPARs can trigger KCa3.1 channels to hyperpolarize neurons and protect them from seizures.


Assuntos
Cálcio , Canais de Potássio Ativados por Cálcio de Condutância Intermediária , Neurônios , Receptores de AMPA , Animais , Potenciais de Ação , Cálcio/metabolismo , Células Cultivadas , Epilepsia/metabolismo , Epilepsia/genética , Epilepsia/fisiopatologia , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/genética , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/antagonistas & inibidores , Neurônios/metabolismo , Receptores de AMPA/metabolismo , Receptores de AMPA/genética
5.
Arch Biochem Biophys ; 759: 110105, 2024 09.
Artigo em Inglês | MEDLINE | ID: mdl-39059600

RESUMO

OBJECTIVES: Potassium channels in the endoplasmic reticulum (ER) are crucial for maintaining calcium balance during calcium fluxes. Disruption in ER calcium balance leads to ER stress, implicated in diseases like diabetes and Alzheimer's disease (AD). However, limited data exists on ER potassium channels in excitable tissues such as the brain. To fill this gap, we aimed to evaluate potassium currents in rat brain rough endoplasmic reticulum (RER). METHODS: Rats were euthanized under deep anesthesia and their brains were immediately removed. The brains were then homogenized in ice-cold sucrose buffer, followed by the extraction of RER microsomes through a series of centrifugation processes. Purity of sample was evaluated using western blotting technique. Single channel recordings were done in voltage steps from +50 to -60 mV following incorporation of rat brain RER vesicles into planar bilayers. RESULTS: We observed a voltage-dependent potassium channel with an approximate conductance of 188 pS. Channel open probability was low at negative voltages, increasing at positive voltages. The channel was blocked by Charybdotoxin but not by Iberiotoxin. Additionally, TRAM-34, a specific KCa3.1 channel blocker, suppressed channel current amplitude and open probability. Western blot analysis revealed specific bands for anti-KCa3.1 antibody, approximately 50 kDa in brain homogenate and RER fraction. CONCLUSION: Our study provides strong evidence for the presence of an KCa3.1 channel on the RER membrane in rat brain, exhibiting distinct electro-pharmacological profile compared to plasma membrane and other organelles.


Assuntos
Encéfalo , Animais , Ratos , Encéfalo/metabolismo , Masculino , Ratos Wistar , Canais de Potássio Cálcio-Ativados/metabolismo , Retículo Endoplasmático/metabolismo
6.
Int J Mol Sci ; 25(10)2024 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-38791278

RESUMO

Recent advancements in understanding the intricate molecular mechanisms underlying immunological responses have underscored the critical involvement of ion channels in regulating calcium influx, particularly in inflammation. Nootkatone, a natural sesquiterpenoid found in Alpinia oxyphylla and various citrus species, has gained attention for its diverse pharmacological properties, including anti-inflammatory effects. This study aimed to elucidate the potential of nootkatone in modulating ion channels associated with calcium signaling, particularly CRAC, KV1.3, and KCa3.1 channels, which play pivotal roles in immune cell activation and proliferation. Using electrophysiological techniques, we demonstrated the inhibitory effects of nootkatone on CRAC, KV1.3, and KCa3.1 channels in HEK293T cells overexpressing respective channel proteins. Nootkatone exhibited dose-dependent inhibition of channel currents, with IC50 values determined for each channel. Nootkatone treatment did not significantly affect cell viability, indicating its potential safety for therapeutic applications. Furthermore, we observed that nootkatone treatment attenuated calcium influx through activated CRAC channels and showed anti-proliferative effects, suggesting its role in regulating inflammatory T cell activation. These findings highlight the potential of nootkatone as a natural compound for modulating calcium signaling pathways by targeting related key ion channels and it holds promise as a novel therapeutic agent for inflammatory disorders.


Assuntos
Sinalização do Cálcio , Canais de Potássio Ativados por Cálcio de Condutância Intermediária , Sesquiterpenos Policíclicos , Linfócitos T , Humanos , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Linfócitos T/imunologia , Sesquiterpenos Policíclicos/farmacologia , Células HEK293 , Sinalização do Cálcio/efeitos dos fármacos , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Proliferação de Células/efeitos dos fármacos , Canais de Cálcio Ativados pela Liberação de Cálcio/metabolismo , Cálcio/metabolismo , Canal de Potássio Kv1.3/metabolismo , Canal de Potássio Kv1.3/antagonistas & inibidores , Sobrevivência Celular/efeitos dos fármacos , Ativação Linfocitária/efeitos dos fármacos , Sesquiterpenos/farmacologia
7.
J Neurosci ; 42(30): 5843-5859, 2022 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-35732494

RESUMO

Temporal lobe epilepsy (TLE), the most common focal seizure disorder in adults, can be instigated in experimental animals by convulsant-induced status epilepticus (SE). Principal hippocampal neurons from SE-experienced epileptic male rats (post-SE neurons) display markedly augmented spike output compared with neurons from nonepileptic animals (non-SE neurons). This enhanced firing results from a cAMP-dependent protein kinase A-mediated inhibition of KCa3.1, a subclass of Ca2+-gated K+ channels generating the slow afterhyperpolarizing Ca2+-gated K+ current (IsAHP). The inhibition of KCa3.1 in post-SE neurons leads to a marked reduction in amplitude of the IsAHP that evolves during repetitive firing, as well as in amplitude of the associated Ca2+-dependent component of the slow afterhyperpolarization potential (KCa-sAHP). Here we show that KCa3.1 inhibition in post-SE neurons is induced by corticotropin releasing factor (CRF) through its Type 1 receptor (CRF1R). Acute application of CRF1R antagonists restores KCa3.1 activity in post-SE neurons, normalizing KCa-sAHP/IsAHP amplitudes and neuronal spike output, without affecting these variables in non-SE neurons. Moreover, pharmacological antagonism of CRF1Rs in vivo reduces the frequency of spontaneous recurrent seizures in post-SE chronically epileptic rats. These findings may provide a new vista for treating TLE.SIGNIFICANCE STATEMENT Epilepsy, a common neurologic disorder, often develops following a brain insult. Identifying key cellular mechanisms underlying acquired epilepsy is critical for developing effective antiepileptic therapies. In an experimental model of acquired epilepsy, principal hippocampal neurons manifest hyperexcitability because of downregulation of KCa3.1, a subtype of Ca2+-gated K+ ion channels. We show that KCa3.1 downregulation is mediated by corticotropin releasing factor (CRF) acting through its Type 1 receptor (CRF1R). Congruently, acute application of selective CRF1R antagonists restores KCa3.1 channel activity, leading to normalization of neuronal excitability. In the same model, injection of a CRF1R antagonist to epileptic animals markedly decreases the frequency of electrographic seizures. Therefore, targeting CRF1Rs may provide a new strategy in the treatment of acquired epilepsy.


Assuntos
Hormônio Liberador da Corticotropina , Epilepsia do Lobo Temporal , Epilepsia , Canais de Potássio Ativados por Cálcio de Condutância Intermediária , Estado Epiléptico , Animais , Hormônio Liberador da Corticotropina/metabolismo , Modelos Animais de Doenças , Epilepsia/tratamento farmacológico , Epilepsia/metabolismo , Epilepsia do Lobo Temporal/metabolismo , Hipocampo/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Masculino , Neurônios/fisiologia , Ratos , Estado Epiléptico/metabolismo
8.
Am J Physiol Cell Physiol ; 324(6): C1249-C1262, 2023 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-37125772

RESUMO

Control of the movement of ions and water across epithelia is essential for homeostasis. Changing the number or activity of ion channels at the plasma membrane is a significant regulator of epithelial transport. In polarized epithelia, the intermediate-conductance calcium-activated potassium channel, KCa3.1 is delivered to the basolateral membrane where it generates and maintains the electrochemical gradients required for epithelial transport. The mechanisms that control the delivery of KCa3.1 to the basolateral membrane are still emerging. Herein, we investigated the role of the highly conserved tethering complex exocyst. In epithelia, exocyst is involved in the tethering of post-Golgi secretory vesicles with the basolateral membrane, which is required before membrane fusion. In our Fisher rat thyroid cell line that stably expresses KCa3.1, siRNA knockdown of either of the exocyst subunits Sec3, Sec6, or Sec8 significantly decreased KCa3.1-specific current. In addition, knockdown of exocyst complex subunits significantly reduced the basolateral membrane protein level of KCa3.1. Finally, co-immunoprecipitation experiments suggest associations between Sec6 and KCa3.1, but not between Sec8 and KCa3.1. Collectively, based on these data and our previous studies, we suggest that components of exocyst complex are crucially important in the tethering of KCa3.1 to the basolateral membrane. After which, Soluble N-ethylmaleimide-sensitive factor (SNF) Attachment Receptors (SNARE) proteins aid in the insertion of KCa3.1-containing vesicles into the basolateral membrane of polarized epithelia.NEW & NOTEWORTHY Our Ussing chamber and immunoblot experiments demonstrate that when subunits of the exocyst complex were transiently knocked down, this significantly reduced the basolateral population and functional expression of KCa3.1. These data suggest, combined with our protein association experiments, that the exocyst complex regulates the tethering of KCa3.1-containing vesicles to the basolateral membrane prior to the SNARE-dependent insertion of channels into the basolateral membrane of epithelial cells.


Assuntos
Células Epiteliais , Fusão de Membrana , Ratos , Animais , Membrana Celular/metabolismo , Epitélio , Células Epiteliais/metabolismo , Proteínas SNARE/genética , Proteínas SNARE/metabolismo
9.
Annu Rev Pharmacol Toxicol ; 60: 219-240, 2020 01 06.
Artigo em Inglês | MEDLINE | ID: mdl-31337271

RESUMO

The three small-conductance calcium-activated potassium (KCa2) channels and the related intermediate-conductance KCa3.1 channel are voltage-independent K+ channels that mediate calcium-induced membrane hyperpolarization. When intracellular calcium increases in the channel vicinity, it calcifies the flexible N lobe of the channel-bound calmodulin, which then swings over to the S4-S5 linker and opens the channel. KCa2 and KCa3.1 channels are highly druggable and offer multiple binding sites for venom peptides and small-molecule blockers as well as for positive- and negative-gating modulators. In this review, we briefly summarize the physiological role of KCa channels and then discuss the pharmacophores and the mechanism of action of the most commonly used peptidic and small-molecule KCa2 and KCa3.1 modulators. Finally, we describe the progress that has been made in advancing KCa3.1 blockers and KCa2.2 negative- and positive-gating modulators toward the clinic for neurological and cardiovascular diseases and discuss the remaining challenges.


Assuntos
Canais de Potássio Ativados por Cálcio de Condutância Intermediária/efeitos dos fármacos , Canais de Potássio Ativados por Cálcio de Condutância Baixa/efeitos dos fármacos , Animais , Sítios de Ligação , Doenças Cardiovasculares/tratamento farmacológico , Doenças Cardiovasculares/fisiopatologia , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Doenças do Sistema Nervoso/tratamento farmacológico , Doenças do Sistema Nervoso/fisiopatologia , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio Ativados por Cálcio de Condutância Baixa/metabolismo
10.
Biol Chem ; 404(4): 339-353, 2023 03 28.
Artigo em Inglês | MEDLINE | ID: mdl-36571487

RESUMO

Ion channels play an important role for regulation of the exocrine and the endocrine pancreas. This review focuses on the Ca2+-regulated K+ channel KCa3.1, encoded by the KCNN4 gene, which is present in both parts of the pancreas. In the islets of Langerhans, KCa3.1 channels are involved in the regulation of membrane potential oscillations characterizing nutrient-stimulated islet activity. Channel upregulation is induced by gluco- or lipotoxic conditions and might contribute to micro-inflammation and impaired insulin release in type 2 diabetes mellitus as well as to diabetes-associated renal and vascular complications. In the exocrine pancreas KCa3.1 channels are expressed in acinar and ductal cells. They are thought to play a role for anion secretion during digestion but their physiological role has not been fully elucidated yet. Pancreatic carcinoma, especially pancreatic ductal adenocarcinoma (PDAC), is associated with drastic overexpression of KCa3.1. For pharmacological targeting of KCa3.1 channels, we are discussing the possible benefits KCa3.1 channel inhibitors might provide in the context of diabetes mellitus and pancreatic cancer, respectively. We are also giving a perspective for the use of a fluorescently labeled derivative of the KCa3.1 blocker senicapoc as a tool to monitor channel distribution in pancreatic tissue. In summary, modulating KCa3.1 channel activity is a useful strategy for exo-and endocrine pancreatic disease but further studies are needed to evaluate its clinical suitability.


Assuntos
Diabetes Mellitus Tipo 2 , Ilhotas Pancreáticas , Neoplasias Pancreáticas , Humanos , Pâncreas , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas
11.
Acta Pharmacol Sin ; 44(2): 259-267, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-35715699

RESUMO

Small- and intermediate-conductance Ca2+-activated K+ (KCa2.x/KCa3.1 also called SK/IK) channels are gated exclusively by intracellular Ca2+. The Ca2+ binding protein calmodulin confers sub-micromolar Ca2+ sensitivity to the channel-calmodulin complex. The calmodulin C-lobe is constitutively associated with the proximal C-terminus of the channel. Interactions between calmodulin N-lobe and the channel S4-S5 linker are Ca2+-dependent, which subsequently trigger conformational changes in the channel pore and open the gate. KCNN genes encode four subtypes, including KCNN1 for KCa2.1 (SK1), KCNN2 for KCa2.2 (SK2), KCNN3 for KCa2.3 (SK3), and KCNN4 for KCa3.1 (IK). The three KCa2.x channel subtypes are expressed in the central nervous system and the heart. The KCa3.1 subtype is expressed in the erythrocytes and the lymphocytes, among other peripheral tissues. The impact of dysfunctional KCa2.x/KCa3.1 channels on human health has not been well documented. Human loss-of-function KCa2.2 mutations have been linked with neurodevelopmental disorders. Human gain-of-function mutations that increase the apparent Ca2+ sensitivity of KCa2.3 and KCa3.1 channels have been associated with Zimmermann-Laband syndrome and hereditary xerocytosis, respectively. This review article discusses the physiological significance of KCa2.x/KCa3.1 channels, the pathophysiology of the diseases linked with KCa2.x/KCa3.1 mutations, the structure-function relationship of the mutant KCa2.x/KCa3.1 channels, and potential pharmacological therapeutics for the KCa2.x/KCa3.1 channelopathy.


Assuntos
Canalopatias , Canais de Potássio Ativados por Cálcio de Condutância Baixa , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Baixa/genética , Canais de Potássio Ativados por Cálcio de Condutância Baixa/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/genética , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Calmodulina/genética , Calmodulina/metabolismo , Mutação
12.
Int J Mol Sci ; 24(22)2023 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-38003471

RESUMO

Many studies highlighted the importance of the IK channel for the proliferation and the migration of different types of cancer cells, showing how IK blockers could slow down cancer growth. Based on these data, we wanted to characterize the effects of IK blockers on melanoma metastatic cells and to understand if such effects were exclusively IK-dependent. For this purpose, we employed two different blockers, namely clotrimazole and senicapoc, and two cell lines: metastatic melanoma WM266-4 and pancreatic cancer Panc-1, which is reported to have little or no IK expression. Clotrimazole and senicapoc induced a decrease in viability and the migration of both WM266-4 and Panc-1 cells irrespective of IK expression levels. Patch-clamp experiments on WM266-4 cells revealed Ca2+-dependent, IK-like, clotrimazole- and senicapoc-sensitive currents, which could not be detected in Panc-1 cells. Neither clotrimazole nor senicapoc altered the intracellular Ca2+ concentration. These results suggest that the effects of IK blockers on cancer cells are not strictly dependent on a robust presence of the channel in the plasma membrane, but they might be due to off-target effects on other cellular targets or to the blockade of IK channels localized in intracellular organelles.


Assuntos
Clotrimazol , Melanoma , Humanos , Clotrimazol/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Acetamidas
13.
Molecules ; 28(2)2023 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-36677942

RESUMO

The Ca2+ ion is used ubiquitously as an intracellular signaling molecule due to its high external and low internal concentration. Many Ca2+-sensing ion channel proteins have evolved to receive and propagate Ca2+ signals. Among them are the Ca2+-activated potassium channels, a large family of potassium channels activated by rises in cytosolic calcium in response to Ca2+ influx via Ca2+-permeable channels that open during the action potential or Ca2+ release from the endoplasmic reticulum. The Ca2+ sensitivity of these channels allows internal Ca2+ to regulate the electrical activity of the cell membrane. Activating these potassium channels controls many physiological processes, from the firing properties of neurons to the control of transmitter release. This review will discuss what is understood about the Ca2+ sensitivity of the two best-studied groups of Ca2+-sensitive potassium channels: large-conductance Ca2+-activated K+ channels, KCa1.1, and small/intermediate-conductance Ca2+-activated K+ channels, KCa2.x/KCa3.1.


Assuntos
Canais de Potássio Ativados por Cálcio de Condutância Intermediária , Canais de Potássio , Canais de Potássio/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Baixa/metabolismo , Membrana Celular/metabolismo , Potenciais da Membrana/fisiologia , Cálcio/metabolismo , Potássio/metabolismo
14.
Am J Physiol Cell Physiol ; 323(4): C1215-C1230, 2022 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-36062876

RESUMO

We previously identified potentiators of KCa3.1 (5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one; DCEBIO) that stimulate Cl- secretion across human bronchial epithelial cells (HBEs) expressing wild-type (WT) cystic fibrosis transmembrane conductance regulator (CFTR). However, these compounds failed to stimulate Cl- secretion in F508del CFTR HBEs. Drug discovery efforts identified CFTR potentiators (VX-770) and correctors (VX-445, VX-661) for cystic fibrosis (CF) disease-causing mutations, including F508del and G551D. Herein, we evaluated the effect of KCa3.1 potentiation on Cl- equivalent current (ICl) across primary HBEs expressing WT, F508del, and G551D CFTR. Transepithelial impedance analysis was used to obtain estimates of apical (Ra) and basolateral membrane (BLM; Rb) resistances. In WT CFTR HBEs, DCEBIO stimulated ICl, which was increased by forskolin. Similarly, forskolin stimulated ICl, and this was increased by DCEBIO. The KCa3.1 blocker, TRAM-34 inhibited ICl. DCEBIO decreased Rb, whereas TRAM-34 increased Rb, consistent with BLM localization of KCa3.1. Following correction of F508del CFTR with VX-445 + VX-661, DCEBIO failed to stimulate ICl, although the subsequent addition of forskolin + VX-770 increased ICl. Importantly, following stimulation of ICl with forskolin + VX-770, DCEBIO induced a further significant increase in ICl. As above, DCEBIO reduced Rb, whereas TRAM-34 increased Rb, consistent with BLM localized KCa3.1. Finally, we assessed KCa3.1 potentiation on ICl in G551D/F508del CFTR HBEs in the absence or presence of VX-445 + VX-661. In both cases, DCEBIO failed to stimulate ICl. However, following stimulation with forskolin + VX-770, DCEBIO nearly doubled ICl. Our results demonstrate that following correction/potentiation of F508del and G551D CFTR, potentiation of KCa3.1 increases the Cl- secretory response, suggesting this class of compounds may represent a novel means of further increasing Cl- secretion across CF airway.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística , Fibrose Cística , Aminofenóis/farmacologia , Colforsina/farmacologia , Fibrose Cística/tratamento farmacológico , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Células Epiteliais , Humanos , Quinolonas
15.
Am J Physiol Cell Physiol ; 323(3): C694-C705, 2022 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-35848620

RESUMO

Red cell volume is a major determinant of HbS concentration in sickle cell disease. Cellular deoxy-HbS concentration determines the delay time, the interval between HbS deoxygenation and deoxy-HbS polymerization. Major membrane transporter protein determinants of sickle red cell volume include the SLC12/KCC K-Cl cotransporters KCC3/SLC12A6 and KCC1/SLC12A4, and the KCNN4/KCa3.1 Ca2+-activated K+ channel (Gardos channel). Among standard inhibitors of KCC-mediated K-Cl cotransport, only [(dihydroindenyl)oxy]acetic acid (DIOA) has been reported to lack inhibitory activity against the related bumetanide-sensitive erythroid Na-K-2Cl cotransporter NKCC1/SLC12A2. DIOA has been often used to inhibit K-Cl cotransport when studying the expression and regulation of other K+ transporters and K+ channels. We report here that DIOA at concentrations routinely used to inhibit K-Cl cotransport can also abrogate activity of the KCNN4/KCa3.1 Gardos channel in human and mouse red cells and in human sickle red cells. DIOA inhibition of A23187-stimulated erythroid K+ uptake (Gardos channel activity) was chloride-independent and persisted in mouse red cells genetically devoid of the principal K-Cl cotransporters KCC3 and KCC1. DIOA also inhibited YODA1-stimulated, chloride-independent erythroid K+ uptake. In contrast, DIOA exhibited no inhibitory effect on K+ influx into A23187-treated red cells of Kcnn4-/- mice. DIOA inhibition of human KCa3.1 was validated (IC50 42 µM) by whole cell patch clamp in HEK-293 cells. RosettaLigand docking experiments identified a potential binding site for DIOA in the fenestration region of human KCa3.1. We conclude that DIOA at concentrations routinely used to inhibit K-Cl cotransport can also block the KCNN4/KCa3.1 Gardos channel in normal and sickle red cells.


Assuntos
Anemia Falciforme , Simportadores , Ácido Acético , Anemia Falciforme/tratamento farmacológico , Animais , Calcimicina , Cloretos/metabolismo , Células HEK293 , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/genética , Camundongos , Potássio/metabolismo , Membro 2 da Família 12 de Carreador de Soluto , Simportadores/metabolismo
16.
Physiol Genomics ; 54(7): 273-282, 2022 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-35658672

RESUMO

Ion channels are potentially exploitable as pharmacological targets to treat asthma. This study evaluated the role of KCa3.1 channels, encoded by Kcnn4, in regulating the gene expression of mouse airway epithelium and the development of asthma traits. We used the ovalbumin (OVA) challenge as an asthma model in wild-type and Kcnn4-/- mice, performed histological analysis, and measured serum IgE to evaluate asthma traits. We analyzed gene expression of isolated epithelial cells of trachea or bronchi using mRNA sequencing and gene ontology and performed Ussing chamber experiments in mouse trachea to evaluate anion secretion. Gene expression of epithelial cells from mouse airways differed between trachea and bronchi, indicating regional differences in the inflammatory and transepithelial transport properties of proximal and distal airways. We found that Kcnn4 silencing reduced mast cell numbers, mucus, and collagen in the airways, and reduced the amount of epithelial anion secretion in the OVA-challenged animals. In addition, gene expression was differentially modified in the trachea and bronchi, with Kcnn4 genetic silencing significantly altering the expression of genes involved in the TNF pathway, supporting the potential of KCa3.1 as a therapeutic target for asthma.


Assuntos
Asma , Traqueia , Animais , Asma/genética , Asma/metabolismo , Asma/patologia , Brônquios/metabolismo , Modelos Animais de Doenças , Expressão Gênica , Camundongos , Camundongos Endogâmicos BALB C , Ovalbumina/metabolismo , Traqueia/metabolismo , Traqueia/patologia
17.
Pflugers Arch ; 474(11): 1147-1157, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36152073

RESUMO

Lung cancer is one of the leading causes of cancer-related deaths worldwide. The Ca2+-activated K+ channel KCa3.1 contributes to the progression of non-small cell lung cancer (NSCLC). Recently, KCa3.1 channels were found in the inner membrane of mitochondria in different cancer cells. Mitochondria are the main sources for the generation of reactive oxygen species (ROS) that affect the progression of cancer cells. Here, we combined Western blotting, immunofluorescence, and fluorescent live-cell imaging to investigate the expression and function of KCa3.1 channels in the mitochondria of NSCLC cells. Western blotting revealed KCa3.1 expression in mitochondrial lysates from different NSCLC cells. Using immunofluorescence, we demonstrate a co-localization of KCa3.1 channels with mitochondria of NSCLC cells. Measurements of the mitochondrial membrane potential with TMRM reveal a hyperpolarization following the inhibition of KCa3.1 channels with the cell-permeable blocker senicapoc. This is not the case when cells are treated with the cell-impermeable peptidic toxin maurotoxin. The hyperpolarization of the mitochondrial membrane potential is accompanied by an increased generation of ROS in NSCLC cells. Collectively, our results provide firm evidence for the functional expression of KCa3.1 channels in the inner membrane of mitochondria of NSCLC cells.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo
18.
Arch Pharm (Weinheim) ; 355(12): e2200388, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36161669

RESUMO

The calcium-activated potassium channel 3.1 (KCa 3.1) is overexpressed in many tumor entities and has predictive power concerning disease progression and outcome. Imaging of the KCa 3.1 channel in vivo using a radiotracer for positron emission tomography (PET) could therefore establish a potentially powerful diagnostic tool. Senicapoc shows high affinity and excellent selectivity toward the KCa 3.1 channel. We have successfully pursued the synthesis of the 18 F-labeled derivative [18 F]3 of senicapoc using the prosthetic group approach with 1-azido-2-[18 F]fluoroethane ([18 F]6) in a "click" reaction. The biological activity of the new PET tracer was evaluated in vitro and in vivo. Inhibition of the KCa 3.1 channel by 3 was demonstrated by patch clamp experiments and the binding pose was analyzed by docking studies. In mouse and human serum, [18 F]3 was stable for at least one half-life of [18 F]fluorine. Biodistribution experiments in wild-type mice were promising, showing rapid and predominantly renal excretion. An in vivo study using A549-based tumor-bearing mice was performed. The tumor signal could be delineated and image analysis showed a tumor-to-muscle ratio of 1.47 ± 0.24. The approach using 1-azido-2-[18 F]fluoroethane seems to be a good general strategy to achieve triarylacetamide-based fluorinated PET tracers for imaging of the KCa 3.1 channel in vivo.


Assuntos
Neoplasias , Canais de Potássio Cálcio-Ativados , Animais , Humanos , Camundongos , Radioisótopos de Flúor/metabolismo , Compostos Radiofarmacêuticos/farmacologia , Compostos Radiofarmacêuticos/metabolismo , Distribuição Tecidual , Canais de Potássio Cálcio-Ativados/metabolismo , Relação Estrutura-Atividade , Tomografia por Emissão de Pósitrons/métodos , Neoplasias/metabolismo
19.
Int J Mol Sci ; 23(15)2022 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-35955737

RESUMO

THP-1-differentiated macrophages are useful for investigating the physiological significance of tumor-associated macrophages (TAMs). In the tumor microenvironment (TME), TAMs with the M2-like phenotype play a critical role in promoting cancer progression and metastasis by inhibiting the immune surveillance system. We examined the involvement of Ca2+-activated K+ channel KCa3.1 in TAMs in expressing pro-tumorigenic cytokines and angiogenic growth factors. In THP-1-derived M2 macrophages, the expression levels of IL-8 and IL-10 were significantly decreased by treatment with the selective KCa3.1 activator, SKA-121, without changes in those of VEGF and TGF-ß1. Furthermore, under in vitro experimental conditions that mimic extracellular K+ levels in the TME, IL-8 and IL-10 levels were both significantly elevated, and these increases were reversed by combined treatment with SKA-121. Among several signaling pathways potentially involved in the transcriptional regulation of IL-8 and IL-10, respective treatments with ERK and JNK inhibitors significantly repressed their transcriptions, and treatment with SKA-121 significantly reduced the phosphorylated ERK, JNK, c-Jun, and CREB levels. These results strongly suggest that the KCa3.1 activator may suppress IL-10-induced tumor immune surveillance escape and IL-8-induced tumorigenicity and metastasis by inhibiting their production from TAMs through ERK-CREB and JNK-c-Jun cascades.


Assuntos
Interleucina-10 , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Neoplasias , Regulação para Baixo , Humanos , Interleucina-10/genética , Interleucina-10/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Macrófagos/metabolismo , Neoplasias/metabolismo , Microambiente Tumoral
20.
Cell Physiol Biochem ; 55(S3): 131-144, 2021 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-34043300

RESUMO

The Kca3.1 channels, previously designated as IK1 or SK4 channels and encoded by the KCNN4 gene, are activated by a rise of the intracellular Ca2+ concentration. These K+ channels are widely expressed in many organs and involved in many pathologies. In particular, Kca3.1 channels have been studied intensively in the context of cancer. They are not only a marker and a valid prognostic tool for cancer patients, but have an important share in driving cancer progression. Their function is required for many characteristic features of the aggressive cancer cell behavior such as migration, invasion and metastasis as well as proliferation and therapy resistance. In the context of cancer, another property of Kca3.1 is now emerging. These channels can be a target for novel small molecule-based imaging probes, as it has been validated in case of fluorescently labeled senicapoc-derivatives. The aim of this review is (i) to give an overview on the role of Kca3.1 channels in cancer progression and in shaping the cancer microenvironment, (ii) discuss the potential of using Kca3.1 targeting drugs for cancer imaging, (iii) and highlight the possibility of combining molecular dynamics simulations to image inhibitor binding to Kca3.1 channels in order to provide a deeper understanding of Kca3.1 channel pharmacology. Alltogether, Kca3.1 is an attractive therapeutic target so that senicapoc, originally developed for the treatment of sickle cell anemia, should be repurposed for the treatment of cancer patients.


Assuntos
Acetamidas/uso terapêutico , Antineoplásicos/uso terapêutico , Cálcio/metabolismo , Neoplasias/tratamento farmacológico , Bloqueadores dos Canais de Potássio/uso terapêutico , Canais de Potássio Ativados por Cálcio de Condutância Baixa/antagonistas & inibidores , Compostos de Tritil/uso terapêutico , Antineoplásicos/química , Antidrepanocíticos/química , Antidrepanocíticos/uso terapêutico , Sítios de Ligação , Sinalização do Cálcio , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Simulação de Dinâmica Molecular , Terapia de Alvo Molecular/métodos , Metástase Neoplásica , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Bloqueadores dos Canais de Potássio/química , Estrutura Secundária de Proteína , Canais de Potássio Ativados por Cálcio de Condutância Baixa/química , Canais de Potássio Ativados por Cálcio de Condutância Baixa/genética , Canais de Potássio Ativados por Cálcio de Condutância Baixa/metabolismo , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/genética
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