Kv1.3 in the spotlight for treating immune diseases.

INTRODUCTION: Kv1.3 is the main voltage-gated potassium channel of leukocytes from both the innate and adaptive immune systems. Channel function is required for common processes such as Ca2+ signaling but also for cell-specific events. In this context, alterations in Kv1.3 are associated with multiple immune disorders. Excessive channel activity correlates with numerous autoimmune diseases, while reduced currents result in increased cancer prevalence and immunodeficiencies. AREAS COVERED: This review offers a general view of the role of Kv1.3 in every type of leukocyte. Moreover, diseases stemming from dysregulations of the channel are detailed, as well as current advances in their therapeutic research. EXPERT OPINION: Kv1.3 arises as a potential immune target in a variety of diseases. Several lines of research focused on channel modulation have yielded positive results. However, among the great variety of specific channel blockers, only one has reached clinical trials. Future investigations should focus on developing simpler administration routes for channel inhibitors to facilitate their entrance into clinical trials. Prospective Kv1.3-based treatments will ensure powerful therapies while minimizing undesired side effects.

A partial human LCK defect causes a T cell immunodeficiency with intestinal inflammation.

Lymphocyte-specific protein tyrosine kinase (LCK) is essential for T cell antigen receptor (TCR)-mediated signal transduction. Here, we report two siblings homozygous for a novel LCK variant (c.1318C>T; P440S) characterized by T cell lymphopenia with skewed memory phenotype, infant-onset recurrent infections, failure to thrive, and protracted diarrhea. The patients' T cells show residual TCR signal transduction and proliferation following anti-CD3/CD28 and phytohemagglutinin (PHA) stimulation. We demonstrate in mouse models that complete (Lck-/-) versus partial (LckP440S/P440S) loss-of-function LCK causes disease with differing phenotypes. While both Lck-/- and LckP440S/P440S mice exhibit arrested thymic T cell development and profound T cell lymphopenia, only LckP440S/P440S mice show residual T cell proliferation, cytokine production, and intestinal inflammation. Furthermore, the intestinal disease in the LckP440S/P440S mice is prevented by CD4+ T cell depletion or regulatory T cell transfer. These findings demonstrate that P440S LCK spares sufficient T cell function to allow the maturation of some conventional T cells but not regulatory T cells-leading to intestinal inflammation.

Dynamics and spatial organization of Kv1.3 at the immunological synapse of human CD4+ T cells.

Formation of the immunological synapse (IS) is a key event during initiation of an adaptive immune response to a specific antigen. During this process, a T cell and an antigen presenting cell form a stable contact that allows the T cell to integrate both internal and external stimuli in order to decide whether to activate. The threshold for T cell activation depends on the strength and frequency of the calcium (Ca2+) signaling induced by antigen recognition, and it must be tightly regulated to avoid undesired harm to healthy cells. Potassium (K+) channels are recruited to the IS to maintain the negative membrane potential required to sustain Ca2+ entry. However, the precise localization of K+ channels within the IS remains unknown. Here, we visualized the dynamic subsynaptic distribution of Kv1.3, the main voltage-gated potassium channel in human T cells. Upon T cell receptor engagement, Kv1.3 polarized toward the synaptic cleft and diffused throughout the F-actin rich distal compartment of the synaptic interface-an effect enhanced by CD2-CD58 corolla formation. As the synapse matured, Kv1.3 clusters were internalized at the center of the IS and released in extracellular vesicles. We propose a model in which specific distribution of Kv1.3 within the synapse indirectly regulates the channel function and that this process is limited through Kv1.3 internalization and release in extracellular vesicles.

Mapping autophagosome contents identifies interleukin-7 receptor-α as a key cargo modulating CD4+ T cell proliferation.

CD4+ T cells are pivotal cells playing roles in the orchestration of humoral and cytotoxic immune responses. It is known that CD4+ T cell proliferation relies on autophagy, but identification of the autophagosomal cargo involved is missing. Here we create a transgenic mouse model, to enable direct mapping of the proteinaceous content of autophagosomes in primary cells by LC3 proximity labelling. Interleukin-7 receptor-α, a cytokine receptor mostly found in naïve and memory T cells, is reproducibly detected in autophagosomes of activated CD4+ T cells. Consistently, CD4+ T cells lacking autophagy show increased interleukin-7 receptor-α surface expression, while no defect in internalisation is observed. Mechanistically, excessive surface interleukin-7 receptor-α sequestrates the common gamma chain, impairing the interleukin-2 receptor assembly and downstream signalling crucial for T cell proliferation. This study shows that key autophagy substrates can be reliably identified in this mouse model and help mechanistically unravel autophagy's contribution to healthy physiology and disease.

Palmitoylation of Voltage-Gated Ion Channels.

Protein lipidation is one of the most common forms of posttranslational modification. This alteration couples different lipids, such as fatty acids, phospho- and glycolipids and sterols, to cellular proteins. Lipidation regulates different aspects of the protein's physiology, including structure, stability and affinity for cellular membranes and protein-protein interactions. In this scenario, palmitoylation is the addition of long saturated fatty acid chains to amino acid residues of the proteins. The enzymes responsible for this modification are acyltransferases and thioesterases, which control the protein's behavior by performing a series of acylation and deacylation cycles. These enzymes target a broad repertoire of substrates, including ion channels. Thus, protein palmitoylation exhibits a pleiotropic role by differential modulation of the trafficking, spatial organization and electrophysiological properties of ion channels. Considering voltage-gated ion channels (VGICs), dysregulation of lipidation of both the channels and the associated ancillary subunits correlates with the development of various diseases, such as cancer or mental disorders. Therefore, a major role for protein palmitoylation is currently emerging, affecting not only the dynamism and differential regulation of a moiety of cellular proteins but also linking to human health. Therefore, palmitoylation of VGIC, as well as related enzymes, constitutes a novel pharmacological tool for drug development to target related pathologies.

The Mitochondrial Routing of the Kv1.3 Channel.

Voltage-gated potassium channels control neuronal excitability and cardiac action potentials. In addition, these proteins are involved in a myriad of cellular processes. The potassium channel Kv1.3 plays an essential role in the immune response mediated by leukocytes. Kv1.3 is functional both at the plasma membrane and the inner mitochondrial membrane. Plasma membrane Kv1.3 mediates cellular activation and proliferation, whereas mitochondrial Kv1.3 participates in cell survival and apoptosis. Therefore, this protein emerges as an important target in cancer therapies. Several forward-traffic motifs target the channel to the plasma membrane in a COPII-dependent manner. However, the mitochondrial import pathway for Kv1.3 is largely unknown. Here, we deciphered the mitochondrial routing of the mitoKv1.3 channel. Kv1.3 uses the TIM23 complex to translocate to the inner mitochondrial membrane. This mechanism is unconventional because the channel is a multimembrane spanning protein without a defined N-terminal presequence. We found that transmembrane domains cooperatively mediate Kv1.3 mitochondrial targeting and identified the cytosolic HSP70/HSP90 chaperone complex as a key regulator of the process. Our results provide insights into the mechanisms mediating the localization of Kv1.3 to mitochondrial membranes, further extending the knowledge of ion channel biogenesis and turnover in mitochondria.

S-acylation-dependent membrane microdomain localization of the regulatory Kvß2.1 subunit.

The voltage-dependent potassium (Kv) channel Kvß family was the first identified group of modulators of Kv channels. Kvß regulation of the α-subunits, in addition to their aldoketoreductase activity, has been under extensive study. However, scarce information about their specific α-subunit-independent biology is available. The expression of Kvßs is ubiquitous and, similar to Kv channels, is tightly regulated in leukocytes. Although Kvß subunits exhibit cytosolic distribution, spatial localization, in close contact with plasma membrane Kv channels, is crucial for a proper immune response. Therefore, Kvß2.1 is located near cell surface Kv1.3 channels within the immunological synapse during lymphocyte activation. The objective of this study was to analyze the structural elements that participate in the cellular distribution of Kvßs. It was demonstrated that Kvß peptides, in addition to the cytoplasmic pattern, targeted the cell surface in the absence of Kv channels. Furthermore, Kvß2.1, but not Kvß1.1, targeted lipid raft microdomains in an S-acylation-dependent manner, which was concomitant with peptide localization within the immunological synapse. A pair of C-terminal cysteines (C301/C311) was mostly responsible for the specific palmitoylation of Kvß2.1. Several insults altered Kvß2.1 membrane localization. Therefore, growth factor-dependent proliferation enhanced surface targeting, whereas PKC activation impaired lipid raft expression. However, PSD95 stabilized Kvß2.1 in these domains. This data shed light on the molecular mechanism by which Kvß2.1 clusters into immunological synapses during leukocyte activation.

Kv1.3 Controls Mitochondrial Dynamics during Cell Cycle Progression.

The voltage-gated potassium channel Kv1.3 is a potential therapeutic target for obesity and diabetes. The genetic ablation and pharmacological inhibition of Kv1.3 lead to a lean phenotype in rodents. The mechanism of regulation of body weight and energy homeostasis involves Kv1.3 expression in different organs, including white and brown adipose tissues. Here, we show that Kv1.3 promotes the proliferation of preadipocytes through the control of mitochondrial dynamics. Kv1.3 is expressed in mitochondria exhibiting high affinity for the perinuclear population. The mitochondrial network is highly dynamic during the cell cycle, showing continuous fusion-fission events. The formation of a hyperfused mitochondrial network at the G1/S phase of the cell cycle is dependent on Kv1.3 expression. Our results demonstrate that Kv1.3 promotes preadipocyte proliferation and differentiation by controlling mitochondrial membrane potential and mitochondrial dynamics at the G1 phase of the cell cycle.

KCNE4-dependent functional consequences of Kv1.3-related leukocyte physiology.

The voltage-dependent potassium channel Kv1.3 plays essential roles in the immune system, participating in leukocyte activation, proliferation and apoptosis. The regulatory subunit KCNE4 acts as an ancillary peptide of Kv1.3, modulates K+ currents and controls channel abundance at the cell surface. KCNE4-dependent regulation of the oligomeric complex fine-tunes the physiological role of Kv1.3. Thus, KCNE4 is crucial for Ca2+-dependent Kv1.3-related leukocyte functions. To better understand the role of KCNE4 in the regulation of the immune system, we manipulated its expression in various leukocyte cell lines. Jurkat T lymphocytes exhibit low KCNE4 levels, whereas CY15 dendritic cells, a model of professional antigen-presenting cells, robustly express KCNE4. When the cellular KCNE4 abundance was increased in T cells, the interaction between KCNE4 and Kv1.3 affected important T cell physiological features, such as channel rearrangement in the immunological synapse, cell growth, apoptosis and activation, as indicated by decreased IL-2 production. Conversely, ablation of KCNE4 in dendritic cells augmented proliferation. Furthermore, the LPS-dependent activation of CY15 cells, which induced Kv1.3 but not KCNE4, increased the Kv1.3-KCNE4 ratio and increased the expression of free Kv1.3 without KCNE4 interaction. Our results demonstrate that KCNE4 is a pivotal regulator of the Kv1.3 channelosome, which fine-tunes immune system physiology by modulating Kv1.3-associated leukocyte functions.

A novel mitochondrial Kv1.3-caveolin axis controls cell survival and apoptosis.

The voltage-gated potassium channel Kv1.3 plays an apparent dual physiological role by participating in activation and proliferation of leukocytes as well as promoting apoptosis in several types of tumor cells. Therefore, Kv1.3 is considered a potential pharmacological target for immunodeficiency and cancer. Different cellular locations of Kv1.3, at the plasma membrane or the mitochondria, could be responsible for such duality. While plasma membrane Kv1.3 facilitates proliferation, the mitochondrial channel modulates apoptotic signaling. Several molecular determinants of Kv1.3 drive the channel to the cell surface, but no information is available about its mitochondrial targeting. Caveolins, which are able to modulate cell survival, participate in the plasma membrane targeting of Kv1.3. The channel, via a caveolin-binding domain (CDB), associates with caveolin 1 (Cav1), which localizes Kv1.3 to lipid raft membrane microdomains. The aim of our study was to understand the role of such interactions not only for channel targeting but also for cell survival in mammalian cells. By using a caveolin association-deficient channel (Kv1.3 CDBless), we demonstrate here that while the Kv1.3-Cav1 interaction is responsible for the channel localization in the plasma membrane, a lack of such interaction accumulates Kv1.3 in the mitochondria. Kv1.3 CDBless severely affects mitochondrial physiology and cell survival, indicating that a functional link of Kv1.3 with Cav1 within the mitochondria modulates the pro-apoptotic effects of the channel. Therefore, the balance exerted by these two complementary mechanisms fine-tune the physiological role of Kv1.3 during cell survival or apoptosis. Our data highlight an unexpected role for the mitochondrial caveolin-Kv1.3 axis during cell survival and apoptosis.

Implication of Voltage-Gated Potassium Channels in Neoplastic Cell Proliferation.

Voltage-gated potassium channels (Kv) are the largest group of ion channels. Kv are involved in controlling the resting potential and action potential duration in the heart and brain. Additionally, these proteins participate in cell cycle progression as well as in several other important features in mammalian cell physiology, such as activation, differentiation, apoptosis, and cell volume control. Therefore, Kv remarkably participate in the cell function by balancing responses. The implication of Kv in physiological and pathophysiological cell growth is the subject of study, as Kv are proposed as therapeutic targets for tumor regression. Though it is widely accepted that Kv channels control proliferation by allowing cell cycle progression, their role is controversial. Kv expression is altered in many cancers, and their participation, as well as their use as tumor markers, is worthy of effort. There is an ever-growing list of Kv that remodel during tumorigenesis. This review focuses on the actual knowledge of Kv channel expression and their relationship with neoplastic proliferation. In this work, we provide an update of what is currently known about these proteins, thereby paving the way for a more precise understanding of the participation of Kv during cancer development.

The Potassium Channel Odyssey: Mechanisms of Traffic and Membrane Arrangement.

Ion channels are transmembrane proteins that conduct specific ions across biological membranes. Ion channels are present at the onset of many cellular processes, and their malfunction triggers severe pathologies. Potassium channels (KChs) share a highly conserved signature that is necessary to conduct K⁺ through the pore region. To be functional, KChs require an exquisite regulation of their subcellular location and abundance. A wide repertoire of signatures facilitates the proper targeting of the channel, fine-tuning the balance that determines traffic and location. These signature motifs can be part of the secondary or tertiary structure of the protein and are spread throughout the entire sequence. Furthermore, the association of the pore-forming subunits with different ancillary proteins forms functional complexes. These partners can modulate traffic and activity by adding their own signatures as well as by exposing or masking the existing ones. Post-translational modifications (PTMs) add a further dimension to traffic regulation. Therefore, the fate of a KCh is not fully dependent on a gene sequence but on the balance of many other factors regulating traffic. In this review, we assemble recent evidence contributing to our understanding of the spatial expression of KChs in mammalian cells. We compile specific signatures, PTMs, and associations that govern the destination of a functional channel.

Caveolar targeting links Kv1.3 with the insulin-dependent adipocyte physiology.

The voltage-dependent potassium channel Kv1.3 participates in peripheral insulin sensitivity. Genetic ablation of Kv1.3 triggers resistance to diet-induced weight gain, thereby pointing to this protein as a pharmacological target for obesity and associated type II diabetes. However, this role is under intense debate because Kv1.3 expression in adipose tissue raises controversy. We demonstrated that Kv1.3 is expressed in white adipose tissue from humans and rodents. Moreover, other channels, such as Kv1.1, Kv1.2, Kv1.4 and especially Kv1.5, from the same Shaker family are also present. Although elevated insulin levels and adipogenesis remodel the Kv phenotype, which could lead to multiple heteromeric complexes, Kv1.3 markedly participates in the insulin-dependent regulation of glucose uptake in mature adipocytes. Adipocyte differentiation increased the expression of Kv1.3, which is targeted to caveolae by molecular interactions with caveolin 1. Using a caveolin 1-deficient 3T3-L1 adipocyte cell line, we demonstrated that the localization of Kv1.3 in caveolar raft structures is important for proper insulin signaling. Insulin-dependent phosphorylation of the channel occurs at the onset of insulin-mediated signaling. However, when Kv1.3 was spatially outside of these lipid microdomains, impaired phosphorylation was exhibited. Our data shed light on the putative role of Kv1.3 in weight gain and insulin-dependent responses contributing to knowledge about adipocyte physiology.

Caveolin interaction governs Kv1.3 lipid raft targeting.

The spatial localization of ion channels at the cell surface is crucial for their functional role. Many channels localize in lipid raft microdomains, which are enriched in cholesterol and sphingolipids. Caveolae, specific lipid rafts which concentrate caveolins, harbor signaling molecules and their targets becoming signaling platforms crucial in cell physiology. However, the molecular mechanisms involved in such spatial localization are under debate. Kv1.3 localizes in lipid rafts and participates in the immunological response. We sought to elucidate the mechanisms of Kv1.3 surface targeting, which govern leukocyte physiology. Kv1 channels share a putative caveolin-binding domain located at the intracellular N-terminal of the channel. This motif, lying close to the S1 transmembrane segment, is situated near the T1 tetramerization domain and the determinants involved in the Kvß subunit association. The highly hydrophobic domain (FQRQVWLLF) interacts with caveolin 1 targeting Kv1.3 to caveolar rafts. However, subtle variations of this cluster, putative ancillary associations and different structural conformations can impair the caveolin recognition, thereby altering channel's spatial localization. Our results identify a caveolin-binding domain in Kv1 channels and highlight the mechanisms that govern the regulation of channel surface localization during cellular processes.

The voltage-gated potassium channel Kv1.3 is a promising multitherapeutic target against human pathologies.

INTRODUCTION: The voltage-dependent potassium channel Kv1.3 is mainly present in the nervous and immune systems. In leukocytes, Kv1.3 fine-tunes the activation and proliferation of the immune response. However, Kv1.3 is also present in other tissues where its physiological role is still under investigation. Thus, Kv1.3 alterations have been related to several human diseases. AREAS COVERED: In this work, the authors highlight the role of Kv1.3 in various pathologies and the potential use of Kv1.3 blockers as safe pharmacological tools. The limited repertoire of K(+) channels in leukocytes and its expression pattern makes Kv1.3 crucial for effector memory T cell physiology and it is therefore a good pharmacological target for chronic inflammatory diseases. Moreover, Kv1.3 has been related to insulin sensitivity, cell proliferation and apoptosis. In this scenario, Kv1.3 activity is also implicated in non-insulin-dependent type II diabetes mellitus, obesity and cancer. EXPERT OPINION: Fortunately, Kv1.3 is characterized by a very selective and potent pharmacology that has been demonstrated to ameliorate autoimmune and metabolic symptoms in disease-animal models without major side effects. Moreover, Kv1.3 blockers are showing positive results in preclinical trials. Considering this evidence, the implication of Kv1.3 in a wide repertoire of human pathologies indicates this channel is an important therapeutic target.

Involvement of potassium channels in the progression of cancer to a more malignant phenotype.

Potassium channels are a diverse group of pore-forming transmembrane proteins that selectively facilitate potassium flow through an electrochemical gradient. They participate in the control of the membrane potential and cell excitability in addition to different cell functions such as cell volume regulation, proliferation, cell migration, angiogenesis as well as apoptosis. Because these physiological processes are essential for the correct cell function, K+ channels have been associated with a growing number of diseases including cancer. In fact, different K+ channel families such as the voltage-gated K+ channels, the ether à-go-go K+ channels, the two pore domain K+ channels and the Ca2+-activated K+ channels have been associated to tumor biology. Potassium channels have a role in neoplastic cell-cycle progression and their expression has been found abnormal in many types of tumors and cancer cells. In addition, the expression and activity of specific K+ channels have shown a significant correlation with the tumor malignancy grade. The aim of this overview is to summarize published data on K+ channels that exhibit oncogenic properties and have been linked to a more malignant cancer phenotype. This article is part of a Special Issue entitled: Membrane channels and transporters in cancers.