RESUMO
Two pore channels are lysosomal cation channels with crucial roles in tumor angiogenesis and viral release from endosomes. Inhibition of the two-pore channel 2 (TPC2) has emerged as potential therapeutic strategy for the treatment of cancers and viral infections, including Ebola and COVID-19. Here, we demonstrate that antagonist SG-094, a synthetic analog of the Chinese alkaloid medicine tetrandrine with increased potency and reduced toxicity, induces asymmetrical structural changes leading to a single binding pocket at only one intersubunit interface within the asymmetrical dimer. Supported by functional characterization of mutants by Ca2+ imaging and patch clamp experiments, we identify key residues in S1 and S4 involved in compound binding to the voltage sensing domain II. SG-094 arrests IIS4 in a downward shifted state which prevents pore opening via the IIS4/S5 linker, hence resembling gating modifiers of canonical VGICs. These findings may guide the rational development of new therapeutics antagonizing TPC2 activity.
Assuntos
Canais de Cálcio , Humanos , Canais de Cálcio/metabolismo , Canais de Cálcio/química , Sítios de Ligação , Lisossomos/metabolismo , Células HEK293 , Ligação Proteica , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Bloqueadores dos Canais de Cálcio/química , Bloqueadores dos Canais de Cálcio/metabolismo , Modelos Moleculares , Canais de Dois PorosRESUMO
Infectious diseases and cancer are among the key medical challenges that humankind is facing today. A growing amount of evidence suggests that ion channels in the endolysosomal system play a crucial role in the pathology of both groups of diseases. The development of advanced patch-clamp technologies has allowed us to directly characterize ion fluxes through endolysosomal ion channels in their native environments. Endolysosomes are essential organelles for intracellular transport, digestion and metabolism, and maintenance of homeostasis. The endolysosomal ion channels regulate the function of the endolysosomal system through four basic mechanisms: calcium release, control of membrane potential, pH change, and osmolarity regulation. In this review, we put particular emphasis on the endolysosomal cation channels, including TPC2 and TRPML2, which are particularly important in monocyte function. We discuss existing endogenous and synthetic ligands of these channels and summarize current knowledge of their impact on channel activity and function in different cell types. Moreover, we summarize recent findings on the importance of TPC2 and TRPML2 channels as potential drug targets for the prevention and treatment of the emerging infectious diseases and cancer.
Assuntos
Doenças Transmissíveis/terapia , Endossomos/metabolismo , Canais Iônicos/metabolismo , Lisossomos/metabolismo , Neoplasias/terapia , Animais , Transporte Biológico , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Cátions/metabolismo , Doenças Transmissíveis/metabolismo , Homeostase , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Monócitos/metabolismo , Neoplasias/metabolismo , Medicina de Precisão/métodos , Canais de Potencial de Receptor Transitório/metabolismoRESUMO
The endo-lysosomal two-pore channel (TPC2) has been established as an intracellular cation channel of significant physiological and pathophysiological relevance in recent years. For example, TPC2-/- mice show defects in cholesterol degradation, leading to hypercholesterinemia; TPC2 absence also results in mature-onset obesity, and a role in glucagon secretion and diabetes has been proposed. Infections with bacterial toxins or viruses e.g., cholera toxin or Ebola virus result in reduced infectivity rates in the absence of TPC2 or after pharmacological blockage, and TPC2-/- cancer cells lose their ability to migrate and metastasize efficiently. Finally, melanin production is affected by changes in hTPC2 activity, resulting in pigmentation defects and hair color variation. Here, we analyzed several publicly available genome variation data sets and identified multiple variations in the TPC2 protein in distinct human populations. Surprisingly, one variation, L564P, was found to be the predominant TPC2 isoform on a global scale. By applying endo-lysosomal patch-clamp electrophysiology, we found that L564P is a prerequisite for the previously described M484L gain-of-function effect that is associated with blond hair. Additionally, other gain-of-function variants with distinct geographical and ethnic distribution were discovered and functionally characterized. A meta-analysis of genome-wide association studies was performed, finding the polymorphisms to be associated with both distinct and overlapping traits. In sum, we present the first systematic analysis of variations in TPC2. We functionally characterized the most common variations and assessed their association with various disease traits. With TPC2 emerging as a novel drug target for the treatment of various diseases, this study provides valuable insights into ethnic and geographical distribution of TPC2 polymorphisms and their effects on channel activity.
Assuntos
Canais de Cálcio/genética , Estudo de Associação Genômica Ampla , Cor de Cabelo/genética , Animais , Fibroblastos/metabolismo , Mutação com Ganho de Função/genética , Genoma Humano/genética , Humanos , Lisossomos/genética , Camundongos , Camundongos Knockout , NADP/genética , Pigmentação/genética , Polimorfismo de Nucleotídeo Único/genética , Transdução de Sinais/genéticaRESUMO
Two pore channels (TPCs) and mucolipins (TRPML) are the most prominent cation channels expressed in endolysosomes. Recently, roles of TPCs and TRPML2 have been revealed in regulating and detecting osmotically-driven changes in the surface-to-volume ratio of endolysosomes to promote endocytic and recycling traffic. TPCs and TRPML2 are highly expressed in macrophages and contribute to immune cell function. Here, we provide an overview of the emerging roles of these channels in innate immune cells, in particular macrophages, and highlight two models for osmo-mechanical regulation of intracellular organelle volume, trafficking, and cell homeostasis involving either TPCs or TRPML2.
Assuntos
Canais de Cálcio/metabolismo , Tamanho Celular , Endossomos/metabolismo , Lisossomos/metabolismo , Canais de Potencial de Receptor Transitório/metabolismo , Animais , Homeostase/imunologia , Humanos , Imunidade Inata , Macrófagos/imunologia , Macrófagos/metabolismo , Pressão OsmóticaRESUMO
Two-pore channels (TPCs) have been a hot topic in recent literature. Their involvement in various diseases such as viral infections and cancer is of great interest for drug research. Due to their localization in the endolysosomal system and the lack of cell-permeable activators, complex techniques were required for studying channel functions. Here, we review the first published lipophilic small-molecule activators of TPCs. In independent high-throughput screens, several new agonists were discovered, which now allow simple and fast investigation of TPCs in more detail in intact cells and in vivo. Zhang et al. identified tricyclic and phenothiazine antidepressants as TPC1 and TPC2 activators by screening a library of approved drugs. In contrast, Gerndt et al. screened an extensive compound library with mostly new chemotypes and drug structures. The latter resulted in two structurally distinct high-affinity agonists, which are able to selectively activate TPC2 in either an NAADP- or PI(3,5)P2 -like manner. Here, we discuss the advantages and drawbacks of the identified molecules and their structural features. The versatility by which TPCs can be activated indicates many opportunities for future studies.
Assuntos
Canais de Cálcio/química , Sinalização do Cálcio , Descoberta de Drogas , Ativação do Canal Iônico/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Canais de Potencial de Receptor Transitório/agonistas , Animais , HumanosRESUMO
Ion selectivity is a defining feature of a given ion channel and is considered immutable. Here we show that ion selectivity of the lysosomal ion channel TPC2, which is hotly debated (Calcraft et al., 2009; Guo et al., 2017; Jha et al., 2014; Ruas et al., 2015; Wang et al., 2012), depends on the activating ligand. A high-throughput screen identified two structurally distinct TPC2 agonists. One of these evoked robust Ca2+-signals and non-selective cation currents, the other weaker Ca2+-signals and Na+-selective currents. These properties were mirrored by the Ca2+-mobilizing messenger, NAADP and the phosphoinositide, PI(3,5)P2, respectively. Agonist action was differentially inhibited by mutation of a single TPC2 residue and coupled to opposing changes in lysosomal pH and exocytosis. Our findings resolve conflicting reports on the permeability and gating properties of TPC2 and they establish a new paradigm whereby a single ion channel mediates distinct, functionally-relevant ionic signatures on demand.
Assuntos
Agonistas dos Canais de Cálcio/farmacologia , Canais de Cálcio/metabolismo , Macrófagos/metabolismo , Cloridrato de Raloxifeno/farmacologia , Animais , Benzilisoquinolinas/farmacologia , Cálcio/metabolismo , Agonistas dos Canais de Cálcio/química , Canais de Cálcio/genética , Flufenazina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Células HeLa , Humanos , Ionomicina/farmacologia , Macrófagos/efeitos dos fármacos , Camundongos , NADP/análogos & derivados , NADP/metabolismo , Fosfatos de Fosfatidilinositol/farmacologia , Imagem Individual de Molécula , Sódio/metabolismoRESUMO
BACKGROUND: The endolysosomal, non-selective cation channels, two-pore channels (TPCs) and mucolipins (TRPMLs), regulate intracellular membrane dynamics and autophagy. While partially compensatory for each other, isoform-specific intracellular distribution, cell-type expression patterns, and regulatory mechanisms suggest different channel isoforms confer distinct properties to the cell. SCOPE OF REVIEW: Briefly, established TPC/TRPML functions and interaction partners ('interactomes') are discussed. Novel TRPML3 interactors are shown, and a meta-analysis of experimentally obtained channel interactomes conducted. Accordingly, interactomes are compared and contrasted, and subsequently described in detail for TPC1, TPC2, TRPML1, and TRPML3. MAJOR CONCLUSIONS: TPC interactomes are well-defined, encompassing intracellular membrane organisation proteins. TRPML interactomes are varied, encompassing cardiac contractility- and chaperone-mediated autophagy proteins, alongside regulators of intercellular signalling. GENERAL SIGNIFICANCE: Comprising recently proposed targets to treat cancers, infections, metabolic disease and neurodegeneration, the advancement of TPC/TRPML understanding is of considerable importance. This review proposes novel directions elucidating TPC/TRPML relevance in health and disease. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.
Assuntos
Canais de Cálcio/metabolismo , Sinalização do Cálcio , Canais de Potencial de Receptor Transitório/metabolismo , Animais , Canais de Cálcio/genética , Humanos , Canais de Potencial de Receptor Transitório/genéticaRESUMO
Cytokines and chemokines are produced and secreted by a broad range of immune cells including macrophages. Remarkably, little is known about how these inflammatory mediators are released from the various immune cells. Here, the endolysosomal cation channel TRPML2 is shown to play a direct role in chemokine trafficking and secretion from murine macrophages. To demonstrate acute and direct involvement of TRPML2 in these processes, the first isoform-selective TRPML2 channel agonist was generated, ML2-SA1. ML2-SA1 was not only found to directly stimulate release of the chemokine CCL2 from macrophages but also to stimulate macrophage migration, thus mimicking CCL2 function. Endogenous TRPML2 is expressed in early/recycling endosomes as demonstrated by endolysosomal patch-clamp experimentation and ML2-SA1 promotes trafficking through early/recycling endosomes, suggesting CCL2 being transported and secreted via this pathway. These data provide a direct link between TRPML2 activation, CCL2 release and stimulation of macrophage migration in the innate immune response.