RESUMEN
The initial activation step in the gating of ubiquitously expressed Orai1 calcium (Ca2+) ion channels represents the activation of the Ca2+-sensor protein STIM1 upon Ca2+ store depletion of the endoplasmic reticulum. Previous studies using constitutively active Orai1 mutants gave rise to, but did not directly test, the hypothesis that STIM1-mediated Orai1 pore opening is accompanied by a global conformational change of all Orai transmembrane domain (TM) helices within the channel complex. We prove that a local conformational change spreads omnidirectionally within the Orai1 complex. Our results demonstrate that these locally induced global, opening-permissive TM motions are indispensable for pore opening and require clearance of a series of Orai1 gating checkpoints. We discovered these gating checkpoints in the middle and cytosolic extended TM domain regions. Our findings are based on a library of double point mutants that contain each one loss-of-function with one gain-of-function point mutation in a series of possible combinations. We demonstrated that an array of loss-of-function mutations are dominant over most gain-of-function mutations within the same as well as of an adjacent Orai subunit. We further identified inter- and intramolecular salt-bridge interactions of Orai subunits as a core element of an opening-permissive Orai channel architecture. Collectively, clearance and synergistic action of all these gating checkpoints are required to allow STIM1 coupling and Orai1 pore opening. Our results unravel novel insights in the preconditions of the unique fingerprint of CRAC channel activation, provide a valuable source for future structural resolutions, and help to understand the molecular basis of disease-causing mutations.
Asunto(s)
Señalización del Calcio , Calcio/metabolismo , Activación del Canal Iónico/genética , Proteínas de Neoplasias/química , Proteína ORAI1/química , Molécula de Interacción Estromal 1/química , Sustitución de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Regulación de la Expresión Génica , Genes Reporteros , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Liposomas/química , Liposomas/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Simulación de Dinámica Molecular , Mutación , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/genética , Proteína ORAI1/metabolismo , Técnicas de Placa-Clamp , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Molécula de Interacción Estromal 1/genética , Molécula de Interacción Estromal 1/metabolismoRESUMEN
Stromal interaction molecule 1 (STIM1) is a ubiquitously expressed Ca2+ sensor protein that induces permeation of Orai Ca2+ channels upon endoplasmic reticulum Ca2+-store depletion. A drop in luminal Ca2+ causes partial unfolding of the N-terminal STIM1 domains and thus initial STIM1 activation. We compared the STIM1 structure upon Ca2+ depletion from our molecular dynamics (MD) simulations with a recent 2D NMR structure. Simulation- and structure-based results showed unfolding of two α-helices in the canonical and in the non-canonical EF-hand. Further, we structurally and functionally evaluated mutations in the non-canonical EF-hand that have been shown to cause tubular aggregate myopathy. We found these mutations to cause full constitutive activation of Ca2+-release-activated Ca2+ currents (ICRAC) and to promote autophagic processes. Specifically, heterologously expressed STIM1 mutations in the non-canonical EF-hand promoted translocation of the autophagy transcription factors microphthalmia-associated transcription factor (MITF) and transcription factor EB (TFEB) into the nucleus. These STIM1 mutations additionally stimulated an enhanced production of autophagosomes. In summary, mutations in STIM1 that cause structural unfolding promoted Ca2+ down-stream activation of autophagic processes.
Asunto(s)
Autofagia , Miopatías Estructurales Congénitas/genética , Proteínas de Neoplasias/genética , Molécula de Interacción Estromal 1/genética , Calcio/metabolismo , Cationes Bivalentes/metabolismo , Motivos EF Hand , Humanos , Simulación de Dinámica Molecular , Mutación , Miopatías Estructurales Congénitas/metabolismo , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Conformación Proteica en Hélice alfa , Desplegamiento Proteico , Molécula de Interacción Estromal 1/química , Molécula de Interacción Estromal 1/metabolismoRESUMEN
The interplay of SK3, a Ca2+ sensitive K+ ion channel, with Orai1, a Ca2+ ion channel, has been reported to increase cytosolic Ca2+ levels, thereby triggering proliferation of breast and colon cancer cells, although a molecular mechanism has remained elusive to date. We show in the current study, via heterologous protein expression, that Orai1 can enhance SK3 K+ currents, in addition to constitutively bound calmodulin (CaM). At low cytosolic Ca2+ levels that decrease SK3 K+ permeation, co-expressed Orai1 potentiates SK3 currents. This positive feedback mechanism of SK3 and Orai1 is enabled by their close co-localization. Remarkably, we discovered that loss of SK3 channel activity due to overexpressed CaM mutants could be restored by Orai1, likely via its interplay with the SK3-CaM binding site. Mapping for interaction sites within Orai1, we identified that the cytosolic strands and pore residues are critical for a functional communication with SK3. Moreover, STIM1 has a bimodal role in SK3-Orai1 regulation. Under physiological ionic conditions, STIM1 is able to impede SK3-Orai1 interplay by significantly decreasing their co-localization. Forced STIM1-Orai1 activity and associated Ca2+ influx promote SK3 K+ currents. The dynamic regulation of Orai1 to boost endogenous SK3 channels was also determined in the human prostate cancer cell line LNCaP.
RESUMEN
Store-operated calcium entry (SOCE) through STIM-gated ORAI channels governs vital cellular functions. In this context, SOCE controls cellular redox signaling and is itself regulated by redox modifications. However, the molecular mechanisms underlying this calcium-redox interplay and the functional outcomes are not fully understood. Here, we examine the role of STIM2 in SOCE redox regulation. Redox proteomics identify cysteine 313 as the main redox sensor of STIM2 in vitro and in vivo. Oxidative stress suppresses SOCE and calcium currents in cells overexpressing STIM2 and ORAI1, an effect that is abolished by mutation of cysteine 313. FLIM and FRET microscopy, together with MD simulations, indicate that oxidative modifications of cysteine 313 alter STIM2 activation dynamics and thereby hinder STIM2-mediated gating of ORAI1. In summary, this study establishes STIM2-controlled redox regulation of SOCE as a mechanism that affects several calcium-regulated physiological processes, as well as stress-induced pathologies.
Asunto(s)
Calcio/metabolismo , Molécula de Interacción Estromal 2/metabolismo , Canales de Calcio/metabolismo , Señalización del Calcio , Línea Celular Tumoral , Cisteína/metabolismo , Humanos , Proteínas Sensoras del Calcio Intracelular/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/metabolismo , Oxidación-Reducción , Estrés Oxidativo/fisiología , Molécula de Interacción Estromal 1/genética , Molécula de Interacción Estromal 1/metabolismo , Molécula de Interacción Estromal 2/genética , Molécula de Interacción Estromal 2/fisiologíaRESUMEN
A primary calcium (Ca2+) entry pathway into non-excitable cells is through the store-operated Ca2+ release activated Ca2+ (CRAC) channel. Ca2+ entry into cells is responsible for the initiation of diverse signalling cascades that affect essential cellular processes like gene regulation, cell growth and death, secretion and gene transcription. Upon depletion of intracellular Ca2+ stores within the endoplasmic reticulum (ER), the CRAC channel opens to refill depleted stores. The two key limiting molecular players of the CRAC channel are the stromal interaction molecule (STIM1) embedded in the ER-membrane and Orai1, residing in the plasma membrane (PM), respectively. Together, they form a highly Ca2+ selective ion channel complex. STIM1 senses the Ca2+ content of the ER and confers Ca2+ store-depletion into the opening of Orai1 channels in the PM for triggering Ca2+-dependent gene transcription, T-cell activation or mast cell degranulation. The interplay of Orai and STIM proteins in the CRAC channel signalling cascade has been the main focus of research for more than twelve years. This chapter focuses on current knowledge and main experimental advances in the understanding of Orai1 activation by STIM1, thereby portraying key mechanistic steps in the CRAC channel signalling cascade.
Asunto(s)
Señalización del Calcio/fisiología , Calcio/metabolismo , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/metabolismo , Molécula de Interacción Estromal 1/metabolismo , Canales de Calcio Activados por la Liberación de Calcio/metabolismo , Humanos , Activación de Linfocitos/fisiología , Linfocitos T/metabolismoRESUMEN
Calcium signalling through store-operated calcium (SOC) entry is of crucial importance for T-cell activation and the adaptive immune response. This entry occurs via the prototypic Ca2+ release-activated Ca2+ (CRAC) channel. STIM1, a key molecular component of this process, is located in the membrane of the endoplasmic reticulum (ER) and is initially activated upon Ca2+ store depletion. This activation signal is transmitted to the plasma membrane via a direct physical interaction that takes place between STIM1 and the highly Ca2+-selective ion channel Orai1. The activation of STIM1 induces an extended cytosolic conformation. This, in turn, exposes the CAD/SOAR domain and leads to the formation of STIM1 oligomers. In this study, we focused on a small helical segment (STIM1 α3, aa 400-403), which is located within the CAD/SOAR domain. We determined this segment's specific functional role in terms of STIM1 activation and Orai1 gating. The STIM1 α3 domain appears not essential for STIM1 to interact with Orai1. Instead, it represents a key domain that conveys STIM1 interaction into Orai1 channel gating. The results of cysteine crosslinking experiments revealed the close proximity of STIM1 α3 to a region within Orai1, which was located at the cytosolic extension of transmembrane helix 3, forming a STIM1-Orai1 gating interface (SOGI). We suggest that the interplay between STIM1 α3 and Orai1 TM3 allows STIM1 coupling to be transmitted into physiological CRAC channel activation.
Asunto(s)
Canales de Calcio Activados por la Liberación de Calcio/metabolismo , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/metabolismo , Molécula de Interacción Estromal 1/metabolismo , Calcio/metabolismo , Células Cultivadas , Clonación Molecular , Células HEK293 , Humanos , Proteínas de Neoplasias/deficiencia , Proteínas de Neoplasias/genética , Proteína ORAI1/deficiencia , Proteína ORAI1/genética , Molécula de Interacción Estromal 1/deficiencia , Molécula de Interacción Estromal 1/genéticaRESUMEN
The stromal interaction molecule 1 (STIM1) has two important functions, Ca2+ sensing within the endoplasmic reticulum and activation of the store-operated Ca2+ channel Orai1, enabling plasma-membrane Ca2+ influx. We combined molecular dynamics (MD) simulations with live-cell recordings and determined the sequential Ca2+-dependent conformations of the luminal STIM1 domain upon activation. Furthermore, we identified the residues within the canonical and noncanonical EF-hand domains that can bind to multiple Ca2+ ions. In MD simulations, a single Ca2+ ion was sufficient to stabilize the luminal STIM1 complex. Ca2+ store depletion destabilized the two EF hands, triggering disassembly of the hydrophobic cleft that they form together with the stable SAM domain. Point mutations associated with tubular aggregate myopathy or cancer that targeted the canonical EF hand, and the hydrophobic cleft yielded constitutively clustered STIM1, which was associated with activation of Ca2+ entry through Orai1 channels. On the basis of our results, we present a model of STIM1 Ca2+ binding and refine the currently known initial steps of STIM1 activation on a molecular level.
Asunto(s)
Calcio/metabolismo , Simulación de Dinámica Molecular , Proteínas de Neoplasias/química , Dominios Proteicos , Desplegamiento Proteico , Molécula de Interacción Estromal 1/química , Algoritmos , Animales , Línea Celular Tumoral , Membrana Celular/metabolismo , Motivos EF Hand , Retículo Endoplásmico/metabolismo , Células HEK293 , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Microscopía Confocal , Mutación , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/química , Proteína ORAI1/metabolismo , Ratas , Molécula de Interacción Estromal 1/genética , Molécula de Interacción Estromal 1/metabolismoRESUMEN
The channel Orai1 requires Ca2+ store depletion in the endoplasmic reticulum and an interaction with the Ca2+ sensor STIM1 to mediate Ca2+ signaling. Alterations in Orai1-mediated Ca2+ influx have been linked to several pathological conditions including immunodeficiency, tubular myopathy, and cancer. We screened large-scale cancer genomics data sets for dysfunctional Orai1 mutants. Five of the identified Orai1 mutations resulted in constitutively active gating and transcriptional activation. Our analysis showed that certain Orai1 mutations were clustered in the transmembrane 2 helix surrounding the pore, which is a trigger site for Orai1 channel gating. Analysis of the constitutively open Orai1 mutant channels revealed two fundamental gates that enabled Ca2+ influx: Arginine side chains were displaced so they no longer blocked the pore, and a chain of water molecules formed in the hydrophobic pore region. Together, these results enabled us to identify a cluster of Orai1 mutations that trigger Ca2+ permeation associated with gene transcription and provide a gating mechanism for Orai1.