RESUMEN
Piezo1 and Piezo2 are mechanically activated ion channels that mediate touch perception, proprioception and vascular development. Piezo proteins are distinct from other ion channels and their structure remains poorly defined, which impedes detailed study of their gating and ion permeation properties. Here we report a high-resolution cryo-electron microscopy structure of the mouse Piezo1 trimer. The detergent-solubilized complex adopts a three-bladed propeller shape with a curved transmembrane region containing at least 26 transmembrane helices per protomer. The flexible propeller blades can adopt distinct conformations, and consist of a series of four-transmembrane helical bundles that we term Piezo repeats. Carboxy-terminal domains line the central ion pore, and the channel is closed by constrictions in the cytosol. A kinked helical beam and anchor domain link the Piezo repeats to the pore, and are poised to control gating allosterically. The structure provides a foundation to dissect further how Piezo channels are regulated by mechanical force.
Asunto(s)
Microscopía por Crioelectrón , Canales Iónicos/química , Canales Iónicos/ultraestructura , Animales , Sitios de Unión , Activación del Canal Iónico , Canales Iónicos/genética , Canales Iónicos/metabolismo , Lípidos , Ratones , Modelos Moleculares , Mutación , Docilidad , Dominios Proteicos , SolubilidadRESUMEN
MicroRNAs (miRNAs) are thought to exert their functions by modulating the expression of hundreds of target genes and each to a small degree, but it remains unclear how small changes in hundreds of target genes are translated into the specific function of a miRNA. Here, we conducted an integrated analysis of transcriptome and translatome of primary B cells from mutant mice expressing miR-17~92 at three different levels to address this issue. We found that target genes exhibit differential sensitivity to miRNA suppression and that only a small fraction of target genes are actually suppressed by a given concentration of miRNA under physiological conditions. Transgenic expression and deletion of the same miRNA gene regulate largely distinct sets of target genes. miR-17~92 controls target gene expression mainly through translational repression and 5'UTR plays an important role in regulating target gene sensitivity to miRNA suppression. These findings provide molecular insights into a model in which miRNAs exert their specific functions through a small number of key target genes.
Asunto(s)
Linfocitos B/metabolismo , Regulación de la Expresión Génica , MicroARNs/genética , Biosíntesis de Proteínas/genética , Transcriptoma/genética , Regiones no Traducidas 5'/genética , Animales , Linfocitos B/citología , Secuencia de Bases , Proteína 11 Similar a Bcl2/genética , Proteína 11 Similar a Bcl2/metabolismo , Células Cultivadas , Citometría de Flujo , Perfilación de la Expresión Génica/métodos , Immunoblotting , Ratones Noqueados , Ratones Transgénicos , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/genética , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ribosomas/genética , Ribosomas/metabolismoRESUMEN
SWELL1 (LRRC8A) is the only essential subunit of the Volume Regulated Anion Channel (VRAC), which regulates cellular volume homeostasis and is activated by hypotonic solutions. SWELL1, together with four other LRRC8 family members, potentially forms a vastly heterogeneous cohort of VRAC channels with different properties; however, SWELL1 alone is also functional. Here, we report a high-resolution cryo-electron microscopy structure of full-length human homo-hexameric SWELL1. The structure reveals a trimer of dimers assembly with symmetry mismatch between the pore-forming domain and the cytosolic leucine-rich repeat (LRR) domains. Importantly, mutational analysis demonstrates that a charged residue at the narrowest constriction of the homomeric channel is an important pore determinant of heteromeric VRAC. Additionally, a mutation in the flexible N-terminal portion of SWELL1 affects pore properties, suggesting a putative link between intracellular structures and channel regulation. This structure provides a scaffold for further dissecting the heterogeneity and mechanism of activation of VRAC.
Asunto(s)
Proteínas de la Membrana/química , Multimerización de Proteína/genética , Relación Estructura-Actividad , Canales Aniónicos Dependientes del Voltaje/química , Aminoácidos/química , Aminoácidos/genética , Células HeLa , Humanos , Proteínas de la Membrana/genética , Familia de Multigenes , Mutación , Estructura Cuaternaria de Proteína , Canales Aniónicos Dependientes del Voltaje/genéticaRESUMEN
The conversion of mechanical force to chemical signals is critical for many biological processes, including the senses of touch, pain, and hearing. Mechanosensitive ion channels play a key role in sensing the mechanical stimuli experienced by various cell types and are present in organisms from bacteria to mammals. Bacterial mechanosensitive channels are characterized thoroughly, but less is known about their counterparts in vertebrates. Piezos have been recently established as ion channels required for mechanotransduction in disparate cell types in vitro and in vivo. Overexpression of Piezos in heterologous cells gives rise to large mechanically activated currents; however, it is unclear whether Piezos are inherently mechanosensitive or rely on alternate cellular components to sense mechanical stimuli. Here, we show that mechanical perturbations of the lipid bilayer alone are sufficient to activate Piezo channels, illustrating their innate ability as molecular force transducers.