Mechanotransduction is the process by which cells convert physical forces into electrochemical responses. On a molecular scale, these forces are detected by mechanically activated ion channels, which ...constitute the basis for hearing, touch, pain, cold, and heat sensation, among other physiological processes. Exciting high-resolution structural details of these channels are currently emerging that will eventually allow us to delineate the molecular determinants of gating and ion permeation. However, our structural–functional understanding across the family remains limited. Piezo1 is one of the largest and least understood of these channels, with various structurally identified features within its trimeric assembly. This study seeks to determine the modularity and function of Piezo1 channels by constructing deletion proteins guided by cryo EM structural knowledge. Our comprehensive functional study identified, for the first time, the minimal amino acid sequence of the full-length Piezo1 that can fold and function as the channel’s pore domain between E2172 and the last residue E2547. While the addition of an anchor region has no effect on permeation properties. The Piezo1 pore domain is not pressure-sensitive and the appending of Piezo Repeat-A did not restore pressure-dependent gating, hence the sensing module must exist between residues 1 to 1952. Our efforts delineating the permeation and gating regions within this complex ion channel have implications in identifying small molecules that exclusively regulate the activity of the channel’s pore module to influence mechanotransduction and downstream processes.
Center for Basic Neuroscience, Department of Physiology, University of Texas Southwestern Medical Center, Dallas, Texas
Submitted 14 December 2005;
accepted in final form 27 January 2006
Fragile X ...syndrome (FXS), a form of human mental retardation, is caused by loss of function mutations in the fragile X mental retardation gene ( FMR1 ). The protein product of FMR1 , fragile X mental retardation protein (FMRP) is an RNA-binding protein and may function as a translational suppressor. Metabotropic glutamate receptordependent long-term depression (mGluR-LTD) in hippocampal area CA1 is a form of synaptic plasticity that relies on dendritic protein synthesis. mGluR-LTD is enhanced in the mouse model of FXS, Fmr1 knockout (KO) mice, suggesting that FMRP negatively regulates translation of proteins required for LTD. Here we examine the synaptic and cellular mechanisms of mGluR-LTD in KO mice and find that mGluR-LTD no longer requires new protein synthesis, in contrast to wild-type (WT) mice. We further show that mGluR-LTD in KO and WT mice is associated with decreases in AMPA receptor (AMPAR) surface expression, indicating a similar postsynaptic expression mechanism. However, like LTD, mGluR-induced decreases in AMPAR surface expression in KO mice persist in protein synthesis inhibitors. These results are consistent with recent findings of elevated protein synthesis rates and synaptic protein levels in Fmr1 KO mice and suggest that these elevated levels of synaptic proteins are available to increase the persistence of LTD without de novo protein synthesis.
Address for reprint requests and other correspondence: K. Huber, Center for Basic Neuroscience, UT Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9111 (E-mail: kimberly.huber{at}utsouthwestern.edu )
Stromal cells in adult bone marrow that express leptin receptor (LEPR) are a critical source of growth factors, including stem cell factor (SCF), for the maintenance of haematopoietic stem cells and ...early restricted progenitors
. LEPR
cells are heterogeneous, including skeletal stem cells and osteogenic and adipogenic progenitors
, although few markers have been available to distinguish these subsets or to compare their functions. Here we show that expression of an osteogenic growth factor, osteolectin
, distinguishes peri-arteriolar LEPR
cells poised to undergo osteogenesis from peri-sinusoidal LEPR
cells poised to undergo adipogenesis (but retaining osteogenic potential). Peri-arteriolar LEPR
osteolectin
cells are rapidly dividing, short-lived osteogenic progenitors that increase in number after fracture and are depleted during ageing. Deletion of Scf from adult osteolectin
cells did not affect the maintenance of haematopoietic stem cells or most restricted progenitors but depleted common lymphoid progenitors, impairing lymphopoiesis, bacterial clearance, and survival after acute bacterial infection. Peri-arteriolar osteolectin
cell maintenance required mechanical stimulation. Voluntary running increased, whereas hindlimb unloading decreased, the frequencies of peri-arteriolar osteolectin
cells and common lymphoid progenitors. Deletion of the mechanosensitive ion channel PIEZO1 from osteolectin
cells depleted osteolectin
cells and common lymphoid progenitors. These results show that a peri-arteriolar niche for osteogenesis and lymphopoiesis in bone marrow is maintained by mechanical stimulation and depleted during ageing.
Salient stimuli that modify behavior induce transcription of activity-regulated cytoskeleton-associated protein (Arc/Arg3.1) and transport
Arc mRNA into dendrites, suggesting that local Arc ...translation mediates synaptic plasticity that encodes such stimuli. Here, we demonstrate that long-term synaptic depression (LTD) in hippocampal neurons induced by group 1 metabotropic glutamate receptors (mGluRs) relies on rapid translation of Arc. mGluR-LTD induction causes long-term increases in AMPA receptor endocytosis rate and dendritic synthesis of Arc, a component of the AMPAR endocytosis machinery. Knockdown of Arc prevents mGluRs from triggering AMPAR endocytosis or LTD, and acute blockade of new Arc synthesis with antisense oligonucleotides blocks mGluR-LTD and AMPAR trafficking. In contrast, LTD induced by NMDA receptors does not persistently alter AMPAR endocytosis rate, induce Arc synthesis, or require Arc protein. These data demonstrate a role for local Arc synthesis specifically in mGluR-LTD and suggest that mGluR-LTD may be one consequence of Arc mRNA induction during experience.
Prune belly syndrome (PBS), also known as Eagle-Barret syndrome, is a rare, multi-system congenital myopathy primarily affecting males. Phenotypically, PBS cases manifest three cardinal pathological ...features: urinary tract dilation with poorly contractile smooth muscle, wrinkled flaccid ventral abdominal wall with skeletal muscle deficiency, and intra-abdominal undescended testes. Genetically, PBS is poorly understood. After performing whole exome sequencing in PBS patients, we identify one compound heterozygous variant in the PIEZO1 gene. PIEZO1 is a cation-selective channel activated by various mechanical forces and widely expressed throughout the lower urinary tract. Here we conduct an extensive functional analysis of the PIEZO1 PBS variants that reveal loss-of-function characteristics in the pressure-induced normalized open probability (NPo) of the channel, while no change is observed in single-channel currents. Furthermore, Yoda1, a PIEZO1 activator, can rescue the NPo defect of the PBS mutant channels. Thus, PIEZO1 mutations may be causal for PBS and the in vitro cellular pathophysiological phenotype could be rescued by the small molecule, Yoda1. Activation of PIEZO1 might provide a promising means of treating PBS and other related bladder dysfunctional states.
The mental retardation, autistic features, and behavioral abnormalities characteristic of the Fragile X mental retardation syndrome result from the loss of function of the RNA-binding protein FMRP. ...The disease is usually caused by a triplet repeat expansion in the 5'UTR of the FMR1 gene. This leads to loss of function through transcriptional gene silencing, pointing to a key function for FMRP, but precluding genetic identification of critical activities within the protein. Moreover, antisense transcripts (FMR4, ASFMR1) in the same locus have been reported to be silenced by the repeat expansion. Missense mutations offer one means of confirming a central role for FMRP in the disease, but to date, only a single such patient has been described. This patient harbors an isoleucine to asparagine mutation (I304N) in the second FMRP KH-type RNA-binding domain, however, this single case report was complicated because the patient harbored a superimposed familial liver disease. To address these issues, we have generated a new Fragile X Syndrome mouse model in which the endogenous Fmr1 gene harbors the I304N mutation. These mice phenocopy the symptoms of Fragile X Syndrome in the existing Fmr1-null mouse, as assessed by testicular size, behavioral phenotyping, and electrophysiological assays of synaptic plasticity. I304N FMRP retains some functions, but has specifically lost RNA binding and polyribosome association; moreover, levels of the mutant protein are markedly reduced in the brain specifically at a time when synapses are forming postnatally. These data suggest that loss of FMRP function, particularly in KH2-mediated RNA binding and in synaptic plasticity, play critical roles in pathogenesis of the Fragile X Syndrome and establish a new model for studying the disorder.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Mechanically activated Piezo1 channels undergo transitions from closed to open-state in response to pressure and other mechanical stimuli. However, the molecular details of these mechanosensitive ...gating transitions are unknown. Here, we used cell-attached pressure-clamp recordings to acquire single channel data at steady-state conditions (where inactivation has settled down), at various pressures and voltages. Importantly, we identify and analyze subconductance states of the channel which were not reported before. Pressure-dependent activation of Piezo1 increases the occupancy of open and subconductance state at the expense of decreased occupancy of shut-states. No significant change in the mean open time of subconductance states was observed with increasing negative pipette pressure or with varying voltages (ranging from −40 to −100 mV). Using Markov-chain modeling, we identified a minimal four-states kinetic scheme, which recapitulates essential characteristics of the single channel data, including that of the subconductance level. This study advances our understanding of Piezo1-gating mechanism in response to discrete stimuli (such as pressure and voltage) and paves the path to develop cellular and tissue level models to predict Piezo1 function in various cell types.
The presynaptic and postsynaptic properties of synapses change over the course of postnatal development. Therefore, synaptic plasticity mechanisms would be expected to adapt to these changes to ...facilitate alterations of synaptic strength throughout ontogeny. Here, we identified developmental changes in long-term depression (LTD) mediated by group 1 metabotropic glutamate receptors (mGluRs) and dendritic protein synthesis in hippocampal CA1 slices (mGluR-LTD). In slices prepared from adolescent rats postnatal day 21 (P21) to P35, mGluR activation induces LTD and a long-term decrease in AMPA receptor (AMPAR) surface expression, both of which require protein synthesis. In neonatal animals (P8-P15), mGluR-LTD is independent of protein synthesis and is not associated with changes in the surface expression of AMPARs. Instead, mGluR-LTD at neonatal synapses results in large decreases in presynaptic function, measured by changes in paired-pulse facilitation and the rate of blockade by the use-dependent NMDA receptor blocker (+)-5-methyl-10,11-dihydro-5H-dibenzoa,dcyclohepten-5,10-imine maleate. Conversely, mGluR-LTD at mature synapses results in little or no change in presynaptic function, suggesting a postsynaptic mechanism of expression. The developmental switch in the synaptic mechanisms of LTD would differentially affect synapse dynamics and perhaps information processing over the course of postnatal development.
CASK is an evolutionarily conserved multidomain protein composed of an N-terminal Ca²⁺/calmodulin-kinase domain, central PDZ and SH3 domains, and a C-terminal guanylate kinase domain. Many potential ...activities for CASK have been suggested, including functions in scaffolding the synapse, in organizing ion channels, and in regulating neuronal gene transcription. To better define the physiological importance of CASK, we have now analyzed CASK "knockdown" mice in which CASK expression was suppressed by ≈70%, and CASK knockout (KO) mice, in which CASK expression was abolished. CASK knockdown mice are viable but smaller than WT mice, whereas CASK KO mice die at first day after birth. CASK KO mice exhibit no major developmental abnormalities apart from a partially penetrant cleft palate syndrome. In CASK-deficient neurons, the levels of the CASK-interacting proteins Mints, Veli/Mals, and neurexins are decreased, whereas the level of neuroligin 1 (which binds to neurexins that in turn bind to CASK) is increased. Neurons lacking CASK display overall normal electrical properties and form ultrastructurally normal synapses. However, glutamatergic spontaneous synaptic release events are increased, and GABAergic synaptic release events are decreased in CASK-deficient neurons. In contrast to spontaneous neurotransmitter release, evoked release exhibited no major changes. Our data suggest that CASK, the only member of the membrane-associated guanylate kinase protein family that contains a Ca²⁺/calmodulin-dependent kinase domain, is required for mouse survival and performs a selectively essential function without being in itself required for core activities of neurons, such as membrane excitability, Ca²⁺-triggered presynaptic release, or postsynaptic receptor functions.
Mechanotransduction is the process by which cells convert physical forces into electro-chemical responses. On a molecular scale these forces are detected by mechanically activated ion channels, which ...constitute the basis for hearing, touch, pain, cold and heat sensation amongst other physiological processes. Exciting high-resolution structural details of these channels are currently emerging that will eventually allow us to delineate the molecular determinants of gating and ion permeation. However, our structural-functional understanding across the family remains limited. Piezo1 is one of the largest and least understood of these channels, with various structurally identified features within its trimeric assembly. This study seeks to determine the modularity and function of Piezo1 channels by constructing deletion proteins guided by cryo EM structural knowledge. Our comprehensive functional study identified, for the first time, the minimal amino acid sequence of the full-length Piezo1 that can fold and function as the channel's pore domain between E2172 and the last residue E2547. While, the addition of an anchor region has no effect on permeation properties. The Piezo1 pore domain is not pressure sensitive and the appending of Piezo Repeat-A did not restore pressure-dependent gating, hence the sensing module must exist between residues 1-1952. Our efforts delineating the permeation and gating regions within this complex ion channel have implications in identifying small molecules that exclusively regulate the activity of the channel's pore module to influence mechanotransduction and downstream processes.
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