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BACKGROUND & AIMS - Inactivating mutations in MYO5B cause microvillus inclusion disease (MVID), but the physiological cause of the diarrhea associated with this disease is unclear. We investigated whether loss of MYO5B results in aberrant expression of apical enterocyte transporters.
METHODS - We studied alterations in apical membrane transporters in MYO5B-knockout mice, as well as mice with tamoxifen-inducible, intestine-specific disruption of Myo5b (VilCre;Myo5b mice) or those not given tamoxifen (controls). Intestinal tissues were collected from mice and analyzed by immunostaining, immunoelectron microscopy, or cultured enteroids were derived. Functions of brush border transporters in intestinal mucosa were measured in Ussing chambers. We obtained duodenal biopsy specimens from individuals with MVID and individuals without MVID (controls) and compared transporter distribution by immunocytochemistry.
RESULTS - Compared to intestinal tissues from littermate controls, intestinal tissues from MYO5B-knockout mice had decreased apical localization of SLC9A3 (also called NHE3), SLC5A1 (also called SGLT1), aquaporin (AQP) 7, and sucrase isomaltase, and subapical localization of intestinal alkaline phosphatase and CDC42. However, CFTR was present on apical membranes of enterocytes from MYO5B knockout and control mice. Intestinal biopsies from patients with MVID had subapical localization of NHE3, SGLT1, and AQP7, but maintained apical CFTR. After tamoxifen administration, VilCre;Myo5b mice lost apical NHE3, SGLT1, DRA, and AQP7, similar to germline MYO5B knockout mice. Intestinal tissues from VilCre;Myo5b mice had increased CFTR in crypts and CFTR localized to the apical membranes of enterocytes. Intestinal mucosa from VilCre;Myo5b mice given tamoxifen did not have an intestinal barrier defect, based on Ussing chamber analysis, but did have decreased SGLT1 activity and increased CFTR activity.
CONCLUSIONS - Although trafficking of many apical transporters is regulated by MYO5B, trafficking of CFTR is largely independent of MYO5B. Decreased apical localization of NHE3, SGLT1, DRA, and AQP7 might be responsible for dysfunctional water absorption in enterocytes of patients with MVID. Maintenance of apical CFTR might exacerbate water loss by active secretion of chloride into the intestinal lumen.
Copyright © 2018 AGA Institute. Published by Elsevier Inc. All rights reserved.
Loss-of-function mutations in the potassium-chloride cotransporter KCC3 lead to Andermann syndrome, a severe sensorimotor neuropathy characterized by areflexia, amyotrophy and locomotor abnormalities. The molecular events responsible for axonal loss remain poorly understood. Here, we establish that global or neuron-specific KCC3 loss-of-function in mice leads to early neuromuscular junction (NMJ) abnormalities and muscular atrophy that are consistent with the pre-synaptic neurotransmission defects observed in patients. KCC3 depletion does not modify chloride handling, but promotes an abnormal electrical activity among primary motoneurons and mislocalization of Na/K-ATPase α1 in spinal cord motoneurons. Moreover, the activity-targeting drug carbamazepine restores Na/K-ATPase α1 localization and reduces NMJ denervation in Slc12a6 mice. We here propose that abnormal motoneuron electrical activity contributes to the peripheral neuropathy observed in Andermann syndrome.
Copyright © 2017 Elsevier Inc. All rights reserved.
Pharmacoresistant seizures and cytotoxic cerebral edema are serious complications of ischemic and traumatic brain injury. Intraneuronal Cl concentration ([Cl]) regulation impacts on both cell volume homeostasis and Cl-permeable GABA receptor-dependent membrane excitability. Understanding the pleiotropic molecular determinants of neuronal [Cl] - cytoplasmic impermeant anions, polyanionic extracellular matrix (ECM) glycoproteins, and plasmalemmal Cl transporters - could help the identification of novel anticonvulsive and neuroprotective targets. The cation/Cl cotransporters and ECM metalloproteinases may be particularly druggable targets for intervention. We establish here a paradigm that accounts for recent data regarding the complex regulatory mechanisms of neuronal [Cl] and how these mechanisms impact on neuronal volume and excitability. We propose approaches to modulate [Cl] that are relevant for two common clinical sequela of brain injury: edema and seizures.
Copyright © 2017 Elsevier Ltd. All rights reserved.
Some forms of long-term synaptic plasticity require docking of Ca/calmodulin-dependent protein kinase II α (CaMKIIα) to residues 1290-1309 within the intracellular C-terminal tail of the N-methyl-d-aspartate (NMDA) receptor GluN2B subunit. The phosphorylation of Ser1303 within this region destabilizes CaMKII binding. Interestingly, Ser1303 is a substrate for CaMKII itself, as well as PKC and DAPK1, but these kinases have been reported to have contradictory effects on the activity of GluN2B-containing NMDA receptors. Here, we re-assessed the effect of CaMKII on NMDA receptor desensitization in heterologous cells, as measured by the ratio of steady-state to peak currents induced during 3s agonist applications. CaMKIIα co-expression or infusion of constitutively active CaMKII limits the extent of desensitization and preserves current amplitude with repeated stimulation of recombinant GluN1A/GluN2B when examined using low intracellular chloride (Cl) levels, characteristic of neurons beyond the first postnatal week. In contrast, CaMKIIα enhances the acute rate and extent of desensitization when intracellular Cl concentrations are high. The apparent dependence of CaMKIIα effects on NMDA receptor desensitization on Cl concentrations is consistent with the presence of a Ca-activated Cl conductance endogenous to HEK 293 cells, which was confirmed by photolysis of caged-Ca. However, Ca-activated Cl conductances are unaffected by CaMKIIα expression, indicating that CaMKII affects agonist-induced whole cell currents via modulation of the NMDA receptor. In support of this idea, CaMKIIα modulation of GluN2B-NMDA receptors is abrogated by the phospho-null mutation of Ser1303 in GluN2B to alanine and occluded by phospho-mimetic mutation of Ser1303 to aspartate regardless of intracellular Cl concentration. Thus, CaMKII-mediated phosphorylation of GluN2B-containing NMDA receptors reduces desensitization at physiological (low) intracellular Cl, perhaps serving as a feed-forward mechanism to sustain NMDA-mediated Ca entry and continued CaMKII activation during learning and memory.
Copyright © 2016 Elsevier Inc. All rights reserved.
This study describes a 13-yr-old girl with orthostatic intolerance, respiratory weakness, multiple endocrine abnormalities, pancreatic insufficiency, and multiorgan failure involving the gut and bladder. Exome sequencing revealed a de novo, loss-of-function allele in , the gene encoding the Na-K-2Cl cotransporter-1. The 11-bp deletion in exon 22 results in frameshift (p.Val1026Phe*2) and truncation of the carboxy-terminal tail of the cotransporter. Preliminary studies in heterologous expression systems demonstrate that the mutation leads to a nonfunctional transporter, which is expressed and trafficked to the plasma membrane alongside wild-type NKCC1. The truncated protein, visible at higher molecular sizes, indicates either enhanced dimerization or misfolded aggregate. No significant dominant-negative effect was observed. K transport experiments performed in fibroblasts from the patient showed reduced total and NKCC1-mediated K influx. The absence of a bumetanide effect on K influx in patient fibroblasts only under hypertonic conditions suggests a deficit in NKCC1 regulation. We propose that disruption in NKCC1 function might affect sensory afferents and/or smooth muscle cells, as their functions depend on NKCC1 creating a Cl gradient across the plasma membrane. This Cl gradient allows the γ-aminobutyric acid (GABA) receptor or other Cl channels to depolarize the membrane affecting processes such as neurotransmission or cell contraction. Under this hypothesis, disrupted sensory and smooth muscle function in a diverse set of tissues could explain the patient's phenotype.
Zinc (Zn) is an essential metal that vertebrates sequester from pathogens to protect against infection. Investigating the opportunistic pathogen Acinetobacter baumannii's response to Zn starvation, we identified a putative Zn metallochaperone, ZigA, which binds Zn and is required for bacterial growth under Zn-limiting conditions and for disseminated infection in mice. ZigA is encoded adjacent to the histidine (His) utilization (Hut) system. The His ammonia-lyase HutH binds Zn very tightly only in the presence of high His and makes Zn bioavailable through His catabolism. The released Zn enables A. baumannii to combat host-imposed Zn starvation. These results demonstrate that A. baumannii employs several mechanisms to ensure bioavailability of Zn during infection, with ZigA functioning predominately during Zn starvation, but HutH operating in both Zn-deplete and -replete conditions to mobilize a labile His-Zn pool.
Copyright © 2016 Elsevier Inc. All rights reserved.
Basement membranes are defining features of the cellular microenvironment; however, little is known regarding their assembly outside cells. We report that extracellular Cl(-) ions signal the assembly of collagen IV networks outside cells by triggering a conformational switch within collagen IV noncollagenous 1 (NC1) domains. Depletion of Cl(-) in cell culture perturbed collagen IV networks, disrupted matrix architecture, and repositioned basement membrane proteins. Phylogenetic evidence indicates this conformational switch is a fundamental mechanism of collagen IV network assembly throughout Metazoa. Using recombinant triple helical protomers, we prove that NC1 domains direct both protomer and network assembly and show in Drosophila that NC1 architecture is critical for incorporation into basement membranes. These discoveries provide an atomic-level understanding of the dynamic interactions between extracellular Cl(-) and collagen IV assembly outside cells, a critical step in the assembly and organization of basement membranes that enable tissue architecture and function. Moreover, this provides a mechanistic framework for understanding the molecular pathobiology of NC1 domains.
© 2016 Cummings et al.
Gold nanorods with varying aspect ratios have been utilized in recent years for a wide range of applications including vaccines, surface enhanced Raman spectroscopy (SERS) substrates, and as medicinal therapeutic agents. The surfactant-directed seed mediated approach is an aqueous based protocol that produces monodisperse nanorods with controlled aspect ratios. However, an inherent problem with this approach is poor efficiency of gold conversion from HAuCl4 into nanorods. In fact only ∼15% of gold is converted, motivating the need for alternate synthetic protocols in order to make the process more scalable and efficient as gold nanorods progress toward commercial applications. In the current study, we have significantly improved this conversion by growing rods in several iterations of supernatant solutions that were previously discarded as waste. Inductively coupled plasma mass spectrometry (ICP-MS) data indicates ∼14% gold conversion per nanorod solution with a total recovery of ∼75%. Gold nanorods prepared in consecutive supernatant solutions generally have slightly increased aspect ratios and maintain stability and monodispersity as measured by UV-vis and TEM. The increased nanorod yield minimizes gold waste and results in a greener synthetic approach.
Copyright © 2015 Elsevier Inc. All rights reserved.
Thiazide diuretics are used to treat hypertension; however, compensatory processes in the kidney can limit antihypertensive responses to this class of drugs. Here, we evaluated compensatory pathways in SPAK kinase-deficient mice, which are unable to activate the thiazide-sensitive sodium chloride cotransporter NCC (encoded by Slc12a3). Global transcriptional profiling, combined with biochemical, cell biological, and physiological phenotyping, identified the gene expression signature of the response and revealed how it establishes an adaptive physiology. Salt reabsorption pathways were created by the coordinate induction of a multigene transport system, involving solute carriers (encoded by Slc26a4, Slc4a8, and Slc4a9), carbonic anhydrase isoforms, and V-type H⁺-ATPase subunits in pendrin-positive intercalated cells (PP-ICs) and ENaC subunits in principal cells (PCs). A distal nephron remodeling process and induction of jagged 1/NOTCH signaling, which expands the cortical connecting tubule with PCs and replaces acid-secreting α-ICs with PP-ICs, were partly responsible for the compensation. Salt reabsorption was also activated by induction of an α-ketoglutarate (α-KG) paracrine signaling system. Coordinate regulation of a multigene α-KG synthesis and transport pathway resulted in α-KG secretion into pro-urine, as the α-KG-activated GPCR (Oxgr1) increased on the PP-IC apical surface, allowing paracrine delivery of α-KG to stimulate salt transport. Identification of the integrated compensatory NaCl reabsorption mechanisms provides insight into thiazide diuretic efficacy.
AIMS - Thymic stromal lymphopoietin (TSLP) plays an important role in inflammatory diseases and is over-expressed in human atherosclerotic artery specimens. The present study investigated the role of TSLP in platelet activation and thrombosis models in vitro and in vivo, as well as the underlying mechanism and signaling pathway.
METHODS AND RESULTS - Western blotting and flow cytometry demonstrated that the TSLP receptor was expressed on murine platelets. According to flow cytometry, platelet stimulation with TSLP induced platelet degranulation and integrin αIIbβ3 activation. A TSLPR deficiency caused defective platelet aggregation, defective platelet secretion and markedly blunted thrombus growth in perfusion chambers at both low and high shear rates. TSLPR KO mice exhibited defective carotid artery thrombus formation after exposure to FeCl3. TSLP increased Akt phosphorylation, an effect that was abrogated by the PI3K inhibitors wortmannin and LY294002. The PI3K inhibitors further diminished TSLP-induced platelet activation. TSLP-mediated platelet degranulation, integrin αIIbβ3 activation and Akt phosphorylation were blunted in platelets that lacked the TSLP receptor.
CONCLUSION - This study demonstrated that the functional TSLPR was surface-expressed on murine platelets. The inflammatory cytokine TSLP triggered platelet activation and thrombus formation via TSLP-dependent PI3K/Akt signaling, which suggests an important role for TSLP in linking vascular inflammation and thrombo-occlusive diseases.
© 2015 S. Karger AG, Basel.