Electron microscopic evidence for the assembly of soluble pentameric extracellular domains of the nicotinic acetylcholine receptor

Exploitation of soluble extracellular domains (ECDs) of the nicotinic acetylcholine receptor may provide a route to crystallographic studies aimed at exploring the structure and function of the intact receptor. The first step towards this goal is to manufacture and isolate soluble fragments that fold and assemble to form a functionally relevant complex. The baculovirus insect cell expression system was used to co-express soluble ECDs of all four muscle-type nicotinic acetylcholine receptor subunits (alpha, beta, gamma & delta-ECD) from Torpedo. Protein complexes were purified using either the conformationally sensitive monoclonal antibody mAb35, specific for a folded alpha subunit, or a NiNTA affinity resin, specific for a polyhistidine tag engineered on the delta-ECD. Western blotting with subunit specific antibodies confirmed the co-expression of each ECD and furthermore, indicated that the alpha, beta and gamma-ECDs were being co-purified with the polyhistidine-tagged delta-ECD. Chemical cross-linking was used to show that these co-purified proteins had indeed interacted specifically to form soluble oligomeric complexes. A low-resolution, three-dimensional image of these purified complexes, composed only of ECDs, was obtained by electron microscopy. They were shown to resemble the extracellular vestibule of the native receptor, having the same pseudo-pentameric symmetry, size and shape. Expression of incomplete sets of the four nicotinic acetylcholine receptor ECDs did not yield detectable complexes.     

The calcineurin inhibitor tacrolimus activates the renal sodium chloride cotransporter to cause hypertension

Calcineurin inhibitors (CNIs) are immunosuppressive drugs that are used widely to prevent rejection of transplanted organs and to treat autoimmune disease. Hypertension and renal tubule dysfunction, including hyperkalemia, hypercalciuria and acidosis, often complicate their use. These side effects resemble familial hyperkalemic hypertension, a genetic disease characterized by overactivity of the renal sodium chloride cotransporter (NCC) and caused by mutations in genes encoding WNK kinases. We hypothesized that CNIs induce hypertension by stimulating NCC. In wild-type mice, the CNI tacrolimus caused salt-sensitive hypertension and increased the abundance of phosphorylated NCC and the NCC-regulatory kinases WNK3, WNK4 and SPAK. We demonstrated the functional importance of NCC in this response by showing that tacrolimus did not affect blood pressure in NCC-knockout mice, whereas the hypertensive response to tacrolimus was exaggerated in mice overexpressing NCC. Moreover, hydrochlorothiazide, an NCC-blocking drug, reversed tacrolimus-induced hypertension. These observations were extended to humans by showing that kidney transplant recipients treated with tacrolimus had a greater fractional chloride excretion in response to bendroflumethiazide, another NCC-blocking drug, than individuals not treated with tacrolimus; renal NCC abundance was also greater. Together, these findings indicate that tacrolimus-induced chronic hypertension is mediated largely by NCC activation, and suggest that inexpensive and well-tolerated thiazide diuretics may be especially effective in preventing the complications of CNI treatment.     

Leaf water relations and anatomy of a tropical rainforest tree species vary with crown position

Summary Leaf water potentials, osmotic properties and structural characteristics were examined in the Australian tropical rainforest tree species, Castanospermum australe. These features were compared for individuals growing in the understorey and canopy of the undisturbed forest and in an open pasture from which the forest had been cleared. Leaf water potentials during the day declined to significantly lower values in the open-grown and canopy trees than in the understorey trees. During most of the day the opengrown tree experienced the lowest water potentials. These differences were paralleled by significant differences in tissue osmotic properties. The tissue osmotic potential at full hydration was lowest in the open-grown tree (-1.80 MPa), intermediate in the canopy trees (-1.38 MPa), and highest in the understorey trees (-0.80 MPa). As a result, the degree to which high and positive turgor pressures were maintained as water potentials declined was highest in the open-grown tree, intermediate in the canopy trees, and lowest in the understorey trees. The differences in tissue osmotic properties between individuals in the three crown positions were paralleled, in turn, by differences in leaf structual characteristics. Relative to leaves of the canopy and open-grown trees, leaves of the understorey trees had significantly larger epidermal cells with thinner cell walls, larger specific leaf areas and turgid weight: dry weight ratios, and a higher proportion of intercellular air space.Abbreviations ₁ Leaf tissue water potential - _min Lowest value of ₁ during the day ( noon) - _{P=0 1} zero turgor - R Relative water content - P Tissue turgor pressure - Tissue osmotic potential - ₀ at full hydration     

The Croonian Lecture 2000. Nicotinic acetylcholine receptor and the structural basis of fast synaptic transmission

Communication in the nervous system takes place at chemical and electrical synapses, where neurotransmitter-gated ion channels, such as the nicotinic acetylcholine (ACh) receptor, and gap junction channels control propagation of electrical signals from one cell to the next. Newly developed electron crystallographic methods have revealed the structures of these channels trapped in open as well as closed states, suggesting how they work. The ACh receptor has large vestibules extending from the membrane which shape the ACh-binding pockets and facilitate selective transport of cations across a narrow membrane-spanning pore. When ACh enters the pockets it triggers a concerted conformational change that opens the pore by destabilizing a gate in the middle of the membrane made by a ring of pore-lining alpha-helical segmets. The alternative 'open' configuration of pore-lining segments reshapes the lumen and creates new surfaces, allowing the ions to pass through. The gap junction channel uses a similar structural mechanism, involving coordinated rearrangements of alpha-helical segments in the plane of the membrane, to open its pore.     

The use of isobaric tag peptide labeling (iTRAQ) and mass spectrometry to examine rare,primitive hematopoietic cells from patients with chronic myeloid leukemia

Chronic Myeloid Leukemia (CML) is a hematopoietic stem cell disease, associated with a t(9, 22) chromosomal translocation leading to formation of the BCR/ABL chimeric protein, which has an intrinsic tyrosine kinase activity. Recently, the BCR/ABL tyrosine kinase inhibitor imatinib mesylate (imatinib) has been successfully used clinically, although, disease relapse can still occur. The precise detail of the mechanism by which CML cells respond to imatinib is still unclear. We therefore systematically examined the effects of imatinib on the primitive CML cell proteome, having first established that the drug inhibits proliferation and induces increased apoptosis and differentiation. To define imatinib-induced effects on the CML proteome, we employed isobaric tag peptide labeling (iTRAQ) coupled to two-dimensional liquid chromatography/tandem mass spectrometry. Given the limited clinical material available, the isobaric tag approach identified a large population of proteins and provided relative quantification on four samples at once. Novel consequences of the action of imatinib were identified using this mass spectrometric approach. DEAD-box protein 3, heat shock protein 105 kDa, and peroxiredoxin-3 were identified as potential protein markers for response to imatinib. Electronic Supplementary Material The online version of this article (doi: ) contains supplementary material, which is available to authorized users. Stephen D. Griffiths and John Burthem contributed equally to this publication. This work is supported by The Leukaemia Research Fund (UK).     

Variation in heat shock proteins within tropical and desert species of poeciliid fishes

The 70-kilodalton heat shock protein (hsp70) family of molecular chaperones, which contains both stress-inducible and normally abundant constitutive members, is highly conserved across distantly related taxa. Analysis of this protein family in individuals from an outbred population of tropical topminnows, Poeciliopsis gracilis, showed that while constitutive hsp70 family members showed no variation in protein isoforms, inducibly synthesized hsp70 was polymorphic. Several species of Poeciliopsis adapted to desert environments exhibited lower levels of inducible hsp70 polymorphism than the tropical species, but constitutive forms were identical to those in P. gracilis, as they were in the confamilial species Gambusia affinis. These differences suggest that inducible and constitutive members of this family are under different evolutionary constraints and may indicate differences in their function within the cell. Also, northern desert species of Poeciliopsis synthesize a subset of the inducible hsp70 isoforms seen in tropical species. This distribution supports the theory that ancestral tropical fish migrated northward and colonized desert streams; the subsequent decrease in variation of inducible hsp70 may have been due to genetic drift or a consequence of adaptation to the desert environment. Higher levels of variability were found when the 30- kilodalton heat shock protein (hsp30) family was analyzed within different strains of two desert species of Poeciliopsis and also in wild-caught individuals of Gambusia affinis. In both cases the distribution of hsp30 isoform diversity was similar to that seen previously with allozyme polymorphisms.