NAD binding induces conformational changes in Rho ADP-ribosylating clostridium botulinum C3 exoenzyme

We have solved the crystal structures of Clostridium botulinum C3 exoenzyme free and complexed to NAD in the same crystal form, at 2.7 and 1.95 A, respectively. The asymmetric unit contains four molecules, which, in the free form, share the same conformation. Upon NAD binding, C3 underwent various conformational changes, whose amplitudes were differentially limited in the four molecules of the crystal unit. A major rearrangement concerns the loop that contains the functionally important ARTT motif (ADP-ribosyltransferase toxin turn-turn). The ARTT loop undergoes an ample swinging motion to adopt a conformation that covers the nicotinamide moiety of NAD. In particular, Gln-212, which belongs to the ARTT motif, flips over from a solvent-exposed environment to a buried conformation in the NAD binding pocket. Mutational experiments showed that Gln-212 is neither involved in NAD binding nor in the NAD-glycohydrolase activity of C3, whereas it plays a critical role in the ADP-ribosyl transfer to the substrate Rho. We observed additional NAD-induced movements, including a crab-claw motion of a subdomain that closes the NAD binding pocket. The data emphasized a remarkable NAD-induced plasticity of the C3 binding pocket and suggest that the NAD-induced ARTT loop conformation may be favored by the C3-NAD complex to bind to the substrate Rho. Our structural observations, together with a number of mutational experiments suggest that the mechanisms of Rho ADP-ribosylation by C3-NAD may be more complex than initially anticipated.     

Mutations in SLC2A2 Gene Reveal hGLUT2 Function in Pancreatic β Cell Development

The structure-function relationships of sugar transporter-receptor hGLUT2 coded by SLC2A2 and their impact on insulin secretion and β cell differentiation were investigated through the detailed characterization of a panel of mutations along the protein. We studied naturally occurring SLC2A2 variants or mutants: two single-nucleotide polymorphisms and four proposed inactivating mutations associated to Fanconi-Bickel syndrome. We also engineered mutations based on sequence alignment and conserved amino acids in selected domains. The single-nucleotide polymorphisms P68L and T110I did not impact on sugar transport as assayed in Xenopus oocytes. All the Fanconi-Bickel syndrome-associated mutations invalidated glucose transport by hGLUT2 either through absence of protein at the plasma membrane (G20D and S242R) or through loss of transport capacity despite membrane targeting (P417L and W444R), pointing out crucial amino acids for hGLUT2 transport function. In contrast, engineered mutants were located at the plasma membrane and able to transport sugar, albeit with modified kinetic parameters. Notably, these mutations resulted in gain of function. G20S and L368P mutations increased insulin secretion in the absence of glucose. In addition, these mutants increased insulin-positive cell differentiation when expressed in cultured rat embryonic pancreas. F295Y mutation induced β cell differentiation even in the absence of glucose, suggesting that mutated GLUT2, as a sugar receptor, triggers a signaling pathway independently of glucose transport and metabolism. Our results describe the first gain of function mutations for hGLUT2, revealing the importance of its receptor versus transporter function in pancreatic β cell development and insulin secretion.     

Influence of the magnetic fields on frog sciatic nerve

The constant magnetic field (1000–7120 gauss) was applied to previously stimulated frog sciatic nerve. The following was observed : a) There is no instantaneous effect of either parallel or perpendicular magnetic field on compound action potential amplitude. b) Parallel magnetic field of 1000–7120 gauss does not change the amplitude of compound action potential significantly with time. c) When perpendicular magnetic field was applied to the nerve, an increase in the amplitude of compound action potential was observed, which can mean that the nerve exhibits some sort of magnetic anisotropy.     

Effects of internal ph on the nonselective cation channel from the mouse collecting tubule

We investigated the effects of internal pH on Ca-activated, nucleotide-inhibited nonselective cation channels in the basolateral membranes of mouse collecting tubules, using the inside-out variant of the patch clamp technique. pH modulated the channel open probability (P _o ), giving a bell-shaped curve peaking at pH 6.8/7.0: P _o at pH 6.0 was 11±6% of P _o at pH 7.2 and 32 ±7% at pH 8.0. The open and closed time distributions, best fitted to the sum of two exponentials, were differently sensitive to acid and alkaline conditions. Low pH reduced the short and long open times to 38 and 24% of their pH 7.2 values, while high pH produced a 4-fold increase in the long closed time. As previously reported, 4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid (SITS) induced a quasi-permanent opening of the channel. The inhibition of the channel produced by high pH disappeared in the presence of SITS, while the inhibition produced by low pH was unaffected. These results suggest that the pH dependence of the channel is due to two separate mechanisms. pH was without effect on the ATP-evoked inhibition of the channel, while high pH profoundly reduced the steepness of the AMP inhibition curve, without altering the half-maximal inhibitory AMP concentration.     

Identification and Functional Expression of a Glutamate- and Avermectin-Gated Chloride Channel from Caligus rogercresseyi,a Southern Hemisphere Sea Louse Affecting Farmed Fish

Parasitic sea lice represent a major sanitary threat to marine salmonid aquaculture, an industry accounting for 7% of world fish production. Caligus rogercresseyi is the principal sea louse species infesting farmed salmon and trout in the southern hemisphere. Most effective control of Caligus has been obtained with macrocyclic lactones (MLs) ivermectin and emamectin. These drugs target glutamate-gated chloride channels (GluCl) and act as irreversible non-competitive agonists causing neuronal inhibition, paralysis and death of the parasite. Here we report the cloning of a full-length CrGluClα receptor from Caligus rogercresseyi. Expression in Xenopus oocytes and electrophysiological assays show that CrGluClα is activated by glutamate and mediates chloride currents blocked by the ligand-gated anion channel inhibitor picrotoxin. Both ivermectin and emamectin activate CrGluClα in the absence of glutamate. The effects are irreversible and occur with an EC₅₀ value of around 200 nM, being cooperative (n_H = 2) for ivermectin but not for emamectin. Using the three-dimensional structure of a GluClα from Caenorabditis elegans, the only available for any eukaryotic ligand-gated anion channel, we have constructed a homology model for CrGluClα. Docking and molecular dynamics calculations reveal the way in which ivermectin and emamectin interact with CrGluClα. Both drugs intercalate between transmembrane domains M1 and M3 of neighbouring subunits of a pentameric structure. The structure displays three H-bonds involved in this interaction, but despite similarity in structure only of two these are conserved from the C. elegans crystal binding site. Our data strongly suggest that CrGluClα is an important target for avermectins used in the treatment of sea louse infestation in farmed salmonids and open the way for ascertaining a possible mechanism of increasing resistance to MLs in aquaculture industry. Molecular modeling could help in the design of new, more efficient drugs whilst functional expression of the receptor allows a first stage of testing of their efficacy.     

Novel 3-(4-piperidinylthio)-1H-indoles as potent nonopioid orally active central analgesics

A series of 3-(4-piperidinylthio)-1H-indoles was synthesized and evaluated in mice in the phenylbenzoquinone(PBQ)-induced writhing and hot plate tests. Most of these compounds are good analgesics with activities comparable to that of morphine. Among them compound 1i (UP 237-61), which has a strong serotonin binding profile, has an interesting antinociceptive activity which is not reversed by naloxone.     

A Na+- and Cl- -activated K+ channel in the thick ascending limb of mouse kidney

This study investigates the presence and properties of Na+-activated K+ (K(Na)) channels in epithelial renal cells. Using real-time PCR on mouse microdissected nephron segments, we show that Slo2.2 mRNA, which encodes for the K(Na) channels of excitable cells, is expressed in the medullary and cortical thick ascending limbs of Henle's loop, but not in the other parts of the nephron. Patch-clamp analysis revealed the presence of a high conductance K+ channel in the basolateral membrane of both the medullary and cortical thick ascending limbs. This channel was highly K+ selective (P(K)/P(Na) approximately 20), its conductance ranged from 140 to 180 pS with subconductance levels, and its current/voltage relationship displayed intermediate, Na+-dependent, inward rectification. Internal Na+ and Cl- activated the channel with 50% effective concentrations (EC50) and Hill coefficients (nH) of 30 +/- 1 mM and 3.9 +/- 0.5 for internal Na+, and 35 +/- 10 mM and 1.3 +/- 0.25 for internal Cl-. Channel activity was unaltered by internal ATP (2 mM) and by internal pH, but clearly decreased when internal free Ca2+ concentration increased. This is the first demonstration of the presence in the epithelial cell membrane of a functional, Na+-activated, large-conductance K+ channel that closely resembles native K(Na) channels of excitable cells. This Slo2.2 type, Na+- and Cl--activated K+ channel is primarily located in the thick ascending limb, a major renal site of transcellular NaCl reabsorption.     

A calcium-permeable non-selective cation channel in the thick ascending limb apical membrane of the mouse kidney

Non-selective cation channels have been described in the basolateral membrane of the renal tubule, but little is known about functional channels on the apical side. Apical membranes of microdissected fragments of mouse cortical thick ascending limbs were searched for ion channels using the cell-free configuration of the patch-clamp technique. A cation channel with a linear current-voltage relationship (19pS) that was permeable both to monovalent cations [P(NH4)(1.7)>P(Na) (1.0)=P(K) (1.0)] and to Ca(2+) (P(Ca)/P(Na)≈0.3) was detected. Unlike the basolateral TRPM4 Ca(2+)-impermeable non-selective cation channel, this non-selective cation channel was insensitive to internal Ca(2+), pH and ATP. The channel was already active after patch excision, and its activity increased after reduced pressure was applied via the pipette. External gadolinium (10(-5)M) decreased the channel-open probability by 70% in outside-out patches, whereas external amiloride (10(-4)M) had no effect. Internal flufenamic acid (10(-4)M) inhibited the channel in inside-out patches. Its properties suggest that the current might be supported by the TRPM7 protein that is expressed in the loop of Henle. The conduction properties of the channel suggest that it could be involved in Ca(2+) signaling.     

Canonical signal recognition particle components can be bypassed for posttranslational protein targeting in chloroplasts

The chloroplast signal recognition particle (cpSRP) and its receptor (cpFtsY) target proteins both cotranslationally and posttranslationally to the thylakoids. This dual function enables cpSRP to utilize its posttranslational activities for targeting a family of nucleus-encoded light-harvesting chlorophyll binding proteins (LHCPs), the most abundant membrane proteins in plants. Previous in vitro experiments indicated an absolute requirement for all cpSRP pathway soluble components. In agreement, a cpFtsY mutant in Arabidopsis thaliana exhibits a severe chlorotic phenotype resulting from a massive loss of LHCPs. Surprisingly, a double mutant, cpftsy cpsrp54, recovers to a great extent from the chlorotic cpftsy phenotype. This establishes that in plants, a new alternative pathway exists that can bypass cpSRP posttranslational targeting activities. Using a mutant form of cpSRP43 that is unable to assemble with cpSRP54, we complemented the cpSRP43-deficient mutant and found that this subunit is required for the alternative pathway. Along with the ability of cpSRP43 alone to bind the ALBINO3 translocase required for LHCP integration, our results indicate that cpSRP43 has developed features to function independently of cpSRP54/cpFtsY in targeting LHCPs to the thylakoid membranes.     

Piezo1‐dependent stretch‐activated channels are inhibited by Polycystin‐2 in renal tubular epithelial cells

Mechanical forces associated with fluid flow and/or circumferential stretch are sensed by renal epithelial cells and contribute to both adaptive or disease states. Non‐selective stretch‐activated ion channels (SACs), characterized by a lack of inactivation and a remarkably slow deactivation, are active at the basolateral side of renal proximal convoluted tubules. Knockdown of Piezo1 strongly reduces SAC activity in proximal convoluted tubule epithelial cells. Similarly, overexpression of Polycystin‐2 (PC2) or, to a greater extent its pathogenic mutant PC2‐740X, impairs native SACs. Moreover, PC2 inhibits exogenous Piezo1 SAC activity. PC2 coimmunoprecipitates with Piezo1 and deletion of its N‐terminal domain prevents both this interaction and inhibition of SAC activity. These findings indicate that renal SACs depend on Piezo1, but are critically conditioned by PC2.