the efficacy at 10 µM in whole-cell and RVD experiments and the corresponding calculated Hill coefficient for all inhibitors. Indeed, the results of qPCR experiments showed that CFTR, TMEM16A and TMEM16B are not expressed in HEK-293 cells. These cells mainly expressed LRRC8A and LRRC8D, which form VRAC, and three members of TMEM16: TMEM16E (ANO5), TMEM16F (ANO6) and TMEM16K (ANO10). Little is known about TMEM16E and TMEM16K ( Pedemonte and Galietta, 2014 ). TMEM16E does not form an ion channel, whereas TMEM16K has been proposed to be a chloridechannel, although it seems to be localized in an intracellular compartment. TMEM16F has been reported to act both as a scramblase and as a chloridechannel that is activated by micromolar concentrations of calcium ( Shimizu et al., 2013 ). Although patch-clamp recording confirmed the absence of CFTR and CaCC conductance in our experiments, we cannot exclude the possibility that TMEM16F functions as a CaCC in HEK-293 RVD measurements. Indeed, Juul et al. (2014) first showed that TMEM16F differs from VRAC but supports volume regulation in the presence of calcium. A recent study confirmed the role of TMEM16F in volume regulation and proposed that it could be an osmosensor that is in close proximity to VRAC ( Sirianant et al., 2016a ).
conﬁrmed the increase in ANO1 channel activity in the cells transfected with ANO1 TSB (P < 0.05; Student’s t-test) (Fig. 4 c). Interestingly, in cells transfected with ANO1 TSB, the ANO1 channel activity was similar to that observed in non-CF cells. The speciﬁcity of ANO1 activity was conﬁrmed in an experiment using speciﬁc siRNA-ANO1 14 and by sh-ANO1 in a CF cell line (Supplementary Fig. 15 ). Finally, the cell migration rate in cells transfected with ANO1 TSB was signiﬁcantly higher than that in TSB control cells (Fig. 4 d). Together, these results indicated that ANO1 TSB could speciﬁcally target ANO1 and modulate its channel activity and that it plays a role in cell migration. TSB increases de ﬁcient parameters in primary human CF cells. To mimic in vivo epithelium, we used primary human bronchial epithelial cells (hAECB) isolated from bronchial biopsies from CF (F508del/F508del) patients and fully differentiated ALI cultures of these cells. We successfully transfected the cells by adding med- ium containing ANO1 TSB or TSB control to the apical face of ALI cultures, without the use of any transfection reagent (Fig. 5 a, b; Supplementary Movie 3 ). After 2 h incubation at 37 °C, the medium was removed from the apical face to restore the ALI conditions. Freshly prepared control oligonucleotides or ANO1 TSB were added on three consecutive days, and the effects of the transfection were observed 24 h post treatment. Western blot results showed a signiﬁcant 68% increase in ANO1 expression in ANO1 TSB-transfected cells as compared to the control (Fig. 5 c), while the chloridechannel activity was increased 1.7-fold in these cells (Fig. 5 d). Further, we noted a 2.2-fold increase in the migration rate of primary CF cells transfected with ANO1 TSB (Fig. 5 e; Supplementary Movies 4 and 5 ). Additionally, we studied the mucus dynamics using the movement of ﬂuorescent beads (Fig. 5 f; Supplementary Movies 6 and 7 ). Interestingly, the average speed of movement of the beads on ANO1 TSB- expressing cells was higher than that on TSB control-treated cells. Thus, the utilization of ANO1 TSB in primary CF cells allowed improving the chloride activity, migration rate, and mucus dynamics of the cells, suggesting that ANO1 TSB has therapeutic potential for patients.
The Rockefeller University Press $30.00 J. Gen. Physiol. 2016 Vol. 148 No. 3 213–226
I N T R O D U C T I O N
It is widely acknowledged that ClC-Kb in humans (ClC- K2 in rodents), in association with the regulatory sub- unit barttin, is the main basolateral chloridechannel in the distal nephron and is therefore of prime impor- tance in NaCl absorption, body salt homeostasis, and possibly long-term blood pressure regulation (Jentsch, 2008; Fahlke and Fischer, 2010; Staruschenko, 2012; Eladari et al., 2014; Andrini et al., 2015; Sepúlveda et al., 2015). Bartter’s syndrome type III, a rare salt- and potassium-losing tubulopathy that targets the thick as- cending limb (TAL) and the distal convoluted tubule (DCT), is caused by loss-of-function CLC KNB muta- tions leading to impairment of NaCl balance and hypo- kalemic metabolic alkalosis (Krämer et al., 2008; Stölting et al., 2014; Andrini et al., 2015). The recent description of a severe Bartter’s syndrome in Clcnk2 −/− mice ascertained that ClC-K2 plays a similar role in the mouse (Hennings et al., 2016).
ly 20 mV more depolarized values of RMP compared to E GABA-A conﬁrm
similar hyperpolarizing effects of GABAergic transmission in electrically mature motoneurons of both genotypes as reported in spinal cord de- velopmental studies ( Delpy et al., 2008; Stil et al., 2009 ). Quantitatively, there is a factor of 10 in the fold change in KCC2 transcripts expression in spinal cord compared to isolated motoneurons, which could be relat- ed to the presence of interneurons in spinal cord and suggests that reg- ulation of KCC2 expression is more moderate in motoneurons than in interneurons or dorsal horn. KCC2 protein expression increases until the end of the ﬁrst post-natal week both in ventral and dorsal horn ( Stil et al., 2009 ) and the chloride switch in rat dorsal horn occurs at P0–P1 in 60% of neurons and is completed by P7 ( Baccei and Fig. 4. Deletion of Slc12a6 does not affect the developmental chloride shift. (A) Quantitative PCR of KCC3 transcript expression in the spinal cord at embryonic day 12.5 (E12.5), postnatal day 1 (P1), P15 and young adult (P30) (n = 3 for each group) shows a signiﬁcant upregulation of KCC3 transcript during spinal cord development (one-way ANOVA; *P b 0.05). (B) Representative RT-PCRs showing the presence of KCC3 transcript isoforms in P30 adult spinal cord (SC), P15 and P30 tibialis anterior muscle and cultured motoneurons at 7 days in vitro (DIV). Spinal cord and motoneurons show preferential expression of the exon 2-deleted KCC3a transcript isoform while both KCC3a transcript isoforms (FL KCC3a = full length KCC3a; Δ2-KCC3a = exon-2-deleted) are similarly expressed within muscle. KCC3b transcript is not expressed in motoneurons. (C) Quantitative PCR of KCC2 and (D) NKCC1 transcripts was performed on the lumbar spinal cord of E12.5, P1 and P30 Slc12a6 +/+ and Slc12a6 −/− mice and expressed relative to E12.5. For KCC2 at P1: 15.9 ± 2.0 fold increase
Fig. 9A shows representative cell-attached current recordings from HEK-293 cells transfected with V166E mClC-K1/mBarttin channels. The mean current-voltage relationship was essentially linear (Fig. 8B) and reversed at -1.4 ± 2.1 mV; a unit conductance of 19.9 ± 1.3 pS was computed (n = 4). Channel activity increased at positive voltages (n = 4) (Fig. 8C). The best fit of our data indicated that the half-maximal activation voltage of the mutant channel was shifted to more positive potentials as compared to WT mClC-K1/mBarttin (V1/2 = 24.9 mV). Voltage dependence persisted in the cell-free mode (not shown). In contrast, two other point mutations introduced at position 166 did not have the same effect as V166E. The V166L mutation did not significantly modify the unit conductance (38.5 ± 4.0 pS, n = 3; not shown), and the V166M only moderately modified the unit conductance (38.5 and 29.5 pS, n = 2; not shown). We can therefore reasonably conclude that the V166E mutation revealed the activity of the channel protopore by introducing de novo protopore gating.
First, Section 3.2 presents the transmission model, then Section 3.3 describes the classical minimum MSE solution under a total power constraint, proposed in [LP76], with an alterna- tive proof involving majorization techniques advocated by [PCL03, PLC04, PJ07], where the source components were assumed all with unit variance. In our LVC case, we extend this re- sult to source components with dierent variances. The solution of this rst problem is then used in Section 3.4 to address the design of the precoding matrix minimizing the MSE under per-subchannel power constraints. In Section 3.6, the advantage of the proposed methods com- paring respectively to [JK10b] under total power constraint and to [LP76] under per-subchannel power constraint is shown. Moreover in Section 3.6.5 the robustness of the proposed scheme to mismatched channel characteristics un has also been analyzed. We consider a multi-user sce- nario, where the transmitter uses a common precoding matrix for the transmission to dierent users.
Among this large enzyme family, one group is allosteri- cally activated by the binding of a chloride ion. These chlo- ride-dependent ␣-amylases are specific to all animals (ver- tebrates and invertebrates) but are also found in some gram- negative bacteria such as Pseudoalteromonas haloplanktis, formerly Alteromonas haloplanctis (D’Amico et al. 2000). Both human salivary and pancreatic ␣-amylases belong to this group, which is subjected to intensive investigations for fundamental, therapeutic, or diagnostic purposes. Although significant advances in the elucidation of the reaction mechanism have been made recently (McCarter and With- ers 1996; Uitdehaag et al. 1999; Brayer et al. 2000; Aghajari et al. 2002), the function of the chloride ion remains unclear. In all chloride-dependent crystal structures solved so far— namely, ␣-amylases from pig pancreas (Qian et al. 1993; Larson et al. 1994), human salivary (Ramasubbu et al. 1996) and pancreatic (Brayer et al. 1995) glands, the beetle Tene-
sidered as the growth of the thickness of the nonbearing zone along the whole length of the tensile specimens. Figure 15 gives a descrip- tion of this approach: due to specimen geometry, the decrease of the bearing area was assumed to be essentially related to the propaga- tion of intergranular corrosion on the large sides of the section. From this description of the corrosion damage and the results given in Fig. 12, it was then possible to calculate the thickness of the corroded zone, called x 共t兲, vs the time of immersion in chloride solution. As a consequence of this type of approach, it was assumed that the stress to failure fail 共ultimate stress兲 was a constant char- acteristic of the material. Thus, for an uncorroded specimen, this constant is given by the following equation
3.2. Influence of cracking on the steady-state migration test
Fig. 4 is a schematic representation of the total increase of chloride concentration in the anode solution, with elapsed time. Two distinct phases with respect to the change of chloride concentration are acknowledged. The first is a phase where chloride ions are in the process of migrating through saturated pores in concrete and hence have not yet reached the anode compartment. This phase is normally called a transition period and the migration of chloride ions is under non steady-state conditions. The second phase is called the steady-state period where the increase of chloride concentration becomes constant with elapsed time, providing a constant flux of chloride ions. In order to calculate the diffusion coefficient of chloride ions for concrete under test, the flux J is calculated with the constant slope of a regression line (Eq. (3)):
b Institut Universitaire de Technologie, Universite´ Paul Sabatier-av. Georges
Pompidou 81104 Castres, France
M unicipal solid waste incinerator by products include fly ash and air pollution control residues. In order to transform these incinerator wastesintoreusablemineralspecies,solublealkalichloridesmustbeseparatedandtoxictraceelementsmustbestabi- lized ininsolubleform.Weshowthatalkalichloridescanbeextractedeﬃcientlyinanaqueousextractionstepcombiningacalcium phosphate gelprecipitation.Insuchaprocess,sodiumandpotassiumchloridesareobtainedfreefromcalciumsalts,andthetrace metal ions are immobilized in the calcium phosphate matrix. M oderate calcination of the chemically treated fly ash leads to the formation ofcristallinehydroxylapatite.Flyashspikedwithcopperionsandtreatedbythisprocessshowsimprovedstabilityof metal ions.LeachingtestswithwaterorEDTArevealasignificantdropinmetaliondissolution.Hydroxylapatitemaytraptoxic metals andalsopreventtheir evaporationduringthermaltreatments.Incineratorflyashtogetherwithairpollution controlresi- dues, treatedbythecombinedchlorideextractionandhydroxylapatiteformationprocessmaybeconsideredsafetouseasamineral filler invalueaddedproductssuchasroadbaseorcementblocks.
1 Unité de Recherche sur les Maladies Cardiovasculaires, le Métabolisme et la Nutrition, Faculté de Médecine
Pitié-Salpêtrière, Sorbonne Universités, UPMC Univ. Paris VI, Inserm, UMRS 1166, Université Pierre et Marie Curie, Paris, France, 2 Department of Cardiovascular Medicine, John Radcliffe Hospital, University of Oxford, Oxford, United Kingdom
The shape of the cardiac action potential (AP) is determined by the contributions of numerous ion channels. Any dysfunction in the proper function or expression of these ion channels can result in a change in effective refractory period (ERP) and lead to arrhythmia. The processes underlying the correct targeting of ion channels to the plasma membrane are complex, and have not been fully characterized in cardiac myocytes. Emerging evidence highlights ion channel trafficking as a potential causative factor in certain acquired and inherited arrhythmias, and therapies which target trafficking as opposed to pore block are starting to receive attention. In this review we present the current evidence for the mechanisms which underlie precise control of cardiac ion channel trafficking and targeting.
Role of cAMP and vasopressin in ADPKD
Increased concentrations of cAMP play a central role in the pro- gression of renal cystic disease in ADPKD ( Fig. 1 ). Stimulation of the V2R by the antidiuretic hormone arginine vasopressin (AVP) is the major source of cAMP production in the collecting ducts [ 101 ]. Hap- loinsu ﬃciency in polycystin-1 in Pkd1 +/− mice is associated with ex- cessive AVP signaling, causing increased phosphorylation and recruit- ment of aquaporin-2 water channels and inappropriate antidiuresis [ 102 ]. The decreased intracellular Ca 2+ concentration secondary to PKD1/2 mutations may downregulate the Ca 2+ -dependent phospho- diesterase PDE1 and stimulate the Ca 2+ inhibitable adenylyl cyclase 6 (AC6), causing an increase in cAMP levels. In turn, increased cAMP stimulates proliferation and growth of ADPKD cells and drives transe- pithelial chloride and ﬂuid secretion, through activation of protein ki- nase A (PKA) and pathways including the mitogen-activated protein kinase (MAPK) kinase MEK and the extracellular signal-regulated ki- nase ERK, mTOR and Wnt-β-catenin, STAT3 and possibly PAX2 sig- naling (25).
Electrochemical Deoxidation of Titanium Foam in Molten Calcium Chloride
PRABHAT K. TRIPATHY, MAXIME GAUTHIER, and DEREK J. FRAY
Titanium foam, prepared by using a patented powder-metallurgy–based process involving a powder blend that was molded, foamed, and sintered using a three-step thermal treatment, was deoxidized in a molten CaCl 2 bath. The polarization experiments were carried out by cathod- ically polarizing the foam (working electrode) against a counter (graphite) electrode. Under constant potential (polarization) mode, the dominant mechanism of deoxidation was the ioni- zation of oxygen, present in the foam, and its subsequent discharge, as CO 2 /CO, at the anode surface. More than ~85 pct oxygen could be eﬀectively removed by carrying out the electro- deoxidation experiments in fresh and pre-electrolyzed melt(s) at an electrolyte temperature of 950 C. Scanning electron microscopy–energy dispersive X-ray (SEM-EDX) detection of the deoxidized foams did not show a presence of any inclusion(s) or secondary phase(s).
These results may explain the higher eﬃciency of ionic plasticizers (compared to glycerol) for starch destructuration/thermoplasticization which leads to the complete disappearance of the native A-type crys- tallinity (Fig. 1b, Table 1) after extrusion. However, in contradiction to the work of Sciarini et al. (Sciarini et al., 2015) on starches in ILs so- lutions, it seems that during extrusion, the presence of chloride anions do not produce a higher chain scission in starches than with acetate anions. Actually the lower molar masses are here observed for the two bio-ILs: [Chol][Ace] and [Chol][Lac] (Table 2). So unexpectedly, the use of [Chol][Cl] allows a better compromise between an e ﬃcient