Cryo-EM structure of the human NKCC1 transporter reveals mechanisms of ion coupling and specificity

The sodium-potassium-chloride transporter NKCC1 (SLC12A2) performs Na+-dependent Cl- and K+ ion uptake across plasma membranes. NKCC1 is important for regulating e.g. cell volume, hearing, blood pressure, and chloride gradients defining GABAergic and glycinergic signaling in brain. Here, we present a 2.6 Å resolution cryo-electron microscopy (cryo-EM) structure of human NKCC1 in the substrate-loaded (Na+, K+, 2 Cl-) and inward-facing conformation adopting an occluded state that has also been observed for the SLC6 type transporters MhsT and LeuT. Cl- binding at the Cl1 site together with the nearby K+ ion provide a crucial bridge between the LeuT-fold scaffold and bundle domains. Cl- ion binding at the Cl2 site seems to undertake a structural role similar to a conserved glutamate of SLC6 transporters and may allow for chloride-sensitive regulation of transport. Supported by functional studies in mammalian cells and computational simulations we describe the Na+ binding site and a putative Na+ release pathway along transmembrane helix 5. The results provide insight into the structure-function relationship of NKCC1 with broader implications for other SLC12 family members.

The beryllium fluoride form of the Na+,K+-ATPase and the intermediates of the functional cycle

The Na+,K+-ATPase generates electrochemical gradients of Na+ and K+ across the plasma membrane. Here, we describe a 4.0 Å resolution crystal structure of the pig kidney Na+,K+-ATPase stabilized by beryllium fluoride (denoted E2-BeFx). The structure shows high resemblance to the E2P phosphoenzyme obtained by phosphorylation from inorganic phosphate (Pi) and stabilised by cardiotonic steroids, and reveals a Mg2+ bound near the ion binding site II. Anomalous Fourier analysis of the crystals soaked in Rb+ (K+ congener) followed by a low resolution rigid-body refinement (6.9-7.5 Å) revealed pre-occlusion transitions leading to activation of the desphosphorylation reaction. Mg2+ location indicates a site of an initial K+ recognition and acceptance upon binding to the outward-open E2P state after Na+ release. Despite the overall structural resemblance to the Pi-induced E2P phosphoform, BeFx inhibited enzyme is able to binds both ADP/ATP and ions, the features that relate E2-BeFx complex to an intermediate of the functional cycle of the Na+,K+-ATPase prior E2P.

Use of clonidine in the management of opiate withdrawal in community patients

AimsClonidine has been used to alleviate symptoms of opiate withdrawal. No validated prescribing schedules exist for the use of Clonidine in opiate detoxification in community patients. We have devised a Clonidine prescribing schedule for adult outpatients seeking opiate detoxification.BackgroundOpiate cessation following prolonged use produces a central noradrenergic (NA) response in the locus coeruleus (LC), causing symptoms that can result in reinstatement of use. Pharmacotherapies for withdrawal are thought to work through decreased NA release in the LC by agonising pre-synaptic alpha-2 adrenoceptors. Clonidine has been used since the 1970s. However, it is off-license in the UK, and superseded by Lofexidine. Though both cause hypotension, this is less marked with Lofexidine, which may be anxiolytic and considered better tolerated. Lofexidine is no longer available in the UK. Specialists may need to resort to Clonidine for those seeking opiate detoxification.MethodWe performed a feasibility study with the primary outcome being tolerability of an outpatient clonidine schedule. Patients (n = 7) were aged between 18 and 65 years (mean 32). Six were prescribed buprenorphine as opiate substitution (OST), and one methadone.Exclusion criteria were in keeping with BNF contraindications.An ECG was obtained for each patient before treatment. A urine drug screen and Clinical Opiate Withdrawal Scale were taken to confirm opiate dependence and withdrawal. Patients self-monitored withdrawal using the Subjective Opiate Withdrawal Scale and daily blood pressure measurements. Standard adjuvants for withdrawal were prescribed.A test dose of 100mcg Clonidine was given to assess for hypotension. If tolerant they received 100mcg QDS, reducing over eight days.Patients were contacted by their recovery worker twice during the period.ResultFive of the seven completed the course, two dropped out due to hypotension. No other adverse effects warranting discontinuation were encountered. Patients reported fatigue and light-headedness as their most troublesome side-effects. Of 3 patients who returned SOWS scores, 2 reported decline by 21/64 and 14/64 respectively. One reported an increase of 49/64 over 8 days. 3 of the 5 subjects who completed the course were not abstinent at completion, citing opiate withdrawal symptoms as causative.ConclusionThere is scope for the safe use of clonidine in the community for motivated individuals. Adequate monitoring of heart rate and blood pressure is required. Starting doses at 100mcg QDS appear well tolerated. Prescribers may wish to reduce this over a longer period to encourage completion and improve tolerability. Further research is needed.

Vitamin B6 deficiency hyperactivates the noradrenergic system, leading to social deficits and cognitive impairment

We have reported that a subpopulation of patients with schizophrenia have lower levels of vitamin B6 (VB6) in peripheral blood than do healthy controls. In a previous study, we found that VB6 level was inversely proportional to the patient’s positive and negative symptom scale (PANSS) score for measuring symptom severity, suggesting that the loss of VB6 might contribute to the development of schizophrenia symptoms. In the present study, to clarify the relationship between VB6 deficiency and schizophrenia, we generated VB6-deficient (VB6(−)) mice through feeding with a VB6-lacking diet as a mouse model for the subpopulation of schizophrenia patients with VB6 deficiency. After feeding for 4 weeks, plasma VB6 level in VB6(−) mice decreased to 3% of that in control mice. The VB6(−) mice showed social deficits and cognitive impairment. Furthermore, the VB6(−) mice showed a marked increase in 3-methoxy-4-hydroxyphenylglycol (MHPG) in the brain, suggesting enhanced noradrenaline (NA) metabolism in VB6(−) mice. We confirmed the increased NA release in the prefrontal cortex (PFC) and the striatum (STR) of VB6(−) mice through in vivo microdialysis. Moreover, inhibiting the excessive NA release by treatment with VB6 supplementation into the brain and α2A adrenoreceptor agonist guanfacine (GFC) suppressed the increased NA metabolism and ameliorated the behavioral deficits. These findings suggest that the behavioral deficits shown in VB6(−) mice are caused by enhancement of the noradrenergic (NAergic) system.

CALICUM, Mg AND Na RELEASE KINETICS FROM SALINE- SODIC SOIL MIXED WITH SOME AMENDMENTS

This study was aimed to investigate the effect of phosphogypsum and humic acids on the leaching and releasing of salts from saline- sodic soil. A laboratory experiment was conducted in polyethylene columns (60.0 cm and 7.1 cm). The columns were filled with 30 cm soil (EC=73.78 dSm-1). The experiment included two factors, phosphogypsum added at levels 0, 5, 10 and 15 mtons ha-1 with symbols PG0,PG1, PG2 and PG3 respectively, and humic acids were added at levels 0, 50, 100 and 150 kg ha-1 with symbols HA0, HA1, HA2 and HA3, mixing them with the top 5 cm of soil column. The electrical conductivity and the concentrations of water soluble cations (Ca, Mg, and Na) in leachate were determined and sodium adsorption ratio (SAR) was calculated. Results showed that accumulative salts (TDS), sodium released from soil columns increased with increasing the level of addition, whether of phosphogypsum or humic acids. The highest value of salts and sodium released was 682.63 g L-1 and 7086.12 mmol L-1 respectively in the treatment PG3HA3, while the lowest value was 455.94 g L-1 and 3899.40 mmol L-1 respectively in the treatment PG0HA0. Calcium (mmol L-1) increased by increasing the level of phosphogypsum, decreased by increasing the level of humic acids, the highest value of accumulated calcium was 1599.0 mmol L-1 in PG3HA0 while the lowest value was 820.53 mmol L-1 in PG0HA3. The results showed that the best equation for describing release kinetics of sodium adsorption ratio in soil is the diffusion equation. Increasing level of phosphogypsum and humic acids increased the release constant velocity (K) of sodium adsorption ratio.

The Optimum Conditions of Carboxymethyl Chitosan Synthesis on Drug Delivery Application and Its Release of Kinetics Study

In this paper, carboxymethyl chitosan (CMC) was synthesized and studied as a carrier to encapsulate vitamin (as drug model) and controlled release. Chitosan (CS) is a polycationic derivated from chitin, which suitable for active substance carrier system on biomedical function. CS has good properties such as non-toxic, biodegradable, and biocompatible. However, CS insoluble in an aqueous solvent so CS was modified chemically into CMC. CMC was formed by reacting CS and monochloroacetic acid with sodium hydroxide (NaOH) as a catalyst. Optimation was performed by varying the NaOH concentration during alkalizing the CS and the temperature reaction. The functional group and crystallinity of CS and CMC were estimated by FTIR and XRD. The degree substitution of carboxymethylation has an average value of 0.60. The results show optimum temperature reaction and NaOH concentration were 60 °C and 40% (w/v). The nicotinamide (NA), a hydrophilic vitamin, was loaded within CMC matrix system through in vitro precipitation method. To confirm the encapsulation of NA in CMC and the release kinetics of NA from CMC in distilled water was studied through UV-Vis spectrophotometry. The release profile of NA from CMC matrix system carried out for 3 h and 12 h. The rate of NA release from CMC increases with increasing time and the follows a zero order, Higuchi, and Korsmeyer-Peppas kinetics rules.