Effect of modern infusion pumps on RBC quality.

BACKGROUND: Mechanical stress on red blood cells is associated with using infusion pumps for blood administration. Current standards in North America leave it to healthcare facilities to consult with manufacturers about infusion pump safety for transfusion; studies on various pumps and red blood cell (RBC) conditions are scarce. STUDY DESIGN AND METHODS: RBC units were pumped through four infusion pumps on d22 (22 days postcollection), d40, d28 after gamma irradiation on d14 (I14d28), and d22 after irradiation on d21 (I21d22). For each experiment, three units were pooled and split among four bags. Samples were collected at gravity and after pumping at clinical nonemergency rates. Hemolysis %, microvesicles, potassium, lactate dehydrogenase, mechanical fragility index levels, and morphology evaluations were performed (n = 5-6). RESULTS: Hemolysis levels of Piston and Linear Peristaltic pump samples were not different from hemolysis of corresponding gravity samples. Peristaltic samples had significantly higher hemolysis compared to gravity, and other pumps, however, maximum mean difference was limited to 0.05%. Pumping at 50 mL/h resulted in the highest hemolysis level. Change in hemolysis % due to pumping was significantly higher in d40 and I21d22 units. No combination of pumps and RBCs conditions led to hemolysis >0.8%. Besides hemolysis, lactate dehydrogenase release was the only marker that demonstrated some differences between infusions via pump versus gravity. CONCLUSION: The pump design affects the degree of hemolysis. However, for all tested pumps and RBC conditions, this increase was minimal. Hemolysis measurement on d40 and I21d22 at 50 mL/h were concluded to be appropriate parameters for pump evaluation.

Risk analysis of transfusion of cryoprecipitate without consideration of ABO group.

BACKGROUND: Transfusion medicine standards in Canada state that adult recipients can be transfused with cryoprecipitate of any ABO group, however, not all hospitals follow this guideline. There is a paucity of data on cryoprecipitate anti-A/B levels to reinforce standards. STUDY DESIGN AND METHODS: Manual tube antibody titration was performed on 7 units of group O plasma and the corresponding cryosupernatant plasma and cryoprecipitate. IgG/IgM levels were determined by nephelometry. Additionally, 10 cryoprecipitate each from groups A, B, and O were similarly assessed. From the antibody titer distribution among these samples, the probability of making a pool of cryoprecipitate with a titer ≥1:100 was calculated using bootstrap analysis. RESULTS: Anti-A/B titers in cryoprecipitate were equivalent to those in corresponding plasma; partitioning of anti-A/B activity into cryoprecipitate was not observed. Average IgM concentration was higher in cryoprecipitate than in plasma (P < .01). However, no correlation between IgM levels and anti-A/B titers was established. Among 30 cryoprecipitates from routine blood bank inventory, the median antibody titer and mode were 1:32 and 1:16, respectively. Of the samples tested, 4 of 30 and 9 of 30 had titers above 1:100 and 1:50, respectively. The probability of transfusing an adult dose of cryoprecipitate (pool of 10 cryoprecipitate) with a titer higher than 1:100 was calculated to be less than 1 in 3 million. CONCLUSIONS: This study provides strong evidence to support current Canadian transfusion medicine standards on the safety of transfusion of cryoprecipitate without the need for blood group matching in adult recipients.

X-ray structure of a mammalian stearoyl-CoA desaturase.

Stearoyl-CoA desaturase (SCD) is conserved in all eukaryotes and introduces the first double bond into saturated fatty acyl-CoAs. Because the monounsaturated products of SCD are key precursors of membrane phospholipids, cholesterol esters and triglycerides, SCD is pivotal in fatty acid metabolism. Humans have two SCD homologues (SCD1 and SCD5), while mice have four (SCD1-SCD4). SCD1-deficient mice do not become obese or diabetic when fed a high-fat diet because of improved lipid metabolic profiles and insulin sensitivity. Thus, SCD1 is a pharmacological target in the treatment of obesity, diabetes and other metabolic diseases. SCD1 is an integral membrane protein located in the endoplasmic reticulum, and catalyses the formation of a cis-double bond between the ninth and tenth carbons of stearoyl- or palmitoyl-CoA. The reaction requires molecular oxygen, which is activated by a di-iron centre, and cytochrome b5, which regenerates the di-iron centre. To understand better the structural basis of these characteristics of SCD function, here we crystallize and solve the structure of mouse SCD1 bound to stearoyl-CoA at 2.6 Å resolution. The structure shows a novel fold comprising four transmembrane helices capped by a cytosolic domain, and a plausible pathway for lateral substrate access and product egress. The acyl chain of the bound stearoyl-CoA is enclosed in a tunnel buried in the cytosolic domain, and the geometry of the tunnel and the conformation of the bound acyl chain provide a structural basis for the regioselectivity and stereospecificity of the desaturation reaction. The dimetal centre is coordinated by a unique spacial arrangement of nine conserved histidine residues that implies a potentially novel mechanism for oxygen activation. The structure also illustrates a possible route for electron transfer from cytochrome b5 to the di-iron centre.

Multiple transcriptomic analyses and characterization of pathogen-related core effectors and LysM family members reveal their differential roles in fungal growth and pathogenicity in Penicillium expansum.

Penicillium expansum is a destructive phytopathogen causing postharvest decay on many stored fruits. To develop effective and safe management strategies, it is important to investigate its pathogenicity-related mechanisms. In this study, a bioinformatic pipeline was constructed and 50 core effector genes were identified in P. expansum using multiple RNA-seq data sets and their putative functions were implicated by comparatively homologous analyses using pathogen-host interaction database. To functionally characterize P. expansum LysM domain proteins during infection, null mutants for the 15 uncharacterized putative LysM effectors were constructed and the fungal growth rate on either PDA or Cazpek medium or lesion expansion rate on the infected apple fruits was evaluated. The results showed the growth rate of knockout mutants from PeLysM5, PeLysM12 and PeLysM15 was retarded on PDA medium. No significant difference in growth rate was observed between wild type and all mutants on solid Cazpek medium. Nevertheless, the hypha of wild type displayed deeper yellow on the back of Cazpek medium than those of knockout mutants. On the infecting apples fruits, the knockout mutants from PeLysM5, PeLysM7, PeLysM8, PeLysM9, PeLysM10, PeLysM11, PeLysM14, PeLysM15, PeLysM16, PeLysM18 and PeLysM19 showed enhanced fungal virulence, with faster decaying on infected fruits than those from wild type. By contrast, the knockout mutation at PeLysM12 locus led to reduced lesion expansion rate on the infected apple fruits. In addition, P. expansum-apple interaction RNA-seq experiment was performed using apple fruit tissues infected by the wild type and knockout mutant ΔPeLysM15, respectively. Transcriptome analyses indicated that deletion of PeLysM15 could activate expression of several core effector genes, such as PEX2_055830, PEX2_036960 and PEX2_108150, and a chitin-binding protein, PEX2_064520. These results suggest PeLysM15 may play pivotal roles in fungal growth and development and involve pathogen-host interaction by modulating other effector genes' expression. Our results could provide solid data reference and good candidates for further pathogen-related studies in P. expansum.

Structural basis of the alternating-access mechanism in a bile acid transporter.

Bile acids are synthesized from cholesterol in hepatocytes and secreted through the biliary tract into the small intestine, where they aid in absorption of lipids and fat-soluble vitamins. Through a process known as enterohepatic recirculation, more than 90% of secreted bile acids are then retrieved from the intestine and returned to the liver for resecretion. In humans, there are two Na(+)-dependent bile acid transporters involved in enterohepatic recirculation, the Na(+)-taurocholate co-transporting polypeptide (NTCP; also known as SLC10A1) expressed in hepatocytes, and the apical sodium-dependent bile acid transporter (ASBT; also known as SLC10A2) expressed on enterocytes in the terminal ileum. In recent years, ASBT has attracted much interest as a potential drug target for treatment of hypercholesterolaemia, because inhibition of ASBT reduces reabsorption of bile acids, thus increasing bile acid synthesis and consequently cholesterol consumption. However, a lack of three-dimensional structures of bile acid transporters hampers our ability to understand the molecular mechanisms of substrate selectivity and transport, and to interpret the wealth of existing functional data. The crystal structure of an ASBT homologue from Neisseria meningitidis (ASBT(NM)) in detergent was reported recently, showing the protein in an inward-open conformation bound to two Na(+) and a taurocholic acid. However, the structural changes that bring bile acid and Na(+) across the membrane are difficult to infer from a single structure. To understand the structural changes associated with the coupled transport of Na(+) and bile acids, here we solved two structures of an ASBT homologue from Yersinia frederiksenii (ASBTYf) in a lipid environment, which reveal that a large rigid-body rotation of a substrate-binding domain gives the conserved 'crossover' region, where two discontinuous helices cross each other, alternating accessibility from either side of the cell membrane. This result has implications for the location and orientation of the bile acid during transport, as well as for the translocation pathway for Na(+).

Improved in vitro quality of stored red blood cells upon oxygen reduction prior to riboflavin/UV light treatment of whole blood.

BACKGROUND: The application of riboflavin/UV-based pathogen inactivation (PI) to whole blood (WB) is currently limited by its negative impact on red blood cell (RBC) quality. The generation of reactive oxidative species in RBC products contributes to increased hemolysis. This study evaluated the impact of deoxygenation of WB prior to riboflavin/UV light treatment versus deoxygenation of RBC concentrates after PI treatment by monitoring RBC in vitro quality parameters. STUDY DESIGN AND METHODS: Six ABO-matched WB units were pooled and split. Within three pairs, one unit was treated with riboflavin/UV light while the other was kept as an untreated control prior to manufacture into red cell concentrates (RCCs). The first pair (Cntr; Cntr-PI) served as the normoxic controls. Deoxygenation was performed at the RCC level for the second pair (RCCdeox; PI-RCCdeox), and at the WB level of the third pair (WBdeox; WBdeox-PI). In vitro qualities of the respective RBC units were assessed throughout storage. RESULTS: The data for the Cntr and Cntr-PI units were comparable to previous reports. The PI-RCCdeox units exhibited worse in vitro quality for most parameters tested compared to Cntr-PI and WBdeox-PI units throughout storage. Hemolysis and microvesicle release was significantly (p < 0.05) higher on Days 21 and 42 in Cntr-PI units compared to WBdeox-PI units. CONCLUSION: WB deoxygenation may help to decrease the accelerated deterioration in RCC in vitro quality caused by treatment with riboflavin/UV light. Treatment of WB under reduced oxygen levels needs to be assessed for PI effectiveness.

Gating of the TrkH ion channel by its associated RCK protein TrkA.

TrkH belongs to a superfamily of K(+) transport proteins required for growth of bacteria in low external K(+) concentrations. The crystal structure of TrkH from Vibrio parahaemolyticus showed that TrkH resembles a K(+) channel and may have a gating mechanism substantially different from K(+) channels. TrkH assembles with TrkA, a cytosolic protein comprising two RCK (regulate the conductance of K(+)) domains, which are found in certain K(+) channels and control their gating. However, fundamental questions on whether TrkH is an ion channel and how it is regulated by TrkA remain unresolved. Here we show single-channel activity of TrkH that is upregulated by ATP via TrkA. We report two structures of the tetrameric TrkA ring, one in complex with TrkH and one in isolation, in which the ring assumes two markedly different conformations. These results suggest a mechanism for how ATP increases TrkH activity by inducing conformational changes in TrkA.

Supernatant reduction of stored gamma-irradiated red blood cells minimizes potentially harmful substances present in transfusion aliquots for neonates.

BACKGROUND: In neonate transfusion, the use of a dedicated red blood cell (RBC) unit decreases donor exposure. A separate safety measure involves gamma irradiation of the RBCs to abrogate the possibility of transfusion-associated graft-versus-host disease. However, in combination, storage of gamma-irradiated RBCs leads to accumulation of potentially harmful substances in the supernatant. STUDY DESIGN AND METHODS: For this study, RBCs were pooled and split into three study arms. Centrifugation or gravity was used to pack RBCs of matched units thereby reducing the amount of supernatant that would be present in neonate transfusion aliquots; these were compared to matched control units. Supernatant measurements of potassium, hemoglobin (Hb), RBC microvesicle (RMV) content, and mannitol were made in aliquots prepared weekly up to 21 days after gamma irradiation. RBC morphology and osmotic fragility were also assessed to determine if supernatant reduction methods affected the storage lesion. RESULTS: Potassium and mannitol were significantly decreased in transfusion aliquots prepared with either of the supernatant reduction methods. On Day 21, potassium levels from supernatant-reduced aliquots were below those of Day 7 control aliquots. A decrease in free Hb was only detected on Day 21 in centrifuged aliquots. RMVs were significantly reduced in centrifuged aliquots and significantly increased in gravity-settled aliquots. The only measurable effect on storage lesion was a small increase in osmotic fragility of the RBCs subjected to supernatant reduction. CONCLUSION: Supernatant reduction by centrifugation effectively reduces potassium, mannitol, and RMVs in aliquots from gamma-irradiated RBCs stored up to 21 days.

Altered timing of riboflavin and ultraviolet light pathogen inactivation improves platelet in vitro quality.

BACKGROUND: The platelet (PLT) storage lesion is in part caused by the collection and/or production process. Pathogen inactivation (PI) further accelerates its development leading to a reduced in vitro PLT functionality and hence quality. Although the treatment of PLT concentrates (PCs) with riboflavin and ultraviolet light PI should occur within 22 hours of collection, in this study the impact of treatment timing on in vitro PLT quality was investigated. STUDY DESIGN AND METHODS: Apheresis PCs were PI treated on the day of production or on Days 1, 3, or 4 of storage or left untreated as control. A panel of in vitro variables was used to monitor quality throughout 7-day storage, including metabolism, PLT activation, and release of microparticles. Changes in phosphorylation profiles of proteins in the lysate and levels of PLT factor 4, thrombospondin, and epidermal growth factor (EGF) in the releasate were analyzed by immunoblots or enzyme-linked immunosorbent assay. RESULTS: By Day 7 of storage, units illuminated on Day 4 showed a smaller impact of the PI process than units treated on the day of production or one day after on PLT quality such as PLT activation; metabolic activity; microvesicle and EGF release; and phosphorylation of p38, ERK, and HSP27. PCs treated on Day 3 of storage displayed an intermediate effect. CONCLUSION: The timing of PI treatment of PCs influences in vitro PLT quality. Based on these results, timing recommendations should be reconsidered. If PI is applied, inventory management in blood banks might improve with a more flexible collection and treatment regime.

Structure of a membrane-embedded prenyltransferase homologous to UBIAD1.

Membrane-embedded prenyltransferases from the UbiA family catalyze the Mg2+-dependent transfer of a hydrophobic polyprenyl chain onto a variety of acceptor molecules and are involved in the synthesis of molecules that mediate electron transport, including Vitamin K and Coenzyme Q. In humans, missense mutations to the protein UbiA prenyltransferase domain-containing 1 (UBIAD1) are responsible for Schnyder crystalline corneal dystrophy, which is a genetic disease that causes blindness. Mechanistic understanding of this family of enzymes has been hampered by a lack of three-dimensional structures. We have solved structures of a UBIAD1 homolog from Archaeoglobus fulgidus, AfUbiA, in an unliganded form and bound to Mg2+ and two different isoprenyl diphosphates. Functional assays on MenA, a UbiA family member from E. coli, verified the importance of residues involved in Mg2+ and substrate binding. The structural and functional studies led us to propose a mechanism for the prenyl transfer reaction. Disease-causing mutations in UBIAD1 are clustered around the active site in AfUbiA, suggesting the mechanism of catalysis is conserved between the two homologs.

Crystal structure of a phosphorylation-coupled saccharide transporter.

Saccharides have a central role in the nutrition of all living organisms. Whereas several saccharide uptake systems are shared between the different phylogenetic kingdoms, the phosphoenolpyruvate-dependent phosphotransferase system exists almost exclusively in bacteria. This multi-component system includes an integral membrane protein EIIC that transports saccharides and assists in their phosphorylation. Here we present the crystal structure of an EIIC from Bacillus cereus that transports diacetylchitobiose. The EIIC is a homodimer, with an expansive interface formed between the amino-terminal halves of the two protomers. The carboxy-terminal half of each protomer has a large binding pocket that contains a diacetylchitobiose, which is occluded from both sides of the membrane with its site of phosphorylation near the conserved His250 and Glu334 residues. The structure shows the architecture of this important class of transporters, identifies the determinants of substrate binding and phosphorylation, and provides a framework for understanding the mechanism of sugar translocation.

Crystal structure of a potassium ion transporter, TrkH.

The TrkH/TrkG/KtrB proteins mediate K(+) uptake in bacteria and probably evolved from simple K(+) channels by multiple gene duplications or fusions. Here we present the crystal structure of a TrkH from Vibrio parahaemolyticus. TrkH is a homodimer, and each protomer contains an ion permeation pathway. A selectivity filter, similar in architecture to those of K(+) channels but significantly shorter, is lined by backbone and side-chain oxygen atoms. Functional studies showed that TrkH is selective for permeation of K(+) and Rb(+) over smaller ions such as Na(+) or Li(+). Immediately intracellular to the selectivity filter are an intramembrane loop and an arginine residue, both highly conserved, which constrict the permeation pathway. Substituting the arginine with an alanine significantly increases the rate of K(+) flux. These results reveal the molecular basis of K(+) selectivity and suggest a novel gating mechanism for this large and important family of membrane transport proteins.

Structural insight into the PTS sugar transporter EIIC.

BACKGROUND: The enzyme IIC (EIIC) component of the phosphotransferase system (PTS) is responsible for selectively transporting sugar molecules across the inner bacterial membrane. This is accomplished in parallel with phosphorylation of the sugar, which prevents efflux of the sugar back across the membrane. This process is a key part of an extensive signaling network that allows bacteria to efficiently utilize preferred carbohydrate sources. SCOPE OF REVIEW: The goal of this review is to examine the current understanding of the structural features of the EIIC and how it mediates concentrative, selective sugar transport. The crystal structure of an N,N'-diacetylchitobiose transporter is used as a structural template for the glucose superfamily of PTS transporters. MAJOR CONCLUSIONS: Comparison of protein sequences in context with the known EIIC structure suggests that members of the glucose superfamily of PTS transporters may exhibit variations in topology. Despite these differences, a conserved histidine and glutamate appear to have roles shared across the superfamily in sugar binding and phosphorylation. In the proposed transport model, a rigid body motion between two structural domains and movement of an intracellular loop provide the substrate binding site with alternating access, and reveal a surface required for interaction with the phosphotransfer protein responsible for catalysis. GENERAL SIGNIFICANCE: The structural and functional data discussed here give a preliminary understanding of how transport in EIIC is achieved. However, given the great sequence diversity between varying glucose-superfamily PTS transporters and lack of data on conformational changes needed for transport, additional structures of other members and conformations are still required. This article is part of a Special Issue entitled: Structural biochemistry and biophysics of membrane proteins.

Platelet concentrate functionality assessed by thromboelastography or rotational thromboelastometry.

BACKGROUND: There is poor correlation between in vivo platelet concentrate (PC) transfusion outcome and in vitro tests, which typically do not test the functional effectiveness of platelets (PLTs), but rather measure PLT characteristics. We hypothesize that the application of thromboelastography (TEG) or rotational thromboelastometry (ROTEM) to evaluate the procoagulant activity of stored PLTs can predict PLT quality and, ultimately, distinguish among hyper- and nonresponsive PCs. Additionally, we hypothesize that due to their procoagulant properties, PLT microvesicles (PMVs) contribute to the clot signature in these methods. STUDY DESIGN AND METHODS: After the TEG assays were validated, buffy coat PCs were evaluated during the storage time and reconstituted with frozen plasma to different PLT concentrations. Poor quality PCs were generated and assessed by TEG and other in vitro tests. The contribution of PMVs to the TEG clot signature was assessed. RESULTS: Hemostatic analysis showed no significant change in maximum amplitude (MA) during storage of PCs up to Day 10. On Day 8 of storage, PCs that had been manipulated to have poor quality showed a significant decrease in MA. PMV-rich samples contributed to a significant increase in MA, and PMV count showed a significant correlation with maximum clot formation (r = 0.51, p < 0.01). CONCLUSION: TEG was optimized for use with buffy coat PCs, although it was found to lack sensitivity to detect normal storage-related quality changes. Hemostatic measurement was sufficiently sensitive to dissect PLT and PMV contributions to clot formation and to detect PCs stored under poor conditions.

Genome, Transcriptome, and Functional Analyses of Penicillium expansum Provide New Insights Into Secondary Metabolism and Pathogenicity.

The relationship between secondary metabolism and infection in pathogenic fungi has remained largely elusive. The genus Penicillium comprises a group of plant pathogens with varying host specificities and with the ability to produce a wide array of secondary metabolites. The genomes of three Penicillium expansum strains, the main postharvest pathogen of pome fruit, and one Pencillium italicum strain, a postharvest pathogen of citrus fruit, were sequenced and compared with 24 other fungal species. A genomic analysis of gene clusters responsible for the production of secondary metabolites was performed. Putative virulence factors in P. expansum were identified by means of a transcriptomic analysis of apple fruits during the course of infection. Despite a major genome contraction, P. expansum is the Penicillium species with the largest potential for the production of secondary metabolites. Results using knockout mutants clearly demonstrated that neither patulin nor citrinin are required by P. expansum to successfully infect apples. Li et al. ( MPMI-12-14-0398-FI ) reported similar results and conclusions in their recently accepted paper.

Whole blood treated with riboflavin and ultraviolet light: quality assessment of all blood components produced by the buffy coat method.

BACKGROUND: Pathogen inactivation (PI) technologies are currently licensed for use with platelet (PLT) and plasma components. Treatment of whole blood (WB) would be of benefit to the blood banking community by saving time and costs compared to individual component treatment. However, no paired, pool-and-split study directly assessing the impact of WB PI on the subsequently produced components has yet been reported. STUDY DESIGN AND METHODS: In a "pool-and-split" study, WB either was treated with riboflavin and ultraviolet (UV) light or was kept untreated as control. The buffy coat (BC) method produced plasma, PLT, and red blood cell (RBC) components. PLT units arising from the untreated WB study arm were treated with riboflavin and UV light on day of production and compared to PLT concentrates (PCs) produced from the treated WB units. A panel of common in vitro variables for the three types of components was used to monitor quality throughout their respective storage periods. RESULTS: PCs derived from the WB PI treatment were of significantly better quality than treated PLT components for most variables. RBCs produced from the WB treatment deteriorated earlier during storage than untreated units. Plasma components showed a 3% to 44% loss in activity for several clotting factors. CONCLUSION: Treatment of WB with riboflavin and UV before production of components by the BC method shows a negative impact on all three blood components. PLT units produced from PI-treated WB exhibited less damage compared to PLT component treatment.

Crystal structure of a bacterial homologue of the kidney urea transporter.

Urea is highly concentrated in the mammalian kidney to produce the osmotic gradient necessary for water re-absorption. Free diffusion of urea across cell membranes is slow owing to its high polarity, and specialized urea transporters have evolved to achieve rapid and selective urea permeation. Here we present the 2.3 A structure of a functional urea transporter from the bacterium Desulfovibrio vulgaris. The transporter is a homotrimer, and each subunit contains a continuous membrane-spanning pore formed by the two homologous halves of the protein. The pore contains a constricted selectivity filter that can accommodate several dehydrated urea molecules in single file. Backbone and side-chain oxygen atoms provide continuous coordination of urea as it progresses through the filter, and well-placed alpha-helix dipoles provide further compensation for dehydration energy. These results establish that the urea transporter operates by a channel-like mechanism and reveal the physical and chemical basis of urea selectivity.

Structure of urea transporters.

Members of the urea transporter (UT) family mediate rapid, selective transport of urea down its concentration gradient. To date, crystal structures of two evolutionarily distant UTs have been solved. These structures reveal a common UT fold involving two structurally homologous domains that encircle a continuous membrane-spanning pore and indicate that UTs transport urea via a channel-like mechanism. Examination of the conserved architecture of the pore, combined with crystal structures of ligand-bound proteins, molecular dynamics simulations, and functional data on permeation and inhibition by a broad range of urea analogs and other small molecules, provides insight into the structural basis of urea permeation and selectivity.

Structure and permeation mechanism of a mammalian urea transporter.

As an adaptation to infrequent access to water, terrestrial mammals produce urine that is hyperosmotic to plasma. To prevent osmotic diuresis by the large quantity of urea generated by protein catabolism, the kidney epithelia contain facilitative urea transporters (UTs) that allow rapid equilibration between the urinary space and the hyperosmotic interstitium. Here we report the first X-ray crystal structure of a mammalian UT, UT-B, at a resolution of 2.36 Å. UT-B is a homotrimer and each protomer contains a urea conduction pore with a narrow selectivity filter. Structural analyses and molecular dynamics simulations showed that the selectivity filter has two urea binding sites separated by an approximately 5.0 kcal/mol energy barrier. Functional studies showed that the rate of urea conduction in UT-B is increased by hypoosmotic stress, and that the site of osmoregulation coincides with the location of the energy barrier.

Oxidation of NADPH on Kvbeta1 inhibits ball-and-chain type inactivation by restraining the chain.

The Kv1 family voltage-dependent K(+) channels assemble with cytosolic ß subunits (Kvß), which are composed of a flexible N terminus followed by a structured core domain. The N terminus of certain Kvßs inactivates the channel by blocking the ion conduction pore, and the core domain is a functional enzyme that uses NADPH as a cofactor. Oxidation of the Kvß-bound NADPH inhibits inactivation and potentiates channel current, but the mechanism behind this effect is unknown. Here we show that after oxidation, the core domain binds to part of the N terminus, thus restraining it from blocking the channel. The interaction is partially mediated by two negatively charged residues on the core domain and three positively charged ones on the N terminus. These results provide a molecular basis for the coupling between the cellular redox state and channel activity, and establish Kvß as a target for pharmacological control of Kv1 channels.