Native Whey Induces Similar Post Exercise Muscle Anabolic Responses as Regular Whey, Despite Greater Leucinemia, in Elderly Individuals.

OBJECTIVE: Elderly muscle seems less sensitive to the anabolic stimulus of a meal. Changes in blood concentrations of leucine are suggested as one important trigger of the anabolic response in muscle. The aim of this study was to investigate whether native whey protein, containing high amounts of leucine, may be a more potent stimulator of muscle protein synthesis (MPS) in elderly than regular whey protein (WPC-80) or milk. DESIGN: Randomized controlled partial crossover. SETTING: Norwegian School of Sport Sciences. PARTICIPANTS: 21 healthy elderly men and women (≥70 years). INTERVENTION: Participants received either 20 g of WPC-80 and native whey (n = 11) on separate days in a crossover design, or milk (n = 10). Supplements were ingested immediately and two hours after a bout of lower body heavy-load resistance exercise. MEASUREMENTS: Blood samples and muscle biopsies were collected to measure blood concentrations of amino acids by gas-chromatography mass spectrometry (GCMS), phosphorylation of p70S6K, 4E-BP1 and eEF-2 by immunoblotting and mixed muscle fractional synthetic rate (FSR) by use of [2H5]phenylalanine-infusion, GCMS and isotope-ratio mass spectrometry. RESULTS: Native whey increased blood leucine concentrations more than WPC-80 (P < 0.05), but not p70S6K phosphorylation or mixed muscle FSR. Both whey supplements increased blood leucine concentrations (P < 0.01) and P70S6K phosphorylation more than milk (P = 0.014). Native whey reached higher mixed muscle FSR values than milk (P = 0.026) 1-3h after exercise. CONCLUSIONS: Despite greater increases in blood leucine concentrations than WPC-80 and milk, native whey was only superior to milk concerning increases in MPS and phosphorylation of P70S6K during a 5-hour post-exercise period in elderly individuals.

Protein leucine content is a determinant of shorter- and longer-term muscle protein synthetic responses at rest and following resistance exercise in healthy older women: a randomized, controlled trial.

Background: Older women may not be consuming enough protein to maintain muscle mass. Augmentation of protein intake with leucine may enhance the muscle protein synthetic response in older women to aid in maintaining muscle mass. Objective: We measured the acute (hourly) and integrated (daily) myofibrillar protein synthesis (myoPS) response to consumption of a high-quality mixed protein beverage compared with an isonitrogenous protein beverage with added leucine. Design: In a parallel design, free-living, healthy older women (aged 65-75 y, n = 11/group) consumed a fixed, weight-maintaining diet with protein at 1.0 g · kg-1 · d-1 and were randomly assigned to twice-daily consumption of either 15 g milk protein beverage containing 4.2 g leucine (LEU) or 15 g mixed protein (milk and soy) beverage containing 1.3 g leucine (CON). Unilateral leg resistance exercise allowed a determination of acute ([13C6]-phenylalanine infusion, hourly rate) and integrated (deuterated water ingestion, daily rate) exercised and rested myoPS responses. Results: Acute myoPS increased in response to feeding in the rested (CON: 13% ± 4%; LEU: 53% ± 5%) and exercised (CON: 30% ± 4%; LEU: 87% ± 7%) leg in both groups, but the increase was greater in LEU (P < 0.001). Integrated myoPS increased during the supplementation period in both legs (rested: 9% ±1%; exercised: 17% ± 2%; P < 0.001) in LEU, but in the exercised leg only (7% ± 2%; P < 0.001) in CON. Conclusions: A 15-g protein-containing beverage with ∼4 g leucine induced greater increases in acute and integrated myoPS than did an isonitrogenous, isoenergetic mixed-protein beverage. Declines in muscle mass in older women may be attenuated with habitual twice-daily consumption of a protein beverage providing 15 g protein and higher (4.2 g/serving) amounts of leucine. This trial was registered at clinicaltrials.gov as NCT02282566.

YY1 DNA binding and interaction with YAF2 is essential for Polycomb recruitment.

Polycomb Group (PcG) proteins are crucial for epigenetic inheritance of cell identity and are functionally conserved from Drosophila to humans. PcG proteins regulate expression of homeotic genes and are essential for axial body patterning during development. Earlier we showed that transcription factor YY1 functions as a PcG protein. YY1 also physically interacts with YAF2, a homolog of RYBP. Here we characterize the mechanism and physiologic relevance of this interaction. We found phenotypic and biochemical correction of dRYBP mutant flies by mouse YAF2 demonstrating functional conservation across species. Further biochemical analysis revealed that YAF2 bridges interaction between YY1 and the PRC1 complex. ChIP assays in HeLa cells showed that YAF2 is responsible for PcG recruitment to DNA, which is mediated by YY1 DNA binding. Knock-down of YY1 abrogated PcG recruitment, which was not compensated by exogenous YAF2 demonstrating that YY1 DNA binding is a priori necessary for Polycomb assembly on chromatin. Finally, we found that although YAF2 and RYBP regulate a similar number of Polycomb target genes, there are very few genes that are regulated by both implying functional distinction between the two proteins. We present a model of YAF2-dependent and independent PcG DNA recruitment by YY1.

Recovery in soccer : part ii-recovery strategies.

In the formerly published part I of this two-part review, we examined fatigue after soccer matchplay and recovery kinetics of physical performance, and cognitive, subjective and biological markers. To reduce the magnitude of fatigue and to accelerate the time to fully recover after completion, several recovery strategies are now used in professional soccer teams. During congested fixture schedules, recovery strategies are highly required to alleviate post-match fatigue, and then to regain performance faster and reduce the risk of injury. Fatigue following competition is multifactorial and mainly related to dehydration, glycogen depletion, muscle damage and mental fatigue. Recovery strategies should consequently be targeted against the major causes of fatigue. Strategies reviewed in part II of this article were nutritional intake, cold water immersion, sleeping, active recovery, stretching, compression garments, massage and electrical stimulation. Some strategies such as hydration, diet and sleep are effective in their ability to counteract the fatigue mechanisms. Providing milk drinks to players at the end of competition and a meal containing high-glycaemic index carbohydrate and protein within the hour following the match are effective in replenishing substrate stores and optimizing muscle-damage repair. Sleep is an essential part of recovery management. Sleep disturbance after a match is common and can negatively impact on the recovery process. Cold water immersion is effective during acute periods of match congestion in order to regain performance levels faster and repress the acute inflammatory process. Scientific evidence for other strategies reviewed in their ability to accelerate the return to the initial level of performance is still lacking. These include active recovery, stretching, compression garments, massage and electrical stimulation. While this does not mean that these strategies do not aid the recovery process, the protocols implemented up until now do not significantly accelerate the return to initial levels of performance in comparison with a control condition. In conclusion, scientific evidence to support the use of strategies commonly used during recovery is lacking. Additional research is required in this area in order to help practitioners establish an efficient recovery protocol immediately after matchplay, but also for the following days. Future studies could focus on the chronic effects of recovery strategies, on combinations of recovery protocols and on the effects of recovery strategies inducing an anti-inflammatory or a pro-inflammatory response.

Tumor progression-related transmembrane protein aspartate-ß-hydroxylase is a target for immunotherapy of hepatocellular carcinoma.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) has a poor survival rate due to recurrent intrahepatic metastases and lack of effective adjuvant therapy. Aspartate-ß-hydroxylase (ASPH) is an attractive cellular target since it is a highly conserved transmembrane protein overexpressed in both murine and human HCC tumors, and promotes a malignant phenotype as characterized by enhanced tumor cell migration and invasion. METHODS: Dendritic cells (DCs), expanded and isolated from the spleen, were incubated with a cytokine cocktail to optimize IL-12 secretion and co-stimulatory molecule expression, then subsequently loaded with ASPH protein for immunization. Mice were injected with syngeneic BNL HCC tumor cells followed by subcutaneous inoculation with 5-10×10(5) ASPH loaded DCs using a prophylactic and therapeutic experimental approach. Tumor infiltrating lymphocytes (TILs) were characterized, and their role in producing anti-tumor effects determined. The immunogenicity of ASPH protein with respect to activating antigen specific CD4+ T cells derived from human peripheral blood mononuclear cells (PBMCs) was also explored. RESULTS: We found that immunotherapy with ASPH-loaded DCs suppressed and delayed established HCC and tumor growth when administered prophylactically. Ex-vivo re-stimulation experiments and in vivo depletion studies demonstrated that both CD4+ and CD8+ cells contributed to anti-tumor effects. Using PBMCs derived from healthy volunteers and HCC patients, we showed that ASPH stimulation led to significant development of antigen-specific CD4+ T-cells. CONCLUSIONS: Immunization with ASPH-loaded DCs has substantial anti-tumor effects which could reduce the risk of HCC recurrence.

Blocking effects of acehytisine on pacemaker currents (I(f)) in sinoatrial node cells and human HCN4 channels expressed in Xenopus laevis oocytes.

ETHNOPHARMACOLOGICAL RELEVANCE: The root of Aconitum coreanum (Levl.) Raipaics has been extensively used to treat various kinds of disorders including cardiovascular disease in China for a long time. According to recent studies, its antiarrhythmic actions are attributable to the active component, acehytisine. However, the underlying mechanism remains poorly understood. AIM OF THE STUDY: The effects of acehytisine on the spontaneous activity in sinoatrial nodes and the electropharmacological action of this drug on I(f) in pacemaker cells and hHCN4 channels in oocytes were to be investigated. MATERIALS AND METHODS: Sinoatrial nodes were cut from rabbit heart, and transmembrane potentials were recorded by standard microelectrode technique. A whole-cell patch clamp technique was employed to record I(f) isolated enzymatically from rabbit sinoatrial node pacemaker cells. Human HCN4 channels were heterologously expressed in Xenopus oocytes and studied using the two-electrode voltage clamp technique. RESULTS: Acehytisine decreased the pacemaker rate of firing and slope of diastolic depolarization, modified the action potential configurations and blocked I(f) in rabbit sinoatrial node cells and hHCN4 channels expressed in Xenopus oocytes in a concentration-dependent, voltage-independent and non-use-dependent manner. Its electropharmacological properties were consistent with those of a close-state blocker. CONCLUSION: Our findings are likely to shed light on the clinical application of acehytisine in the treatment of cardiovascular disorders.

Effect of carbohydrate-protein supplementation postexercise on rat muscle glycogen synthesis and phosphorylation of proteins controlling glucose storage.

To examine whether addition of protein to a carbohydrate supplement enhances muscle glycogen synthesis, we compared the muscle glycogen concentrations of rats that had been depleted of their muscle glycogen stores with a 3-hour swim and immediately supplemented with a placebo (Con), carbohydrate (CHO), or carbohydrate plus protein supplement (C+P). Rats were given either 0.9 g carbohydrate per kilogram body mass for the CHO group or 0.9 g carbohydrate + 0.3 g protein per kilogram body mass for the C+P groups. Muscle samples of the red and white quadriceps were excised immediately, 30 minutes, or 90 minutes postexercise. Glycogen concentration of the C+P group was greater than that of the CHO group at 90 minutes postexercise in both red (C+P, 28.3 ± 2.6 µmol/g vs CHO, 22.4 ± 2.0 µmol/g; P < .05) and white (C+P, 24.9 ± 2.4 µmol/g vs CHO, 17.64 ± 1.5 µmol/g; P < .01) quadriceps. Protein kinase B phosphorylation was greater in the C+P-30 group (the number following treatment group abbreviation refers to time [in minutes] of euthanasia following exercise) than the sedentary control and exercised control groups in red quadriceps at 30 minutes and in white quadriceps at 90 minutes postexercise. This difference was not observed in the CHO group. Phosphorylation of glycogen synthase was significantly reduced 30 minutes postexercise and returned to baseline levels by 90 minutes postexercise in both CHO- and C+P-supplemented groups, with no difference between supplements. These results demonstrated that the addition of protein to a carbohydrate supplement will enhance the rate of muscle glycogen restoration postexercise and may involve facilitation of the glucose transport process.

Local anesthetics disrupt energetic coupling between the voltage-sensing segments of a sodium channel.

Local anesthetics block sodium channels in a state-dependent fashion, binding with higher affinity to open and/or inactivated states. Gating current measurements show that local anesthetics immobilize a fraction of the gating charge, suggesting that the movement of voltage sensors is modified when a local anesthetic binds to the pore of the sodium channel. Here, using voltage clamp fluorescence measurements, we provide a quantitative description of the effect of local anesthetics on the steady-state behavior of the voltage-sensing segments of a sodium channel. Lidocaine and QX-314 shifted the midpoints of the fluorescence-voltage (F-V) curves of S4 domain III in the hyperpolarizing direction by 57 and 65 mV, respectively. A single mutation in the S6 of domain IV (F1579A), a site critical for local anesthetic block, abolished the effect of QX-314 on the voltage sensor of domain III. Both local anesthetics modestly shifted the F-V relationships of S4 domain IV toward hyperpolarized potentials. In contrast, the F-V curve of the S4 domain I was shifted by 11 mV in the depolarizing direction upon QX-314 binding. These antagonistic effects of the local anesthetic indicate that the drug modifies the coupling between the voltage-sensing domains of the sodium channel. Our findings suggest a novel role of local anesthetics in modulating the gating apparatus of the sodium channel.

Preclinical results with I(f) current inhibition by ivabradine.

Ivabradine, a highly selective I(f) current inhibitor acting directly on the sinoatrial node, induces a rapid, sustained and dose-dependent reduction of heart rate at rest and during exercise, without significant effects on atrioventricular conduction, left ventricular (LV) contraction-relaxation or vascular tissues. These properties, associated with an improvement in LV loading related to bradycardia, resulted in an increase in stroke volume and preservation of cardiac output at rest and during exercise. Reducing myocardial oxygen consumption and improving oxygen supply, ivabradine reduced the severity of ischaemia and associated regional contractile dysfunction of the stunned myocardium. Long-term administration of ivabradine in rats with chronic heart failure improved cardiac haemodynamics associated with a progressive remodelling of LV structure. In dyslipidaemic mice, ivabradine prevented the renal and cerebrovascular endothelial dysfunction associated with atherosclerosis. These preclinical data suggest that long-term reduction in heart rate with ivabradine might interact with multiple a priori unexpected mechanisms involved in cardiac and vascular remodelling processes associated with chronic heart diseases.

Molecular and cellular events in alcohol-induced muscle disease.

Alcohol consumption induces a dose-dependent noxious effect on skeletal muscle, leading to progressive functional and structural damage of myocytes, with concomitant reductions in lean body mass. Nearly half of high-dose chronic alcohol consumers develop alcoholic skeletal myopathy. The pathogenic mechanisms that lie between alcohol intake and loss of muscle tissue involve multiple pathways, making the elucidation of the disease somewhat difficult. This review discusses the recent advances in basic and clinical research on the molecular and cellular events involved in the development of alcohol-induced muscle disease. The main areas of recent research interest on this field are as follows: (i) molecular mechanisms in alcohol exposed muscle in the rat model; (ii) gene expression changes in alcohol exposed muscle; (iii) the role of trace elements and oxidative stress in alcoholic myopathy; and (iv) the role of apoptosis and preapoptotic pathways in alcoholic myopathy. These aforementioned areas are crucial in understanding the pathogenesis of this disease. For example, there is overwhelming evidence that both chronic alcohol ingestion and acute alcohol intoxication impair the rate of protein synthesis of myofibrillar proteins, in particular, under both postabsorptive and postprandial conditions. Perturbations in gene expression are contributory factors to the development of alcoholic myopathy, as ethanol-induced alterations are detected in over 400 genes and the protein profile (i.e., the proteome) of muscle is also affected. There is supportive evidence that oxidative damage is involved in the pathogenesis of alcoholic myopathy. Increased lipid peroxidation is related to muscle fibre atrophy, and reduced serum levels of some antioxidants may be related to loss of muscle mass and muscle strength. Finally, ethanol induces skeletal muscle apoptosis and increases both pro- and antiapoptotic regulatory mechanisms.

Properties and microstructure of protein-based film from round scad (Decapterus maruadsi) muscle as affected by palm oil and chitosan incorporation.

The properties of protein-based film prepared from round scad (Decapterus maruadsi) muscle in the absence and the presence of palm oil and/or chitosan were investigated. Films added with 25% palm oil (as glycerol substitiution) had the slight decrease in water vapor permeability (WVP) and elongation at break (EAB) (p<0.05). WVP and tensile strength (TS) of films increased but EAB decreased when 10-40% chitosan (as protein substitution) was incorporated (p<0.05). Hydrophobic interactions and hydrogen bonds, together with disulfide and non-disulfide covalent bonds, played an important role in stabilizing the film matrix. The a* and b*-values increased with increasing chitosan levels (p<0.05). Films added with chitosan were less transparent and had the lowered transmission in the visible range. The incorporation of 25% palm oil and 40% chitosan yielded the films with the improved TS but decreased water vapor barrier property. Apart from film strengthening effect, chitosan inconjunction with Tween-20 most likely functioned as the emulsifier/stabilizer in film forming solution containing palm oil.

Targeted overexpression of sarcolipin in the mouse heart decreases sarcoplasmic reticulum calcium transport and cardiac contractility.

The role of sarcolipin (SLN) in cardiac physiology was critically evaluated by generating a transgenic (TG) mouse model in which the SLN to sarco(endoplasmic)reticulum (SR) Ca(2+) ATPase (SERCA) ratio was increased in the ventricle. Overexpression of SLN decreases SR calcium transport function and results in decreased calcium transient amplitude and rate of relaxation. SLN TG hearts exhibit a significant decrease in rates of contraction and relaxation when assessed by ex vivo work-performing heart preparations. Similar results were also observed with muscle preparations and myocytes from SLN TG ventricles. Interestingly, the inhibitory effect of SLN was partially relieved upon high dose of isoproterenol treatment and stimulation at high frequency. Biochemical analyses show that an increase in SLN level does not affect PLB levels, monomer to pentamer ratio, or its phosphorylation status. No compensatory changes were seen in the expression of other calcium-handling proteins. These studies suggest that the SLN effect on SERCA pump is direct and is not mediated through increased monomerization of PLB or by a change in PLB phosphorylation status. We conclude that SLN is a novel regulator of SERCA pump activity, and its inhibitory effect can be reversed by beta-adrenergic agonists.

Differentially transcribed genes in skeletal muscle of Duroc and Taoyuan pigs.

The objective of this study was to compare gene transcription profiles of LM between two pig breeds, Duroc and Taoyuan, which display dramatically different postnatal muscle growth. We isolated LM from neonatal pigs, and the Duroc muscle length and mass were greater (P < 0.01) than for Taoyuan pigs; however, insignificant differences in the muscle fiber area and the percentage of fiber types were found. A human high-density complementary DNA (cDNA) microarray consisting of 9,182 probes was used to compare gene transcription profiles of LM between the two breeds. The results showed that the transcription level of 73 genes and 44 genes in Duroc LM were upregulated and down-regulated by at least 1.75-fold (P < 0.05) compared with Taoyuan, respectively. The strongly upregulated genes in Duroc pigs included those encoding the complex of myofibrillar proteins (e.g., myosin light and heavy chains, and troponin), ribosomal proteins, transcription regulatory proteins (e.g., skeletal muscle LIM protein 1 [SLIM1] and high-mobility group proteins), and energy metabolic enzymes (e.g., electron-transferring flavo-protein dehydrogenase, NADH dehydrogenase, malate dehydrogenase, and ATP synthases). The highly transcribed genes that encode energy metabolic enzymes indicate a more glycolytic metabolism in Duroc LM, thereby favoring carbohydrates rather than lipids for use as energy substrates in this tissue. The over-transcribed genes that encode skeletal muscle-predominant proteins or transcription regulators that control myogenesis and/or muscle growth suggest a general mechanism for the observed higher rate of postnatal muscle growth in Duroc pigs. The transcription of one such gene, SLIM1, was more highly transcribed (P < 0.01) in Duroc LM at birth and at postnatal d 7 than in Taoyuan. The transcription of SLIM1 increased (P < 0.05) in Duroc LM from neonate through 7 d of age, whereas its transcription remained essentially constant in Taoyuan during this period. These results suggest that SLIM1 may be useful for the development of markers associated with the postnatal muscle growth of pigs.

Altered hepatic and muscle substrate utilization provoked by GLUT4 ablation.

Studies were conducted to explore altered substrate utilization and metabolism in GLUT4 null mice. Liver fatty acid synthase mRNA and fatty acid synthesis rates were dramatically increased in GLUT4 null mice compared with control mice and were supported by increased rates of the pentose phosphate pathway oxidative phase and sterol regulatory binding protein mRNA expression. Increased GLUT2 protein content, glucokinase mRNA, and glucose-6-phosphate in GLUT4 null mice may provide substrate for the enhanced fatty acid synthesis. Increased fatty acid synthesis, however, did not lead to hepatic triglyceride accumulation in GLUT4 null mice because of increased hepatic triglyceride secretion rates. GLUT4 null mice rapidly cleared orally administered olive oil, had reduced serum triglyceride concentrations in the fed and the fasted state, and increased skeletal muscle lipoprotein lipase when compared with controls. Oleate oxidation rates were increased in GLUT4 null skeletal muscle in association with mitochondrial hyperplasia/hypertrophy. This study demonstrated that GLUT4 null mice had increased hepatic glucose uptake and conversion into triglyceride for subsequent use by muscle. The ability of GLUT4 null mice to alter hepatic carbohydrate and lipid metabolism to provide proper nutrients for peripheral tissues may explain (in part) their ability to resist diabetes when fed a normal diet.

Differential actin binding along the PEVK domain of skeletal muscle titin.

Parts of the PEVK (Pro-Glu-Val-Lys) domain of the skeletal muscle isoform of the giant intrasarcomeric protein titin have been shown to bind F-actin. However, the mechanisms and physiological function of this are poorly understood. To test for actin binding along PEVK, we expressed contiguous N-terminal (PEVKI), middle (PEVKII), and C-terminal (PEVKIII) PEVK segments of the human soleus muscle isoform. We found a differential actin binding along PEVK in solid-state binding, cross-linking and in vitro motility assays. The order of apparent affinity is PEVKII>PEVKI>PEVKIII. To explore which sequence motifs convey the actin-binding property, we cloned and expressed PEVK fragments with different motif structure: PPAK, polyE-rich and pure polyE fragments. The polyE-containing fragments had a stronger apparent actin binding, suggesting that a local preponderance of polyE motifs conveys an enhanced local actin-binding property to PEVK. The actin binding of PEVK may serve as a viscous bumper mechanism that limits the velocity of unloaded muscle shortening towards short sarcomere lengths. Variations in the motif structure of PEVK might be a method of regulating the magnitude of the viscous drag.

Recreating the biological pacemaker.

In recent years, several groups have reported a variety of strategies for developing biological pacemakers whose ultimate function would be to supplement/replace electronic pacemakers. Strategies have included gene therapy using naked plasmids or viral vectors and cell therapy for which both adult human mesenchymal stem cells (hMSCs) and human embryonic stem cells have been employed. This article reviews the various approaches and summarizes our own research in which the pacemaker gene, HCN2, is administered via viral vector or in an hMSC platform to produce pacemaker function in the intact canine heart.

State-dependent block of voltage-gated Na+ channels by amitriptyline via the local anesthetic receptor and its implication for neuropathic pain.

Amitriptyline is a tricyclic antidepressant, which also alleviates various pain syndromes at its therapeutic plasma concentration (0.36-0.90 microM). Accumulated evidence suggests that such efficacy may be due to block of voltage-gated Na(+) channels. The Na(+) channel alpha-subunit protein consists of four homologous domains (D1-D4), each with six transmembrane segments (S1-S6). The aims of this study were to locate the amitriptyline receptor in the Na(+) channel alpha-subunit and to compare the amitriptyline affinity in open, inactivated, and resting states of the Na(+) channel. Wild-type and mutant rat skeletal muscle alpha-subunit Na(+) channels were expressed in human embryonic kidney cells and assayed under whole-cell voltage clamp conditions. Our results indicate that the amitriptyline receptor overlaps with the local anesthetic receptor to a great extent in Na(+) channels. Residues N434 (at D1-S6), L1280 (D3-S6), and F1579 (D4-S6) may jointly form parts of the amitriptyline/local anesthetic receptor, with residue L1280 being most critical for amitriptyline binding. Open-channel block by amitriptyline was assessed in inactivation-deficient Na(+) channels and compared with the resting- and inactivated-channel block in wild-type channels. The open-channel block by amitriptyline has the highest affinity, with a 50% inhibitory concentration (IC(50)) of 0.26 microM. The inactivated-channel block by amitriptyline had a weaker affinity (0.51 microM), whereas the resting-channel displayed the weakest affinity (33 microM). We hypothesize that selective block of both persistent late openings and the inactivated state of neuronal Na(+) channel isoforms by amitriptyline also occurs at its therapeutic concentration and likely contributes to its efficacy in pain syndromes.

Hyperpolarization-activated, cyclic nucleotide-gated HCN2 cation channel forms a protein assembly with multiple neuronal scaffold proteins in distinct modes of protein-protein interaction.

Hyperpolarization-activated cation currents, termed Ih, are non-uniformly distributed along dendritic arbors with current density increasing with increasing distance from the soma. The non-uniform distribution of Ih currents contributes to normalization of location-dependent variability in temporal integration of synaptic input, but the molecular basis for the graded HCN distribution remains to be investigated. The hyperpolarization-activated, cyclic nucleotide-gated cation channels (HCNs) underlie Ih currents and consist of four members (HCN1-HCN4) of the gene family in mammals. In this investigation, we report that HCN2 forms a protein assembly with tamalin, S-SCAM and Mint2 scaffold proteins, using several different approaches including immunoprecipitation of rat brain and heterologously expressing cell extracts and glutathione S-transferase pull-down assays. The PDZ domain of tamalin interacts with HCN2 at both the PDZ-binding motif and the internal carboxy-terminal tail of HCN2, whereas binding of the PDZ domain of S-SCAM occurs at the cyclic nucleotide-binding domain (CNBD) and the CNBD-downstream sequence of the carboxy-terminal tail of HCN2. A protein assembly between HCN2 and Mint2 is formed by the interaction of the munc18-interacting domain of Mint2 with the CNBD-downstream sequence of HCN2. The results demonstrate that HCN2 forms a protein complex with multiple neuronal scaffold proteins in distinct modes of protein-protein interaction.

Sarcolipin regulates sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) by binding to transmembrane helices alone or in association with phospholamban.

Phospholamban (PLN), a regulator of sarco(endo)plasmic reticulum Ca(2+)-ATPases (SERCAs), interacts with both the cytosolic N domain and transmembrane helices M2, M4, M6, and M9 of SERCA. Amino acids in the transmembrane domain of PLN that are predicted to interact with SERCA1a are conserved in sarcolipin (SLN), a functional PLN homologue. Accordingly, the effects of critical mutations in SERCA1a, PLN, and NF-SLN (SLN tagged N-terminally with a FLAG epitope) on NF-SLN/SERCA1a and PLN/NF-SLN/SERCA1a interactions were compared. Critical mutations in SERCA1a and NF-SLN diminished functional interactions between SERCA1a and NF-SLN, indicating that NF-SLN and PLN interact with some of the same amino acids in SERCA1a. Mutations in PLN or NF-SLN affected the amount of SERCA1a that was coimmunoprecipitated in each complex with antibodies against either PLN or SLN, but not the pattern of coimmunoprecipitation. PLN mutations had more dramatic effects on SERCA1a coimmunoprecipitation than SLN mutations, suggesting that PLN dominates in the primary interaction with SERCA1a. Coimmunoprecipitation also confirmed that PLN and NF-SLN form a heterodimer that interacts with SERCA1a in a regulatory fashion to form a very stable PLN/NF-SLN/SERCA1a complex. Modeling showed that the SLN/SERCA1a complex closely resembles the PLN/SERCA1a complex, but with the luminal end of SLN extending to the loop connecting M1 and M2, where Tyr-29 and Tyr-31 interact with aromatic residues in SERCA1a. Modeling of the PLN/SLN/SERCA1a complex predicts that the regulator binding cavity in the E(2) conformation of SERCA1a can accommodate both SLN and PLN helices, but not two PLN helices.