Local and global interpretations of a disease-causing mutation near the ligand entry path in hyperpolarization-activated cAMP-gated channel.

Hyperpolarization-activated, cAMP-gated (HCN) channels sense membrane potential and intracellular cAMP levels. A mutation identified in the cAMP binding domain (CNBD) of the human HCN4 channel, S672R, severely reduces the heart rate, but the molecular mechanism has been unclear. Our biochemical binding assays on isolated CNBD and patch-clamp recordings on the functional channel show that S672R reduces cAMP binding. The crystal structure of the mutant CNBD revealed no global changes except a disordered loop on the cAMP entry path. To address this localized structural perturbation at a whole protein level, we studied the activity-dependent dynamic interaction between cAMP and the functional channel using the patch-clamp fluorometry technique. S672R reduces the binding of cAMP to the channels in the resting state and significantly increases the unbinding rate during channel deactivation. This study on a disease-causing mutation illustrates the important roles played by the structural elements on the ligand entry-exit path in stabilizing the bound ligand in the binding pocket.

Normal-mode-analysis-guided investigation of crucial intersubunit contacts in the cAMP-dependent gating in HCN channels.

Protein structures define a complex network of atomic interactions in three dimensions. Direct visualization of the structure and analysis of the interaction potential energy are not straightforward approaches to pinpoint the atomic contacts that are crucial for protein function. We used the tetrameric hyperpolarization-activated cAMP-regulated (HCN) channel as a model system to study the intersubunit contacts in cAMP-dependent gating. To obtain a systematic survey of the contacts between each pair of residues, we used normal-mode analysis, a computational approach for studying protein dynamics, and constructed the covariance matrix for C-α atoms. The significant contacts revealed by covariance analysis were further investigated by means of mutagenesis and functional assays. Among the mutant channels that show phenotypes different from those of the wild-type, we focused on two mutant channels that express opposite changes in cAMP-dependent gating. Subsequent biochemical assays on isolated C-terminal fragments, including the cAMP binding domain, revealed only minimal effects on cAMP binding, suggesting the necessity of interpreting the cAMP-dependent allosteric regulation at the whole-channel level. For this purpose, we applied the patch-clamp fluorometry technique and observed correlated changes in the dynamic, state-dependent cAMP binding in the mutant channels. This study not only provides further understanding of the intersubunit contacts in allosteric coupling in the HCN channel, it also illustrates an effective strategy for delineating important atomic contacts within a structure.

Inner activation gate in S6 contributes to the state-dependent binding of cAMP in full-length HCN2 channel.

Recently, applications of the patch-clamp fluorometry (PCF) technique in studies of cyclic nucleotide-gated (CNG) and hyperpolarization-activated, cyclic nucleotide-regulated (HCN) channels have provided direct evidence for the long-held notion that ligands preferably bind to and stabilize these channels in an open state. This state-dependent ligand-channel interaction involves contributions from not only the ligand-binding domain but also other discrete structural elements within the channel protein. This insight led us to investigate whether the pore of the HCN channel plays a role in the ligand-whole channel interaction. We used three well-characterized HCN channel blockers to probe the ion-conducting passage. The PCF technique was used to simultaneously monitor channel activity and cAMP binding. Two ionic blockers, Cs(+) and Mg(2+), effectively block channel conductance but have no obvious effect on cAMP binding. Surprisingly, ZD7288, an open channel blocker specific for HCN channels, significantly reduces the activity-dependent increase in cAMP binding. Independent biochemical assays exclude any nonspecific interaction between ZD7288 and isolated cAMP-binding domain. Because ZD7228 interacts with the inner pore region, where the activation gate is presumably located, we did an alanine scanning of the intracellular end of S6, from T426 to A435. Mutations of three residues, T426, M430, and H434, which are located at regular intervals on the S6 α-helix, enhance cAMP binding. In contrast, mutations of two residues in close proximity, F431A and I432A, dampen the response. Our results demonstrate that movements of the structural elements near the activation gate directly affect ligand binding affinity, which is a simple mechanistic explanation that could be applied to the interpretation of ligand gating in general.

Intracellular Ca(2+)- and PKC-dependent upregulation of T-type Ca(2+) channels in LPC-stimulated cardiomyocytes.

Lysophosphatidylcholine (LPC) accumulation in intracellular and/or interstitial space in cardiomyocytes may underlie as a mechanism for tachycardia and various arrhythmias during cardiac ischemia, which is usually accompanied by elevation of intracellular Ca(2+) concentration ([Ca(2+)](i)). The present study was therefore designed to investigate possible mechanisms responsible for [Ca(2+)](i) elevation by LPC focusing on T-type Ca(2+) channel current (I(Ca.T)). LPC as well as phorbol 12-myristate 13-acetate (PMA) significantly accelerated the beating rates of neonatal rat cardiomyocytes. Augmentation of I(Ca.T) by LPC was dependent on the intracellular Ca(2+) concentration: an increase of I(Ca.T) was significantly larger in high [Ca(2+)](i) condition (pCa=7) than those in low [Ca(2+)](i) condition (pCa=11). In heterologous expression system by use of human cardiac Ca(V)3.1 and Ca(V)3.2 channels expressed in HEK293 cells, LPC augmented Ca(V)3.2 channel current (I(Cav3.2)) in a concentration-dependent manner but not Ca(V)3.1 channel current (I(Cav3.1)). Augmentation of I(Cav3.2) by LPC was highly [Ca(2+)](i) dependent: I(Cav3.2) was unchanged when pCa was 11 but was markedly increased when [Ca(2+)](i) was higher than 10(-10) M (pCa

Effect of aqueous extracts of black and green teas in arsenic-induced toxicity in rabbits.

Arsenic causes oxidative stress in the body. Its administration (3 mg/kg/day) for 14 days in rabbits resulted in a significant reduction of whole blood glutathione (GSH), and elevation of thiobarbituric acid reactive substances (TBARS) and the index of nitrite/nitrate (NOx) levels. These are the markers of oxidative stress. Both black tea (BT) and green tea (GT) (Camellia sinensis), when administered to the arsenic-treated rabbits for 14 days, caused a significant elevation of the depleted GSH level to 53.12% and 57.47%, respectively. On the contrary, in the placebo group the level was 26.59%. The BT and GT reduced the elevated TBARS level to 43.27% and 62.28%, respectively, whereas the corresponding level in the placebo groups was 21.24%. The NOx levels were also reduced to 63.62%, 67.67% and 58.94% in BT, GT and the placebo groups, respectively. When arsenic and black tea were given concurrently to another group the results were even more pronounced. The polyphenol components of black and green tea were 27.69% and 29.71% of the dry weight of the total extracts, respectively. These results indicated that arsenic-induced toxicities in rabbits were significantly reversed by the black and green tea polyphenols. The greater activity of green tea than that of black tea correlates with the slightly higher content of polyphenols in green tea.

Green banana reduces clinical severity of childhood shigellosis: a double-blind, randomized, controlled clinical trial.

BACKGROUND AND AIMS: Mature green banana (GB) fruit is rich in amylase-resistant starch that stimulates colonic production of short-chain fatty acids (referred to as fatty acid) and is useful in treating diarrheal diseases. We studied therapeutic effects of GB in childhood shigellosis by determining colonic fatty acid production in a double-blind, randomized, controlled, clinical trial. METHODS: Seventy-three children aged 6 to 60 months with severe bloody dysentery caused by Shigella infection were either given a rice-based diet (54 kcal/dL), with cooked GB (250 g/L) (n = 34) or without GB (n = 39) for 5 days; all given ciprofloxacin (15 mg/kg, q12 hours). Stool volume, frequency, excretion of blood/mucus, and relevant clinical and laboratory indices were determined. RESULTS: On day 5 (post-treatment), 59% children in GB group had no mucus compared with 36% in controls, fecal blood was completely cleared from 96% in GB group compared with 60% without GB (P < 0.05). GB treatment significantly reduced (P < 0.01) numbers of stools/day compared with controls (70% vs. 50%, P < 0.05). GB-specific reductions of mean fecal volumes (mL/kg) ranged from 25% to 40%; (P < 0.05) during the 5-day observations. Clinical success rates were 85% in GB group compared with 67% in controls (P < 0.05). GB significantly (P < 0.01) reduced fecal myeloperoxidase activity and increased fecal fatty acid concentrations (P < 0.01). CONCLUSIONS: GB diet improves clinical severity in childhood shigellosis and could be a simple and useful adjunct for dietary management of this illness.

17 beta-estradiol modulates expression of low-voltage-activated Ca(V)3.2 T-type calcium channel via extracellularly regulated kinase pathway in cardiomyocytes.

T-type Ca(2+) channel current (I(Ca,T)) plays an important role for spontaneous pacemaker activity and is involved in the progression of structural heart diseases. Estrogens are of importance for the regulation of growth and differentiation and function in a wide array of target tissues, including those in the cardiovascular system. The aim of this study was to elucidate the short-term and long-term effects of 17beta-estradiol (E(2)) on I(Ca,T) in cardiomyocytes. We employed in vivo and in vitro techniques to clarify E(2)-mediated modulation of heart rate (HR) in ovariectomized rats and I(Ca,T) in cardiomyocytes. Ovariectomy increased HR and E(2) supplement reduced HR in ovariectomized rats. Slowing of E(2)-induced HR was consistent with the deceleration of automaticity in E(2)-treated neonatal cardiomyocytes. Short-term application of E(2) did not have significant effects on I(Ca,T), whereas in cardiomyocytes treated with 10 nm E(2) for 24 h, estrogen receptor-independent down-regulation of peak I(Ca,T) and declination of Ca(V)3.2 mRNA were observed. Expression of a cardiac-specific transcription factor Csx/Nkx2.5 was also suppressed by E(2) treatment for 24 h. On the other hand, expression of Ca(V)3.1 mRNA was unaltered by E(2) treatment in this study. An ERK-1/2, 5 inhibitor, PD-98059, abolished the effects of E(2) on I(Ca,T) and Ca(V)3.2 mRNA as well as Csx/Nkx2.5 mRNA. These findings indicate that E(2) decreases Ca(V)3.2 I(Ca,T) through activation of ERK-1/2, 5, which is mediated by the suppression of Csx/Nkx2.5-dependent transcription, suggesting a genomic effect of E(2) as a negative chronotropic factor in the heart.

Antidiarrheal effects of L-histidine-supplemented rice-based oral rehydration solution in the treatment of male adults with severe cholera in Bangladesh: a double-blind, randomized trial.

BACKGROUND: Because of the antisecretory potential of L-histidine in the intestinal tract, its antidiarrheal effects were determined in cholera. METHODS: In a double-blind trial of 126 adult male patients with cholera, L-histidine (2.5 g/L) was mixed with a rice-based oral rehydration solution (ORS) and administered to 62 patients; 64 patients received the same ORS without L-histidine. All patients received ciprofloxacin at a dosage of 500 mg every 12 h for 72 h. Fluid output (of stool, urine, and vomit) and intake (of ORS, water, and intravenous fluid) were determined every 8 h for 72 h. RESULTS: Administration of ORS with L-histidine significantly (P<.05) reduced the frequency of stool output during 32-64 h after initiation of ORS treatment, compared with that in patients given ORS without L-histidine ([all data are means+/-SD] 32-48 h, 11.5+/-6.9 mL/kg vs. 18.8+/-16.0 mL/kg; 40-48 h, 6.7+/-4.4 mL/kg vs. 11.5+/-9.7 mL/kg; and 56-64 h, 6.3+/-5.8 mL/kg vs. 7.8+/-4.1 mL/kg). An overall reduction of 22% in the volume of stool was observed in patients given ORS without L-histidine. The amount of required unscheduled intravenous fluid was lower in patients given ORS with L-histidine, compared with that in patients given ORS without L-histidine (0-24 h, 82.5+/-44.4 mL/kg vs. 158.6+/-72.2 mL/kg [P<.01]; and 24-48 h, 41.6+/-40.4 mL/kg vs. 52.5+/-22.1 mL/kg [P>.05]). Administration of ORS with L-histidine also significantly reduced (P<.05) the intake of ORS and the duration of illness. No adverse effects were observed in these patients. CONCLUSIONS: L-histidine reduces the weight of stool and the frequency of stool output in cholera and could be a useful and safe adjunct treatment that will increase the success rate of ORS and antibiotic therapy in cholera.

Antioxidants in detoxification of arsenic-induced oxidative injury in rabbits: preliminary results.

To assess the oxidative injuries caused by arsenic toxicity in rabbits and evaluate the detoxifying effects of exogenous antioxidants, we administered arsenic trioxide (3-5 mg/kg/day) in rabbits through a feeding tube for seven days. These rabbits were then treated with a recipe of vitamins, zinc, selenium (VZS) or a plant polyphenol or a placebo for the next seven days. Blood samples were collected from ear vein for spectrophotometric assay of reduced glutathione (GSH), thiobarbituric acid reactive substances (TBARS), and nitrite/nitrate (NOx; index of nitric oxide formation) before arsenic administration, seven days after arsenic administration, and seven days after antioxidant treatment. The total arsenic concentrations in hair and spot urine samples of rabbits before arsenic administration were 0.6 +/- 0.21 microg/g and 34.0 +/- 5.9 microg/L, respectively. Administration of arsenic trioxide significantly increased arsenic concentrations in hair and in urine to 2.8 +/- 0.40 microg/g (p<0.001) and 7372 +/- 1392.0 microg/L (p<0.001), respectively. Arsenic administration to rabbits significantly reduced GSH concentration (post-arsenic, 17.5 +/- 0.81 mg/dL vs. pre-arsenic, 32.0 +/- 0.76 mg/dL, p<0.001), increased TBARS concentration (post-arsenic, 8 +/- 1.1 microM vs. pre-arsenic, 5 +/- 0.7 microM, p<0.05), and NOx concentration (post-arsenic, 465 +/- 38.5 microM vs. pre-arsenic, 320 +/- 24.7 microM, p<0.001) as compared to the pre-arsenic levels. There was a negative correlation between TBARS and GSH concentrations (r=-0.464, p<0.01) and between NOx and GSH concentrations (r=-0.381, p<0.05) of intoxicated rabbits. The recovery of the depleted GSH was significantly greater in the polyphenols (77.0 +/- 12.0%) or VZS (67.0 +/- 17.0%) treatment groups compared with the placebo group (36.0 +/- 7.0%). The decrease in NOx level of arsenic-treated rabbits was significantly greater in polyphenols treatment group than the placebo group (60.0 +/- 9.0% vs. 17.0 +/- 6.0%, p<0.001). These results indicate that arsenic induces toxicity in rabbits associated with an increase in lipid peroxidation. Arsenic toxicity increases nitric oxide production in the body. Exogenous antioxidants such as polyphenols and recipe of vitamins, zinc, and selenium are useful for arsenic detoxification.