Regulation of PKD2 channel function by TACAN.

Autosomal dominant polycystic kidney disease is caused by mutations in the membrane receptor PKD1 or the cation channel PKD2. TACAN (also termed TMEM120A), recently reported as an ion channel in neurons for mechanosensing and pain sensing, is also distributed in diverse non-neuronal tissues, such as kidney, heart and intestine, suggesting its involvement in other functions. In this study, we found that TACAN is in a complex with PKD2 in native renal cell lines. Using the two-electrode voltage clamp in Xenopus oocytes, we found that TACAN inhibits the channel activity of PKD2 gain-of-function mutant F604P. TACAN fragments containing the first and last transmembrane domains interacted with the PKD2 C- and N-terminal fragments, respectively. The TACAN N-terminus acted as a blocking peptide, and TACAN inhibited the function of PKD2 by the binding of PKD2 with TACAN. By patch clamping in mammalian cells, we found that TACAN inhibits both the single-channel conductance and the open probability of PKD2 and mutant F604P. PKD2 co-expressed with TACAN, but not PKD2 alone, exhibited pressure sensitivity. Furthermore, we found that TACAN aggravates PKD2-dependent tail curvature and pronephric cysts in larval zebrafish. In summary, this study revealed that TACAN acts as a PKD2 inhibitor and mediates mechanosensitivity of the PKD2-TACAN channel complex. KEY POINTS: TACAN inhibits the function of PKD2 in vitro and in vivo. TACAN N-terminal S1-containing fragment T160X interacts with the PKD2 C-terminal fragment N580-L700, and its C-terminal S6-containing fragment L296-D343 interacts with the PKD2 N-terminal A594X. TACAN inhibits the function of the PKD2 channel by physical interaction. The complex of PKD2 with TACAN, but not PKD2 alone, confers mechanosensitivity.

Do Dipeptidyl Peptidase-4 Inhibitors Increase the Risk of Heart Failure in Patients with Type 2 Diabetes?

Dipeptidyl peptidase-4 inhibitors (DDP-4Is) or gliptins have been extensively studied in recent years. These studies have shown the safety and efficacy of gliptins in managing hyperglycemia in diabetic patients. However, there is an ongoing debate on whether DDP-4Is are associated with a higher risk for developing heart failure. It is expected that long-term data from patients who are currently prescribed DDP-4Is will provide a clearer understanding of their potential benefits. This should also help guide the development of future guidelines. The focus of this perspective is on associations between the "use of DPP-4Is" and "increased risk of heart failure". Thus, we examine several key publications and reviews on clinical trials on this class of oral antidiabetic medications. For this communication, the pertinent literature has been critically analyzed to provide an evidence-based overview of the evolving concept of DPP-4Is-induced risk of heart failure.

Cardiac Late Sodium Channel Current Is a Molecular Target for the Sodium/Glucose Cotransporter 2 Inhibitor Empagliflozin.

BACKGROUND: SGLT2 (sodium/glucose cotransporter 2) inhibitors exert robust cardioprotective effects against heart failure in patients with diabetes, and there is intense interest to identify the underlying molecular mechanisms that afford this protection. Because the induction of the late component of the cardiac sodium channel current (late-INa) is involved in the etiology of heart failure, we investigated whether these drugs inhibit late-INa. METHODS: Electrophysiological, in silico molecular docking, molecular, calcium imaging, and whole heart perfusion techniques were used to address this question. RESULTS: The SGLT2 inhibitor empagliflozin reduced late-INa in cardiomyocytes from mice with heart failure and in cardiac Nav1.5 sodium channels containing the long QT syndrome 3 mutations R1623Q or ΔKPQ. Empagliflozin, dapagliflozin, and canagliflozin are all potent and selective inhibitors of H2O2-induced late-INa (half maximal inhibitory concentration = 0.79, 0.58, and 1.26 µM, respectively) with little effect on peak sodium current. In mouse cardiomyocytes, empagliflozin reduced the incidence of spontaneous calcium transients induced by the late-INa activator veratridine in a similar manner to tetrodotoxin, ranolazine, and lidocaine. The putative binding sites for empagliflozin within Nav1.5 were investigated by simulations of empagliflozin docking to a three-dimensional homology model of human Nav1.5 and point mutagenic approaches. Our results indicate that empagliflozin binds to Nav1.5 in the same region as local anesthetics and ranolazine. In an acute model of myocardial injury, perfusion of isolated mouse hearts with empagliflozin or tetrodotoxin prevented activation of the cardiac NLRP3 (nuclear-binding domain-like receptor 3) inflammasome and improved functional recovery after ischemia. CONCLUSIONS: Our results provide evidence that late-INa may be an important molecular target in the heart for the SGLT2 inhibitors, contributing to their unexpected cardioprotective effects.

Vitamin D is an endogenous partial agonist of the transient receptor potential vanilloid 1 channel.

KEY POINTS: 25-Hydroxyvitamin D (25OHD) is a partial agonist of TRPV1 whereby 25OHD can weakly activate TRPV1 yet antagonize the stimulatory effects of the full TRPV1 agonists capsaicin and oleoyl dopamine. 25OHD binds to TRPV1 within the same vanilloid binding pocket as capsaicin. 25OHD inhibits the potentiating effects of PKC-mediated TRPV1 activity. 25OHD reduces T-cell activation and trigeminal neuron calcium signalling mediated by TRPV1 activity. These results provide evidence that TRPV1 is a novel receptor for the biological actions of vitamin D in addition to the well-documented effects of vitamin D upon the nuclear vitamin D receptor. The results may have important implications for our current understanding of certain diseases where TRPV1 and vitamin D deficiency have been implicated, such as chronic pain and autoimmune diseases, such as type 1 diabetes. ABSTRACT: The capsaicin receptor TRPV1 plays an important role in nociception, inflammation and immunity and its activity is regulated by exogenous and endogenous lipophilic ligands. As vitamin D is lipophilic and involved in similar biological processes as TRPV1, we hypothesized that it directly regulates TRPV1 activity and function. Our calcium imaging and electrophysiological data demonstrate that vitamin D (25-hydroxyvitamin D (25OHD) and 1,25-hydroxyvitamin D (1,25OHD)) can weakly activate TRPV1 at physiologically relevant concentrations (100 nM). Furthermore, both 25OHD and 1,25OHD can inhibit capsaicin-induced TRPV1 activity (IC50  = 34.3 ± 0.2 and 11.5 ± 0.9 nM, respectively), but not pH-induced TRPV1 activity, suggesting that vitamin D interacts with TRPV1 in the same region as the TRPV1 agonist capsaicin. This hypothesis is supported by our in silico TRPV1 structural modelling studies, which place 25OHD in the same binding region as capsaicin. 25OHD also attenuates PKC-dependent TRPV1 potentiation via interactions with a known PKC phospho-acceptor residue in TRPV1. To provide evidence for a physiological role for the interaction of vitamin D with TRPV1, we employed two different cellular models known to express TRPV1: mouse CD4+ T-cells and trigeminal neurons. Our results indicate that 25OHD reduces TRPV1-induced cytokine release from T-cells and capsaicin-induced calcium activity in trigeminal neurons. In summary, we provide evidence that vitamin D is a novel endogenous regulator of TRPV1 channel activity that may play an important physiological role in addition to its known effects through the canonical nuclear vitamin D receptor pathway.

Androgen Therapy in Male Patients Suffering from Type 2 Diabetes: A Review of Benefits and Risks.

BACKGROUND: The current estimated numbers of patients with Type 2 Diabetes (T2D) is believed to be close to 10% of the whole populations of many geographical regions, causing serious concerns over the resulting elevated morbidity and mortality as well as the impact on health care systems around the world. In addition to negatively affecting the quality of life, diabetes is associated with cardiovascular and cerebrovascular complications, indicating that appropriate drug therapy should not only deal with metabolic dysfunction but also protect the vascular system, kidney function and skeletal muscle mass from the effects of the epigenetic changes induced by hyperglycaemia. OBJECTIVE: To provide an insight into the management of hypogonadism associated with T2D, this review focuses on clinical observations related to androgen therapy in qualified diabetic patients, and discusses the lines of evidence for its benefits and risks. The potential interactions of testosterone with medicines used by patients with T2D will also be discussed. CONCLUSION: From recent clinical findings, it became evident that a considerable percentage of patients suffering from T2D manifested low serum testosterone and experienced diminished sexual activity, as well as reduced skeletal muscle mass and lower bone density. Although there are some controversies, Testosterone Replacement Therapy (TRT) for this particular population of patients appears to be beneficial overall only if it is implemented carefully and monitored regularly.

Current Views on Dopaminergic Drugs Affecting Glucose Homeostasis.

BACKGROUND: For more than three decades, it has been known that manipulation of dopaminergic system could affect glucose homesotasis in experimental animals. The notion that glucose homeostasis in human might be influenced by dopaminergic drugs has attracted a great deal of attention in the past two decades. In spite of rapid advancements in revealing involvement of dopaminergic neurotransmission in insulin release, glucose up-take and pancreatic beta cell function in general through centrally and peripherally controlled mechanisms, there are discrepancies among observations on experimental animals and human subjects. CONCLUSION: With the expansion of pharmacotherapy in psychotic conditions, depression and endocrine abnormalities along with a sharp increase in prevalence of type two diabetes and disturbances of glucose homeostasis as a major risk factor for many cardiovascular complications and associated mortalities; it seems a critical analysis of recent investigations on drugs which act as agonists or antagonists of dopaminergic receptors in various tissues and organs may provide better insight into how safe and efficient these medicines could be prescribed. Furthermore, the other main objective of present review is to compare clinical data on significance of changes in blood glucose and insulin levels during short term and after long term treatment with these agents. This in turn would be beneficial for determining adequate strategies to combat or to avoid adverse effects associated with dopaminergic drug therapy.

Direct Binding between Pre-S1 and TRP-like Domains in TRPP Channels Mediates Gating and Functional Regulation by PIP2.

Transient receptor potential (TRP) channels are regulated by diverse stimuli comprising thermal, chemical, and mechanical modalities. They are also commonly regulated by phosphatidylinositol-4,5-bisphosphate (PIP2), with underlying mechanisms largely unknown. We here revealed an intramolecular interaction of the TRPP3 N and C termini (N-C) that is functionally essential. The interaction was mediated by aromatic Trp81 in pre-S1 domain and cationic Lys568 in TRP-like domain. Structure-function analyses revealed similar N-C interaction in TRPP2 as well as TRPM8/-V1/-C4 via highly conserved tryptophan and lysine/arginine residues. PIP2 bound to cationic residues in TRPP3, including K568, thereby disrupting the N-C interaction and negatively regulating TRPP3. PIP2 had similar negative effects on TRPP2. Interestingly, we found that PIP2 facilitates the N-C interaction in TRPM8/-V1, resulting in channel potentiation. The intramolecular N-C interaction might represent a shared mechanism underlying the gating and PIP2 regulation of TRP channels.

Identification and characterization of hydrophobic gate residues in TRP channels.

Transient receptor potential (TRP) channels, subdivided into 6 subfamilies in mammals, have essential roles in sensory physiology. They respond to remarkably diverse stimuli, comprising thermal, chemical, and mechanical modalities, through opening or closing of channel gates. In this study, we systematically substituted the hydrophobic residues within the distal fragment of pore-lining helix S6 with hydrophilic residues and, based on Xenopus oocyte and mammalian cell electrophysiology and a hydrophobic gate theory, identified hydrophobic gates in TRPV6/V5/V4/C4/M8. We found that channel activity drastically increased when TRPV6Ala616 or Met617 or TRPV5Ala576 or Met577, but not any of their adjacent residues, was substituted with hydrophilic residues. Channel activity strongly correlated with the hydrophilicity of the residues at those sites, suggesting that consecutive hydrophobic residues TRPV6Ala616-Met617 and TRPV5Ala576-Met577 form a double-residue gate in each channel. By the same strategy, we identified a hydrophobic single-residue gate in TRPV4Iso715, TRPC4Iso617, and TRPM8Val976. In support of the hydrophobic gate theory, hydrophilic substitution at the gate site, which removes the hydrophobic gate seal, substantially increased the activity of TRP channels in low-activity states but had little effect on the function of activated channels. The double-residue gate channels were more sensitive to small changes in the gate's hydrophobicity or size than single-residue gate channels. The unconventional double-reside gating mechanism in TRP channels may have been evolved to respond especially to physiologic stimuli that trigger relatively small gate conformational changes.-Zheng, W., Hu, R., Cai, R., Hofmann, L., Hu, Q., Fatehi, M., Long, W., Kong, T., Tang, J., Light, P., Flockerzi, V., Cao, Y., Chen, X.-Z. Identification and characterization of hydrophobic gate residues in TRP channels.

Subcutaneous white adipocytes express a light sensitive signaling pathway mediated via a melanopsin/TRPC channel axis.

Subcutaneous white adipose tissue (scWAT) is the major fat depot in humans and is a central player in regulating whole body metabolism. Skin exposure to UV wavelengths from sunlight is required for Vitamin D synthesis and pigmentation, although it is plausible that longer visible wavelengths that penetrate the skin may regulate scWAT function. In this regard, we discovered a novel blue light-sensitive current in human scWAT that is mediated by melanopsin coupled to transient receptor potential canonical cation channels. This pathway is activated at physiological intensities of light that penetrate the skin on a sunny day. Daily exposure of differentiated adipocytes to blue light resulted in decreased lipid droplet size, increased basal lipolytic rate and alterations in adiponectin and leptin secretion. Our results suggest that scWAT function may be directly under the influence of ambient sunlight exposure and may have important implications for our current understanding of adipocyte biology. (150 words).

The mechano-sensitivity of cardiac ATP-sensitive potassium channels is mediated by intrinsic MgATPase activity.

Cardiac ATP-sensitive K+ (KATP) channel activity plays an important cardio-protective role in regulating excitability in response to metabolic stress. Evidence suggests that these channels are also mechano-sensitive and therefore may couple KATP channel activity to increased cardiac workloads. However, the molecular mechanism that couples membrane stretch to channel activity is not currently known. We hypothesized that membrane stretch may alter the intrinsic MgATPase activity of the cardiac KATP channel resulting in increased channel activation. The inside-out patch-clamp technique was used to record single-channel and macroscopic recombinant KATP channel activity in response to membrane stretch elicited by negative pipette pressure. We found that stretch activation requires the presence of the SUR subunit and that inhibition of MgATPase activity with either the non-hydrolysable ATP analog AMP-PNP or the ATPase inhibitor BeFx significantly reduced the stimulatory effect of stretch. We employed a point mutagenic approach to determine that a single residue (K1337) in the hairpin loop proximal to the major MgATPase catalytic site in the SUR2A subunit is responsible for the difference in mechano-sensitivity between SUR2A and SUR1 containing KATP channels. Moreover, using a double cysteine mutant substitution in the hairpin loop region revealed the importance of a key residue-residue interaction in this region that transduces membrane mechanical forces into KATP channel stimulation via increases in channel MgATPase activity. With respect to KATP channel pharmacology, glibenclamide, but not glicalizide or repaglinide, was able to completely inhibit KATP channel mechano-sensitivity. In summary, our results provide a highly plausible molecular mechanism by which mechanical membrane forces are rapidly converted in changes in KATP channel activity that have implications for our understanding of cardiac KATP channels in physiological or pathophysiological settings that involve increased workload.

Molecular determinants of ATP-sensitive potassium channel MgATPase activity: diabetes risk variants and diazoxide sensitivity.

ATP-sensitive K(+) (KATP) channels play an important role in insulin secretion. KATP channels possess intrinsic MgATPase activity that is important in regulating channel activity in response to metabolic changes, although the precise structural determinants are not clearly understood. Furthermore, the sulfonylurea receptor 1 (SUR1) S1369A diabetes risk variant increases MgATPase activity, but the molecular mechanisms remain to be determined. Therefore, we hypothesized that residue-residue interactions between 1369 and 1372, predicted from in silico modelling, influence MgATPase activity, as well as sensitivity to the clinically used drug diazoxide that is known to increase MgATPase activity. We employed a point mutagenic approach with patch-clamp and direct biochemical assays to determine interaction between residues 1369 and 1372. Mutations in residues 1369 and 1372 predicted to decrease the residue interaction elicited a significant increase in MgATPase activity, whereas mutations predicted to possess similar residue interactions to wild-type (WT) channels elicited no alterations in MgATPase activity. In contrast, mutations that were predicted to increase residue interactions resulted in significant decreases in MgATPase activity. We also determined that a single S1369K substitution in SUR1 caused MgATPase activity and diazoxide pharmacological profiles to resemble those of channels containing the SUR2A subunit isoform. Our results provide evidence, at the single residue level, for a molecular mechanism that may underlie the association of the S1369A variant with type 2 diabetes. We also show a single amino acid difference can account for the markedly different diazoxide sensitivities between channels containing either the SUR1 or SUR2A subunit isoforms.

A review on natural products for controlling type 2 diabetes with an emphasis on their mechanisms of actions.

The use of natural products is very common among non-industrialized societies because these remedies are more accessible and affordable than modern pharmaceuticals. In developed countries, use of herbal products has recently increased as scientific evidence about their effectiveness has become broadly available. For the past two decades many research articles in the field of ethno-pharmacology have focused on the anti-diabetic effects of some natural products. This dramatic increase of interest was partly due to the fact that type 2 diabetes mellitus (T2DM) was considered as becoming a global epidemic health problem which imposed high cost to national health services around the world. We have no intention to advocate for replacing conventional pharmacotherapy with natural products to prevent and control T2DM. However, the fact that a lack of highly effective drug-therapy with existing synthetic agents and their resulting adverse effects motivated further search into traditional medicine in order to re-evaluate old remedies as well as screening to find new natural entities to be used as anti-diabetic products cannot be ignored. Some recent reports on the natural products with anti-diabetic effects have provided evidence for possible mechanisms of action. Nonetheless, the majority of investigators only speculated on a wide range of possible mechanisms or simply demonstrated an anti-hyperglycemic effect for the crude plant extracts or the isolated compounds of interest. A few reviews with less attention paid to mechanisms of action have been published on medicinal plants and diabetes. This article reviews publications on anti-diabetic natural products that have appeared in PUBMED or other research-related literature found on the Internet (from 1990 to present) to categorize them based on their mechanisms of action. We hope that this communication will be beneficial as a starting point to consider the discussed products for further investigations to identify and develop new remedies with potential alternative or complementary use in controlling T2DM.

Late sodium current inhibition alone with ranolazine is sufficient to reduce ischemia- and cardiac glycoside-induced calcium overload and contractile dysfunction mediated by reverse-mode sodium/calcium exchange.

Excessive reverse-mode (RM) sodium/calcium exchanger 1.1 (NCX1.1) activity, resulting from intracellular sodium accumulation caused by reduced Na+/K+-ATPase activity, increased Na-H exchanger 1 activity. The induction of the voltage-gated sodium channel late current component (late INa), is a major pathway for intracellular calcium (Ca2+i) loading in cardiac ischemia-reperfusion (IR) injury and cardiac glycoside toxicity. Inhibition of late INa with the antianginal agent ranolazine is protective in models of IR injury and cardiac glycoside toxicity. However, whether inhibition of late INa alone is sufficient to provide maximal protection or additional inhibition of RM NCX1.1 provides further benefit remains to be determined conclusively. Therefore, the effects of ranolazine were compared with the INa inhibitor lidocaine in models of IR injury and ouabain toxicity, RM NCX1.1-mediated Ca2+ overload, and patch-clamp assays of RM NCX1.1 currents. Ranolazine and lidocaine (10 µM) similarly reduced Ca2+i overload and improved left ventricle work recovery in whole-heart models of IR injury or exposure to ouabain (80 µM). Ranolazine (10 µM), but not lidocaine (10 µM), reduced RM NCX1.1-mediated Ca2+i overload in ventricular myocytes. Furthermore, ranolazine inhibited RM NCX1.1 currents (IC50 1.7 µM), without affecting forward mode currents, revealing that ranolazine has novel RM NCX1.1 inhibitory actions. However, because lidocaine provides similar protection to ranolazine in whole-heart models but does not inhibit RM NCX1.1, we conclude that induction of late INa is upstream of RM NCX1.1 activity and selective inhibition of late INa alone is sufficient to reduce Ca2+i overload and contractile dysfunction in IR injury and cardiac glycoside toxicity.

Pharmacogenomic analysis of ATP-sensitive potassium channels coexpressing the common type 2 diabetes risk variants E23K and S1369A.

OBJECTIVES: The common ATP-sensitive potassium (KATP) channel variants E23K and S1369A, found in the KCNJ11 and ABCC8 genes, respectively, form a haplotype that is associated with an increased risk for type 2 diabetes. Our previous studies showed that KATP channel inhibition by the A-site sulfonylurea gliclazide was increased in the K23/A1369 haplotype. Therefore, we studied the pharmacogenomics of seven clinically used sulfonylureas and glinides to determine their structure-activity relationships in KATP channels containing either the E23/S1369 nonrisk or K23/A1369 risk haplotypes. RESEARCH DESIGN AND METHODS: The patch-clamp technique was used to determine sulfonylurea and glinide inhibition of recombinant human KATP channels containing either the E23/S1369 or the K23/A1369 haplotype. RESULTS: KATP channels containing the K23/A1369 risk haplotype were significantly less sensitive to inhibition by tolbutamide, chlorpropamide, and glimepiride (IC50 values for K23/A1369 vs. E23/S1369=1.15 vs. 0.71 µmol/l; 4.19 vs. 3.04 µmol/l; 4.38 vs. 2.41 nmol/l, respectively). In contrast, KATP channels containing the K23/A1369 haplotype were significantly more sensitive to inhibition by mitiglinide (IC50=9.73 vs. 28.19 nmol/l for K23/A1369 vs. E23/S1369) and gliclazide. Nateglinide, glipizide, and glibenclamide showed similar inhibitory profiles in KATP channels containing either haplotype. CONCLUSION: Our results demonstrate that the ring-fused pyrrole moiety in several A-site drugs likely underlies the observed inhibitory potency of these drugs on KATP channels containing the K23/A1369 risk haplotype. It may therefore be possible to tailor existing therapy or design novel drugs that display an increased efficacy in type 2 diabetes patients homozygous for these common KATP channel haplotypes.

The ATP-sensitive K(+) channel ABCC8 S1369A type 2 diabetes risk variant increases MgATPase activity.

Pancreatic ß-cell ATP-sensitive K(+) (K(ATP)) channels are composed of Kir6.2 and SUR1 subunits encoded by the KCNJ11 and ABCC8 genes, respectively. Although rare monogenic activating mutations in these genes cause overt neonatal diabetes, the common variants E23K (KCNJ11) and S1369A (ABCC8) form a tightly heritable haplotype that is associated with an increased susceptibility to type 2 diabetes (T2D) risk. However, the molecular mechanism(s) underlying this risk remain to be elucidated. A homology model of the SUR1 nucleotide-binding domains (NBDs) indicates that residue 1369 is in close proximity to the major MgATPase site. Therefore, we investigated the intrinsic MgATPase activity of K(ATP) channels containing these variants. Electrophysiological and biochemical techniques were used to study the MgATPase activity of recombinant human K(ATP) channels or glutathione S-transferase and NBD2 fusion proteins containing the E23/S1369 (nonrisk) or K23/A1369 (risk) variant haplotypes. K(ATP) channels containing the K23/A1369 haplotype displayed a significantly increased stimulation by guanosine triphosphate compared with the E23/S1369 haplotype (3.2- vs. 1.8-fold). This effect was dependent on the presence of the A1369 variant and was lost in the absence of Mg(2+) ions or in the presence of the MgATPase inhibitor beryllium fluoride. Direct biochemical assays also confirmed an increase in MgATPase activity in NBD2 fusion proteins containing the A1369 variant. Our findings demonstrate that the A1369 variant increases K(ATP) channel MgATPase activity, providing a plausible molecular mechanism by which the K23/A1369 haplotype increases susceptibility to T2D in humans homozygous for these variants.

Intravitreal triamcinolone for acute branch retinal vein occlusion: a randomized clinical trial.

PURPOSE: To evaluate the therapeutic effect of intravitreal triamcinolone (IVT) injection for recent branch retinal vein occlusion (BRVO). METHODS: In a randomized controlled clinical trial, 30 phakic eyes with recent (less than 10 weeks' duration) BRVO were assigned to two groups. The treatment group (16 eyes) received 4 mg IVT and the control group (14 eyes) received subconjunctival sham injections. Changes in visual acuity (VA) were the main outcome measure. RESULTS: VA and central macular thickness (CMT) changes were not significantly different between the study groups at any time point. Within group analysis showed significant VA improvement from baseline in the IVT group up to three months (P < 0.05); the amount of this change was -0.53 ± 0.46, -0.37 ± 0.50, -0.46 ± 0.50, and -0.29 ± 0.45 logMAR at 1, 2, 3, and 4 months, respectively. Corresponding VA improvements in the control group were -0.20 ± 0.37, -0.11 ± 0.46, -0.25 ± 0.58, and -0.05 ± 0.50 logMAR (all P values > 0.05). Significant reduction in CMT was noticed only in the treatment group (-172 ± 202 µm, P = 0.029) and at 4 months. Ocular hypertension occurred in 4 (25%) and 2 (14.3%) eyes in the IVT and control groups, respectively. CONCLUSION: A single IVT injection had a non-significant beneficial effect on VA and CMT in acute BRVO as compared to the natural history of the condition. The 3-month deferred treatment protocol advocated by the Branch Vein Occlusion Study Group may be a safer option than IVT injection considering its potential side effects.

Novel residues lining the CFTR chloride channel pore identified by functional modification of introduced cysteines.

Substituted cysteine accessibility mutagenesis (SCAM) has been used widely to identify pore-lining amino acid side chains in ion channel proteins. However, functional effects on permeation and gating can be difficult to separate, leading to uncertainty concerning the location of reactive cysteine side chains. We have combined SCAM with investigation of the charge-dependent effects of methanethiosulfonate (MTS) reagents on the functional permeation properties of cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channels. We find that cysteines substituted for seven out of 21 continuous amino acids in the eleventh and twelfth transmembrane (TM) regions can be modified by external application of positively charged [2-(trimethylammonium)ethyl] MTS bromide (MTSET) and negatively charged sodium [2-sulfonatoethyl] MTS (MTSES). Modification of these cysteines leads to changes in the open channel current-voltage relationship at both the macroscopic and single-channel current levels that reflect specific, charge-dependent effects on the rate of Cl(-) permeation through the channel from the external solution. This approach therefore identifies amino acid side chains that lie within the permeation pathway. Cysteine mutagenesis of pore-lining residues also affects intrapore anion binding and anion selectivity, giving more information regarding the roles of these residues. Our results demonstrate a straightforward method of screening for pore-lining amino acids in ion channels. We suggest that TM11 contributes to the CFTR pore and that the extracellular loop between TMs 11 and 12 lies close to the outer mouth of the pore.

Mechanism of direct bicarbonate transport by the CFTR anion channel.

BACKGROUND: CFTR contributes to HCO(3)(-) transport in epithelial cells both directly (by HCO(3)(-) permeation through the channel) and indirectly (by regulating Cl(-)/HCO(3)(-) exchange proteins). While loss of HCO(3)(-) transport is highly relevant to cystic fibrosis, the relative importance of direct and indirect HCO(3)(-) transport it is currently unknown. METHODS: Patch clamp recordings from membrane patches excised from cells heterologously expressing wild type and mutant forms of human CFTR were used to isolate directly CFTR-mediated HCO(3)(-) transport and characterize its functional properties. RESULTS: The permeability of HCO(3)(-) was approximately 25% that of Cl(-) and was invariable under all ionic conditions studied. CFTR-mediated HCO(3)(-) currents were inhibited by open channel blockers DNDS, glibenclamide and suramin, and these inhibitions were affected by mutations within the channel pore. Cystic fibrosis mutations previously associated with disrupted cellular HCO(3)(-) transport did not affect direct HCO(3)(-) permeability. CONCLUSIONS: Cl(-) and HCO(3)(-) share a common transport pathway in CFTR, and selectivity between Cl(-) and HCO(3)(-) is independent of ionic conditions. The mechanism of transport is therefore effectively identical for both ions. We suggest that mutations in CFTR that cause cystic fibrosis by selectively disrupting HCO(3)(-) transport do not impair direct CFTR-mediated HCO(3)(-) transport, but may predominantly alter CFTR regulation of other HCO(3)(-) transport pathways.

Identification of positive charges situated at the outer mouth of the CFTR chloride channel pore.

We have used site-directed mutagenesis and functional analysis to identify positively charged amino acid residues in the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel that interact with extracellular anions. Mutation of two positively charged arginine residues in the first extracellular loop (ECL) of CFTR, R104, and R117, as well as lysine residue K335 in the sixth transmembrane region, leads to inward rectification of the current-voltage relationship and decreased single channel conductance. These effects are dependent on the charge of the substituted side chain and on the Cl(-) concentration, suggesting that these positive charges normally act to concentrate extracellular Cl(-) ions near the outer mouth of the pore. Side chain charge-dependent effects are mimicked by manipulating charge in situ by mutating these amino acids to cysteine followed by covalent modification with charged cysteine-reactive reagents, confirming the location of these side chains within the pore outer vestibule. State-independent modification of R104C and R117C suggests that these residues are located at the outermost part of the pore. We suggest that ECL1 contributes to the CFTR pore external vestibule and that positively charged amino acid side chains in this region act to attract Cl(-) ions into the pore. In contrast, we find no evidence that fixed positive charges in other ECLs contribute to the permeation properties of the pore.

State-dependent access of anions to the cystic fibrosis transmembrane conductance regulator chloride channel pore.

The cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel is gated by intracellular factors; however, conformational changes in the channel pore associated with channel activation have not been identified. We have used patch clamp recording to investigate the state-dependent accessibility of substituted cysteine residues in the CFTR channel pore to a range of cysteine-reactive reagents applied to the extracellular side of the membrane. Using functional modification of the channel current-voltage relationship as a marker of modification, we find that several positively charged reagents are able to penetrate deeply into the pore from the outside irrespective of whether or not the channels have been activated. In contrast, access of three anionic cysteine-reactive reagents, the methanesulfonate sodium (2-sulfonatoethyl)methanesulfonate, the organic mercurial p-chloromercuriphenylsulfonic acid, and the permeant anion Au(CN)(2)(-), to several different sites in the pore is strictly limited prior to channel activation. This suggests that in nonactivated channels some ion selectivity mechanism exists to exclude anions yet permit cations into the channel pore from the extracellular solution. We suggest that activation of CFTR channels involves a conformational change in the pore that removes a strong selectivity against anion entry from the extracellular solution. We propose further that this conformational change occurs in advance of channel opening, suggesting that multiple distinct closed pore conformations exist.