ABSTRACT
The Ca2+-activated Cl- channel regulator CLCA1 potentiates the activity of the Ca2+-activated Cl- channel (CaCC) TMEM16A by directly engaging the channel at the cell surface, inhibiting its reinternalization and increasing Ca2+-dependent Cl- current (ICaCC) density. We now present evidence of functional pairing between two other CLCA and TMEM16 protein family members, namely CLCA4 and the CaCC TMEM16B. Similar to CLCA1, (i) CLCA4 is a self-cleaving metalloprotease, and the N-terminal portion (N-CLCA4) is secreted; (ii) the von Willebrand factor type A (VWA) domain in N-CLCA4 is sufficient to potentiate ICaCC in HEK293T cells; and (iii) this is mediated by the metal ion-dependent adhesion site motif within VWA. The results indicate that, despite the conserved regulatory mechanism and homology between CLCA1 and CLCA4, CLCA4-dependent ICaCC are carried by TMEM16B, rather than TMEM16A. Our findings show specificity in CLCA/TMEM16 interactions and suggest broad physiological and pathophysiological links between these two protein families.
Subject(s)
Anoctamins , Chloride Channels , Humans , Anoctamin-1/metabolism , Anoctamin-1/genetics , Anoctamins/metabolism , Anoctamins/genetics , Anoctamins/chemistry , Calcium/metabolism , Chloride Channels/metabolism , Chloride Channels/genetics , Chlorides/metabolism , HEK293 Cells , Neoplasm Proteins/metabolism , Neoplasm Proteins/genetics , Protein DomainsABSTRACT
Goblet cells (GCs) are specialized cells of the intestinal epithelium contributing critically to mucosal homeostasis. One of the functions of GCs is to produce and secrete MUC2, the mucin that forms the scaffold of the intestinal mucus layer coating the epithelium and separates the luminal pathogens and commensal microbiota from the host tissues. Although a variety of ion channels and transporters are thought to impact on MUC2 secretion, the specific cellular mechanisms that regulate GC function remain incompletely understood. Previously, we demonstrated that leucine-rich repeat-containing protein 26 (LRRC26), a known regulatory subunit of the Ca2+-and voltage-activated K+ channel (BK channel), localizes specifically to secretory cells within the intestinal tract. Here, utilizing a mouse model in which MUC2 is fluorescently tagged, thereby allowing visualization of single GCs in intact colonic crypts, we show that murine colonic GCs have functional LRRC26-associated BK channels. In the absence of LRRC26, BK channels are present in GCs, but are not activated at physiological conditions. In contrast, all tested MUC2- cells completely lacked BK channels. Moreover, LRRC26-associated BK channels underlie the BK channel contribution to the resting transepithelial current across mouse distal colonic mucosa. Genetic ablation of either LRRC26 or BK pore-forming α-subunit in mice results in a dramatically enhanced susceptibility to colitis induced by dextran sodium sulfate. These results demonstrate that normal potassium flux through LRRC26-associated BK channels in GCs has protective effects against colitis in mice.
Subject(s)
Colitis/genetics , Large-Conductance Calcium-Activated Potassium Channels/genetics , Mucin-2/genetics , Animals , Colitis/pathology , Colitis/prevention & control , Colitis/therapy , Colon/metabolism , Colon/pathology , Disease Models, Animal , Goblet Cells/metabolism , Goblet Cells/pathology , Humans , Intestinal Mucosa/metabolism , Intestinal Mucosa/pathology , Membrane Potentials/genetics , Mice , Patch-Clamp TechniquesABSTRACT
The complex disorder Cantu syndrome (CS) arises from gain-of-function mutations in either KCNJ8 or ABCC9, the genes encoding the Kir6.1 and SUR2 subunits of ATP-sensitive potassium (KATP) channels, respectively. Recent reports indicate that such mutations can increase channel activity by multiple molecular mechanisms. In this study, we determined the mechanism by which KATP function is altered by several substitutions in distinct structural domains of SUR2: D207E in the intracellular L0-linker and Y985S, G989E, M1060I, and R1154Q/R1154W in TMD2. We engineered substitutions at their equivalent positions in rat SUR2A (D207E, Y981S, G985E, M1056I, and R1150Q/R1150W) and investigated functional consequences using macroscopic rubidium (86Rb+) efflux assays and patch-clamp electrophysiology. Our results indicate that D207E increases KATP channel activity by increasing intrinsic stability of the open state, whereas the cluster of Y981S/G985E/M1056I substitutions, as well as R1150Q/R1150W, augmented Mg-nucleotide activation. We also tested the responses of these channel variants to inhibition by the sulfonylurea drug glibenclamide, a potential pharmacotherapy for CS. None of the D207E, Y981S, G985E, or M1056I substitutions had a significant effect on glibenclamide sensitivity. However, Gln and Trp substitution at Arg-1150 significantly decreased glibenclamide potency. In summary, these results provide additional confirmation that mutations in CS-associated SUR2 mutations result in KATP gain-of-function. They help link CS genotypes to phenotypes and shed light on the underlying molecular mechanisms, including consequences for inhibitory drug sensitivity, insights that may inform the development of therapeutic approaches to manage CS.
Subject(s)
Cardiomegaly/genetics , Gain of Function Mutation , Hypertrichosis/genetics , Mutation, Missense , Osteochondrodysplasias/genetics , Sulfonylurea Receptors/chemistry , Sulfonylurea Receptors/genetics , Animals , Cardiomegaly/metabolism , Glyburide/chemistry , Glyburide/metabolism , Humans , Hypertrichosis/metabolism , KATP Channels/chemistry , KATP Channels/genetics , KATP Channels/metabolism , Osteochondrodysplasias/metabolism , Protein Domains , Rats , Sulfonylurea Receptors/metabolismABSTRACT
KEY POINTS: The goal was to determine the importance of the sodium-glucose cotransporter SGLT1 and the glucose uniporter GLUT2 in intestinal glucose absorption during oral glucose tolerance tests (OGTTs) in mice. Glucose absorption was determined in mice using positron emission tomography and three non-metabolizable glucose probes: one specific for SGLTs, one specific for GLUTs, and one a substrate for both SGLTs and GLUTs. Absorption was determined in wild-type, Sglt1-/- and Glut2-/- mice. Gastric emptying was a rate-limiting step in absorption. SGLT1, but not GLUT2, was important in fast glucose absorption. In the absence of SGLT1 or GLUT2, the oral glucose load delivered to the small intestine was slowly absorbed. Oral phlorizin only inhibited the fast component of glucose absorption, but it contributed to decreasing blood glucose levels by inhibiting renal reabsorption. ABSTRACT: The current model of intestinal absorption is that SGLT1 is responsible for transport of glucose from the lumen into enterocytes across the brush border membrane, and GLUT2 for the downhill transport from the epithelium into blood across the basolateral membrane. Nevertheless, questions remain about the importance of these transporters in vivo. To address these questions, we have developed a non-invasive imaging method, positron emission tomography (PET), to monitor intestinal absorption of three non-metabolized glucose tracers during standard oral glucose tolerance tests (OGTTs) in mice. One tracer is specific for SGLTs (α-methyl-4-[18 F]fluoro-4-deoxy-d-glucopyranoside; Me-4FDG), one is specific for GLUTs (2-deoxy-2-[18 F]fluoro-d-glucose; 2-FDG), and one is a substrate for both SGLTs and GLUTs (4-deoxy-4-[18 F]fluoro-d-glucose; 4-FDG). OGTTs were conducted on adult wild-type, Sglt1-/- and Glut2-/- mice. In conscious mice, OGTTs resulted in the predictable increase in blood glucose that was blocked by phlorizin in both wild-type and Glut2-/- animals. The blood activity of both Me-4FDG and 4-FDG, but not 2-FDG, accompanied the changes in glucose concentration. PET imaging during OGTTs further shows that: (i) intestinal absorption of the glucose load depends on gastric emptying; (ii) SGLT1 is important for the fast absorption; (iii) GLUT2 is not important in absorption; and (iv) oral phlorizin reduces absorption by SGLT1, but is absorbed and blocks glucose reabsorption in the kidney. We conclude that in standard OGTTs in mice, SGLT1 is essential in fast absorption, GLUT2 does not play a significant role, and in the absence of SGLT1 the total load of glucose is slowly absorbed.
Subject(s)
Glucose/metabolism , Intestinal Absorption , Intestine, Small/metabolism , Positron-Emission Tomography/methods , Animals , Biological Transport , Female , Glucose Tolerance Test , Glucose Transporter Type 2/metabolism , Intestine, Small/diagnostic imaging , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Sodium-Glucose Transporter 1/metabolismABSTRACT
Calcium-activated chloride channels (CaCCs) are key players in transepithelial ion transport and fluid secretion, smooth muscle constriction, neuronal excitability, and cell proliferation. The CaCC regulator 1 (CLCA1) modulates the activity of the CaCC TMEM16A/Anoctamin 1 (ANO1) by directly engaging the channel at the cell surface, but the exact mechanism is unknown. Here we demonstrate that the von Willebrand factor type A (VWA) domain within the cleaved CLCA1 N-terminal fragment is necessary and sufficient for this interaction. TMEM16A protein levels on the cell surface were increased in HEK293T cells transfected with CLCA1 constructs containing the VWA domain, and TMEM16A-like currents were activated. Similar currents were evoked in cells exposed to secreted VWA domain alone, and these currents were significantly knocked down by TMEM16A siRNA. VWA-dependent TMEM16A modulation was not modified by the S357N mutation, a VWA domain polymorphism associated with more severe meconium ileus in cystic fibrosis patients. VWA-activated currents were significantly reduced in the absence of extracellular Mg2+, and mutation of residues within the conserved metal ion-dependent adhesion site motif impaired the ability of VWA to potentiate TMEM16A activity, suggesting that CLCA1-TMEM16A interactions are Mg2+- and metal ion-dependent adhesion site-dependent. Increase in TMEM16A activity occurred within minutes of exposure to CLCA1 or after a short treatment with nocodazole, consistent with the hypothesis that CLCA1 stabilizes TMEM16A at the cell surface by preventing its internalization. Our study hints at the therapeutic potential of the selective activation of TMEM16A by the CLCA1 VWA domain in loss-of-function chloride channelopathies such as cystic fibrosis.
Subject(s)
Chloride Channels/metabolism , Magnesium/metabolism , Mutation, Missense , Neoplasm Proteins/metabolism , Amino Acid Substitution , Anoctamin-1 , Cell Line , Chloride Channels/genetics , Humans , Neoplasm Proteins/genetics , Protein Domains , Protein StabilityABSTRACT
KEY POINTS: Glucose transporters are central players in glucose homeostasis. There are two major classes of glucose transporters in the body, the passive facilitative glucose transporters (GLUTs) and the secondary active sodium-coupled glucose transporters (SGLTs). In the present study, we report the use of a non-invasive imaging technique, positron emission tomography, in mice aiming to evaluate the role of GLUTs and SGLTs in controlling glucose distribution and utilization. We show that GLUTs are most significant for glucose uptake into the brain and liver, whereas SGLTs are important in glucose recovery in the kidney. This work provides further support for the use of SGLT imaging in the investigation of the role of SGLT transporters in human physiology and diseases such as diabetes and cancer. ABSTRACT: The importance of sodium-coupled glucose transporters (SGLTs) and facilitative glucose transporters (GLUTs) in glucose homeostasis was studied in mice using fluorine-18 labelled glucose molecular imaging probes and non-invasive positron emission tomography (PET) imaging. The probes were: α-methyl-4-[F-18]-fluoro-4-deoxy-d-glucopyranoside (Me-4FDG), a substrate for SGLTs; 4-deoxy-4-[F-18]-fluoro-d-glucose (4-FDG), a substrate for SGLTs and GLUTs; and 2-deoxy-2-[F-18]-fluoro-d-glucose (2-FDG), a substrate for GLUTs. These radiolabelled imaging probes were injected i.v. into wild-type, Sglt1(-/-) , Sglt2(-/-) and Glut2(-/-) mice and their dynamic whole-body distribution was determined using microPET. The distribution of 2-FDG was similar to that reported earlier (i.e. it accumulated in the brain, heart, liver and kidney, and was excreted into the urinary bladder). There was little change in the distribution of 2-FDG in Glut2(-/-) mice, apart from a reduction in the rate of uptake into liver. The major differences between Me-4FDG and 2-FDG were that Me-4FDG did not enter the brain and was not excreted into the urinary bladder. There was urinary excretion of Me-4FDG in Sglt1(-/-) and Sglt2(-/-) mice. However, Me-4FDG was not reabsorbed in the kidney in Glut2(-/-) mice. There were no differences in Me-4FDG uptake into the heart of wild-type, Sglt1(-/-) and Sglt2(-/-) mice. We conclude that GLUT2 is important in glucose liver transport and reabsorption of glucose in the kidney along with SGLT2 and SGLT1. Complete reabsorption of Me-4FDG from the glomerular filtrate in wild-type mice and the absence of reabsorption in the kidney in Glut2(-/-) mice confirm the importance of GLUT2 in glucose absorption across the proximal tubule.
Subject(s)
Glucose Transport Proteins, Facilitative/metabolism , Sodium-Glucose Transport Proteins/metabolism , Animals , Brain/diagnostic imaging , Brain/metabolism , Deoxyglucose/analogs & derivatives , Female , Fluorine Radioisotopes , Glucose/pharmacokinetics , Glucose Transport Proteins, Facilitative/genetics , Glucosides , Heart/diagnostic imaging , Kidney/diagnostic imaging , Kidney/metabolism , Liver/diagnostic imaging , Liver/metabolism , Male , Mice, Inbred C57BL , Mice, Knockout , Muscles/diagnostic imaging , Muscles/metabolism , Myocardium/metabolism , Positron-Emission Tomography , Sodium-Glucose Transport Proteins/genetics , Urinary Bladder/diagnostic imaging , Urinary Bladder/metabolismABSTRACT
Organic cation transporter 3 (OCT3, SLC22A3) is a polyspecific, facilitative transporter expressed in astrocytes and in placental, intestinal, and blood-brain barrier epithelia, and thus elucidating the molecular mechanisms underlying OCT3 substrate recognition is critical for the rational design of drugs targeting these tissues. The pharmacology of OCT3 is distinct from that of other OCTs, and here we investigated the role of a hydrophobic cavity tucked within the translocation pathway in OCT3 transport properties. Replacement of an absolutely conserved Asp by charge reversal (D478E), neutralization (D478N), or even exchange (D478E) abolished MPP(+) uptake, demonstrating this residue to be obligatory for OCT3-mediated transport. Mutations at non-conserved residues lining the putative binding pocket of OCT3 to the corresponding residue in OCT1 (L166F, F450L, and E451Q) reduced the rate of MPP(+) transport, but recapitulated the higher sensitivity pharmacological profile of OCT1. Thus, interactions of natural polyamines (putrescine, spermidine, spermine) and polyamine-like potent OCT1 blockers (1,10-diaminodecane, decamethonium, bistriethylaminodecane, and 1,10-bisquinuclidinedecane) with wild-type OCT3 were weak, but were significantly potentiated in the mutant OCT3s. Conversely, a reciprocal mutation in OCT1 (F161L) shifted the polyamine-sensitivity phenotype toward that of OCT3. Further analysis indicated that OCT1 and OCT3 can recognize essentially the same substrates, but the strength of substrate-transporter interactions is weaker in OCT3, as informed by the distinct makeup of the hydrophobic cleft. The residues identified here are key contributors to both the observed differences between OCT3 and OCT1 and to the mechanisms of substrate recognition by OCTs in general.
Subject(s)
Hydrophobic and Hydrophilic Interactions , Organic Cation Transport Proteins/chemistry , Polyamines/chemistry , Amino Acid Sequence , Amino Acid Substitution , Animals , Diamines/chemistry , Humans , Molecular Sequence Data , Mutation , Organic Cation Transport Proteins/genetics , Organic Cation Transporter 1/chemistry , Organic Cation Transporter 1/genetics , Protein Structure, Secondary , Putrescine/chemistry , Rats , Spermidine/chemistryABSTRACT
Chloride transport proteins play critical roles in inflammatory airway diseases, contributing to the detrimental aspects of mucus overproduction, mucus secretion, and airway constriction. However, they also play crucial roles in contributing to the innate immune properties of mucus and mucociliary clearance. In this review, we focus on the emerging novel roles for a chloride channel regulator (CLCA1), a calcium-activated chloride channel (TMEM16A), and two chloride exchangers (SLC26A4/pendrin and SLC26A9) in chronic inflammatory airway diseases.
Subject(s)
Antiporters/physiology , Asthma/etiology , Chloride Channels/physiology , Membrane Transport Proteins/physiology , Neoplasm Proteins/physiology , Pulmonary Disease, Chronic Obstructive/etiology , Anoctamin-1 , Cystic Fibrosis Transmembrane Conductance Regulator/physiology , Humans , STAT6 Transcription Factor/physiology , Sulfate TransportersABSTRACT
The sulfonylurea receptor SUR1 associates with Kir6.2 or Kir6.1 to form K(ATP) channels, which link metabolism to excitability in multiple cell types. The strong physical coupling of SUR1 with Kir6 subunits appears exclusive, but recent studies argue that SUR1 also modulates TRPM4, a member of the transient receptor potential family of non-selective cation channels. It has been reported that, following stroke, brain, or spinal cord injury, SUR1 is increased in neurovascular cells at the site of injury. This is accompanied by up-regulation of a non-selective cation conductance with TRPM4-like properties and apparently sensitive to sulfonylureas, leading to the postulation that post-traumatic non-selective cation currents are determined by TRPM4/SUR1 channels. To investigate the mechanistic hypothesis for the coupling between TRPM4 and SUR1, we performed electrophysiological and FRET studies in COSm6 cells expressing TRPM4 channels with or without SUR1. TRPM4-mediated currents were Ca(2+)-activated, voltage-dependent, underwent desensitization, and were inhibited by ATP but were insensitive to glibenclamide and tolbutamide. These properties were not affected by cotransfection with SUR1. When the same SUR1 was cotransfected with Kir6.2, functional K(ATP) channels were formed. In cells cotransfected with Kir6.2, SUR1, and TRPM4, we measured K(ATP)-mediated K(+) currents and Ca(2+)-activated, sulfonylurea-insensitive Na(+) currents in the same patch, further showing that SUR1 controls K(ATP) channel activity but not TRPM4 channels. FRET signal between fluorophore-tagged TRPM4 subunits was similar to that between Kir6.2 and SUR1, whereas there was no detectable FRET efficiency between TRPM4 and SUR1. Our data suggest that functional or structural association of TRPM4 and SUR1 is unlikely.
Subject(s)
ATP-Binding Cassette Transporters/metabolism , Potassium Channels, Inwardly Rectifying/metabolism , Receptors, Drug/metabolism , TRPM Cation Channels/metabolism , ATP-Binding Cassette Transporters/chemistry , ATP-Binding Cassette Transporters/genetics , Amino Acid Motifs , Animals , Cell Line , Kinetics , Mice , Potassium Channels/chemistry , Potassium Channels/genetics , Potassium Channels/metabolism , Potassium Channels, Inwardly Rectifying/chemistry , Potassium Channels, Inwardly Rectifying/genetics , Protein Binding , Receptors, Drug/chemistry , Receptors, Drug/genetics , Sulfonylurea Receptors , TRPM Cation Channels/chemistry , TRPM Cation Channels/geneticsABSTRACT
The chloride channel calcium-activated (CLCA) family are secreted proteins that regulate both chloride transport and mucin expression, thus controlling the production of mucus in respiratory and other systems. Accordingly, human CLCA1 is a critical mediator of hypersecretory lung diseases, such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis, that manifest mucus obstruction. Despite relevance to homeostasis and disease, the mechanism of CLCA1 function remains largely undefined. We address this void by showing that CLCA proteins contain a consensus proteolytic cleavage site recognized by a novel zincin metalloprotease domain located within the N terminus of CLCA itself. CLCA1 mutations that inhibit self-cleavage prevent activation of calcium-activated chloride channel (CaCC)-mediated chloride transport. CaCC activation requires cleavage to unmask the N-terminal fragment of CLCA1, which can independently gate CaCCs. Gating of CaCCs mediated by CLCA1 does not appear to involve proteolytic cleavage of the channel because a mutant N-terminal fragment deficient in proteolytic activity is able to induce currents comparable with that of the native fragment. These data provide both a mechanistic basis for CLCA1 self-cleavage and a novel mechanism for regulation of chloride channel activity specific to the mucosal interface.
Subject(s)
Chloride Channels/metabolism , Ion Channel Gating/physiology , Metalloproteases/metabolism , Proteolysis , Cell Line , Chloride Channels/genetics , Humans , Ion Transport/physiology , Metalloproteases/genetics , Protein Structure, TertiaryABSTRACT
Channel replacement therapy, based on synthetic channel-forming peptides (CFPs) with the ability to supersede defective endogenous ion channels, is a novel treatment modality that may augment existing interventions against multiple diseases. Previously, we derived CFPs from the second transmembrane segment of the α-subunit of the glycine receptor, M2GlyR, which forms chloride-selective channels in its native form. The best candidate, NK4-M2GlyR T19R, S22W (p22-T19R, S22W), was water-soluble, incorporated into cell membranes and was nonimmunogenic, but lacked the structural properties for high conductance and anion selectivity when assembled into a pore. Further studies suggested that the threonine residues at positions 13, 17, and 20 line the pore of assembled p22-T19R, S22W, and here we used 2,3-diaminopropionic acid (Dap) substitutions to introduce positive charges to the pore-lining interface of the predicted p22-T19R, S22W channel. Dap-substituted p22-T19R, S22W peptides retained the α-helical secondary structure characteristic of their parent peptide, and induced short-circuit transepithelial currents when exposed to the apical membrane of Madin-Darby canine kidney (MDCK) cells; the sequences containing multiple Dap-substituted residues induced larger currents than the peptides with single or no Dap substitutions. To gain further insights into the effects of Dap residues on the properties of the putative pore, we performed two-electrode voltage clamp electrophysiology on Xenopus oocytes exposed to p22-T19R, S22W or its Dap-modified analogues. We observed that Dap-substituted peptides also induced significantly larger voltage-dependent currents than the parent compound, but there was no apparent change in reversal potential upon replacement of external Na+, Cl- or K+, indicating that these currents remained nonselective. These results suggest that the introduction of positively charged side chains in predicted pore-lining residues does not improve anion-to-cation selectivity, but results in higher conductance, perhaps due to higher oligomerization numbers.
Subject(s)
Peptides/chemistry , beta-Alanine/analogs & derivatives , Ion Channels/chemistry , Protein Structure, Secondary , Receptors, Glycine/chemistry , beta-Alanine/chemistryABSTRACT
Polyamines are ubiquitous organic cations implicated in many physiological processes. Because they are positively charged at physiological pH, carrier-mediated systems are necessary for effective membrane permeation, but the identity of specific polyamine transporter proteins in eukaryotic cells remains unclear. Polyspecific organic cation transporters (OCTs) interact with many natural and xenobiotic monovalent cations and have been reported to transport dicationic compounds, including the short polyamine putrescine. In this study, we used Xenopus oocytes expressing mammalian OCT1 (SLC22A1), OCT2 (SLC22A2), or OCT3 (SLC22A3) to assess binding and transport of longer-chain polyvalent polyamines. In OCT-expressing oocytes, [(3)H]MPP(+) uptake rates were 15- to 35-fold higher than in noninjected oocytes, whereas those for [(3)H]spermidine increased more modestly above the background, up to 3-fold. This reflected up to 20-fold lower affinity for spermidine than for MPP(+); thus, K(0.5) for MPP(+) was ~50 µM in OCT1, ~170 µM in OCT2, and ~60 µM in OCT3, whereas for spermidine, K(0.5) was ~1 mM in OCT1, OCT2, and OCT3. J(max) values for MPP(+) and spermidine were within the same range, suggesting that both compounds are transported at a similar turnover rate. To gain further insight into OCT substrate specificity, we screened a selection of structural polyamine analogues for effect on [(3)H]MPP(+) uptake. In general, blocking potency increased with overall hydrophobic character, which indicates that, as for monovalent cations, hydrophobicity is a major requirement for recognition in polyvalent OCT substrates and inhibitors. Our results demonstrate that the natural polyamines are low affinity, but relatively high turnover, substrates for OCTs. The identification of OCTs as polyamine transport systems may contribute to further understanding of the mechanisms involved in polyamine homeostasis and aid in the design of polyamine-like OCT-targeted drugs.
Subject(s)
Octamer Transcription Factor-2/metabolism , Octamer Transcription Factor-3/metabolism , Oocytes/metabolism , Organic Cation Transporter 1/metabolism , Polyamines/metabolism , Animals , Biological Transport , Cations , Crystallography, X-Ray , Female , Homeostasis , Humans , Hydrogen-Ion Concentration , Oocytes/drug effects , Putrescine/metabolism , Spermidine/metabolism , Substrate Specificity , Xenopus laevisABSTRACT
The Na(+)-glucose cotransporter hSGLT1 is a member of a class of membrane proteins that harness Na(+) electrochemical gradients to drive uphill solute transport. Although hSGLT1 belongs to one gene family (SLC5), recent structural studies of bacterial Na(+) cotransporters have shown that Na(+) transporters in different gene families have the same structural fold. We have constructed homology models of hSGLT1 in two conformations, the inward-facing occluded (based on vSGLT) and the outward open conformations (based on Mhp1), mutated in turn each of the conserved gates and ligand binding residues, expressed the SGLT1 mutants in Xenopus oocytes, and determined the functional consequences using biophysical and biochemical assays. The results establish that mutating the ligand binding residues produces profound changes in the ligand affinity (the half-saturation concentration, K(0.5)); e.g., mutating sugar binding residues increases the glucose K(0.5) by up to three orders of magnitude. Mutation of the external gate residues increases the Na(+) to sugar transport stoichiometry, demonstrating that these residues are critical for efficient cotransport. The changes in phlorizin inhibition constant (K(i)) are proportional to the changes in sugar K(0.5), except in the case of F101C, where phlorizin K(i) increases by orders of magnitude without a change in glucose K(0.5). We conclude that glucose and phlorizin occupy the same binding site and that F101 is involved in binding to the phloretin group of the inhibitor. Substituted-cysteine accessibility methods show that the cysteine residues at the position of the gates and sugar binding site are largely accessible only to external hydrophilic methanethiosulfonate reagents in the presence of external Na(+), demonstrating that the external sugar (and phlorizin) binding vestibule is opened by the presence of external Na(+) and closes after the binding of sugar and phlorizin. Overall, the present results provide a bridge between kinetics and structural studies of cotransporters.
Subject(s)
Sodium-Glucose Transporter 1/chemistry , Sodium-Glucose Transporter 1/metabolism , Amino Acid Sequence , Animals , Humans , Kinetics , Molecular Sequence Data , Patch-Clamp Techniques , Protein Conformation , Protein Structure, Tertiary , Sequence Homology, Amino Acid , Structure-Activity Relationship , Xenopus laevisABSTRACT
Inwardly rectifying potassium channel Kir4.1 is critical for glial function, control of neuronal excitability, and systemic K(+) homeostasis. Novel mutations in Kir4.1 have been associated with EAST/SeSAME syndrome, characterized by mental retardation, ataxia, seizures, hearing loss, and renal salt waste. Patients are homozygous for R65P, G77R, C140R or T164I; or compound heterozygous for A167V/R297C or R65P/R199Stop, a deletion of the C-terminal half of the protein. We investigated the functional significance of these mutations by radiotracer efflux and inside-out membrane patch clamping in COSm6 cells expressing homomeric Kir4.1 or heteromeric Kir4.1/Kir5.1 channels. All of the mutations compromised channel function, but the underlying mechanisms were different. R65P, T164I, and R297C caused an alkaline shift in pH sensitivity, indicating that these positions are crucial for pH sensing and pore gating. In R297C, this was due to disruption of intersubunit salt bridge Glu(288)-Arg(297). C140R breaks the Cys(108)-Cys(140) disulfide bond essential for protein folding and function. A167V did not affect channel properties but may contribute to decreased surface expression in A167V/R297C. In G77R, introduction of a positive charge within the bilayer may affect channel structure or gating. R199Stop led to a dramatic decrease in surface expression, but channel activity was restored by co-expression with intact subunits, suggesting remarkable tolerance for truncation of the cytoplasmic domain. These results provide an explanation for the molecular defects that underlie the EAST/SeSAME syndrome.
Subject(s)
Abnormalities, Multiple/genetics , Ion Channel Gating/genetics , Mutation , Potassium Channels, Inwardly Rectifying/genetics , Abnormalities, Multiple/pathology , Amino Acid Sequence , Animals , Ataxia/pathology , Cell Line , Hearing Loss, Sensorineural/pathology , Humans , Hydrogen-Ion Concentration , Intellectual Disability/pathology , Ion Channel Gating/physiology , Membrane Potentials , Models, Molecular , Molecular Sequence Data , Patch-Clamp Techniques , Potassium/metabolism , Potassium Channels, Inwardly Rectifying/chemistry , Potassium Channels, Inwardly Rectifying/physiology , Protein Multimerization , Protein Structure, Tertiary , Rats , Seizures/pathology , Sequence Homology, Amino Acid , Syndrome , Transfection , Kir5.1 ChannelABSTRACT
Cidofovir (HPMPC), a broad spectrum antiviral agent, cannot be administered orally due to ionization of its phosphonic acid group at physiological pH. One prodrug approach involves conversion to the cyclic form (cHPMPC, 1) and esterification by the side chain hydroxyl group of a peptidomimetic serine. Transport studies in a rat model have shown enhanced levels of total cidofovir species in the plasma after oral dosing with L-Val-L-Ser-OMe cHPMPC, 2a. To explore the possibility that 2a and its three L/D stereoisomers 2b-d undergo active transport mediated by the peptide-specific intestinal transporter PEPT1, we performed radiotracer uptake and electrophysiology experiments applying the two-electrode voltage clamp technique in Xenopus laevis oocytes overexpressing human PEPT1 (hPEPT1, SLC15A1). 2a-d did not induce inward currents, indicating that they are not transported, but the stereoisomers with an L-configuration at the N-terminal valine (2a and 2b) potently inhibited transport of the hPEPT1 substrate glycylsarcosine (Gly-Sar). A "reversed" dipeptide conjugate, L-Ser-L-Ala-OiPr cHPMPC (4), also did not exhibit detectable transport, but completely abolished the Gly-Sar signal, suggesting that affinity of the transporter for these prodrugs is not impaired by a proximate linkage to the drug in the N-terminal amino acid of the dipeptide. Single amino acid conjugates of cHPMPC (3a and 3b) or cHPMPA (5, 6a and 6b) were not transported and only weakly inhibited Gly-Sar transport. The known hPEPT1 prodrug substrate valacyclovir (7) and its L-Val-L-Val dipeptide analogue (8) were used to verify coupled transport by the oocyte model. The results indicate that the previously observed enhanced oral bioavailability of 2a relative to the parent drug is unlikely to be due to active transport by hPEPT1. Syntheses of the novel compounds 2b-d and 3-6 are described, including a convenient solid-phase method to prepare 5, 6a and 6b.
Subject(s)
Adenine/analogs & derivatives , Cytosine/analogs & derivatives , Dipeptides/metabolism , Organophosphonates/chemical synthesis , Organophosphonates/metabolism , Organophosphorus Compounds/chemical synthesis , Organophosphorus Compounds/metabolism , Serine/chemistry , Symporters/metabolism , Adenine/chemical synthesis , Adenine/chemistry , Adenine/metabolism , Adenine/pharmacology , Cytosine/chemical synthesis , Cytosine/chemistry , Cytosine/metabolism , Cytosine/pharmacology , Dipeptides/chemical synthesis , Dipeptides/chemistry , Dipeptides/pharmacology , Humans , Molecular Structure , Organophosphonates/chemistry , Organophosphonates/pharmacology , Organophosphorus Compounds/chemistry , Organophosphorus Compounds/pharmacology , Peptide Transporter 1 , Prodrugs/chemistry , Prodrugs/metabolism , Prodrugs/pharmacology , Serine/metabolism , StereoisomerismABSTRACT
The ATP-sensitive potassium (K(ATP)) channel is expressed in most excitable tissues and plays a critical role in numerous physiological processes by coupling intracellular energetics to electrical activity. The channel is comprised of four Kir6.x subunits associated with four regulatory sulfonylurea receptors (SUR). Intracellular ATP acts on Kir6.x to inhibit channel activity, while MgADP stimulates channel activity through SUR. Changes in the cytosolic [ATP] to [ADP] ratio thus determine channel activity. Multiple mutations in Kir6.x and SUR genes have implicated K(ATP) channels in various diseases ranging from diabetes and hyperinsulinism to cardiac arrhythmias and cardiovascular disease. Continuing studies of channel physiology and pathology will bring new insights to the molecular basis of K(ATP) channel function, leading to a better understanding of the role that K(ATP) channels play in both health and disease.
Subject(s)
Adenosine Triphosphate/physiology , Disease , Health , KATP Channels , Molecular Biology , ATP-Binding Cassette Transporters/genetics , Adenosine Diphosphate/physiology , Animals , Diabetes Mellitus/genetics , Humans , Hyperinsulinism/genetics , KATP Channels/chemistry , KATP Channels/genetics , KATP Channels/metabolism , KATP Channels/physiology , Mutation , Potassium Channels, Inwardly Rectifying/genetics , Receptors, Drug/genetics , Sulfonylurea ReceptorsABSTRACT
Mutations in genes encoding KATP channel subunits have been reported for pancreatic disorders and Cantú syndrome. Here, we report a syndrome in six patients from two families with a consistent phenotype of mild intellectual disability, similar facies, myopathy, and cerebral white matter hyperintensities, with cardiac systolic dysfunction present in the two oldest patients. Patients are homozygous for a splice-site mutation in ABCC9 (c.1320 + 1 G > A), which encodes the sulfonylurea receptor 2 (SUR2) subunit of KATP channels. This mutation results in an in-frame deletion of exon 8, which results in non-functional KATP channels in recombinant assays. SUR2 loss-of-function causes fatigability and cardiac dysfunction in mice, and reduced activity, cardiac dysfunction and ventricular enlargement in zebrafish. We term this channelopathy resulting from loss-of-function of SUR2-containing KATP channels ABCC9-related Intellectual disability Myopathy Syndrome (AIMS). The phenotype differs from Cantú syndrome, which is caused by gain-of-function ABCC9 mutations, reflecting the opposing consequences of KATP loss- versus gain-of-function.
Subject(s)
Adenosine Triphosphate/metabolism , Channelopathies/metabolism , Genetic Predisposition to Disease/genetics , Intellectual Disability/metabolism , Muscular Diseases/metabolism , Mutation , Sulfonylurea Receptors/genetics , Sulfonylurea Receptors/metabolism , Adolescent , Adult , Amino Acid Sequence , Animals , Cardiomegaly/genetics , Cardiomegaly/metabolism , Cell Line , Child , Disease Models, Animal , Facies , Female , Genetic Diseases, X-Linked/genetics , Heart , Heart Diseases/genetics , Heart Diseases/metabolism , Homozygote , Humans , Hypertrichosis/genetics , Hypertrichosis/metabolism , Intellectual Disability/parasitology , Male , Mediator Complex/metabolism , Membrane Proteins/metabolism , Mice , Muscular Diseases/genetics , Neurodevelopmental Disorders/genetics , Neurodevelopmental Disorders/metabolism , Neurodevelopmental Disorders/physiopathology , Osteochondrodysplasias/genetics , Osteochondrodysplasias/metabolism , Pedigree , Phenotype , Rubidium , Whole Genome Sequencing , Young Adult , ZebrafishABSTRACT
The transient receptor potential (TRP) channel TRPV4 participates in multiple biological processes, and numerous TRPV4 mutations underlie several distinct and devastating diseases. Here we present the cryo-EM structure of Xenopus tropicalis TRPV4 at 3.8-Å resolution. The ion-conduction pore contains an intracellular gate formed by the inner helices, but lacks any extracellular gate in the selectivity filter, as observed in other TRPV channels. Anomalous X-ray diffraction analyses identify a single ion-binding site in the selectivity filter, thus explaining TRPV4 nonselectivity. Structural comparisons with other TRP channels and distantly related voltage-gated cation channels reveal an unprecedented, unique packing interface between the voltage-sensor-like domain and the pore domain, suggesting distinct gating mechanisms. Moreover, our structure begins to provide mechanistic insights to the large set of pathogenic mutations, offering potential opportunities for drug development.
Subject(s)
TRPV Cation Channels/chemistry , Xenopus Proteins/chemistry , Animals , Channelopathies/genetics , Cryoelectron Microscopy , Crystallography, X-Ray , Humans , Ion Channel Gating , Ions/chemistry , Ions/metabolism , Models, Molecular , Mutation , Permeability , Protein DomainsABSTRACT
Apicomplexan parasites contain a conserved protein CelTOS that, in malaria parasites, is essential for traversal of cells within the mammalian host and arthropod vector. However, the molecular role of CelTOS is unknown because it lacks sequence similarity to proteins of known function. Here, we determined the crystal structure of CelTOS and discovered CelTOS resembles proteins that bind to and disrupt membranes. In contrast to known membrane disruptors, CelTOS has a distinct architecture, specifically binds phosphatidic acid commonly present within the inner leaflet of plasma membranes, and potently disrupts liposomes composed of phosphatidic acid by forming pores. Microinjection of CelTOS into cells resulted in observable membrane damage. Therefore, CelTOS is unique as it achieves nearly universal inner leaflet cellular activity to enable the exit of parasites from cells during traversal. By providing novel molecular insight into cell traversal by apicomplexan parasites, our work facilitates the design of therapeutics against global pathogens.