Nucleotide-binding Fold Research Articles

Alternative Splicing of sur2 Exon 17 Regulates Nucleotide Sensitivity of the ATP-sensitive Potassium Channel

ATP-sensitive potassium channels (KATP) are implicated in a diverse array of physiological functions. Previous work has shown that alternative usage of exons 14, 39, and 40 of the muscle-specific KATP channel regulatory subunit, sur2, occurs in tissue-specific patterns. Here, we show that exon 17 of the first nucleotide binding fold of sur2 is also alternatively spliced. RNase protection demonstrates that SUR2(Delta17) predominates in skeletal muscle and gut and is also expressed in bladder, fat, heart, lung, liver, and kidney. Polymerase chain reaction and restriction digest analysis of sur2 cDNA demonstrate the existence of at least five sur2 splice variants as follows: SUR2(39), SUR2(40), SUR2(Delta17/39), SUR2(Delta17/40), and SUR2(Delta14/39). Electrophysiological recordings of excised, inside-out patches from COS cells cotransfected with Kir6.2 and the sur2 variants demonstrated that exon 17 splicing alters KATP sensitivity to ATP block by 2-fold from approximately 40 to approximately 90 microM for exon 17 and Delta17, respectively. Single channel kinetic analysis of SUR2(39) and SUR2(Delta17/39) demonstrated that both exhibited characteristic KATP kinetics but that SUR2(Delta17/39) exhibited longer mean burst durations and shorter mean interburst dwell times. In sum, alternative splicing of sur2 enhances the observed diversity of KATP and may contribute to tissue-specific modulation of ATP sensitivity.

Cooperative binding of ATP and MgADP in the sulfonylurea receptor is modulated by glibenclamide.

The ATP-sensitive potassium (KATP) channels in pancreatic beta cells are critical in the regulation of glucose-induced insulin secretion. Although electrophysiological studies provide clues to the complex control of KATP channels by ATP, MgADP, and pharmacological agents, the molecular mechanism of KATP-channel regulation remains unclear. The KATP channel is a heterooligomeric complex of SUR1 subunits of the ATP-binding-cassette superfamily with two nucleotide-binding folds (NBF1 and NBF2) and the pore-forming Kir6.2 subunits. Here, we report that MgATP and MgADP, but not the Mg salt of gamma-thio-ATP, stabilize the binding of prebound 8-azido-[alpha-32P]ATP to SUR1. Mutation in the Walker A and B motifs of NBF2 of SUR1 abolished this stabilizing effect of MgADP. These results suggest that SUR1 binds 8-azido-ATP strongly at NBF1 and that MgADP, either by direct binding to NBF2 or by hydrolysis of bound MgATP at NBF2, stabilizes prebound 8-azido-ATP binding at NBF1. The sulfonylurea glibenclamide caused release of prebound 8-azido-[alpha-32P]ATP from SUR1 in the presence of MgADP or MgATP in a concentration-dependent manner. This direct biochemical evidence of cooperative interaction in nucleotide binding of the two NBFs of SUR1 suggests that glibenclamide both blocks this cooperative binding of ATP and MgADP and, in cooperation with the MgADP bound at NBF2, causes ATP to be released from NBF1.

Walker mutations reveal loose relationship between catalytic and channel-gating activities of purified CFTR (cystic fibrosis transmembrane conductance regulator).

The cystic fibrosis transmembrane conductance regulator (CFTR) functions as an ATPase and as a chloride channel. It has been hypothesized, on the basis of electrophysiological findings, that the catalytic activity of CFTR is tightly coupled to the opening and closing of the channel gate. In the present study, to determine the structural basis for the ATPase activity of CFTR, we assessed the effect of mutations within the "Walker A" consensus motifs on ATP hydrolysis by the purified, intact protein. Mutation of the lysine residue in the "Walker A" motif of either the first nucleotide binding fold (CFTRK464A) or the second nucleotide binding fold (CFTRK1250A) inhibited the ATPase activity of the purified intact CFTR protein significantly, by greater than 50%. This finding suggests that the two nucleotide binding folds of CFTR are functioning cooperatively in catalysis. However, the rate of channel gating was only significantly inhibited in one of these purified mutants, CFTRK1250A, suggesting that ATPase activity may not be tightly coupled to channel gating as previously hypothesized.

The CDK-activating Kinase (Cak1p) from Budding Yeast Has an Unusual ATP-binding Pocket

Cak1p is an essential protein kinase that phosphorylates and thereby activates the major cyclin-dependent kinase in budding yeast, Cdc28p. The sequence of Cak1p differs from other members of the protein kinase superfamily in several conserved regions. Cak1p lacks the highly conserved glycine loop motif (GXGXXG) that is found in the nucleotide binding fold of virtually all protein kinases and also lacks a number of conserved amino acids found at sites throughout the protein kinase core sequence. We have used kinetic and mutagenic analyses to investigate whether these sequence differences affect the nucleotide-binding properties of Cak1p. Although Cak1p differs dramatically from other protein kinases, it binds ATP with a reasonable affinity, with a KM of 4.8 microM. Mutations of the putative invariant lysine in Cak1p (Lys-31), homologous to a residue required for activity in virtually all protein kinases and that interacts with the ATP phosphates, moderately reduced the ability of Cak1p to bind ATP but did not dramatically affect the catalytic rate of the kinase. Similarly, Cak1p is insensitive to the ATP analog 5'-fluorosulfonylbenzoyladenosine, which inhibits most protein kinases through covalent modification of the invariant lysine. We found that Cak1p is tolerant of mutations within its glycine loop region. Remarkably, Cak1p remains functional even following truncation of its first 31 amino acids, including the glycine loop region and the invariant lysine. We conclude that the Cak1p nucleotide-binding pocket differs significantly from those of most other protein kinases and therefore might provide a specific target for an inhibitory drug.

Mapping of ATP binding regions in poly(A) polymerases by photoaffinity labeling and by mutational analysis identifies a domain conserved in many nucleotidyltransferases.

We have identified regions in poly(A) polymerases that interact with ATP. Conditions were established for efficient cross-linking of recombinant bovine and yeast poly(A) polymerases to 8-azido-ATP. Mn2+ strongly stimulated this reaction due to a 50-fold lower Ki for 8-azido-ATP in the presence of Mn2+. Mutations of the highly conserved Asp residues 113, 115, and 167, critical for metal binding in the catalytic domain of bovine poly(A) polymerase, led to a strong reduction of cross-linking efficiency, and Mn2+ no longer stimulated the reaction. Sites of 8-azido-ATP cross-linking were mapped in different poly(A) polymerases by CNBr-cleavage and analysis of tryptic peptides by mass spectroscopy. The main cross-link in Schizosaccharomyces pombe poly(A) polymerase could be assigned to the peptide DLELSDNNLLK (amino acids 167-177). Database searches with sequences surrounding the cross-link site detected significant homologies to other nucleotidyltransferase families, suggesting a conservation of the nucleotide-binding fold among these families of enzymes. Mutations in the region of the "helical turn motif" (a domain binding the triphosphate moiety of the nucleotide) and in the suspected nucleotide-binding helix of bovine poly(A) polymerase impaired ATP binding and catalysis. The results indicate that ATP is bound in part by the helical turn motif and in part by a region that may be a structural analog to the fingers domain found in many polymerases.

Motional dynamics of the catalytic loop in OMP synthase.

In de novo pyrimidine biosynthesis, orotate phosphoribosyltransferase catalyzes the formation of orotidine 5'-monophosphate (OMP) from orotic acid and alpha-D-5-phosphoribosyl-1-pyrophosphate (PRPP). The known three-dimensional structure of the dimeric enzyme from Salmonella typhimurium is similar to that of other Type I phosphoribosyltransferases (nucleotide synthases) with a solvent-exposed active site atop a Rossman-type nucleotide binding fold. The three-dimensional structure of an enzyme-inhibitor complex [Henriksen et al. (1996) Biochemistry 35, 3803-3809] indicates that one of the two identical solvent-exposed loops can descend to cover the active site of the adjacent subunit of the dimeric enzyme. Catalytically essential residues are known to reside on this loop. In the present work, sensitivity toward limited proteolysis by trypsin confirms that the loop is solvent-exposed. Protection by PRPP and, to a lesser extent, by OMP demonstrates the existence of a second, trypsin-inaccessible, loop position. Two-dimensional 1H-15N NMR relaxation experiments on [alpha-15N]histidine-labeled WT OPRTase yielded backbone 15N T1 and T2 relaxation times and 15N[1H] NOE for His-105 (a loop residue) that are characteristic of small peptides. These results document that the surface loop is highly flexible in the unliganded enzyme. Addition of a hydrolytically stable PRPP analogue to the enzyme resulted in a significant reduction of His-105 peak intensity, indicating a dramatic change in the dynamic properties of the loop backbone in the analogue-ligated enzyme. 1H NMR titrations on histidine C2 protons, coupled with 1H and 31P titrations monitoring the C1H and 5-phosphate PRPP resonances, allowed the quantitation of the rates of loop movement during product release, and relate protein motion to enzymatic catalysis. These results suggest that loop opening and PRPP release is a two-step process, whose overall rate is partially rate-limiting in the reverse pyrophosphorolysis reaction.

Potassium channel openers require ATP to bind to and act through sulfonylurea receptors.

KATP channels are composed of a small inwardly rectifying K+ channel subunit, either KIR6.1 or KIR6.2, plus a sulfonylurea receptor, SUR1 or SUR2 (A or B), which belong to the ATP-binding cassette superfamily. SUR1/KIR6.2 reconstitute the neuronal/pancreatic beta-cell channel, whereas SUR2A/KIR6.2 and SUR2B/KIR6.1 (or KIR6.2) are proposed to reconstitute the cardiac and the vascular-smooth-muscle-type KATP channels, respectively. We report that potassium channel openers (KCOs) bind to and act through SURs and that binding to SUR1, SUR2A and SUR2B requires ATP. Non-hydrolysable ATP-analogues do not support binding, and Mg2+ or Mn2+ are required. Point mutations in the Walker A motifs or linker regions of both nucleotide-binding folds (NBFs) abolish or weaken [3H]P1075 binding to SUR2B, rendering reconstituted SUR2B/KIR6.2 channels insensitive towards KCOs. The C-terminus of SUR affects KCO affinity with SUR2B approximately SUR1 > SUR2A. KCOs belonging to different structural classes inhibited specific [3H]P1075 binding to SUR2B in a monophasic manner, with the exception of minoxidil sulfate, which induced a biphasic displacement. The affinities of KCO binding to SUR2B were 3.5-8-fold higher than their potencies for activation of SUR2B/KIR6.2 channels. The results establish that SURs are the KCO receptors of KATP channels and suggest that KCO binding requires a conformational change induced by ATP hydrolysis in both NBFs.

ABC50, a Novel Human ATP-Binding Cassette Protein Found in Tumor Necrosis Factor-α-Stimulated Synoviocytes

We have used the recently developed technique of differential display polymerase chain reaction to seek for new genes modulated by tumor necrosis factor-α (TNF-α) in cultured synoviocytes. One PCR fragment was shown to correspond to a new gene that was mapped by high-resolution fluorescencein situhybridization to band 6p21.33. The cDNA of this gene was cloned, and the deduced amino acid sequence revealed consensus motifs for the nucleotide binding folds of the ATP-binding cassette (ABC) family of proteins. However, a hydropathy curve showed that the polypeptide does not contain the transmembrane domains that are typical of the subfamily of ABC transporters and are associated with transporter/channel functions. The new gene, called ABC50, is the first human and mammalian ABC protein found to lack transmembrane domains. Homology with some yeast ABC proteins suggests that ABC50 codes for a new human ribosomal protein involved in translation of mRNA. It could therefore play a role in the enhancement of protein synthesis that follows TNF-α treatment of synoviocytes and thus participate in the inflammatory processes mediated by this cytokine. Furthermore, since TNF-α also modulates the expression of MHC class I genes, and these genes are known to map to 6p21.33, it is hypothesized that ABC50 and MHC class I are part of the same chromatin expression domain.

Phosphorylation of the periplasmic binding protein in two transport systems for arginine incorporation in Escherichia coli K-12 is unrelated to the function of the transport system.

In Escherichia coli K-12, the accumulation of arginine is mediated by two distinct periplasmic binding protein-dependent transport systems, one common to arginine and ornithine (AO system) and one for lysine, arginine, and ornithine (LAO system). Each of these systems includes a specific periplasmic binding protein, the AO-binding protein for the AO system and the LAO-binding protein for the LAO system. The two systems include a common inner membrane transport protein which is able to hydrolyze ATP and also phosphorylate the two periplasmic binding proteins. Previously, a mutant resistant to the toxic effects of canavanine, with low levels of transport activities and reduced levels of phosphorylation of the two periplasmic binding proteins, was isolated and characterized (R. T. F. Celis, J. Biol. Chem. 265:1787-1793, 1990). The gene encoding the transport ATPase enzyme (argK) has been cloned and sequenced. The gene possesses an open reading frame with the capacity to encode 268 amino acids (mass of 29.370 Da). The amino acid sequence of the protein includes two short sequence motifs which constitute a well-defined nucleotide-binding fold (Walker sequences A and B) present in the ATP-binding subunits of many transporters. We report here the isolation of canavanine-sensitive derivatives of the previously characterized mutant. We describe the properties of these suppressor mutations in which the transport of arginine, ornithine, and lysine has been restored. In these mutants, the phosphorylation of the AO- and LAO-binding proteins remains at a low level. This information indicates that whereas hydrolysis of ATP by the transport ATPase is an obligatory requirement for the accumulation of these amino acids in E. coli K-12, the phosphorylation of the periplasmic binding protein is not related to the function of the transport system.

Non-equivalent cooperation between the two nucleotide-binding folds of P-glycoprotein

To identify the roles of the two nucleotide-binding folds (NBFs) in the function of human P-glycoprotein, a multidrug transporter, we mutated the key lysine residues to methionines and the cysteine residues to alanines in the Walker A (WA) motifs (the core consensus sequence) in the NBFs. We examined the effects of these mutations on N-ethylmaleimide (NEM) and ATP binding, as well as on the vanadate-induced nucleotide trapping with 8-azido-[α-32P]ATP. Mutation of the WA lysine or NEM binding cysteine in either of the NBFs blocked vanadate-induced nucleotide trapping of P-glycoprotein. These results suggest that if one NBF is non-functional, there is no ATP hydrolysis even if the other functional NBF contains a bound nucleotide, further indicating the strong cooperation between the two NBFs of P-glycoprotein. However, we found that the effect of NEM modification at one NBF on ATP binding at the other NBF was not equivalent, suggesting a non-equivalency of the role of the two NBFs in P-glycoprotein function.

Identification of the Domains of Photoincorporation of the 3‘- and 7-Benzophenone Analogues of Taxol in the Carboxyl-Terminal Half of Murine mdr1b P-glycoprotein

P-glycoprotein is an ATP-dependent drug-efflux pump that can transport a diverse range of structurally and functionally unrelated hydrophobic compounds across the plasma membrane. The transporter is composed of two homologous halves, each containing a nucleotide binding fold and six putative transmembrane spanning segments. The contact domains between the murine mdr1b P-glycoprotein and two photoreactive Taxol analogues have been mapped by a combination of CNBr digestion and immunoprecipitation studies. We had demonstrated previously that the 3'-p-benzoyldihydrocinnamoyl (BzDC) analogue of Taxol specifically photolabeled mdr1b P-glycoprotein and now show that the corresponding C-7 analogue likewise specifically photoincorporates into the transporter. CNBr digestion of both photolabeled P-glycoproteins gave rise to an approximate 10 kDa tritium-labeled peptide, each of which was a distinct polypeptide. The CNBr fragment generated from the 3'-BzDC-Taxol-photolabeled P-glycoprotein was immunoprecipitated by a polyclonal antibody (Ab7) raised against amino acid residues 1008-1019 of the mdr1b isoform. In contrast, the CNBr fragment generated from the 7-BzDC-Taxol-photolabeled P-glycoprotein was immunoprecipitated by a polyclonal antibody (Ab4) raised against amino acid residues 740-750. The specificity of these reactions was demonstrated by showing that the presence of the appropriate synthetic peptide blocked the immunoprecipitation. Moreover when the antibodies were reversed, no immunoprecipitation occurred. Based on the deduced amino acid sequence of mdr1b P-glycoprotein, and its hydropathy plot analysis, our data indicated that the 3'-BzDC group photoincorporates into amino acid residues 985-1088, a region of the transporter that includes half of TM 12 and terminates just after the Walker A motif in the second nucleotide binding fold. The 7-BzDC group photoincorporates into amino acid residues 683-760, a region of the transporter that includes all of TM 7 and half of TM 8 plus the intervening extracellular loop.

Functional analyses of novel mutations in the sulfonylurea receptor 1 associated with persistent hyperinsulinemic hypoglycemia of infancy.

The ATP-sensitive potassium channel, K(ATP) channel, a functional complex of the sulfonylurea receptor 1, SUR1, and an inward rectifier potassium channel subunit, Kir6.2, regulates insulin secretion in the pancreas. Mutations in both the Kir6.2 and SUR1 genes are associated with persistent hyperinsulinemic hypoglycemia of infancy (PHHI), a disorder of pancreatic beta-cell function characterized by excess insulin secretion and hypoglycemia. We have studied the functional properties of novel SUR1 mutations identified in PHHI patients, including H125Q, N188S, F591L, T1139M, R1215Q, G1382S, and R1394H. R1394H and deltaF1388 SUR1, a previously identified PHHI mutation, resulted in no functional channels when coexpressed with Kir6.2 in COS cells, while H125Q, N188S, F591L, T1139M, R1215Q, and G1382S SUR1 generated functional channels in the absence of ATP. With the exception of N188S and H125Q, all mutants had reduced response to stimulation by MgADP. These results indicate that lack of, or reduction of, K(ATP) channel sensitivity to MgADP is a common molecular defect associated with the disease. The mutant channels also showed varied response to activation by the potassium channel opener diazoxide. Because these mutations are distributed throughout the molecule, our data have new implications for structure-function relationships of the K(ATP) channel, suggesting that structural elements in SUR1 outside of the two nucleotide-binding folds are also important in regulating channel activity.

A model for the nucleotide‐binding domains of ABC transporters based on the large domain of aspartate aminotransferase

ABC transporters are a large superfamily of integral membrane proteins involved in ATP-dependent transport across biological membranes. Members of this superfamily play roles in a number of phenomena of biomedical interest, including cystic fibrosis (CFTR) and multidrug resistance (P-glycoprotein, MRP). Most ABC transporters are predicted to consist of four domains, two membrane-spanning domains and two cytoplasmic domains. The latter contain conserved nucleotide-binding motifs. Attempts to determine the structure of ABC transporters and of their separate domains are in progress but have not yet been successful. To aid structure determination and possibly learn more about the domain boundaries, we set out to model nucleotide-binding domains (NBDs) of ABC transporters based on a known structure. Previous attempts to predict the 3D structure of NBDs were based solely on sequence similarity with known nucleotide-binding folds. We have analyzed the sequences of a number of nucleotide-binding domains with the algorithm THREADER, developed by D.T. Jones, and a possible fold was found in the structure of aspartate aminotransferase. We present a model for the N-terminal NBD of CFTR, based on the large domain of the A chain of aspartate aminotransferase. The model is refined using multiple sequence alignment, secondary structure prediction, and 3D-1D profiles. Our model seems to be in good agreement with known properties of nucleotide-binding domains and has some appealing characteristics compared with the previous models. Proteins 30:275–286, 1998. © 1998 Wiley-Liss, Inc.

A model for the nucleotide-binding domains of ABC transporters based on the large domain of aspartate aminotransferase

ABC transporters are a large superfamily of integral membrane proteins involved inATP-dependent transport across biological membranes. Members of this superfamily play roles in a number of phenomena of biomedical interest, including cystic fibrosis (CFTR) and multidrug resistance (P-glycoprotein, MRP). Most ABC transporters are predicted to consist of four domains, two membrane-spanning domains and two cytoplasmic domains. The latter contain conserved nucleotide-binding motifs. Attempts to determine the structure of ABC transporters and of their separate domains are in progress but have not yet been successful. To aid structure determination and possibly learn more about the domain boundaries, we set out to model nucleotide-binding domains (NBDs) of ABC transporters based on a known structure. Previous attempts to predict the 3D structure of NBDs were based solely on sequence similarity with known nucleotide-binding folds. We have analyzed the sequences of a number of nucleotide-binding domains with the algorithm THREADER, developed by D.T. Jones, and a possible fold was found in the structure of aspartate aminotransferase. We present a model for the N-terminal NBD of CFTR, based on the large domain of the A chain of aspartate aminotransferase. The model is refined using multiple sequence alignment, secondary structure prediction, and 3D-1D profiles. Our model seems to be in good agreement with known properties of nucleotide-binding domains and has some appealing characteristics compared with the previous models.

Clinical and genetic aspects of X-linked adrenoleukodystrophy.

X-linked adrenoleukodystrophy (ALD), a leukodystrophy characterized by abnormal accumulation of saturated very long chain fatty acids in brain white matter and adrenal cortex, is the most common inherited peroxisomal disorder. The biochemical defect is localized to the level of lignoceroyl-CoA synthesis, a step in the peroxisomal beta-oxidation of very long chain fatty acids. The responsible gene encodes a peroxisomal integral membrane protein of as yet unknown function which is a member of the ATP-binding cassette transporter protein superfamily. The patient gene mutations are heterogeneously distributed over the functional protein domains with a tendency to clustering in the nucleotide-binding fold. The mechanisms by which these mutations cause a loss of protein function is unknown. Diagnosis of patients and carriers, including prenatal testing, is mainly based on the clinical picture, the demonstration of increased levels of saturated very long chain fatty acids in tissues and body fluids as well as on DNA mutation analyses. There are at least six distinct clinical phenotypes ranging from the severe childhood cerebral form to asymptomatic persons. The various phenotypes commonly occur within the same kindred. Modifying genes and/or environmental factors may contribute to this phenomenon. At present, there is no proven therapy for the prevention or cure of the neurological disabilities. Several approaches are under investigation including diets, immunosuppression, bone marrow transplantation and gene therapy.

Structure of Escherichia coli ribokinase in complex with ribose and dinucleotide determined to 1.8 A resolution: insights into a new family of kinase structures.

D-ribose must be phosphorylated at O5' before it can be used in either anabolism or catabolism. This reaction is catalysed by ribokinase and requires the presence of ATP and magnesium. Ribokinase is a member of a family of carbohydrate kinases of previously unknown structure. The crystal structure of ribokinase from Escherichia coli in complex with ribose and dinucleotide was determined at 1.84 A resolution by multiple isomorphous replacement. There is one 33 kDa monomer of ribokinase in the asymmetric unit but the protein forms a dimer around a crystallographic twofold axis. Each subunit consists of a central alpha/beta unit, with a new type of nucleotide-binding fold, and a distinct beta sheet that forms a lid over the ribose-binding site. Contact between subunits involves orthogonal packing of beta sheets, in a novel dimer interaction that we call a beta clasp. Inspection of the complex indicates that ribokinase utilises both a catalytic base for activation of the ribose in nucleophilic attack and an anion hole that stabilises the transition state during phosphoryl transfer. The structure suggests an ordered reaction mechanism, similar to those proposed for other carbohydrate kinases that probably involves conformational changes. We propose that the beta-clasp structure acts as a lid, closing and opening upon binding and release of ribose. From these observations, an understanding of the structure and catalytic mechanism of related sugar kinases can be obtained.

P-glycoprotein structure and evolutionary homologies.

Analysis of multidrug resistant cell lines has led to the identification of the P-glycoprotein multigene family. Two of the three classes of mammalian P-glycoproteins have the ability to confer cellular resistance to a broad range of structurally and functionally diverse cytotoxic agents. P-glycoproteins are integral membrane glycoproteins comprised of two similar halves, each consisting of six membrane spanning domains followed by a cytoplasmic domain which includes a nucleotide binding fold. The P-glycoprotein is a member of a large superfamily of transport proteins which utilize ATP to translocate a wide range of substrates across biological membranes. This superfamily includes transport complexes comprised of multicomponent systems, half P-glycoproteins and P-glycoprotein-like homologs which appear to require approximately 12 alpha-helical transmembrane domains and two nucleotide binding folds for substrate transport. P-glycoprotein homologs have been isolated and characterized from a wide range of species. Amino acid sequences, the similarities between the halves and intron/exon boundaries have been compared to understand the evolutionary origins of the P-glycoprotein.

ATP-sensitive K+ channels in pancreatic, cardiac, and vascular smooth muscle cells

ATP-sensitive K+ (KATP) channels are therapeutic targets for several diseases, including angina, hypertension, and diabetes. This is because stimulation of KATP channels is thought to produce vasorelaxation and myocardial protection against ischemia, whereas inhibition facilitates insulin secretion. It is well known that native KATP channels are inhibited by ATP and sulfonylurea (SU) compounds and stimulated by nucleotide diphosphates and K+ channel-opening drugs (KCOs). Although these characteristics can be shared with KATP channels in different tissues, differences in properties among pancreatic, cardiac, and vascular smooth muscle (VSM) cells do exist in terms of the actions produced by such regulators. Recent molecular biology and electrophysiological studies have provided useful information toward the better understanding of KATP channels. For example, native KATP channels appear to be a complex of a regulatory protein containing the SU-binding site [sulfonylurea receptor (SUR)] and an inward-rectifying K+ channel (Kir) serving as a pore-forming subunit. Three isoforms of SUR (SUR1, SUR2A, and SUR2B) have been cloned and found to have two nucleotide-binding folds (NBFs). It seems that these NBFs play an essential role in conferring the MgADP and KCO sensitivity to the channel, whereas the Kir channel subunit itself possesses the ATP-sensing mechanism as an intrinsic property. The molecular structure of KATP channels is thought to be a heteromultimeric (tetrameric) assembly of these complexes: Kir6.2 with SUR1 (SUR1/Kir6.2, pancreatic type), Kir6.2 with SUR2A (SUR2A/ Kir6.2, cardiac type), and Kir6.1 with SUR2B (SUR2B/Kir6.1, VSM type) [i.e., (SUR/Kir6.x)4]. It remains to be determined what are the molecular connections between the SUR and Kir subunits that enable this unique complex to work as a functional KATP channel.

50] Overexpression, purification, and function of first nucleotide-binding fold of cystic fibrosis transmembrane conductance regulator

Cystic fibrosis (CF) is the most common lethal autosomal recessive genetic disease in the Caucasian population. The disease results from mutations in the gene that codes for the cystic fibrosis transmembrane conductance regulator (CFTR) protein comprised of 1480 amino acid residues. CFTR embraces two transmembrane domains, each having six putative membrane-spanning segments; two nucleotide-binding folds, NBF1 and NBF2, each possessing a Walker A and B nucleotide-binding consensus sequence, and a regulatory (R) domain having numerous acidic and basic amino acid residues as well as serine and threonine residues that can be phosphorylated by some cAMP dependent protein kinases. Mutations in CFTR occur within or near the two NBFs. Most patients with cystic fibrosis have at least one allele in which the codon for phenylalanine-508 in NBF1 is deleted. The interaction of intracellular adenosine triphosphate (ATP) with NBF1 (or NBF2) may be essential for chloride channel activation.

11] Patch-clamp studies of cystic fibrosis transmembrane conductance regulator chloride channel

Publisher Summary The cystic fibrosis transmembrane conductance regulator (CFTR) has two sets of six membrane-spanning regions (TM1-TM12), two nucleotidebinding folds (NBF1, NBF2), and a regulatory domain containing numerous potential sites for phosphorylation by protein kinases. It belongs to an important superfamily of ATP-binding cassette (ABC) transport proteins that has members in bacteria, yeast, and higher eukaryotes. Although it may have multiple functions, it is generally accepted that CFTR functions as an ATP-dependent, phosphorylation-activated CI channel. This chapter describes the methods that have been found useful for studying the channel activity of CFTR. Functional studies of endogenous CFTR have been carried out using epithelial and cardiac cells. However, because it is often difficult to obtain giga-ohm seals on native epithelial cells, heterologous expression systems are generally preferred for biophysical studies. Many cell lines have been used successfully for transient and stable transfection, including Chinese hamster ovary (CHO) cells, fibroblasts, baby hamster kidney cells (BHK), and human enbryonic kidney (HEK 293) cells. The choice of preparations depends on the purpose of the experiment.