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Frances M Ashcroft

Publications and source records attributed to Frances M Ashcroft.

51 records · Page 3Linked to original sources
PubMed2003

Orexin excites GABAergic neurons of the arcuate nucleus by activating the sodium--calcium exchanger.

The neuropeptides orexins/hypocretins are essential for normal wakefulness and energy balance, and disruption of their function causes narcolepsy and obesity. Although much is known of the role of orexins in sleep/wake behavior, it remains unclear how they stimulate feeding and metabolism. One of the main targets of orexinergic neurons is the arcuate nucleus (ARC) of the hypothalamus, which plays a key role in feeding and energy homeostasis. By combining patch-clamp and RT-multiplex PCR analysis of individual neurons in mouse brain slices, we show that an electrophysiologically distinct subset of ARC neurons coexpress orexin receptors and glutamate decarboxylase-67 and are excited by orexin. Acting on postsynaptic orexin type 2 receptors, orexin activates a sodium-calcium exchange current, thereby depolarizing the cell and increasing its firing frequency. Because GABA is a potent stimulus for feeding, in both the ARC and its main projection site, these results suggest a mechanism for how orexin may control appetite.

Animals↗
PubMed2003

Pyridine nucleotide regulation of the KATP channel Kir6.2/SUR1 expressed in Xenopus oocytes.

The pancreatic beta-cell type of ATP-sensitive potassium (KATP) channel (Kir6.2/SUR1) is inhibited by intracellular ATP and ADP, which bind to the Kir6.2 subunit, and is activated by Mg-nucleotide interaction with the regulatory sulphonylurea receptor subunits (SUR1). The nicotinamide adenine dinucleotides NAD and NADP consist of an ADP molecule with a ribose group and a nicotinamide moiety attached to the terminal phosphate. Both these molecules block native KATP channels in pancreatic beta-cells at concentrations above 500 microM, and activate them at lower concentrations. We therefore investigated whether NAD and NADP interact with both Kir6.2 and SUR1 subunits of the KATP channel by comparing the potency of these agents on recombinant Kir6.2DeltaC and Kir6.2/SUR1 channels expressed in Xenopus oocytes. Our results show that, at physiological concentrations, NAD and NADP interact with the nucleotide inhibitory site of Kir6.2 to inhibit Kir6.2/SUR1 currents. They may therefore contribute to the resting level of channel inhibition in the intact cell. Importantly, our data also reveal that this interaction is dependent on the presence of SUR1, which may act by increasing the width of the nucleotide-binding pocket of Kir6.2.

Adenosine Diphosphate↗
PubMed2003

Crystal structure of the potassium channel KirBac1.1 in the closed state.

The KirBac1.1 channel belongs to the inward-rectifier family of potassium channels. Here we report the structure of the entire prokaryotic Kir channel assembly, in the closed state, refined to a resolution of 3.65 angstroms. We identify the main activation gate and structural elements involved in gating. On the basis of structural evidence presented here, we suggest that gating involves coupling between the intracellular and membrane domains. This further suggests that initiation of gating by membrane or intracellular signals represents different entry points to a common mechanistic pathway.

Amino Acid Sequence↗
PubMed2003

A new subtype of autosomal dominant diabetes attributable to a mutation in the gene for sulfonylurea receptor 1.

BACKGROUND: ATP-sensitive potassium (KATP) channels are major regulators of glucose-induced insulin secretion in pancreatic beta cells. We have described a dominant heterozygous mutation--E1506K--in the sulfonylurea receptor 1 (SUR1) gene (ABCC8) in a Finnish family, which leads to congenital hyperinsulinaemia due to reduction of K(ATP)-channel activity. We aimed to characterise glucose metabolism in adults heterozygous for the E1506K mutation. METHODS: Glucose tolerance was assessed by an oral glucose tolerance test, insulin secretion by the intravenous glucose tolerance test and hyperglycaemic clamp, and insulin sensitivity by hyperinsulinaemic euglycaemic clamp in 11 people heterozygous for the E1506K mutation and 19 controls. FINDINGS: Four people who were heterozygous for the SUR1 E1506K mutation had diabetes, five had impaired glucose tolerance, one had impaired fasting glucose, and one had normal glucose tolerance. Although glucose-induced, first-phase insulin secretion was normal in children younger than 10 years of age who were heterozygous for the SUR1 E1506K mutation (n=2; 66 and 334 pmol/L), it fell rapidly after puberty (n=3; 12-32 pmol/L), and was almost completely lost in adulthood (n=11; 12-32 pmol/L). Furthermore, these heterozygous people had a substantial reduction in maximum glucose-stimulated insulin secretion during hyperglycemic clamp (carriers without diabetes 422 pmol/L; carriers with diabetes 97 pmol/L). By contrast, insulin sensitivity (M/I value) was normal in carriers of the E1506K mutation who did not have diabetes and was reduced by 15% in those who were heterozygous with diabetes (0.07 in those without diabetes and 0.05 in those with the disorder; not significantly different from controls). INTERPRETATION: Heterozygous E1506K substitution in the SUR1 gene causes congenital hyperinsulinism in infancy, loss of insulin secretory capacity in early adulthood, and diabetes in middle-age. This variant represents a new subtype of autosomal dominant diabetes.

Adult↗
PubMed2003

Analysis of the differential modulation of sulphonylurea block of beta-cell and cardiac ATP-sensitive K+ (K(ATP)) channels by Mg-nucleotides.

Sulphonylureas stimulate insulin secretion by binding with high-affinity to the sulphonylurea receptor (SUR) subunit of the ATP-sensitive potassium (K(ATP)) channel and thereby closing the channel pore (formed by four Kir6.2 subunits). In the absence of added nucleotides, the maximal block is around 60-80 %, indicating that sulphonylureas act as partial antagonists. Intracellular MgADP modulated sulphonylurea block, enhancing inhibition of Kir6.2/SUR1 (beta-cell type) and decreasing that of Kir6.2/SUR2A (cardiac-type) channels. We examined the molecular basis of the different response of channels containing SUR1 and SUR2A, by recording currents from inside-out patches excised from Xenopus oocytes heterologously expressing wild-type or chimeric channels. We used the benzamido derivative meglitinide as this drug blocks Kir6.2/SUR1 and Kir6.2/SUR2A currents, reversibly and with similar potency. Our results indicate that transfer of the region containing transmembrane helices (TMs) 8-11 and the following 65 residues of SUR1 into SUR2A largely confers a SUR1-like response to MgADP and meglitinide, whereas the reverse chimera (SUR128) largely endows SUR1 with a SUR2A-type response. This effect was not specific for meglitinide, as tolbutamide was also unable to prevent MgADP activation of Kir6.2/SUR128 currents. The data favour the idea that meglitinide binding to SUR1 impairs either MgADP binding or the transduction pathway between the NBDs and Kir6.2, and that TMs 8-11 are involved in this modulatory response. The results provide a basis for understanding how beta-cell K(ATP) channels show enhanced sulphonylurea inhibition under physiological conditions, whereas cardiac K(ATP) channels exhibit reduced block in intact cells, especially during metabolic inhibition.

ATP-Binding Cassette Transporters↗
PubMed2003

Filter flexibility in a mammalian K channel: models and simulations of Kir6.2 mutants.

The single-channel conductance varies significantly between different members of the inward rectifier (Kir) family of potassium channels. Mutations at three sites in Kir6.2 have been shown to produce channels with reduced single-channel conductance, the largest reduction (to 40% of wild-type) being for V127T. We have used homology modeling (based on a KcsA template) combined with molecular dynamics simulations in a phosphatidycholine bilayer to explore whether changes in structural dynamics of the filter were induced by three such mutations: V127T, M137C, and G135F. Overall, 12 simulations of Kir6.2 models, corresponding to a total simulation time of 27 ns, have been performed. In these simulations we focused on distortions of the selectivity filter, and on the presence/absence of water molecules lying behind the filter, which form interactions with the filter and the remainder of the protein. Relative to the wild-type simulation, the V127T mutant showed significant distortion of the filter such that approximately 50% of the simulation time was spent in a closed conformation. While in this conformation, translocation of K(+) ions between sites S1 and S2 was blocked. The distorted filter conformation resembles that of the bacterial channel KcsA when crystallized in the presence of a low [K(+)]. This suggests filter distortion may be a possible general model for determining the conductance of K channels.

Animals↗
PubMed2003

The ligand-sensitive gate of a potassium channel lies close to the selectivity filter.

Potassium channels selectively conduct K(+) ions across cell membranes and have key roles in cell excitability. Their opening and closing can be spontaneous or controlled by membrane voltage or ligand binding. We used Ba(2+) as a probe to determine the location of the ligand-sensitive gate in an inwardly rectifying K(+) channel (Kir6.2). To a K(+) channel, Ba(2+) and K(+) are of similar sizes, but Ba(2+) blocks the pore by binding within the selectivity filter. We found that internal Ba(2+) could still access its binding site when the channel was shut, which indicates that the ligand-sensitive gate lies above the Ba(2+)-block site, and thus within or above the selectivity filter. This is in marked contrast to the voltage-dependent gate of K(V) channels, which is located at the intracellular mouth of the pore.

Adenosine Triphosphate↗
PubMed2003

Potassium channel regulation.

The sulphonylurea receptor (SUR) is a member of the ATP-binding cassette (ABC) family of membrane proteins. It functions as the regulatory subunit of the ATP-sensitive potassium (KATP) channel, which comprises SUR and Kir6.x proteins. Here, we review data demonstrating functional differences between the two nucleotide binding domains (NBDs) of SUR1. In addition, to explain the structural basis of these functional differences, we have constructed a molecular model of the NBD dimer of human SUR1. We discuss the experimental data in the context of this model, and show how the model can be used to design experiments aimed at elucidating the relationship between the structure and function of the KATP channel.

ATP-Binding Cassette Transporters↗
PubMed2002

Mutations in the linker domain of NBD2 of SUR inhibit transduction but not nucleotide binding.

ATP-sensitive potassium (K(ATP)) channels are composed of an ATP-binding cassette (ABC) protein (SUR1, SUR2A or SUR2B) and an inwardly rectifying K(+) channel (Kir6.1 or Kir6.2). Like other ABC proteins, the nucleotide binding domains (NBDs) of SUR contain a highly conserved "signature sequence" (the linker, LSGGQ) whose function is unclear. Mutation of the conserved serine to arginine in the linker of NBD1 (S1R) or NBD2 (S2R) did not alter the ability of ATP or ADP (100 microM) to displace 8-azido-[(32)P]ATP binding to SUR1, or abolish ATP hydrolysis at NBD2. We co-expressed Kir6.2 with wild-type or mutant SUR in Xenopus oocytes and recorded the resulting currents in inside-out macropatches. The S1R mutation in SUR1, SUR2A or SUR2B reduced K(ATP) current activation by 100 microM MgADP, whereas the S2R mutation in SUR1 or SUR2B (but not SUR2A) abolished MgADP activation completely. The linker mutations also reduced (S1R) or abolished (S2R) MgATP-dependent activation of Kir6.2-R50G co-expressed with SUR1 or SUR2B. These results suggest that the linker serines are not required for nucleotide binding but may be involved in transducing nucleotide binding into channel activation.

ATP-Binding Cassette Transporters↗
PubMed2002

Cholecystokinin tunes firing of an electrically distinct subset of arcuate nucleus neurons by activating A-Type potassium channels.

The physiological activity of hypothalamic arcuate nucleus (ARC) neurons is critical for dynamic maintenance of body energy homeostasis, and its malfunction can result in common metabolic disorders, such as obesity. It is therefore of interest to determine which set of ion channels shapes electrical activity in the ARC. Whole-cell patch clamp of ARC neurons in mouse brain slices identified three electrophysiologically distinct types of neurons. These were distinguished by their rebound "signatures" after hyperpolarizing current injection in current clamp and by the presence of transient inward (Type-B neurons) or outward (Type-A and Type-C neurons) subthreshold voltage-gated currents in voltage-clamp recordings. In turn, the transient outward current (A-current) of Type-C neurons had a lower activation threshold and different time and voltage dependence of inactivation than that of Type-A neurons. The brain-gut peptide cholecystokinin (CCK) has long been recognized to control food intake, but how endogenous CCK modulates the activity of central appetite-regulating networks remains unresolved. Here, we show that low (picomolar) concentrations of CCK rapidly and reversibly slow the firing of ARC Type-C neurons. This effect is mediated by postsynaptic CCK-B receptors and is attributable to potentiation of the A-current. Our study thus identifies several fundamental biophysical mechanisms underlying the physiological activity of ARC neurons and suggests a novel mechanism by which endogenous CCK may control appetite.

Action Potentials↗
PubMed2002

Characterization of two novel forms of the rat sulphonylurea receptor SUR1A2 and SUR1BDelta31.

1. The ATP-sensitive potassium channel (K(ATP)) of pancreatic beta-cells is composed of the sulphonylurea-binding protein, SUR1, and the inwardly rectifying K(+) channel subunit, Kir6.2. We have characterized two novel isoforms of rat SUR1 in the RINm5F insulin-secreting cell line. 2. SUR1A2 is an allelic variant with a single amino acid change in the first nucleotide-binding domain. Coinjection of SUR1A2 plus Kir6.2 into Xenopus oocytes or expression of a SUR1A2-Kir6.2 tandem in HEK-293 cells yielded large currents with characteristics similar to the wild-type K(ATP) channel. 3. SUR1BDelta31, detected in several human tissues, is a splice variant of the rat SUR1 gene that lacks exon 31 of the corresponding human SUR1 gene. SUR1BDelta31 lacks the TM16-TM17 transmembrane-spanning helices leading to a protein with a different transmembrane topology. Coinjection of SUR1BDelta31 plus Kir6.2 into Xenopus oocytes or expression of the Kir6.2/SUR1BDelta31 tandem construct in HEK-293 cells did not result in any current, and a surface expression assay indicated that this channel does not reach the plasma membrane. 4. SUR1A2 and SUR1A1 proteins expressed in HEK-293 cells display similar binding affinities for [(3)H]-glibenclamide, while SUR1BDelta31 shows a 500-fold lower affinity. 5. These findings confirm that TM16-TM17 of SUR1 are important for high-affinity glibenclamide binding and that their deletion impairs trafficking of the K(ATP) channel to the surface membrane.

ATP-Binding Cassette Transporters↗
PubMed2002

Interaction of the cytosolic domains of the Kir6.2 subunit of the K(ATP) channel is modulated by sulfonylureas.

The NH(2)- and COOH-termini of the ATP-sensitive potassium (K(ATP)) channel pore-forming subunit, Kir6.2, both lie intracellularly and interact with one another. To study this interaction, cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) were fused to the NH(2)- and COOH-termini of Kir6.2, respectively (CFP-Kir6.2-YFP). These fluorescent proteins have sufficient spectral overlap to allow distance-dependent fluorescence resonance energy transfer (FRET). When CFP-Kir6.2-YFP was expressed in human embryonic kidney cells and illuminated at 440 nm to excite CFP, significant fluorescence was recorded at 535 nm, the peak of the YFP emission spectrum. This indicated that FRET was occurring and thus that the NH(2)- and COOH-termini of Kir6.2 lie in close proximity to one another. The emission ratio, F(535)/F(480), was increased by co-expression of SUR2A, but not SUR1, suggesting that SUR2A but not SUR1 influences the Kir6.2 NH(2)- and COOH-terminal interaction. This interaction was reduced by the sulfonylureas tolbutamide and gliclazide, but not by the pore blocker barium. The properties of the tolbutamide response indicate that the drug disrupts the interaction between the NH(2)- and COOH-termini of Kir6.2 by binding to a low-affinity site on Kir6.2.

Animals↗
PubMed2002

The novel diazoxide analog 3-isopropylamino-7-methoxy-4H-1,2,4-benzothiadiazine 1,1-dioxide is a selective Kir6.2/SUR1 channel opener.

ATP-sensitive K(+) (K(ATP)) channels are activated by a diverse group of compounds known as potassium channel openers (PCOs). Here, we report functional studies of the Kir6.2/SUR1 Selective PCO 3-isopropylamino-7-methoxy-4H-1,2,4-benzothiadiazine 1,1-dioxide (NNC 55-9216). We recorded cloned K(ATP) channel currents from inside-out patches excised from Xenopus laevis oocytes heterologously expressing Kir6.2/SUR1, Kir6.2/SUR2A, or Kir6.2/SUR2B, corresponding to the beta-cell, cardiac, and smooth muscle types of the K(ATP) channel. NNC 55-9216 reversibly activated Kir6.2/SUR1 currents (EC(50) = 16 micromol/l). This activation was dependent on intracellular MgATP and was abolished by mutation of a single residue in the Walker A motifs of either nucleotide-binding domain of SUR1. The drug had no effect on Kir6.2/SUR2A or Kir6.2/SUR2B currents. We therefore used chimeras of SUR1 and SUR2A to identify regions of SUR1 involved in the response to NNC 55-9216. Activation was completely abolished and significantly reduced by swapping transmembrane domains 8-11. The reverse chimera consisting of SUR2A with transmembrane domains 8-11 and NBD2 consisting SUR1 was activated by NNC 55-9216, indicating that these SUR1 regions are important for drug activation. [(3)H]glibenclamide binding to membranes from HEK293 cells transfected with SUR1 was displaced by NNC 55-9216 (IC(50) = 105 micromol/l), and this effect was impaired when NBD2 of SUR1 was replaced by that of SUR2A. These results suggest NNC 55-9216 is a SUR1-selective PCO that requires structural determinants, which differ from those needed for activation of the K(ATP) channel by pinacidil and cromakalim. The high selectivity of NNC 55-9216 may prove to be useful for studies of the molecular mechanism of PCO action.

Adenosine Triphosphate↗
PubMed2002

Differential interactions of nateglinide and repaglinide on the human beta-cell sulphonylurea receptor 1.

Repaglinide and nateglinide represent a new class of insulin secretagogues, structurally unrelated to sulphonylureas, that were developed for the treatment of type 2 diabetes. The inhibitory effect of these drugs was investigated on recombinant wild-type and mutant Kir6.2/SUR1 channels expressed in HEK293 cells. Nateglinide and repaglinide dose-dependently inhibited whole-cell Kir6.2/SUR1 currents with half-maximal inhibitory concentration (IC(50)) values of 800 and 21 nmol/l, respectively. Mutation of serine 1237 in SUR1 to tyrosine (S1237Y) abolished tolbutamide and nateglinide block, suggesting that these drugs share a common point of interaction on the SUR1 subunit of the ATP-sensitive K(+) channel. In contrast, repaglinide inhibition was unaffected by the S1237Y mutation (IC(50) = 23 nmol/l). Radioligand binding studies revealed a single high-affinity binding site for [(3)H]repaglinide on membranes prepared from HEK293 cells expressing wild-type (equilibrium dissociation constant [K(D)] = 0.40 nmol/l) or mutant (K(D) = 0.31 nmol/l) Kir6.2/SUR1 channels. Nateglinide and tolbutamide displaced [(3)H]repaglinide binding to wild-type channels with IC(50) values of 0.7 and 26 micro mol/l, respectively, but produced <10% displacement of [(3)H]repaglinide bound to mutant channels. This is consistent with the idea that binding of nateglinide and tolbutamide, but not repaglinide, is abolished by the SUR1[S1237Y] mutation and that the binding site for repaglinide is not identical to that of nateglinde/tolbutamide. These results are discussed in terms of a conformational analysis of the drug molecules.

ATP-Binding Cassette Transporters↗