ATP binding without hydrolysis switches sulfonylurea receptor 1 (SUR1) to outward-facing conformations that activate KATP channels.

Neuroendocrine-type ATP-sensitive K+ (KATP) channels are metabolite sensors coupling membrane potential with metabolism, thereby linking insulin secretion to plasma glucose levels. They are octameric complexes, (SUR1/Kir6.2)4, comprising sulfonylurea receptor 1 (SUR1 or ABCC8) and a K+-selective inward rectifier (Kir6.2 or KCNJ11). Interactions between nucleotide-, agonist-, and antagonist-binding sites affect channel activity allosterically. Although it is hypothesized that opening these channels requires SUR1-mediated MgATP hydrolysis, we show here that ATP binding to SUR1, without hydrolysis, opens channels when nucleotide antagonism on Kir6.2 is minimized and SUR1 mutants with increased ATP affinities are used. We found that ATP binding is sufficient to switch SUR1 alone between inward- or outward-facing conformations with low or high dissociation constant, KD , values for the conformation-sensitive channel antagonist [3H]glibenclamide ([3H]GBM), indicating that ATP can act as a pure agonist. Assembly with Kir6.2 reduced SUR1's KD for [3H]GBM. This reduction required the Kir N terminus (KNtp), consistent with KNtp occupying a "transport cavity," thus positioning it to link ATP-induced SUR1 conformational changes to channel gating. Moreover, ATP/GBM site coupling was constrained in WT SUR1/WT Kir6.2 channels; ATP-bound channels had a lower KD for [3H]GBM than ATP-bound SUR1. This constraint was largely eliminated by the Q1179R neonatal diabetes-associated mutation in helix 15, suggesting that a "swapped" helix pair, 15 and 16, is part of a structural pathway connecting the ATP/GBM sites. Our results suggest that ATP binding to SUR1 biases KATP channels toward open states, consistent with SUR1 variants with lower KD values causing neonatal diabetes, whereas increased KD values cause congenital hyperinsulinism.

A brachytherapy photon radiation quality index Q(BT) for probe-type dosimetry.

INTRODUCTION: In photon brachytherapy (BT), experimental dosimetry is needed to verify treatment plans if planning algorithms neglect varying attenuation, absorption or scattering conditions. The detector's response is energy dependent, including the detector material to water dose ratio and the intrinsic mechanisms. The local mean photon energy E¯(r) must be known or another equivalent energy quality parameter used. We propose the brachytherapy photon radiation quality indexQ(BT)(E¯), to characterize the photon radiation quality in view of measurements of distributions of the absorbed dose to water, Dw, around BT sources. MATERIALS AND METHODS: While the external photon beam radiotherapy (EBRT) radiation quality index Q(EBRT)(E¯)=TPR10(20)(E¯) is not applicable to BT, the authors have applied a novel energy dependent parameter, called brachytherapy photon radiation quality index, defined as Q(BT)(E¯)=Dprim(r=2cm,θ0=90°)/Dprim(r0=1cm,θ0=90°), utilizing precise primary absorbed dose data, Dprim, from source reference databases, without additional MC-calculations. RESULTS AND DISCUSSION: For BT photon sources used clinically, Q(BT)(E¯) enables to determine the effective mean linear attenuation coefficient µ¯(E) and thus the effective energy of the primary photons Eprim(eff)(r0,θ0) at the TG-43 reference position Pref(r0=1cm,θ0=90°), being close to the mean total photon energy E¯tot(r0,θ0). If one has calibrated detectors, published E¯tot(r) and the BT radiation quality correction factor [Formula: see text] for different BT radiation qualities Q and Q0, the detector's response can be determined and Dw(r,θ) measured in the vicinity of BT photon sources. CONCLUSIONS: This novel brachytherapy photon radiation quality indexQ(BT) characterizes sufficiently accurate and precise the primary photon's penetration probability and scattering potential.

Reinterpreting the action of ATP analogs on K(ATP) channels.

Neuroendocrine-type K(ATP) channels, (SUR1/Kir6.2)4, couple the transmembrane flux of K(+), and thus membrane potential, with cellular metabolism in various cell types including insulin-secreting ß-cells. Mutant channels with reduced activity are a cause of congenital hyperinsulinism, whereas hyperactive channels are a cause of neonatal diabetes. A current regulatory model proposes that ATP hydrolysis is required to switch SUR1 into post-hydrolytic conformations able to antagonize the inhibitory action of nucleotide binding at the Kir6.2 pore, thus coupling enzymatic and channel activities. Alterations in SUR1 ATPase activity are proposed to contribute to neonatal diabetes and type 2 diabetes risk. The regulatory model is partly based on the reduced ability of ATP analogs such as adenosine 5'-(ß,γ-imino)triphosphate (AMP-PNP) and adenosine 5'-O-(thiotriphosphate) (ATPγS) to stimulate channel activity, presumably by reducing hydrolysis. This study uses a substitution at the catalytic glutamate, SUR(1E1507Q), with a significantly increased affinity for ATP, to probe the action of these ATP analogs on conformational switching. ATPγS, a slowly hydrolyzable analog, switches SUR1 conformations, albeit with reduced affinity. Nonhydrolyzable AMP-PNP and adenosine 5'-(ß,γ-methylenetriphosphate) (AMP-PCP) alone fail to switch SUR1, but do reverse ATP-induced switching. AMP-PCP displaces 8-azido-[(32)P]ATP from the noncanonical NBD1 of SUR1. This is consistent with structural data on an asymmetric bacterial ABC protein that shows that AMP-PNP binds selectively to the noncanonical NBD to prevent conformational switching. The results imply that MgAMP-PNP and MgAMP-PCP (AMP-PxP) fail to activate K(ATP) channels because they do not support NBD dimerization and conformational switching, rather than by limiting enzymatic activity.

Two neonatal diabetes mutations on transmembrane helix 15 of SUR1 increase affinity for ATP and ADP at nucleotide binding domain 2.

K(ATP) channels, (SUR1/Kir6.2)(4) (sulfonylurea receptor type 1/potassium inward rectifier type 6.2) respond to the metabolic state of pancreatic ß-cells, modulating membrane potential and insulin exocytosis. Mutations in both subunits cause neonatal diabetes by overactivating the pore. Hyperactive channels fail to close appropriately with increased glucose metabolism; thus, ß-cell hyperpolarization limits insulin release. K(ATP) channels are inhibited by ATP binding to the Kir6.2 pore and stimulated, via an uncertain mechanism, by magnesium nucleotides at SUR1. Glibenclamide (GBC), a sulfonylurea, was used as a conformational probe to compare nucleotide action on wild type versus Q1178R and R1182Q SUR1 mutants. GBC binds with high affinity to aporeceptors, presumably in the inward facing ATP-binding cassette configuration; MgATP reduces binding affinity via a shift to the outward facing conformation. To determine nucleotide affinities under equilibrium, non-hydrolytic conditions, Mg(2+) was eliminated. A four-state equilibrium model describes the allosteric linkage. The K(D) for ATP(4-) is ~1 versus 12 mM, Q1178R versus wild type, respectively. The linkage constant is ~10, implying that outward facing conformations bind GBC with a lower affinity, 9-10 nM for Q1178R. Thus, nucleotides cannot completely inhibit GBC binding. Binding of channel openers is reported to require ATP hydrolysis, but diazoxide, a SUR1-selective agonist, concentration-dependently augments ATP(4-) action. An eight-state model describes linkage between diazoxide and ATP(4-) binding; diazoxide markedly increases the affinity of Q1178R for ATP(4-) and ATP(4-) augments diazoxide binding. NBD2, but not NBD1, has a higher affinity for ATP (and ADP) in mutant versus wild type (with or without Mg(2+)). Thus, the mutants spend more time in nucleotide-bound conformations, with reduced affinity for GBC, that activate the pore.

Role of the amino-terminal transmembrane domain of sulfonylurea receptor SUR2B for coupling to K(IR)6.2, ligand binding, and oligomerization.

ATP-sensitive K(+) (K(ATP)) channels consist of two types of subunits, K(IR)6.x that form the pore, and sulfonylurea receptors (SURs) that serve as regulatory subunits. SURs are ATP-binding cassette (ABC) proteins and contain, in addition to two nucleotide binding folds, the binding sites for channel openers such as diazoxide and P1075 and channel inhibitors such as glibenclamide (GBC) and repaglinide. Structurally, SURs differ from most eukaryotic ABC proteins by an additional amino-terminal transmembrane domain (TMD0); in case of SUR1, the subunit of the pancreatic K(ATP) channel, TMD0 serves as a major domain for association with K(IR). In this study we sought to elucidate the roles of TMD0 in SUR2B, the smooth muscle gating subunit, in the coupling between SUR2B and K(IR)6.2, in the self-association of SUR2B and in channel modulator binding to SUR2B. SUR2B has a weaker affinity for sulfonylureas thus SUR2B(Y1206S), with a higher affinity for GBC, but an equivalent opener binding was used. Association of SUR2B(YS)Δ, lacking TMD0, with K(IR)6.2 was shown by immunoprecipitation; however, no evidence for formation of functional channels was obtained. SUR2B(YS)Δ self-associates like SUR2B(YS) and binds GBC, repaglinide, and P1075 with slightly reduced affinities. The binding profile of the SUR2B(YS)Δ/K(IR)6.2 complex differs slightly but significantly from that of SUR2B(YS)Δ alone showing impaired allosteric coupling of binding sites. We conclude that TMD0 is not required for oligomerization of SUR2B, is of only minor importance in ligand binding, but is essential for both functional and allosteric coupling of SUR2B to K(IR)6.2.

Importance of the Kir6.2 N-terminus for the interaction of glibenclamide and repaglinide with the pancreatic K(ATP) channel.

The pancreatic K(ATP) channel, SUR1/Kir6.2, couples insulin secretion to the plasma glucose level. The channel is an octamer with four Kir6.2 subunits forming the pore and four sulphonylurea receptors (SUR1) regulating channel activity. SUR1 is an ABC protein with adenosine triphosphate (ATP)ase activity which activates the channel. It also contains the binding site for antidiabetic drugs like glibenclamide and repaglinide which close the channel by disrupting the stimulatory effect SUR-ATPase (MgATP-dependent) and by stabilising a long-lived closed channel state (MgATP-independent). In this study, we examined the effects of progressive truncation of the Kir6.2 N-terminus up to 20 amino acids on equilibrium binding and channel closure by glibenclamide and repaglinide, on the channel activating effect of the opener, 6-chloro-3-(1-methylcyclobutyl)amino-4H-thieno[3,2-e]-1,2,4thiadiazine 1,1-dioxide (NNC 55-0462), and on the binding kinetics of [(3)H]glibenclamide. Kir and SUR were transiently coexpressed in HEK cells and [(3)H]glibenclamide binding and patch-clamp experiments were performed in whole cells at 37°C and in isolated inside/out patches at 22°C. Truncation of the first 5 N-terminal amino acids abolished most of the affinity increase for glibenclamide and repaglinide that is produced by the association of Kir6.2 with SUR1. Progressive truncation continuously reduced the potency and efficacy of these drugs in closing the channel and impaired the ability to stabilise the closed state more than the ability to disrupt channel stimulation by SUR-ATPase. The effects of NNC 55-0462 were unchanged. Progressive truncation also speeded up dissociation of [(3)H]glibenclamide from the channel when dissociation was induced by an excess of (unlabelled) glibenclamide. This suggests the existence of a putative low affinity glibenclamide site on the channel whose affinity increases upon truncation. The data show that progressive truncation of the Kir6.2 N-terminus impairs the transduction of drug binding into channel closure more strongly than drug binding but leaves the effect of the opener NNC 55-0462 unchanged.

Substitution of the Walker A lysine by arginine in the nucleotide-binding domains of sulphonylurea receptor SUR2B: effects on ligand binding and channel activity.

Sulphonylurea receptors (SURs) serve as regulatory subunits of ATP-sensitive K(+) channels. SURs are members of the ATP-binding cassette (ABC) protein superfamily and contain two conserved nucleotide-binding domains (NBDs) which bind and hydrolyse MgATP; in addition, they carry the binding sites for the sulphonylureas like glibenclamide (GBC) which close the channel and for the K(ATP) channel openers such as P1075. Here we have exchanged the conserved Lys in the Walker A motif by Arg in both NBDs of SUR2B, the regulatory subunit of the vascular K(ATP) channel. Then the effect of the mutation on the ATPase-dependent binding of GBC and P1075 to SUR2B and on the activity of the recombinant vascular (Kir6.1/SUR2B) channel was assessed. Surprisingly, in the absence of MgATP, the mutation weakened binding of P1075 and the extent of allosteric inhibition of GBC binding by P1075. The mutation abolished most, but not all, of the MgATP effects on the binding of GBC and P1075 and prevented nucleotide-induced activation of the channel which relies on SUR reaching the posthydrolytic (MgADP-bound) state; the mutant channel was, however, opened by P1075 at higher concentrations. The data provide evidence that mutant SUR2B binds MgATP but that the posthydrolytic state is insufficiently populated. This suggests that the mutation locks SUR2B in an MgATP-binding prehydrolytic-like state; binding of P1075 may induce a posthydrolytic-like conformation to open the channel.

Analysis of two KCNJ11 neonatal diabetes mutations, V59G and V59A, and the analogous KCNJ8 I60G substitution: differences between the channel subtypes formed with SUR1.

beta-Cell-type K(ATP) channels are octamers assembled from Kir6.2/KCNJ11 and SUR1/ABCC8. Adenine nucleotides play a major role in their regulation. Nucleotide binding to Kir6.2 inhibits channel activity, whereas ATP binding/hydrolysis on sulfonylurea receptor 1 (SUR1) opposes inhibition. Segments of the Kir6.2 N terminus are important for open-to-closed transitions, form part of the Kir ATP, sulfonylurea, and phosphoinositide binding sites, and interact with L0, an SUR cytoplasmic loop. Inputs from these elements link to the pore via the interfacial helix, which forms an elbow with the outer pore helix. Mutations that destabilize the interfacial helix increase channel activity, reduce sensitivity to inhibitory ATP and channel inhibitors, glibenclamide and repaglinide, and cause neonatal diabetes. We compared Kir6.x/SUR1 channels carrying the V59G substitution, a cause of the developmental delay, epilepsy, and neonatal diabetes syndrome, with a V59A substitution and the equivalent I60G mutation in the related Kir6.1 subunit from vascular smooth muscle. The substituted channels have increased P(O) values, decreased sensitivity to inhibitors, and impaired stimulation by phosphoinositides but retain sensitivity to Ba(2+)-block. The V59G and V59A channels are either not, or poorly, stimulated by phosphoinositides, respectively. Inhibition by sequestrating phosphatidylinositol 4,5-bisphosphate with neomycin and polylysine is reduced in V59A, and abolished in V59G channels. Stimulation by SUR1 is intact, and increasing the concentration of inhibitory ATP restores the sensitivity of Val-59-substituted channels to glibenclamide. The I60G channels, strongly dependent on SUR stimulation, remain sensitive to sulfonylureas. The results suggest the interfacial helix dynamically links inhibitory inputs from the Kir N terminus to the gate and that sulfonylureas stabilize an inhibitory configuration.

Cyclic AMP increases cytoplasmic free calcium in renin-secreting cells from rat kidney.

The renin-secreting juxtaglomerular cells (JGC) in the media of the afferent arteriole at the vessel pole are the major source of circulating renin. The control of renin secretion is complex with increases in cAMP being the major stimulus and increases in intracellular free Ca2+ concentration ([Ca2+]i) being inhibitory. We measured [Ca2+]i in the afferent arteriole from mostly JGC. Manoeuvres that increase cAMP (e.g. isoproterenol) or dibutyryl-cAMP elicited an increase in [Ca2+]i which was approximately 40% of that induced by angiotensin II (3 nmol/l). The Ca2+ response occurred in 50-90% of the cases, and increasing the stimulus increased responder frequency but not response size. The response was (almost) abolished by removal of extracellular Ca2+, prevented by inhibitors of store-operated Ca2+ channels (Gd3+ and 2-aminoethoxydiphenyl-borate), but was unaffected by isradipine or protein kinase A inhibitors. It was not produced by an activator of EPACs (exchange protein activated by cAMP) and was not accompanied by changes in membrane potential. The data suggest that in rat JGC, cAMP, perhaps directly, activates store-operated Ca2+ channels to increase [Ca2+]i. One could speculate that this increase in [Ca2+]i serves to finely adjust the stimulating effect cAMP-increasing signals on the renin-angiotensin system.

Effect of adenosine on membrane potential and Ca2+ in juxtaglomerular cells. Comparison with angiotensin II.

BACKGROUND: Renin is mainly secreted from the juxtaglomerular cells (JGC) in the kidney situated in the afferent arteriole close to the vessel pole. Angiotensin II (ANG II) and adenosine inhibit renin secretion and synergistically constrict the afferent arteriole. ANG II depolarises JGC and increases the cytoplasmic free Ca2+ concentration [Ca2+]i. The responses of JGC to adenosine are less known. METHODS: Effects of adenosine on membrane potential and [Ca2+]i were studied in afferent arterioles from NaCl-depleted rats and mice. RESULT: Stimulation of A1 adenosine receptors (A1AR) by adenosine (10 microM) or cyclohexyladenosine (1 microM) increased the spiking frequency of JGC, slightly depolarised the cells and, in < or =50% of the cases, increased [Ca2+]i. These effects were much smaller than those of ANG II (3 nM). Simultaneous application of cyclohexyladenosine and ANG II gave only additive effects on [Ca2+]i; in addition, responses to ANG II in JGC from A1AR knockout mice were similar to those from control mice. CONCLUSION: The small changes in membrane potential and [Ca2+]i in response to A1AR stimulation as compared to those of ANG II may suggest that these 2 tissue hormones use different signal transduction mechanisms to affect JGC function, including the inhibition of renin release.

Testing the bipartite model of the sulfonylurea receptor binding site: binding of A-, B-, and A + B-site ligands.

ATP-sensitive K(+) (K(ATP)) channels are composed of pore-forming subunits (Kir6.x) and of regulatory subunits, the sulfonylurea receptors (SURx). Subtypes of K(ATP) channels are expressed in different organs. The sulfonylureas and glinides (insulinotropes) close the K(ATP) channel in pancreatic beta-cells and stimulate insulin secretion. The insulinotrope binding site of the pancreatic channel (Kir6.2/SUR1) consists of two overlapping (sub)-sites, site A, located on SUR1 and containing Ser1237 (which in SUR2 is replaced by Tyr1206), and site B, formed by SUR1 and Kir6.2. Insulinotropes bind to the A-, B-, or A + B-site(s) and are grouped accordingly. A-ligands are highly selective in closing the pancreatic channel, whereas B-ligands are nonselective and insensitive to the mutation S1237Y. We have examined the binding of insulinotropes representative of the three groups in [(3)H]glibenclamide competition experiments to determine the contribution of Kir6.x to binding affinity, the effect of the mutation Y1206S in site A of SUR2, and the subtype selectivity of the compounds. The results show that the bipartite nature of the SUR1 binding site applies also to SUR2. Kir6.2 as part of the B-site may interact directly or allosterically with structural elements common to all insulinotropes, i.e., the negative charge and/or the adjacent phenyl ring. The B-site confers a moderate subtype selectivity on B-ligands. The affinity of B-ligands is altered by the mutation SUR2(Y1206S), suggesting that the mutation affects the binding chamber of SUR2 as a whole or subsite A, including the region where the subsites overlap.

Whole body radiotherapy: A TBI-guideline.

Total Body Irradiation (TBI) is one main component in the interdisciplinary treatment of widely disseminated malignancies predominantly of haematopoietic diseases. Combined with intensive chemotherapy, TBI enables myeloablative high dose therapy and immuno-ablative conditioning treatment prior to subsequent transplantation of haematopoietic stem cells: bone marrow stem cells or peripheral blood progenitor stem cells. Jointly prepared by DEGRO and DGMP, the German Society of Radio-Oncology, and the German Association of Medical Physicists, this DEGRO/DGMP-Leitlinie Ganzkoerper-Strahlenbehandlung - DEGRO/DGMP Guideline Whole Body Radiotherapy, summarises the concepts, principles, facts and common methods of Total Body Irradiation and poses a set of recommendations for reliable and successful application of high dose large-field radiotherapy as essential part of this interdisciplinary, multi-modality treatment concept. The guideline is geared towards radio-oncologists, medical physicists, haematooncolo-gists, and all contributing to Whole Body Radiotherapy. To guide centres intending to start or actualise TBI criteria are included. The relevant treatment parameters are defined and a sample of a form is given for reporting TBI to international registries.

Lipids modulate ligand binding to sulphonylurea receptors.

ATP-sensitive K(+) channels (K(ATP) channels) are complexes of inwardly rectifying K(+) channels (Kir6.x) and sulphonylurea receptors (SURs). Kir6.2-containing channels are closed by ATP binding to Kir6.2, and opened by MgADP binding to SUR. Channel activity is modulated by synthetic compounds such as the channel-blocking sulphonylureas and the K(ATP) channel openers, which both act by binding to SUR. By interacting with Kir6.2, phosphatidylinositol-4,5-bisphosphate (PIP(2)) and oleoyl-coenzyme A (OCoA) decrease the ATP-sensitivity of the channel and abolish the effect of the synthetic channel modulators. Here, we have investigated whether lipids and related compounds interfered with the binding of the sulphonylurea, glibenclamide (GBC) and of the opener, N-cyano-N'-(1,1-dimethylpropyl)-N''-3-pyridylguanidine (P1075), to the SUR subtypes. Lipids (100-300 microM) inhibited binding of [(3)H]GBC and [(3)H]P1075 to SUR subtypes in the rank order OCoA>dioleylglycerol-succinyl-nitriloacetic acid (DOGS-NTA)>oleate>malonyl-CoA>PIP(2.)OCoA inhibited radioligand binding to SUR completely, with IC(50) values ranging from 6 to 44 microM. Inhibition was reversed by increasing the concentration of the radioligands in agreement with an essentially competitive mechanism. MgATP and coexpression with Kir6.2 decreased the potency of OCoA. DOGS-NTA inhibited radioligand binding to SUR by 40-88%, with IC(50) values ranging from 38 to 120 microM. Poly-lysine increased radioligand binding to SUR by up to 30% but did not affect much the inhibition of ligand binding by OCoA and DOGS-NTA. Radioligand binding to SUR2A but not to the other SUR subtypes was slightly (10-20%) stimulated by lipids at concentrations approximately 10 x lower than required for inhibition. The data show that certain lipids, at high concentrations, interact with SUR and inhibit the binding of GBC and P1075; with SUR2A, a modest stimulation of ligand binding precedes inhibition. Regarding K(ATP) channel activity, these effects will be overruled by the interaction of the lipids with Kir6.2 at lower (physiological) concentrations. They are, however, of pharmacological importance and must be taken into account if high concentrations of these compounds (e.g. OCoA>10 microM) are used to interfere with the action of sulphonylureas and openers on K(ATP) channel activity.

The mutation Y1206S increases the affinity of the sulphonylurea receptor SUR2A for glibenclamide and enhances the effects of coexpression with Kir6.2.

1. ATP-sensitive K(+) channels (K(ATP) channels) are tetradimeric complexes of inwardly rectifying K(+) channels (Kir6.x) and sulphonylurea receptors (SURs). The SURs SUR2A (cardiac) and SUR2B (smooth muscle) differ only in the last 42 amino acids. In SUR2B, the mutation Y1206S, located at intracellular loop 8, increases the affinity for glibenclamide (GBC) about 10-fold. Here, we examined whether the mutation Y1206S in SUR2A had effects similar to those in SUR2B.2. GBC bound to SUR2A with K(D)=20 nM; the mutation increased affinity approximately 5 x. 3. In cells, coexpression of SUR2A with Kir6.2 increased the affinity for GBC approximately 3 x; with the mutant, the increase was 9 x. 4. The mutation did not affect the affinity of SUR2A for openers; coexpression with Kir6.2 reduced opener affinity of wild-type and mutant SUR2A by about 2 x. 5. The negative allosteric interaction between the opener, P1075, and GBC at wild-type and mutant SUR2A was markedly affected by the presence of MgATP and by coexpression with Kir6.2. 6. In inside-out patches, GBC inhibited the wild-type Kir6.2/SUR2A and 2B channels with IC(50) values of 27 nM; the mutation shifted the IC(50) values to approximately 1 nM. 7. The data show that the mutation Y1206S increased the affinity of SUR2A for GBC and modulated the effects of coexpression. Overall, the changes were similar to those observed with SUR2B(Y1206S), suggesting that the differences in the last 42 carboxy-terminal amino acids of SUR2A and 2B are of limited influence on the binding of GBC and P1075 to the SUR2 isoforms.

The impact of ATP-sensitive K+ channel subtype selectivity of insulin secretagogues for the coronary vasculature and the myocardium.

Insulin secretagogues (sulfonylureas and glinides) increase insulin secretion by closing the ATP-sensitive K+ channel (KATP channel) in the pancreatic beta-cell membrane. KATP channels subserve important functions also in the heart. First, KATP channels in coronary myocytes contribute to the control of coronary blood flow at rest and in hypoxia. Second, KATP channels in the sarcolemma of cardiomyocytes (sarcKATP channels) are required for adaptation of the heart to stress. In addition, the opening of sarcKATP channels and of KATP channels in the inner membrane of mitochondria (mitoKATP channels) plays a central role in ischemic preconditioning. Opening of sarcKATP channels also underlies the ST-segment elevation of the electrocardiogram, the primary diagnostic tool for initiation of lysis therapy in acute myocardial infarction. Therefore, inhibition of cardiovascular KATP channels by insulin secretagogues is considered to increase cardiovascular risk. Electrophysiological experiments have shown that the secretagogues differ in their selectivity for the pancreatic over the cardiovascular KATP channels, being either highly selective (approximately 1,000x; short sulfonylureas such as nateglinide and mitiglinide), moderately selective (10-20x; long sulfonylureas such as glibenclamide [glyburide]), or essentially nonselective (<2x; repaglinide). New binding studies presented here give broadly similar results. In clinical studies, these differences are not yet taken into account. The hypothesis that the in vitro selectivity of the insulin secretagogues is of importance for the cardiovascular outcome of diabetic patients with coronary artery disease needs to be tested.

Electrophysiological and molecular characterization of the inward rectifier in juxtaglomerular cells from rat kidney.

Renin, the key element of the renin-angiotensin-aldosterone system, is mainly produced by and stored in the juxtaglomerular cells in the kidney. These cells are situated in the media of the afferent arteriole close to the vessel pole and can transform into smooth muscle cells and vice versa. In this study, the electrophysiological properties and the molecular identity of the K+ channels responsible for the resting membrane potential (approximately -60 mV) of the juxtaglomerular cells were examined. In order to increase the number of juxtaglomerular cells, afferent arterioles from NaCl-depleted rats were used, and > 90% of the afferent arterioles were renin positive at the distal end of the arteriole. Whole-cell and cell-attached single-channel patch-clamp experiments showed that juxtaglomerular cells are endowed with a strongly inwardly rectifying K+ channel (Kir). The channel was highly sensitive to inhibition by Ba2+ (inhibition constant 37 microM at 0 mV), but relatively insensitive to Cs+ and, with 142 mM K+ in the pipette, had a single-channel conductance of 31.5 pS. Immunocytochemical studies showed the presence of Kir2.1 but no signal for Kir2.2 in the media of the afferent arteriole. In PCR analyses using isolated juxtaglomerular cells, the mRNA for Kir2.1 and Kir2.2 was detected. Collectively, the results show that Kir2.1 is the dominant component of the channel. The current carried by these channels plays a decisive role in setting the membrane potential of juxtaglomerular cells.

Interaction of a novel dihydropyridine K+ channel opener, A-312110, with recombinant sulphonylurea receptors and KATP channels: comparison with the cyanoguanidine P1075.

1. ATP-sensitive K(+) channels (K(ATP) channels) are composed of pore-forming subunits (Kir6.x) and of regulatory subunits, the sulphonylurea receptors (SURx). Synthetic openers of K(ATP) channels form a chemically heterogeneous class of compounds that are of interest in several therapeutic areas. We have investigated the interaction of a novel dihydropyridine opener, A-312110 ((9R)-9-(4-fluoro-3-iodophenyl)-2,3,5,9-tetrahydro-4H-pyrano[3,4-b]thieno [2,3-e]pyridin-8(7H)-one-1,1-dioxide), with SURs and Kir6/SUR channels in comparison to the cyanoguanidine opener P1075. 2. In the presence of 1 mM MgATP, A-312110 bound to SUR2A (the SUR in cardiac and skeletal muscle) and to SUR2B (smooth muscle) with K(i) values of 14 and 18 nM; the corresponding values for P1075 were 16 and 9 nM, respectively. Decreasing the MgATP concentration reduced the affinity of A312110 binding to SUR2A significantly more than that to SUR2B; for P1075, the converse was true. At SUR1 (pancreatic beta-cell), both openers showed little binding up to 100 microM. 3. In the presence of MgATP, both openers inhibited [(3)H]glibenclamide binding to the SUR2 subtypes in a biphasic manner. In the absence of MgATP, the high-affinity component of the inhibition curves was absent. 4. In inside-out patches, the two openers activated the Kir6.2/SUR2A and Kir6.2/SUR2B channels with similar potency (approximately 50 nm). Both were almost 2 x more efficacious in opening the Kir6.2/SUR2B than the Kir6.2/SUR2A channel. 5. The results show that the novel dihydropyridine A-312110 is a potent K(ATP) channel opener with binding and channel-opening properties similar to those of P1075.

Virtual 3D IVUS vessel model for intravascular brachytherapy planning. I. 3D segmentation, reconstruction, and visualization of coronary artery architecture and orientation.

Intravascular brachytherapy (IVB) can significantly reduce the risk of restenosis after interventional treatment of stenotic arteries, if planned and applied correctly. To facilitate computer-based IVB planning, a three-dimensional vessel model has been derived from information on coronary artery segments acquired by intravascular ultrasound (IVUS) and biplane angiography. Part I describes the approach of model construction and presents possibilities of visualization. The vessel model is represented by a voxel volume. Polygonal information about the vessel wall structure is derived by segmentation from a sequence of IVUS images automatically acquired ECG gated during pull back of the IVUS transducer. To detect horizontal, vertical, and radial contours, modified Canny-Edge and Shen-Castan filters are applied on Cartesian and polar coordinate representations of the IVUS tomograms as edge detectors. The spatial course of the vessel wall layers is traced in reconstructed longitudinal IVUS scans. By resampling the sequence of IVUS frames the voxel volume is obtained. For this purpose the frames are properly located in space and augmented with additional intermediate frames generated by interpolation. Their spatial location and orientation is derived from biplane X-ray angiography which is performed simultaneously. For resampling, two approaches are proposed: insertion of the vertices of the rectangular goal grid into the cells of a deformed hexahedral mesh derived from the IVUS sequence, and insertion of the vertices of the hexahedral mesh into the cells of the rectangular grid. Finally, the vessel model is visualized by methods of combined volume and polygon rendering. The segmentation process is verified as being in good agreement with results obtained by manual contour tracing with a commercial system. Our approach of construction of the vessel model has been implemented into an interactive software system, 3D IVUS-View, serving as the basis of a future system for intracoronary brachytherapy treatment planning being currently under development (Part II).

Binding and effect of K ATP channel openers in the absence of Mg2+.

1 Openers of ATP-sensitive K(+) channels (K(ATP) channels) are thought to act by enhancing the ATPase activity of sulphonylurea receptors (SURs), the regulatory channel subunits. At higher concentrations, some openers activate K(ATP) channels also in the absence of MgATP. Here, we describe binding and effect of structurally diverse openers in the absence of Mg(2+) and presence of EDTA. 2 Binding of openers to SUR2B was measured using a mutant with high affinity for [(3)H]glibenclamide ([(3)H]GBC). In the absence of Mg(2+), 'typical' openers (benzopyrans, cyanoguanidines and aprikalim) inhibited [(3)H]GBC binding with K(i) values approximately 200 x higher than in the presence of MgATP. Minoxidil sulphate and nicorandil were inactive, whereas binding of diazoxide was unaffected by MgATP. 3 In the absence/presence of MgATP, N-cyano-N'-(1,1-dimethylpropyl)-N"-3-pyridylguanidine (P1075) activated the Kir6.2/SUR2B channel in inside-out patches with EC(50)=2000/67nM and E(max)=32/134%. In the absence of Mg(2+), responses were variable with only a small part of the variability being explained by a decrease in channel responsiveness with time after patch excision and to differences in the ATP sensitivity between patches. 4 The rank order of efficacy of the openers was P1075>rilmakalim approximately nicorandil>diazoxide>minoxidil sulphate. 5 The data show that structurally diverse openers are able to bind to, and to activate the Kir6.2/SUR2B channel by a pathway independent of ATP hydrolysis. These effects are observed at concentrations used to define the biochemical mechanism of the openers in the presence of MgATP and allow the openers to be classified into 'typical' and 'atypical' KCOs with diazoxide standing apart.

Mg2+ sensitizes KATP channels to inhibition by DIDS: dependence on the sulphonylurea receptor subunit.

1. ATP-sensitive potassium channels (K(ATP) channels) consist of pore-forming Kir6.x subunits and of sulphonylurea receptors (SURs). In the absence of Mg(2+), the stilbene disulphonate, DIDS, irreversibly inhibits K(ATP) channels by binding to the Kir subunit. Here, the effects of Mg(2+) on the interaction of DIDS with recombinant K(ATP) channels were studied in electrophysiological and [(3)H]-glibenclamide binding experiments. 2. In inside-out macropatches, Mg(2+) (0.7 mM) increased the sensitivity of K(ATP) channels towards DIDS up to 70 fold (IC(50)=2.7 micro M for Kir6.2/SUR2B). Inhibition of current at DIDS concentrations > or =10 micro M was irreversible. 3. Mg(2+) sensitized the truncated Kir6.2Delta26 channel towards inhibition by DIDS only upon coexpression with a SUR subunit (SUR2B). The effect of Mg(2+) did not require the presence of nucleotides. 4. [(3)H]-glibenclamide binding to SUR2B(Y1206S), a mutant with improved affinity for glibenclamide, was inhibited by DIDS. The potency of inhibition was increased by Mg(2+) and by coexpression with Kir6.2. 5. In the presence of Mg(2+), DIDS inhibited binding of [(3)H]-glibenclamide to Kir6.2/SUR2B(Y1206S) with IC(50)=7.9 micro M by a non-competitive mechanism. Inhibition was fully reversible. 6. It is concluded that the binding site of DIDS on SUR that is sensed by glibenclamide does not mediate channel inhibition. Instead, Mg(2+) binding to SUR may allosterically increase the accessibility and/or reactivity of the DIDS site on Kir6.2. The fact that the Mg(2+) effect does not require the presence of nucleotides underlines the importance of this ion in modulating the properties of the K(ATP) channel.