No abstract

1 The binding of modulators of the ATP-sensitive K + channel (K ATP channel) to the murine sulphonylurea receptor, SUR2B, was investigated. SUR2B, a proposed subunit of the vascular K ATP channel, was expressed in HEK 293 cells and binding assays were performed in membranes at 378C using the tritiated K ATP channel opener, .4 mM at 3 mM and 1 mM free Mg 2+ , respectively. Glibenclamide enhanced the dissociation of the [ 3 H]-P1075-SUR2B complex suggesting a negative allosteric coupling between the binding sites for P1075 and the sulphonylureas. 6 It is concluded that an MgATP site on SUR2B with mM a nity must be occupied to allow opener binding whereas Mg 2+ concentrations 510 mM decrease the a nities for openers and glibenclamide. The properties of the [ 3 H]-P1075 site strongly suggest that SUR2B represents the drug receptor of the openers in vascular smooth muscle.

No abstract

ATP-dependent K(+) channels are composed of pore-forming subunits of the Kir6.x family and of sulfonylurea receptors (SURs). SUR1, expressed in pancreatic beta-cells, has a higher affinity for sulfonylureas, such as glibenclamide, than SUR2B, expressed in smooth muscle. This difference is mainly caused by serine 1237 in SUR1 corresponding to tyrosine 1206 in SUR2B. To increase the affinity of SUR2B for glibenclamide, the mutant SUR2B(Y1206S) was constructed. In whole-cell patch-clamp experiments, glibenclamide inhibited the channel formed by coexpression of mutant SUR2B with Kir6.1 or 6.2 in human embryonic kidney cells with IC(50) values of 2.7 and 13 nM, respectively (wild-type, 43 and 167 nM). In intact cells, [(3)H]glibenclamide bound to mutant SUR2B with a K(D) value of 4.7 nM (wild-type, 32 nM); coexpression with Kir6.1 or 6.2 increased affinity by 4- and 8-fold, respectively. Binding of the opener [(3)H]P1075 to SUR2B(Y1206S) was the same as to wild-type and was unaffected by coexpression. In cells, the ratio of glibenclamide:P1075 sites was approximately 1:1; in membranes, it varied with the MgATP concentration. Heterologous competition curves were generally biphasic; the shape of the curve depended on the Kir-subtype. The effects of coexpression were weakened or abolished when binding assays were conducted in membranes. It is concluded that the mutation Y1206S increases the affinity of SUR2B for and the channel sensitivity toward glibenclamide by 7- to 15-fold. The interaction of glibenclamide (but not opener) with mutant SUR2B is modified by coexpression with Kir6.x in a manner depending on the Kir subtype and on the integrity of the cell.

No abstract

A time-dependent increase in ligand affinity has been studied in cholinergic ligand binding to Torpedocalifornica acetylcholine receptor by inhibition of the kinetics of of [125I]-alpha-bungarotoxin-receptor complex formation. The conversion of the acetylcholine receptor from low to high affinity form was induced by both agonists and antagonists of acetylcholine and was reversible upon removal of the ligand. The slow ligand induced affinity change in vitro resembled electrophysiological desensitization observed at the neuromuscular junction and described by a two-state model (Katz, B., & Thesleff, S. (1957) J. Physiol. 138, 63). A quantitative treatment of the rate and equilibrium constants determined for binding of the agonist carbamoylcholine to membrane bound acetylcholine receptor indicated that the two-state model is not compatible with the in vitro results.

We have developed dosemeters based on plastic scintillators for a variety of applications in radiation therapy. The dosemeters consist basically of a tissue-substituting scintillator probe, an optical fiber light guide, and a photomultiplier tube. The background light generated in the light guide can be compensated by a simultaneous measurement of the light from a blind fiber. Plastic scintillator dosemeters combine several advantageous properties which render them superior to other dosemeter types for many applications: minimal disturbance of the radiation field because of the homogeneous detector volume and the approximate water equivalence; no dependence on temperature and pressure (under standard clinical conditions) and angle of radiation incidence; no high voltage in the probe; high spatial resolution due to small detector volumes; direct reading of absorbed doses; and a large dynamical range. The high spatial resolution together with direct reading make these detectors suitable for real-time 3-D dosimetry using multi-channel detector systems. Such a system has been developed for eye plaque dosimetry and successfully employed for dosimetric treatment optimization. The plaque optimization can be performed by dosimetric measurements for the individual patient ("dosimetric treatment planning"). The time consumption for this procedure is less than for a physically correct computer-based therapy planning, e.g., by means of a Monte Carlo simulation.

1 ATP-sensitive K + channels are composed of pore-forming subunits Kir6.2 and of sulphonylurea receptors (SURs); the latter are the target of the hypoglycaemic sulphonylureas like glibenclamide. Here, we report on the negative allosteric modulation by MgATP and MgADP of glibenclamide binding to SUR1 and to SUR2 mutants with high glibenclamide anity, SUR2A(Y1206S) and SUR2B(Y1206S). 2 ATP, in the presence of an ATP-regenerating system to oppose hydrolysis during incubation, inhibited glibenclamide binding to SUR1 and SUR2B(Y1206S) by *60%, to SUR2A(Y1206S) by 21%). Inhibition curves for the SUR2(Y1206S) isoforms were monophasic with IC 50 values of 5 ± 10 mM; the curve for SUR1 was biphasic (IC 50 values 4.7 and 1300 mM). 3 Glibenclamide inhibition curves for ADP, performed in the presence of an ATP-consuming system to oppose ATP formation from ADP, were generally shifted rightwards and showed positive cooperativity, in particular with the SUR2(Y1206S) isoforms. 4 In the absence of the coupled enzyme systems, inhibition curves of MgATP or MgADP were generally shifted leftwards. This indicated synergy of MgATP and MgATP in acting together. 5 Coexpression of SUR1 and SUR2B(Y1206S) with Kir6.2 reduced both potency and ecacy of ATP in inhibiting glibenclamide binding; this was particularly marked for Kir6.2/SUR1. 6 The data show (a) that the inhibitory eects of ATP and ADP on glibenclamide binding dier from one another, (b) that they depend on the SUR subtype, and (c) that they are weakened by coexpression with Kir6.2.