[Canine Elbow Humeroulnar Incongruity Measurements Using Computed Tomography]. / Merení humeroulnární inkongruity loketního kloubu u psu pomocí výpocetní tomografie.

PURPOSE OF THE STUDY The purpose of the study is to compare the repeatability and reproducibility of quantitative and subjective evaluation of elbow humeroulnar incongruity (HUI) using computed tomography (CT) on an in vivo canine model. MATERIAL AND METHODS HUI was evaluated on canine (n = 50) elbow joints (n = 100). The computed tomography of elbow joints was performed under intravenous sedation. Multiplanar reconstructions (MPR) were produced. HUI was evaluated on sagittal MPR images subjectively and by measuring the subluxation index (SI). The SI was defined by measuring the distance between the centres of two circles, the shape of which corresponded the most with the shape of the trochlear notch of the ulna at sagittal crest and the shape of humeral trochlea. This distance was divided by the radius of the circle (r) defining the humeral trochlea. HUI was subjectively evaluated based on the width of the joint space at the greatest caudal convexity of the trochlear notch of the ulna. Three categories of HUI were established: 0 (congruent), 1 (moderately incongruent), 2 (strongly incongruent). Measurement and evaluation of HUI was conducted by two evaluators twice at a one-month interval between the first and second measurement. The statistical analysis was carried out using the repeated measures ANOVA and the Cohen s kappa coefficient. RESULTS The mean SI was 11.14 (SD 8.703). The SI values measured by two evaluators were statistically significantly different (p < 0.05). Contrarily, there was no statistically significant difference between the two measurements of the same evaluator (p > 0.05). The subjective evaluation of HUI done by two evaluators showed a mean to substantial agreement (Kappa = 0.53-0.79). There was a substantial to almost perfect agreement between the results of two evaluations carried out by a single evaluator (Kappa = 0.79-0.83). DISCUSSION The radiographic detection of moderate incongruity is unreliable, especially on account of wrongly positioned elbow joint, superposition of bone structures and due to the evaluation of three-dimensional bone structure through a two-dimensional image. Evaluation of humeroulnar congruity by computed tomography (CT) enables to assess the congruity of joints without the superposition of neighbouring bone structures. The quantification of humeroulnar incongruity using the SI does not show a higher degree of agreement between two evaluators as against the subjective evaluation of HUI. On the contrary, the agreement between two measurements of a single evaluator was high in both the cases. CONCLUSIONS Dog is a suitable model animal for evaluation of HUI of elbow joints due to frequent incidence of elbow dysplasia associated with HUI. The quantification of HUI at the expense of subjective evaluation of HUI is often overrated in radiological studies. Key words: dog, elbow, humeroulnar incongruity.

[Treatment of a bone bridge by transplantation of mesenchymal stem cells and chondrocytes in a composite scaffold in pigs: experimental study]. / Lécba kostního mustku transplantací mezenchymových kmenových bunek a chondrocytu v kompozitním nosici u prasat - experimentální studie.

PURPOSE OF THE STUDY: The presented experimental study describes the results of using a combination of allogeneic mesenchymal cells (MSCs) with chondrocytes (CHCs) and a novel scaffold based on type I collagen and chitosan fibres. This biocomposite was transplanted into a defect produced by excision of a bone bridge to induce new cartilaginous tissue formation. The left femur was treated by transplantation into a defect of distal epiphysis; the right femur with implantation of the scaffold only served as control. A better therapeutic result was therefore expected in the left femur - the reduction of growth and angular deformities, and the histological finding of a tissue similar to the cartilage excised from the left femur.. MATERIAL AND METHODS: The miniature pig was selected as an experimental model and 10 pigs were used. Mesenchymal stem cells derived from femoral bone marrow and chondrocytes derived from a sample harvested from the non-weight-bearing articular surface of the distal end of the femur were cultured in medium. The novel scaffold was based on collagen containing chitosan nanofibres. To make manipulation during implantation easier, the cilindrical scaffolds after lyophilisation were again placed in 96-well plates for seeding. The scaffolds before implantation were seeded with 2x106 allogeneic MSCs and 1x106 allogeneic CHCs. The outcomes of treatment were assessed by measuring the length of bone and the degree of distal femoral valgus deformity, and by the histological findings obtained (properties and maturity of the newly-formed tissue, detection of type II collagen, PAS reaction). RESULTS: The right and left legs were examined for longitudinal bone growth and the valgus angle and compared. The treated left leg showed a higher average value for longitudinal growth than the untreated right leg (p = 0.004). The average degree of angular deformity was lower in the left leg than in the right leg (p = 0.008). The microscopic findings showed that a tissue similar to hyaline cartilage was more frequently present in the femoral bone defect of the left leg, as compared with that of the right leg. Type II collagen was detected more frequently and at higher amounts on the left than the right side (p = 0.033). The PAS reaction was positive in all left limbs, with a high degree of positivity in 80 % of them, while this was not achieved in any of the right limbs (p = 0.001). DISCUSSION: The use of stem cells in the indication reported here has only been the matter of time since the information on encouraging results in neurology and cardiology was published. First studies with positive results have soon been reported. The initial hydrogel scaffolds were based on tissue adhesives. However, they were not stable enough and were difficult to handle during surgery. In further studies, therefore, the use was made of a three-dimensional scaffold with a self-supporting structure of collagen fibres. This structure also facilitated its hydrodynamic seeding with MSCs and CHCs, which is an effective and sparing procedure for the transplanted cells. Studies concerned with MSCs and/or CHCs transplantation for re - pair of a physeal defect following bone bridge excision, i.e. for bone bridge treatment, in a broader experimental design, however, are still missing. CONCLUSION: Transplantation of a composite scaffold seeded with mesenchymal stem cells and chondrocytes into a physeal defect following bone bridge excision prevented growth disturbance and angular deformity development in the distal femoral epi - physis. In comparison with the control group, it resulted in a more frequent production of a tissue similar to hyaline cartilage, with a cell formation reminiscent of a typical columnar arrangement of the growth plate. Key words: mesenchymal stem cells, growth plate, bone bridge, scaffold.

Mechanism of disopyramide-induced hypoglycaemia in a patient with Type 2 diabetes.

BACKGROUND: Disopyramide, an antiarrhythmia drug, has been reported to cause hypoglycaemia. Pre-existing factors that increase the concentration of the drug in the blood increase the risk of hypoglycaemia. Furthermore, other factors can also increase the risk of hypoglycaemia even when disopyramide levels are in the therapeutic range. It has been proposed that disopyramide-induced hypoglycaemia is caused by inhibition of the pancreatic B-cell K(ATP) channels. CASE REPORT: We report a case of severe disopyramide-induced hypoglycaemia in a 62-year-old woman with Type 2 diabetes taking low-dose glimepiride treatment. She had not experienced hypoglycaemia prior to the start of disopyramide therapy. No further hypoglycaemic episodes occurred following withdrawal of disopyramide therapy. FUNCTIONAL STUDY: Current recordings of K(ATP) channels expressed in Xenopus oocytes showed that at their estimated therapeutic concentrations, disopyramide and glimepiride inhibited K(ATP) channels by about 50-60%. However, when both drugs were applied together, K(ATP) channels were almost completely closed (approximately 95%). Such dramatic inhibition of K(ATP) channels is sufficient to cause B-cell membrane depolarization and stimulate insulin secretion. CONCLUSIONS: Disopyramide therapy is not recommended for patients treated with K(ATP) channel inhibitors.

Vertebral formula and congenital abnormalities of the vertebral column in rabbits.

The aim of this retrospective study of 330 rabbits (164 males, 166 females) was to determine different vertebral formulas and prevalence of congenital vertebral anomalies in rabbits from radiographs of the cervical (C), thoracic (Th), lumbar (L) and sacral (S) segments of the vertebral column. The number of vertebrae in each segment of vertebral column, position of anticlinal vertebra and localisation and type of congenital abnormalities were recorded. In 280/330 rabbits (84.8%) with normal vertebral morphology, seven vertebral formulas were identified: C7/Th12/L7/S4 (252/330, 76.4%), C7/Th12/L6/S4 (11/330, 3.3%), C7/Th13/L7/S4 (8/330, 2.4%), C7/Th12/L7/S5 (4/330, 1.2%), C7/Th12/L8/S4 (3/330, 0.9%), C7/Th12/L7/S6 (1/330, 0.3%) and C7/Th11/L7/S4 (1/330, 0.3%). The anticlinal vertebra was identified as Th10 in 56.4% of rabbits and Th11 in 42.4% of rabbits. Congenital spinal abnormalities were identified in 50/330 (15.2%) rabbits, predominantly as a single pathology (n=44). Transitional vertebrae represented the most common abnormalities (n=41 rabbits) in the thoracolumbar (n=35) and lumbosacral segments (n=6). Five variants of thoracolumbar transitional vertebrae were identified. Cervical butterfly vertebrae were detected in three rabbits. One rabbit exhibited three congenital vertebral anomalies: cervical block vertebra, thoracic hemivertebra and thoracolumbar transitional vertebra. Five rabbits exhibited congenital vertebral abnormalities with concurrent malalignment, specifically cervical kyphosis/short vertebra (n=1), thoracic lordoscoliosis/thoracolumbar transitional vertebrae (n=1), thoracic kyphoscoliosis/wedge vertebrae (n=2) and thoracolumbar lordoscoliosis/thoracolumbar transitional vertebrae/lumbosacral transitional vertebrae (n=1). These findings suggest that vertebral columns in rabbits display a wide range of morphologies, with occasional congenital malformations.

Use of 3D geometry modeling of osteochondrosis-like iatrogenic lesions as a template for press-and-fit scaffold seeded with mesenchymal stem cells.

Computed tomography (CT) is an effective diagnostic modality for three-dimensional imaging of bone structures, including the geometry of their defects. The aim of the study was to create and optimize 3D geometrical and real plastic models of the distal femoral component of the knee with joint surface defects. Input data included CT images of stifle joints in twenty miniature pigs with iatrogenic osteochondrosis-like lesions in medial femoral condyle of the left knee. The animals were examined eight and sixteen weeks after surgery. Philips MX 8000 MX and View workstation were used for scanning parallel plane cross section slices and Cartesian discrete volume creation. On the average, 100 slices were performed in each stifle joint. Slice matrices size was 512 x 512 with slice thickness of 1 mm. Pixel (voxel) size in the slice plane was 0.5 mm (with average accuracy of +/-0.5 mm and typical volume size 512 x 512 x 100 voxels). Three-dimensional processing of CT data and 3D geometrical modelling, using interactive computer graphic system MediTools formerly developed here, consisted of tissue segmentation (raster based method combination and 5 % of manual correction), vectorization by the marching-cubes method, smoothing and decimation. Stifle- joint CT images of three individuals of different body size (small, medium and large) were selected to make the real plastic models of their distal femurs from plaster composite using rapid prototyping technology of Zcorporation. Accuracy of the modeling was +/- 0.5 mm. The real plastic models of distal femurs can be used as a template for developing custom made press and fit scaffold implants seeded with mesenchymal stem cells that might be subsequently implanted into iatrogenic joint surface defects for articular cartilage-repair enhancement.

Membrane ion channels and diabetes.

Type-2, or non-insulin-dependent diabetes mellitus is a serious disease that is now widespread throughout Western society. Glucose intolerance, or failure of glucose to stimulate insulin secretion, is a primary factor in the manifestation of this disease and is likely to be due to the failure of glucose metabolism to stimulate pancreatic beta-cell electrical activity, calcium influx, and insulin secretion. In this review we describe how ion channels regulate the electrical behaviour of the beta-cell and how the membrane potential depolarises in response to a rise in glucose metabolism. Central to these electrical events is the inhibition of ATP-sensitive potassium channel by ATP, and we summarise recent advances in our understanding of the properties of this ion channel in coupling beta-cell metabolism to electrical activity. We discuss the mechanism, specificity, and clinical implications of the pharmacological inhibition of KATP channels by sulphonyureas and other antidiabetic drugs. The roles of other ion channels in regulating electrical activity are considered, and also their potential use as targets for drug action in treating beta-cell disorders.

Effect of peroxisome proliferator-activated receptor alpha ligand fenofibrate on K(v) channels in the insulin-secreting cell line HIT-T15.

Ligands for peroxisome proliferator-activated receptors alpha (PPARalpha) are clinically used for the treatment of patients with hyperlipidemia. As we have previously shown, a synthetic ligand of PPARalpha, fenofibrate, has a stimulatory effect on insulin secretion in clonal hamster insulinoma beta-cell line HIT-T15 cells. We have also demonstrated that fenofibrate directly inhibits ATP-sensitive potassium (K(ATP)) channels, an effect independent of PPARalpha. In this study, fenofibrate was shown to be able to reduce voltage-dependent K(+) (K(v)) channel currents in voltage-independent manner. Therefore, fenofibrate may modulate insulin secretion not only via inhibition of K(ATP) channels but also via reduction of the K(v) channel current.

Electrophysiological and metabolic characterization of single beta-cells and islets from diabetic GK rats.

We have used the whole-cell recording technique to determine whether ATP-sensitive potassium (K[ATP]) currents, voltage-dependent Ca2+ currents, and exocytosis are different in single beta-cells from pancreatic islets of Goto-Kakizaki (GK) rats, a novel model of NIDDM, and normal rats. In addition, we have also measured the insulin secretory responses, ATP content, and the rate of glucose metabolism in intact islets. Although the glucose sensitivity of the K(ATP) current was similar between GK rats and controls, in the absence of glucose, K(ATP) current density was larger in GK rats, which resulted in a more hyperpolarized membrane potential. Whole-cell Ca2+ currents were similar. By monitoring the cell capacitance with a fixed intracellular solution, no difference was detected in the exocytotic responses of beta-cells from normal and GK rats. In islets from GK rats, the rates of glucose utilization ([3H]H2O production from 5-[3H]glucose) and oxidation ([14C]CO2 production from U-[14C]glucose) were not significantly different from controls. Insulin secretion, however, was impaired (by 50%), and this was paralleled by a smaller increase in ATP content in response to stimulation by 10 mmol/l glucose in islets from GK rats when compared with controls. Under conditions in which K(ATP) channels were held open and the effects of glucose were independent of membrane potential, insulin release was still significantly lower in GK rat islets than in controls. These findings suggest that the impaired insulin secretion in islets from GK rats does not simply result from a failure to close K(ATP) channels, nor does it result from an impairment in calcium secretion coupling.

Molecular analysis of ATP-sensitive K channel gating and implications for channel inhibition by ATP.

The beta cell KATP channel is an octameric complex of four pore-forming subunits (Kir6.2) and four regulatory subunits (SUR1). A truncated isoform of Kir6.2 (Kir6.2DeltaC26), which expresses independently of SUR1, shows intrinsic ATP sensitivity, suggesting that this subunit is primarily responsible for mediating ATP inhibition. We show here that mutation of C166, which lies at the cytosolic end of the second transmembrane domain, to serine (C166S) increases the open probability of Kir6.2DeltaC26 approximately sevenfold by reducing the time the channel spends in a long closed state. Rundown of channel activity is also decreased. Kir6.2DeltaC26 containing the C166S mutation shows a markedly reduced ATP sensitivity: the Ki is reduced from 175 microM to 2.8 mM. Substitution of threonine, alanine, methionine, or phenylalanine at position C166 also reduced the channel sensitivity to ATP and simultaneously increased the open probability. Thus, ATP does not act as an open channel blocker. The inhibitory effects of tolbutamide are reduced in channels composed of SUR1 and Kir6.2 carrying the C166S mutation. Our results are consistent with the idea that C166 plays a role in the intrinsic gating of the channel, possibly by influencing a gate located at the intracellular end of the pore. Kinetic analysis suggests that the apparent decrease in ATP sensitivity, and the changes in other properties, observed when C166 is mutated is largely a consequence of the impaired transition from the open to the long closed state.

Mutations within the P-loop of Kir6.2 modulate the intraburst kinetics of the ATP-sensitive potassium channel.

The ATP-sensitive potassium (K(ATP)) channel exhibits spontaneous bursts of rapid openings, which are separated by long closed intervals. Previous studies have shown that mutations at the internal mouth of the pore-forming (Kir6.2) subunit of this channel affect the burst duration and the long interburst closings, but do not alter the fast intraburst kinetics. In this study, we have investigated the nature of the intraburst kinetics by using recombinant Kir6.2/SUR1 K(ATP) channels heterologously expressed in Xenopus oocytes. Single-channel currents were studied in inside-out membrane patches. Mutations within the pore loop of Kir6.2 (V127T, G135F, and M137C) dramatically affected the mean open time (tau(o)) and the short closed time (tauC1) within a burst, and the number of openings per burst, but did not alter the burst duration, the interburst closed time, or the channel open probability. Thus, the V127T and M137C mutations produced longer tau(o), shorter tauC1, and fewer openings per burst, whereas the G135F mutation had the opposite effect. All three mutations also reduced the single-channel conductance: from 70 pS for the wild-type channel to 62 pS (G135F), 50 pS (M137C), and 38 pS (V127T). These results are consistent with the idea that the K(ATP) channel possesses a gate that governs the intraburst kinetics, which lies close to the selectivity filter. This gate appears to be able to operate independently of that which regulates the long interburst closings.

Expression of functionally active ATP-sensitive K-channels in insect cells using baculovirus.

We have expressed active ATP-sensitive K-channels (K(ATP) channels) in Spodoptera frugiperda (Sf9) cells using a baculovirus vector. A high yield of active channels was obtained on co-infection with SUR1 and Kir6.2 engineered to contain N- and/or C-terminal tags to permit detection by Western blotting. Channel activity was sensitive to ATP, glibenclamide and diazoxide. Channel activity was also obtained on expression of a C-terminally truncated Kir6.2 (Kir6.2 deltaC26): these channels were blocked by ATP but were insensitive to sulphonylureas. In contrast to Xenopus oocytes and mammalian cells the full length Kir6.2 also gave rise to active channels in Sf9 cells when expressed alone. The highest yield of active K(ATP) channels was obtained on infection with a fusion protein containing SUR1 linked to Kir6.2 deltaC26 via a 6-amino acid linker.

Cloning and functional expression of the cDNA encoding an inwardly-rectifying potassium channel expressed in pancreatic beta-cells and in the brain.

A cDNA clone encoding an inwardly-rectifying K-channel (BIR1) was isolated from insulinoma cells. The predicted amino acid sequence shares 72% identity with the cardiac ATP-sensitive K-channel rcKATP (KATP-1;[6]). The mRNA is expressed in the brain and insulinoma cells. Heterologous expression in Xenopus oocytes produced currents which were K(+)-selective, time-independent and showed inward rectification. The currents were blocked by external barium and caesium, but insensitive to tolbutamide and diazoxide. In inside-out patches, channel activity was not blocked by 1 mM internal ATP. The sequence homology with KATP-1 suggests that BIR1 is a subunit of a brain and beta-cell KATP channel. However, pharmacological differences and the lack of ATP-sensitivity, suggest that if, this is the case, heterologous subunits must exert strong modulatory influences on the native channel.

Inhibition of the ATP-sensitive potassium channel from mouse pancreatic beta-cells by surfactants.

1. We have used patch-clamp methods to study the effects of the detergents, Cremophor, Tween 80 and Triton X100 on the K(ATP) channel in the pancreatic beta-cell from mouse. 2. All three detergents blocked K(ATP) channel activity with the following order of potency: Tween 80 (Ki< approximately 83 nM)>Triton X100 (Ki=350 nM)>Cremophor. In all cases the block was poorly reversible. 3. Single-channel studies suggested that at low doses, the detergents act as slow blockers of the K(ATP) channel. 4. Unlike the block produced by tolbutamide, that produced by detergent was not affected by intracellular Mg2+-nucleotide, diazoxide or trypsin treatment, nor did it involve an acceleration of rundown or increase in ATP sensitivity of the chanel. 5. The detergents could block the pore-forming subunit, Kir6.2deltaC26, which can be expressed independently of SUR1 (the regulatory subunit of the K(ATP) channel). These data suggest that the detergents act on Kir6.2 and not SUR1. 6. The detergents had no effect on another member of the inward rectifier family: Kir1.1a (ROMK1). 7. Voltage-dependent K-currents in the beta-cell were reversibly blocked by the detergents with a far lower potency than that found for the K(ATP) channel. 8. Like other insulin secretagogues that act by blocking the K(ATP) channel, Cremophor elevated intracellular Ca2+ in single beta-cells to levels that would be expected to elicit insulin secretion. 9. Given the role of the K(ATP) channel in many physiological processes, we conclude that plasma borne detergent may have pharmacological actions mediated through blockage of the K(ATP) channel.

Effects of mitiglinide (S 21403) on Kir6.2/SUR1, Kir6.2/SUR2A and Kir6.2/SUR2B types of ATP-sensitive potassium channel.

1. We have investigated the mechanism of action of the novel anti-diabetic agent mitiglinide (S 21403) on Kir6.2/SUR1, Kir6.2/SUR2A and Kir6.2/SUR2B types of ATP-sensitive potassium (K(ATP)) channel. These possess a common pore-forming subunit, Kir6.2, and different regulatory sulphonylurea receptor (SUR) subunits. It is believed that they correspond to native K(ATP) channels in pancreatic beta-cells, heart and non-vascular smooth muscle, respectively. 2. Kir6.2 was coexpressed with SUR1, SUR2A or SUR2B in Xenopus oocytes and macroscopic currents were recorded in giant inside-out membrane patches. Mitiglinide was added to the intracellular membrane surface. 3. Mitiglinide inhibited Kir6.2/SUR currents at two sites: a low-affinity site on Kir6.2 and a high-affinity site on SUR. Low-affinity inhibition was similar for all three types of K(ATP) channel but high-affinity inhibition was greater for Kir6.2/SUR1 currents (IC(50), 4 nM) than for Kir6.2/SUR2A or Kir6.2/SUR2B currents (IC(50), 3 and 5 microM, respectively). 4. Inhibition of Kir6.2/SUR1 currents was only slowly reversible on the time scale of electrophysiological experiments. 5. Kir6.2/SUR1-S1237Y currents, which previously have been shown to lack high affinity tolbutamide inhibition, resembled Kir6.2/SUR2 currents in being unaffected by 100 nM but blocked by 10 microM mitiglinide. 6. Our results show that mitiglinide is a high-affinity drug that shows a 1000 fold greater affinity for the beta-cell type than the cardiac and smooth muscle types of K(ATP) channel, when measured in excised patches.

Interaction of stilbene disulphonates with cloned K(ATP) channels.

In this study, we tested the effects of the stilbene disulphonates DIDS and SITS on three different types of cloned K(ATP) channel (Kir6.2/SUR1, Kir6.2/SUR2A and Kir6.2DeltaC) heterologously expressed in Xenopus oocytes, with the aim of identifying the part of the channel which is involved in mediating disulphonate inhibition. We found that the inhibitory site(s) for these drugs lies within the Kir6.2 subunit of the channel, although its properties are further modulated by the sulphonylurea (SUR) subunit. In particular, SUR2A reduces both the rate and extent of block, by impairing the ability of DIDS binding to produce channel closure. The disulphonate-binding site interacts with the ATP inhibitory site on Kir6.2 because ATP is able to protect against irreversible channel inhibition by disulphonates. This effect is not mimicked by tolbutamide (at a concentration that interacts with Kir6.2) and is abolished by mutations that render the channel ATP insensitive. A number of point mutations in both the N and C termini of Kir6.2 reduced the extent and reversibility of channel inhibition by SITS. The results are consistent with the idea that residue C42 of Kir6.2 is likely to be involved in covalently linking of SITS to the channel. Other types of Kir channel (Kir1.1, Kir2.1 and Kir4.1) were also irreversibly blocked by DIDS, suggesting that these channels may share common binding sites for these stilbene disulphonates.

Comparison of 9-aminoacridine and atebrine induced changes in optical, electrical and mechanical characteristics of lipid bilayers.

The effects of fluorescent probes 9-aminoacridine (9AA) and atebrine (AT) on physical properties of liposomes and planar bilayer lipid membranes (BLM) were studied. The method of fluorescence spectroscopy and the electrostriction method based on measurement of higher current harmonics were used. At low concentrations (10(-5)-5 x 10(-5) mol/l), 9AA increased fluorescence intensity, while in liposomes from soybean phosphatidylcholine fluorescence quenching occurred at higher probe concentration. Fluorescence quenching occurred over the entire concentration range tested (10(-5)-10(-4) mol/l) in liposomes made from a mixture of egg phosphatidylcholine and cardiolipin. In contrast to 9AA, AT, thanks to its hydrophobic chain, penetrates deeper into the hydrophobic membrane moiety; thus, immobilization of the molecule and an increase in fluorescence intensity was always observed. Probes adsorbed to membranes, leaving their electric capacitance effectively unchanged. Adsorption of charged dye particles induced small changes in transmembrane potential. In the presence of 10(-5) mol/l AT, the modulus of elasticity E perpendicular increased somewhat for soft membranes (E perpendicular approximately 2.5 x 10(7) Pa), whereas it decreased for hard membranes (E perpendicular approximately 5 x 10(7) Pa). pH gradient present on the membrane affected the ability of the dyes to incorporate into the membranes. Our results provide evidence against the proposed model of the quenching mechanism introduced by Rottenberg and Lee (1975).

Low-conductance chloride channel from crayfish skeletal muscle incorporated into planar lipid bilayers.

Low-conductance chloride channel from skeletal muscle SR vesicles of the crayfish Astacus fluviatilis was incorporated into planar lipid bilayers and its basic characteristics were investigated. The channel has a relatively low unitary conductance of 26 pS in symmetrical 160 mmol/l choline-chloride. The dependence of the channel conductance on Cl- concentration shows saturating behavior with a maximum conductance of 37 pS and an ionic activity for half-maximum conductance Km = 75 mmol/l. The channel exhibits a complex kinetics with several modes of activity. Open state probability slightly decreases with the increasing absolute value of voltage. The channel activity does not appear to be dependent on the presence of Ca2+ ions. The channel is effectively inhibited by DIDS, a stilbene derivative. The permeability properties of the channel are similar to the specific behavior of the "double-barrelled" channel from Torpedo electroplax described by Miller and White (1984).