A hydrophobic area of the GABA ρ1 receptor containing phenylalanine 124 influences both receptor activation and deactivation.

Experimental evidence suggests that GABA ρ1 receptors are potential therapeutic targets for the treatment of a range of neurological conditions, including anxiety and sleep disorders. Homology modelling of the GABA ρ1 extracellular N-terminal domain has revealed a novel hydrophobic area that extends beyond, but not including the GABA-binding site. Phenylalanine 124 (F124) is predicted to be involved in maintaining the structural integrity of the orthosteric-binding site. We have assessed the activity of a series of GABA ρ1 receptors that incorporate a mutation at F124. Wild-type and mutant human GABA ρ1 subunits were expressed in Xenopus laevis oocytes and AD293 cells, and the pharmacology and kinetic properties of the receptors were measured using electrophysiological analysis. Mutation of F124 had minimal effect on receptor pharmacology. However, the rate of deactivation was significantly increased compared to wild type. This study provides further information about the role of residues within a novel hydrophobic area of the GABA ρ1 receptor. This knowledge can help future studies into the design of potent and subtype-selective ligands with therapeutic value.

Training in surgical oncology - the role of VR simulation.

There have been dramatic changes in surgical training over the past two decades which have resulted in a number of concerns for the development of future surgeons. Changes in the structure of cancer services, working hour restrictions and a commitment to patient safety has led to a reduction in training opportunities that are available to the surgeon in training. Simulation and in particular virtual reality (VR) simulation has been heralded as an effective adjunct to surgical training. Advances in VR simulation has allowed trainees to practice realistic full length procedures in a safe and controlled environment, where mistakes are permitted and can be used as learning points. There is considerable evidence to demonstrate that the VR simulation can be used to enhance technical skills and improve operating room performance. Future work should focus on the cost effectiveness and predictive validity of VR simulation, which in turn would increase the uptake of simulation and enhance surgical training.

Compound heterozygosity and nonsense mutations in the alpha(1)-subunit of the inhibitory glycine receptor in hyperekplexia.

The alpha(1)-inhibitory glycine receptor is a ligand-gated chloride channel composed of three ligand-binding alpha1-subunits and two structural beta-subunits that are clustered on the postsynaptic membrane of inhibitory glycinergic neurons. Dominant and recessive mutations in GLRA1 subunits have been associated with a proportion of individuals and families with startle disease or hyperekplexia (MIM: 149400). Following SSCP and bi-directional di-deoxy fingerprinting mutational analysis of 22 unrelated individuals with hyperekplexia and hyperekplexia-related conditions, we report further novel missense mutations and the first nonsense point mutations in GLRA1, the majority of which localise outside the regions previously associated with dominant, disease-segregating mutations. Population studies reveal the unique association of each mutation with disease, and reveals that a proportion of sporadic hyperekplexia is accounted for by the homozygous inheritance of recessive GLRA1 mutations or as part of a compound heterozygote.

Identification of a new ligand binding domain in the alpha1 subunit of the inhibitory glycine receptor.

Four discontinuous extracellular sequence domains have been proposed to form the ligand binding sites of the ligand-gated ion channel receptor superfamily. In this study, we investigated the role of 12 contiguous residues of the inhibitory glycine receptor that define the proposed "loop A" ligand binding domain. Using the techniques of site-directed mutagenesis and patch-clamp electrophysiology, four of the 12 residues were shown to have impaired ligand binding. Three mutants, 193A, A101H, and N102A, resulted in significant (17-44-fold) increases in the agonist EC50 values as compared with the wild-type glycine receptor, whereas Hill coefficients, ImaX values, and antagonist affinity remained largely unaffected. Consideration of receptor efficacy values indicates that these residues are involved in ligand binding rather than channel activation. A fourth mutant, W94A, failed to give rise to any glycine-activated currents, although cell-surface expression was observed, suggesting that this residue may also be involved in agonist binding. These data provide the most extensive characterization of the loop A ligand binding domain available to date and define two new residue locations, Ile93 and Asn102, as contributing to the four-loop model of ligand binding.

Properties of human glycine receptors containing the hyperekplexia mutation alpha1(K276E), expressed in Xenopus oocytes.

1. Inherited defects in human glycine receptors give rise to hyperekplexia (startle disease). We expressed human glycine receptors in Xenopus oocytes, in order to examine the pharmacological and single-channel properties of receptors that contain a mutation, alpha1(K276E), associated with an atypical form of hyperekplexia. 2. Equilibrium concentration-response curves showed that recombinant human alpha1(K276E)beta receptors had a 29-fold lower glycine sensitivity than wild-type alpha1beta receptors, and a greatly reduced Hill coefficient. The maximum response to glycine also appeared much reduced, whereas the equilibrium constant for the glycine receptor antagonist strychnine was unchanged. 3. Both wild-type and mutant channels opened to multiple conductance levels with similar main conductance levels (33 pS) and weighted mean conductances (41.5 versus 49.8 pS, respectively). 4. Channel openings were shorter for the alpha1(K276E)beta mutant than for the wild-type alpha1beta, with mean overall apparent open times of 0.82 and 6.85 ms, respectively. 5. The main effect of the alpha1(K276E) mutation is to impair the opening of the channel rather than the binding of glycine. This is shown by the results of fitting glycine dose-response curves with particular postulated mechanisms, the shorter open times of mutant channels, the properties of single-channel bursts, and the lack of an effect of the mutation on the strychnine-binding site.

Recombinant nicotinic receptors, expressed in Xenopus oocytes, do not resemble native rat sympathetic ganglion receptors in single-channel behaviour.

1. In order to establish the subunit composition of neuronal nicotinic receptors in rat superior cervical ganglia (SCG), their single-channel properties were compared with those of recombinant receptors expressed in Xenopus oocytes, using outside-out excised patch recording. 2. The mean main conductance of SCG channels from adult and 1-day-old rats was 34.8 and 36.6 pS, respectively. Less frequent openings to lower conductances occurred both as isolated bursts and as events connected to the main level by direct transitions. There was considerable interpatch variability in the values of the lower conductances. 3. Nicotinic receptors from oocytes expressing alpha3beta4 and alpha4beta4 subunits had chord conductances lower than that of SCG neurones (22 pS for alpha3beta4 and 29 pS for alpha4beta4). 4. Prolonged recording from both native and recombinant channels was precluded by 'run-down', i.e. channel activity could be elicited for only a few minutes after excision. Nevertheless, SCG channel openings were clearly seen to occur as short bursts (slowest component, 38 ms), whereas recombinant channels opened in very prolonged bursts of activity, the major component being the slowest (480 ms). 5. Addition of the alpha5 subunit to the alpha3beta4 pair produced channels with a higher conductance than those observed after injection of the pair alone (24.9 vs. 22 pS), suggesting incorporation of alpha5 into the channel. Addition of the beta2 subunit did not change alpha3beta4 single-channel properties. In one out of fourteen alpha3alpha5beta4 patches, both ganglion-like, high conductance, short burst openings and recombinant-type, low conductance, slow burst openings were observed. 6. Channels produced by expression in Xenopus oocytes of neuronal nicotinic subunits present in rat SCG as a rule differ from native ganglion receptors in single-channel conductance and gross kinetics. While it is possible that an essential nicotinic subunit remains to be cloned, it is perhaps more likely that oocytes either cannot assemble neuronal nicotinic subunits efficiently into channels with the correct composition and stoichiometry, or that they produce post-translational channel modifications which differ from those of mammalian neurones.

The ion channel properties of a rat recombinant neuronal nicotinic receptor are dependent on the host cell type.

1. A stable mammalian cell line (L-alpha 3 beta 4) has been established which expresses the cloned rat neuronal nicotinic acetylcholine receptor (nAChR) subunits alpha 3 and beta 4, which are the most abundant in autonomic ganglia. Ion channel properties of nAChRs expressed in L-alpha 3 beta 4 cells were investigated by single-channel and whole-cell recording techniques, and compared with both rat alpha 3 beta 4 nAChRs expressed in Xenopus oocytes, and endogenous nicotinic receptors in rat superior cervical ganglion (SCG) neurones, using identical solutions for all cell types. 2. Acetylcholine (ACh) caused activation of single ion channel currents with a range of amplitudes. Some channels had high conductances (30-40 pS), and relatively brief lifetimes; these resembled the predominant native channel from SCG. Other channels had low conductances (20-26 pS) and long bursts of openings which were quite unlike native channels, but which were similar to channels formed by alpha 3 beta 4 in oocytes. Both types often occurred in the same patch. 3. Cytisine was about 3 times more potent than ACh (low-concentration potency ratio) in L-alpha 3 beta 4 cells, which is not dissimilar to the 5-fold potency ratio found in both SCG and oocytes, whereas 1,1-dimethyl-4-phenylpiperazinium (DMPP) was less potent than ACh in some cells (as in the oocyte), but more potent in others (as in SCG). 4. While the channels expressed in L-alpha 3 beta 4 cells do not mimic exactly those expressed in rat SCG, they differ considerably from the same subunit combination expressed in oocytes. Larger conductance, SCG-like channels were detected frequently in L-alpha 3 beta 4, but were rarely, if ever, seen in oocytes injected with alpha 3 and beta 4 mRNA. Our results indicate that ion channel properties such as single-channel conductance can be influenced by the choice of heterologous expression system.

Immunolabelling for VDAC, the mitochondrial voltage-dependent anion channel, on sarcoplasmic reticulum from amphibian skeletal muscle.

Patch-clamp studies of ion channels in the sarcoball membrane, a relatively pure preparation of sarcoplasmic reticulum, had earlier revealed a high-conductance anion channel with some properties similar to the mitochondrial voltage-dependent anion channel (VDAC). Using post-embedding immunolabelling, the presence of VDAC was investigated in sarcoball preparations from the semitendinosus muscle of the cane toad Bufo marinus. As expected, the outer membrane of mitochondria found within the interior of skinned fibres was decorated with gold label. Surprisingly, sarcoplasmic reticulum membrane was also labelled. The sarcoball membranes, which could arise from either the sarcoplasmic reticulum or from mitochondria, were also labelled. These results indicate the presence of a VDAC-like protein in the sarcoplasmic reticulum.

Ultrastructure of sarcoballs on the surface of skinned amphibian skeletal muscle fibres.

The formation of sarcoballs on the surface of skinned fibres from semitendinosus muscles of Xenopus laevis, and the sarcoplasmic reticulum content of the structures, have been studied using conventional electron microscopic techniques and immunoelectron microscopy. Examination of the fibres showed many membrane-bound blebs projecting from the surface in areas where vesicles of internal membranes (including sarcoplasmic reticulum, T-tubules and mitochondria) were clustered in interfilament spaces. The blebs varied in size from 1 micron to 150 microns and those with diameters > 10 microns are referred to as sarcoballs. Small blebs were often seen in close association with each other and might have fused during sarcoball formation. The interior of the sarcoball was filled with foam-like material made up of vesicles with diameters of 100 nm to 1.0 microns. The sarcoplasmic reticulum membrane content of the sarcoballs was evaluated using two monoclonal antibodies, one to the Ca2+ ATPase of the sarcoplasmic reticulum and the second to ryanodine receptor calcium release channels in the junctional-face membrane. The antibodies bound to some components of the surface and interior of the sarcoball, but not to mitochondrial-like structures and tubular vesicles. The results show that a large component of the sarcoball and its surface is derived from sarcoplasmic reticulum and suggest that mitochondria and T-tubules might also contribute membranes to the structures. Our hypothesis is that (a) blebs bud out from the surface of the skinned fibre following fusion of internal vesicles that are extruded along interfilament channels during unrestrained contractures, (b) blebs grow into sarcoballs by additional fusion of internal membrane vesicles and fusion of adjacent blebs, and (c) the sarcoball is a foam-like structure composed of bathing medium and membrane lipid (containing membrane proteins).

Adenocarcinoma of rete testis.

Adenocarcinoma of the rete testis is a rare tumor of the genital tract. An advanced case of adenocarcinoma thought to arise from the rete testis is presented. We believe this is the eighteenth reported case.