We have cloned and sequenced cDNAs of the strychnine-binding subunit of the rat glycine receptor, a neurotransmitter-gated chloride channel protein of the CNS. The deduced polypeptide shows significant structural and amino-acid sequence homology with nicotinic acetylcholine receptor proteins, indicating that there is a family of genes encoding neurotransmitter-gated ion channels.

Amino Acid Sequence↗

PubMedDate not supplied

Glycine vs GABA receptors.

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Amino Acid Sequence↗

PubMedDate not supplied

Gephyrin antisense oligonucleotides prevent glycine receptor clustering in spinal neurons.

Each neuron in the mammalian brain carries many postsynaptic membrane specializations containing high densities of receptors that mediate signal transduction upon neurotransmitter release from the apposed nerve terminal. Little is known about the mechanisms by which receptors are transported to and anchored at postsynaptic sites, but extracellular as well as intracellular components may be involved. Ultrastructural studies have shown that the peripheral membrane protein gephyrin, which co-purifies with the postsynaptic inhibitory glycine receptor (GlyR) upon affinity chromatography, is situated on the cytoplasmic face of glycinergic postsynaptic membranes. Moreover, gephyrin binds with high affinity to polymerized tubulin and has been postulated to link the GlyR to the subsynaptic cytoskeleton. Here we report that treatment of rat spinal neurons in culture with gephyrin antisense oligonucleotides prevents the formation of GlyR clusters in the dendritic plasma membrane. Thus, gephyrin is essential for localizing the GlyR to presumptive postsynaptic plasma membrane specializations.

Animals↗

PubMedDate not supplied

Affinity labeling of cysteine-mutants evidences contact residues in modeled receptor binding sites.

To investigate the topology of binding sites in two ionotropic receptors, we have initiated a strategy combining affinity labeling with cysteine-scanning mutagenesis. For the GABAA receptor we have used reactive derivatives of non-competitive blockers (NCBs) to explore interacting positions in its channel. The polypeptide positions of the M2 segment of the alpha1 subunit which we mutated into cysteine were selected for their established accessibility, as determined by the substituted-cysteine accessibility method (SCAM). Using the Xenopus oocyte expression system, we show that receptors containing mutations V257C and S272C are inactivated by several reactive NCBs. These position-selective inactivations lead to an analysis of NCB binding in the channel. For the NMDA receptor glycine-binding site, the prototype antagonist L-701,324 was derivatized at different positions with different reactive groups. The receptor positions to mutate into cysteine were selected after a 3-D homology model. The observed receptor inactivations are mutant- and probe-selective, leading to an unambiguous chemical docking of the antagonist pharmacophore and supporting the model. The site-specificity of the inactivating reactions is assessed by protection experiments and by mutant to wild-type (WT) comparisons. The scope and limitations of the method are briefly discussed.

Affinity Labels↗

PubMedDate not supplied

Directional C-W Doppler sonography in the diagnosis of basilar artery disease.

Using directional continuous-wave Doppler sonography of the vertebral arteries, we have investigated 1,540 patients during the past 5 years. On the basis of unilateral and bilateral retrograde brachial angiograms in the same patients (a total of 1,989 angiograms) we have developed sonographic criteria for demonstrating a significant increase in the peripheral resistance of both distal vertebral arteries and/or the basilar artery. All 11 cases of basilar artery stenosis of at least 60% reduction in lumen diameter (as shown by angiography) exhibited an approximately 40% or higher reduction in the sum of the modified Pourcelot indices of both vertebral arteries with respect to age-matched controls. All 3 stenoses of less than 60% reduction in lumen diameter were not detected by sonography. Even a good collateral circulation through rete mirabile anastomoses did not normalize the modified Pourcelot indices. One case of a persistent primitive trigeminal artery with thin-calibered vertebral arteries was also detected by sonography. The high percent of patients with one hypoplastic vertebral artery in the group with basilar artery stenoses indicates an increased risk for atherosclerosis of the basilar and/or distal vertebral artery in these patients. All 14 angiographically verified occlusions of the basilar artery were detected by sonographic criteria independent of the occlusion localization. Thus, we believe that directional continuous-wave Doppler sonography is a reliable technique for detecting basilar artery stenoses of at least 60% reduction in lumen diameter and basilar artery occlusions.

Arterial Occlusive Diseases↗