Subject(s)
Angiodysplasia/therapy , Esophagoscopy/methods , Gastrointestinal Hemorrhage/therapy , Angiodysplasia/complications , Angiodysplasia/diagnosis , Esophageal and Gastric Varices/pathology , Esophageal and Gastric Varices/therapy , Follow-Up Studies , Gastrointestinal Hemorrhage/diagnosis , Gastrointestinal Hemorrhage/etiology , Humans , Ligation/methods , Male , Middle Aged , Treatment OutcomeSubject(s)
Gastrointestinal Hemorrhage/therapy , Hemostasis, Endoscopic/methods , Rectal Diseases/therapy , Aged , Female , Humans , Ligation/methods , Male , Middle AgedABSTRACT
Hyperkalemic periodic paralysis (HYPP), one of several inheritable myotonic diseases, results from genetic defects in the human skeletal muscle Na+ channel. In some pedigrees, HYPP is correlated with a single base pair substitution resulting in a Met replacing Thr704 in the fifth transmembrane segment of the second domain. This region is totally conserved between the human and rat channels. We have introduced the human mutation into the corresponding region of the rat muscle Na+ channel cDNA and expressed it in human embryonic kidney 293 cells. Patch-clamp recordings show that this mutation shifts the voltage dependence of activation by 10-15 mV in the negative direction. The shift results in a persistent Na+ current that activates near -70 mV; this phenomenon could underlie the abnormal muscle activity observed in patients with HYPP.
Subject(s)
Hyperkalemia/genetics , Mutation , Paralysis/genetics , Periodicity , Sodium Channels/genetics , Amino Acid Sequence , Base Sequence , Cell Line, Transformed , Electrophysiology , Extracellular Space/metabolism , Homeostasis , Humans , Hydrogen-Ion Concentration , Kinetics , Molecular Sequence Data , Oligonucleotide Probes/genetics , Potassium/metabolism , Sodium Channels/physiologyABSTRACT
We describe the transient expression of the rat skeletal muscle muI Na+ channel in human embryonic kidney (HEK 293) cells. Functional channels appear at a density of approximately 30 in a 10 microns 2 patch, comparable to those of native excitable cells. Unlike muI currents in oocytes, inactivation gating is predominantly (approximately 97%) fast, although clear evidence is provided for noninactivating gating modes, which have been linked to anomalous behavior in the inherited disorder hyperkalemic periodic paralysis. Sequence-specific antibodies detect a approximately 230 kd glycopeptide. The majority of molecules acquire only neutral oligosaccharides and are retained within the cell. Electrophoretic mobility on SDS gels suggests the molecules may acquire covalently attached lipid. The channel is readily phosphorylated by activation of the protein kinase A and protein kinase C second messenger pathways.
Subject(s)
Sodium Channels/physiology , Animals , Cell Membrane/physiology , Cells, Cultured , Cloning, Molecular , Glycoproteins/physiology , Glycosylation , Hexosaminidases/pharmacology , Humans , In Vitro Techniques , Ion Channel Gating , Molecular Weight , Muscles/physiology , Neuraminidase/pharmacology , Phosphoproteins/physiology , Phosphorylation , Protein Kinases/metabolism , Rats , Transfection , Tunicamycin/pharmacologyABSTRACT
In summary, the voltage-sensitive sodium channel from eel electroplax provides an optimal preparation for biochemical and biophysical studies of molecular structure and gating. We have demonstrated that the purified and reconstituted protein is capable of functioning normally, exhibiting, among other properties, voltage-dependent activation and inactivation gating mechanisms. We have been able to recreate the classical electrophysiological studies in which inactivation gating can be removed by proteolytic modification of the cytoplasmic surface of the molecule, and have mapped the probable site of modification to the peptide segment lying between subunit domains III and IV. We have demonstrated that the reconstituted protein undergoes interactions with the lidocaine derivative QX-314 which, at low concentrations, results in paradoxical activation of the channel and a facilitation of modification by oxidizing reagents that remove inactivation gating.
Subject(s)
Anesthetics, Local/pharmacology , Electric Organ/physiology , Ion Channel Gating/drug effects , Lidocaine/analogs & derivatives , Neurotoxins/pharmacology , Sodium Channels/physiology , Amino Acid Sequence , Animals , Bungarotoxins/pharmacology , Electrophorus , Lidocaine/pharmacology , Membrane Potentials/drug effects , Molecular Sequence Data , Peptide Mapping , Sodium Channels/drug effectsABSTRACT
The activity of protein kinase C (Ca2+/phospholipid-dependent enzyme) in the presence of phosphatidylserine and its physiological regulator, diacylglycerol, could be suppressed by a mixture of brain gangliosides. Half-maximal inhibition was observed at 30 microM and was nearly complete at 100 microM. Inhibition was observed at all concentrations of Ca2+ between 10(-8) and 10(-4) M. Inhibition of protein kinase C activity could not be reversed by increasing the concentration of diacylglycerol or the substrate, histone. Inhibition was also observed when myelin basic protein or a synthetic myelin basic protein peptide was used as substrate. Among the individual gangliosides, the rank order of potency was GT1b greater than GD1a = GD1b greater than GM3 = GM1. Our results suggest that gangliosides may regulate the responsiveness of protein kinase C to diacylglycerol.
