ABSTRACT
Using the method of organotypical cell culture, experimental data was obtained supporting the hypothesis that chelate complex ouabain-Ca2+ modulates the transducer function of Na+, K+- ATPase. Quantum-chemical calculations helped to elucidate two principally distinct modes for chelation of Ca2+ ions by ouabain molecule. It is predicted that ligang-receptor complex of ouabain-Ca2+--Na+,K+-ATPase is formed due to ion-ionic bonds. The forming of this complex switches on the transducer function of the sodium pump. Our data also show that removal of free Ca2+ ions from the cell culture media using EGTA has no effect on binding of ouabain with pumping control site of Na+K+-ATPase.
Subject(s)
Calcium/metabolism , Cardiotonic Agents/pharmacology , Ouabain/pharmacology , Sodium-Potassium-Exchanging ATPase/metabolism , Animals , Chelating Agents/pharmacology , Chick Embryo , Organ Culture Techniques , Organ Specificity/drug effectsABSTRACT
A local membrane potential clamping method was used to study the effects of defensin NP-1 on the membranes of rat spinal ganglion neurons. NP-1 led to decreases in the effective charge for the activation gating system. This process depended on the NP-1 concentration. Use of the Hill equation showed that Kd was 2.10(-12) M and the Hill coefficient was 0.9. The structure of the defensin molecule was optimized using quantum chemical calculations based on a molecular mechanics method. The results obtained from these calculations suggested that a single hydroxyl group directed towards the outer part of thedefensin molecule and forming the carboxyl group of amino acid Glu14 could form a hydrogen bond with the active center of the membrane receptor. This explains the experimentally observed 1:1 stoichiometry of the ligand-receptor binding interaction between the defensin and the sensory neuron membrane.
Subject(s)
Defensins/physiology , Neurons, Afferent/physiology , Algorithms , Animals , Animals, Newborn , Cell Membrane/physiology , Defensins/chemistry , Defensins/pharmacology , Electrophysiology , Ganglia, Spinal/cytology , Ganglia, Spinal/physiology , Hydrogen-Ion Concentration , Ion Channel Gating/drug effects , Kinetics , Membrane Potentials/physiology , Models, Molecular , Molecular Conformation , Patch-Clamp Techniques , Quantum Theory , Rabbits , Rats , Sodium Channels/drug effects , Sodium Channels/physiology , Tetrodotoxin/pharmacologyABSTRACT
NP-1 defensin decreased effective charge transfer in the activation gating system of TTX-resistant (slow) sodium channels in a dose-dependent manner. The dissociation constant and Hill coefficient values were KD = 2 pM and X--0.9. Geometry of the NP-1 defensin molecule was built using its primary structure with three S-S briges and fully optimised in the framework of molecular mechanics method. The data obtained explain experimental results of stechiometry 1:1 due to ligand-receptor interaction by the only outward directed carboxyl group of Glu 14 which might form a hydrogen bond with a single binding site of non-identified defensin membrane receptor.
