ABSTRACT
Tok1p is a highly specific yeast plasma membrane potassium channel with strong outward directionality. Its opening is induced by membrane depolarization. Although the biophysical properties of Tok1p are well-described, its potentially important physiological role is currently largely unexplored. To address this issue, we examined the Tok1p activity following chemically-induced depolarization by measuring changes of plasma membrane potential (ΔΨ) using the diS-C3(3) fluorescence assay in a Tok1p-expressing and a Tok1p-deficient strain. We report that Tok1p channel activity in response to chemical stress does not depend solely on the extent of depolarization, as might have been expected, but may also be negatively influenced by accompanying effects of the used compound. The stressors may interact with the plasma membrane or the channel itself, or cause cytosolic acidification. All of these effects may negatively influence the Tok1p channel opening. While ODDC-induced depolarization exhibits the cleanest Tok1p activation, restoring an astonishing 75% of lost ΔΨ, higher BAC concentrations reduce Tok1p activity, probably because of direct interactions with the channel and/or its lipid microenvironment. This is not only the first study of the physiological role of Tok1p in ΔΨ maintenance under chemical stress, but also the first estimate of the extent of depolarization the channel is able to counterbalance.
Subject(s)
Fungal Proteins/metabolism , Membrane Potentials/physiology , Potassium Channels/metabolism , Stress, Physiological/physiology , Yeasts/metabolism , Cell MembraneABSTRACT
We have developed a novel screening method that measures the kinetics and potencies of inhibitors of the yeast multidrug resistance pumps Pdr5p and Snq2p. The assay uses the potentiometric fluorescent probe diS-C(3)(3) (as a benchmark substrate of both pumps) to distinguish drugs with minimal effects on plasma membrane potential as a marker of side-effects on membrane function and integrity. Using FK506, its structural analog rapamycin and enniatin B, we showed that our assay can also be used to determine the minimum drug concentration causing an immediate inhibitory effect and to compare the inhibitory potencies of the drug on the two pumps. We found that the protonophore CCCP effectively inhibits the transport of diS-C(3)(3) by both pumps and confirmed the activation of membrane H(+)-ATPase by CCCP.
Subject(s)
ATP-Binding Cassette Transporters/chemistry , Saccharomyces cerevisiae Proteins/chemistry , ATP-Binding Cassette Transporters/antagonists & inhibitors , Carbocyanines/pharmacology , Carbonyl Cyanide m-Chlorophenyl Hydrazone/pharmacology , Kinetics , Saccharomyces cerevisiae Proteins/antagonists & inhibitors , Spectrometry, Fluorescence , Tacrolimus/pharmacology , beta-Alanine/analogs & derivatives , beta-Alanine/pharmacologyABSTRACT
Physostigmine (Phy), a reversible inhibitor of acetylcholine (ACh) esterase (AChE), may also act as a low potency agonist and a modulator of the nicotinic receptor. The actions of Phy on mouse muscle nicotinic receptors in the COS-7 cell line were studied by the patch-clamp technique. Currents were recorded in the whole-cell mode 3-7 days after cell transfection by plasmids coding alphabetagammadelta combination of receptor subunits. The application of ACh to cells clamped at -10 mV produced inward currents which displayed desensitization. The application of Phy in concentrations up to 1 x 10(-3) M did not give reliable specific whole-cell membrane responses. The application of Phy in concentrations of 10(-6)-10(-4) M together with ACh modulated the amplitude; accelerated desensitization of currents induced by ACh and increased the final extent of desensitization in a concentration-dependent manner. This finding is in contrast to the suppression and slowing down of desensitization by Phy and 1-methyl-galanthamine observed in Torpedo receptors.
Subject(s)
Acetylcholine/metabolism , Muscle, Skeletal/metabolism , Neuromuscular Junction/metabolism , Physostigmine/pharmacology , Receptors, Nicotinic/metabolism , Synaptic Transmission/genetics , Acetylcholine/pharmacology , Animals , COS Cells , Cell Membrane/drug effects , Cell Membrane/genetics , Cell Membrane/metabolism , Chlorocebus aethiops , Cholinesterase Inhibitors/pharmacology , Dose-Response Relationship, Drug , Drug Synergism , Genetic Vectors/genetics , Ion Channels/drug effects , Ion Channels/genetics , Ion Channels/metabolism , Membrane Potentials/drug effects , Membrane Potentials/genetics , Mice , Muscle, Skeletal/innervation , Neuromuscular Junction/drug effects , Patch-Clamp Techniques , Receptors, Nicotinic/drug effects , Receptors, Nicotinic/genetics , Synaptic Transmission/drug effects , TransfectionABSTRACT
The action of physostigmine on mouse muscle nicotinic acetylcholine receptor expressed in the COS-7 cell line was studied by the patch-clamp technique. Physostigmine accelerated, by allosteric modulation, the rate of desensitization of whole cell currents induced by acetylcholine and decreased the maximal amplitude in concentration-dependent manner.
