In Vitro Selection of Short DNA Aptamers that Can Inhibit or Alleviate Cocaine and MK-801 Inhibition of Muscle-Type Nicotinic Acetylcholine Receptors.

Ligands of high specificity and selectivity have been selected for biological molecules of interest including nicotinic acetylcholine receptor (nAChR) using combinatorial libraries of nucleic acids. The nAChR belongs to a group of structurally related proteins that regulate signal transmission between ~ 10 cells of the mammalian nervous system. It is inhibited by both therapeutic agents and abused drugs, including cocaine.

Caged neurotransmitters and other caged compounds: design and application.

The approaches using caged neurotransmitters described here enable transient kinetic investigations to be made with membrane-bound proteins (receptors) on a cell surface with the same time resolution as was previously possible only with proteins in solution.

Cell-flow technique.

Various devices have been used to flow neurotransmitter solutions over cells containing receptors (e.g., ligand-gated ion channels) for whole-cell current recordings.

Use of multicomponent reactions in developing small-molecule tools to study GABAA receptor mechanism and function.

We discuss the potential use of multicomponent reactions in developing small-molecule probes of GABA(A) receptor function. Two examples that illustrate this approach are presented: the synthesis of a class of compounds that specifically modulate the function of GABA(A) receptors containing the δ-subunit, and also 'caged' GABA derivatives. A caged GABA is a photolabile precursor of GABA that releases GABA upon photolysis.

Mechanism of potentiation of a dysfunctional epilepsy-linked mutated GABA(A) receptor by a neurosteroid (3alpha, 21-dihydroxy-5alpha-pregnan-20-one): transient kinetic investigations.

The malfunction of a mutated GABA(A) receptor (alpha1beta2gamma2L(K289M)) in an inheritable form of epilepsy (GEFS+, generalized epilepsy with febrile seizures plus) in humans [Baulac, S., Huberfeld, G., Gourfinkel-An, I.

Dihydropyrimidinone positive modulation of delta-subunit-containing gamma-aminobutyric acid type A receptors, including an epilepsy-linked mutant variant.

Gamma-aminobutyric acid type A receptors (GABA(A) receptors) are ligand-gated chloride channels that play a central role in signal transmission within the mammalian central nervous system. Compounds that modulate specific GABA(A) receptor subtypes containing the delta-subunit are scarce but would be valuable research tools and starting points for potential therapeutic agents. Here we report a class of dihydropyrimidinone (DHPM) heterocycles that preferentially potentiate peak currents of recombinant GABA(A) receptor subtypes containing the delta-subunit expressed in HEK293T cells.

Minimal RNA aptamer sequences that can inhibit or alleviate noncompetitive inhibition of the muscle-type nicotinic acetylcholine receptor.

Combinatorially synthesized nucleotide polymers have been used during the last decade to find ligands that bind to specific sites on biological molecules, including membrane-bound proteins such as the nicotinic acetylcholine receptors (nAChRs). The neurotransmitter receptors belong to a group of four structurally related proteins that regulate signal transmission between ~10(11) neurons of the mammalian nervous system. The nAChRs are inhibited by compounds such as the anticonvulsant MK-801 [(+)-dizocilpine] and abused drugs such as cocaine.

A new synthesis of caged GABA compounds for studying GABAA receptors.

A short and convergent synthetic approach to new photoactivatable precursors of gamma-aminobutyric acid (GABA) is described. When photolyzed, the 'caged' GABA precursor efficiently releases GABA, as judged by depolarization measurements on the mammalian GABA(A) receptor.

Coumarin-caged glycine that can be photolyzed within 3 microseconds by visible light.

The synthesis and characterization of a new photolabile precursor of glycine (coumarin-caged glycine) are reported. The new compound is suitable for rapid chemical kinetic investigations of the membrane-bound neurotransmitter receptor activated by glycine. Unlike previously used caging groups for glycine, this precursor can be photolyzed rapidly and efficiently in the visible wavelength region.

On the mechanism of alleviation by phenobarbital of the malfunction of an epilepsy-linked GABA(A) receptor.

A mechanism for the alleviation of the malfunction of a mutated (gamma2(K289M)) epilepsy-linked gamma-aminobutyric acid (GABA) neurotransmitter receptor by phenobarbital is presented. Compared to the wild-type receptor, the GABA-induced current is considerably reduced in the mutated (alpha1beta2gamma2(K289M)) epilepsy-linked GABA(A) receptor [Baulac, S., Huberfeld, G.

Picrotoxin inhibition mechanism of a gamma-aminobutyric acid A receptor investigated by a laser-pulse photolysis technique.

The gamma-aminobutyric acid(A) (GABA(A)) receptor, a major inhibitory neurotransmitter receptor, belongs to a family of membrane-bound proteins that regulate signal transmission between approximately 10(12) cells of the nervous system. It plays a major role in many neurological disorders, including epilepsy. It is the target of many pharmacological agents, including the convulsant picrotoxin.

A protecting group for carboxylic acids that can be photolyzed by visible light.

We report on a photolabile protecting (caging) group that is new for carboxylic acids. Unlike previously used caging groups for carboxylic acids, it can be photolyzed rapidly and efficiently in the visible wavelength region. The caging group 7-N,N-diethyl aminocoumarin (DECM) was used to cage the gamma-carboxyl group of glutamic acid, which is also a neurotransmitter.

Selection of stable RNA molecules that can regulate the channel-opening equilibrium of the membrane-bound gamma-aminobutyric acid receptor.

The gamma-aminobutyric acid (GABA(A)) receptor belongs to a superfamily of membrane-bound proteins that regulate signal transmission between cells in the nervous system. It is the target of convulsants such as picrotoxin and is mutated in some forms of epilepsy, a disease affecting approximately 50 million people worldwide. In picrotoxin inhibition and in one form of epilepsy, a decrease in the channel-opening equilibrium of a GABA(A) receptor is responsible for receptor dysfunction.

Mechanism-based discovery of small molecules that prevent noncompetitive inhibition by cocaine and MK-801 mediated by two different sites on the nicotinic acetylcholine receptor.

The nicotinic acetylcholine receptor (nAChR) belongs to a group of five structurally related membrane proteins that play a major role in the communication between approximately 10(12) cells of the mammalian nervous system. The receptor is inhibited by both abused drugs and therapeutic agents. During the past two decades, many attempts have been made to find compounds that prevent cocaine inhibition of this protein.

On the mechanism of a mutated and abnormally functioning gamma-aminobutyric acid (A) receptor linked to epilepsy.

A recent report indicates that a lysine-to-methionine mutation (K289M) in the gamma2 subunit of a human gamma-aminobutyric acid neurotransmitter receptor, the GABA(A) receptor, is linked to generalized epilepsy with febrile seizures [Baulac et al. (2001) Nat. Genet.

Mechanism-based approach to the successful prevention of cocaine inhibition of the neuronal (alpha 3 beta 4) nicotinic acetylcholine receptor.

The nicotinic acetylcholine receptor (nAChR) belongs to a family of five channel-forming proteins that regulate communication between the approximately 10(12) cells of the nervous system. A minimum mechanism of inhibition of the muscle-type nAChR (1) by the noncompetitive inhibitors cocaine and MK-801 [(+)-dizocilpine, an anticonvulsant] indicated they bind to a regulatory site, with higher affinity for the closed-channel form than for the open-channel form, thus shifting the equilibrium toward the closed-channel form and inhibiting receptor function. The mechanism predicts that compounds that bind to this regulatory site with equal or higher affinity for the open-channel conformation than for the closed-channel conformation will prevent receptor inhibition (1).

Toward the development of new photolabile protecting groups that can rapidly release bioactive compounds upon photolysis with visible light.

The synthesis and characterization of a new photolabile protecting group (caging group) for carboxylic acids, the 2-(dimethylamino)-5-nitrophenyl (DANP) group, is described. This compound has a major absorption band in the visible wavelength region with a maximum near 400 nm (epsilon400 = 9077 M(-1) cm(-1) at pH 7.4 and 21 degrees C).

Reversing the action of noncompetitive inhibitors (MK-801 and cocaine) on a protein (nicotinic acetylcholine receptor)-mediated reaction.

The nicotinic acetylcholine receptor (nAChR) is one of five structurally related membrane proteins required for communication between approximately 10(12) cells of the mammalian nervous system. The receptor is inhibited by both therapeutic agents and abused drugs. Understanding the mechanism of noncompetitive allosteric inhibitors of the nicotinic acetylcholine receptor is a long-standing and intensely investigated problem.

Rapid chemical reaction techniques developed for use in investigations of membrane-bound proteins (neurotransmitter receptors).

New techniques for investigating chemical reactions on cell surfaces in the microsecond-to-millisecond time region are described. Reactions mediated by membrane-bound neurotransmitter receptors that control signal transmission between approximately 10(12) cells of the nervous system are taken as an example. Cells with receptors on their plasma membrane are equilibrated with photolabile, biologically inactive precursors of the neurotransmitters.

Synthesis and characterization of photolabile o-nitrobenzyl derivatives of urea.

We present here the synthesis and characterization of four photolabile derivatives of urea in which alpha-substituted 2-nitrobenzyl groups are covalently attached to the urea nitrogen. These derivatives photolyze readily in aqueous solution to release free urea. The alpha-substituents of the 2-nitrobenzyl group strongly influence the rate of the photolysis reaction measured with transient absorption spectroscopy.

Mechanism of glutamate receptor-channel function in rat hippocampal neurons investigated using the laser-pulse photolysis (LaPP) technique.

Ionotropic glutamate receptors are members of a large family of plasma membrane proteins expressed by cells of the nervous system. Upon binding glutamate, the receptors transiently open transmembrane channels that allow the entry of sodium ions. The resulting changes in the transmembrane potential of the cell initiates a process that is involved in signal transmission to another cell.