Nicotinic acetylcholine receptors (AChR) belong to a family of proteins that form ligand-gated transmembrane ion channels. They are involved in the fast transmission of signals between cells and the control of intercellular communication in the nervous system. A variety of therapeutic agents and abused drugs, including cocaine, inhibit the AChR and monoamine transporters and interfere with nervous system function. Here we describe a mechanism-based approach to prevent this inhibition. We had previously developed presteady-state kinetic (transient kinetic) techniques, with microsecond-to-millisecond time resolutions, for investigations of reactions on cell surfaces that allow one to determine the effects of inhibitors not only on the channel-opening probability but also on the opening and closing rates of the AChR channel. The transient kinetic measurements led to two predictions. ( i ) Ligands that bind to a regulatory site on the closed-channel conformation of the AChR with higher affinity than to the site on the open-channel form shift the equilibrium toward the closed-channel form, thereby inhibiting the receptor. ( ii ) Ligands that bind to a regulatory site with an affinity for the open conformation equal to or higher than their affinity for the closed conformations are expected not to inhibit the receptor and to displace inhibitors. The identification of such ligands in a combinatorial library of RNA ligands is reported. The implication of this approach to other protein-mediated reactions in which an inhibitor changes the equilibrium between active and inactive conformations is discussed.
The effects of cocaine and of phencyclidine and procaine on acetylcholine receptor-controlled ion flux were measured in the millisecond-to-minute time region. Chemical kinetic measurements of ion flux were made in membrane vesicles prepared from the electric organ of Electrophorus electricus and in PC-12 cells, a sympathetic neuronal cell line. A quench-flow technique was used to measure ion flux in the millisecond-to-second range in membrane vesicles. Cocaine and phencyclidine both inhibit acetylcholine receptor-controlled ion flux, but by different mechanisms. Both compounds decrease the initial rate of ion flux, an effect observed with the local anesthetic procaine. This inhibition cannot be prevented by saturating concentrations of acetylcholine (1 mM). These results from chemical kinetic experiments are consistent with electrophysiological measurements which indicate that local anesthetics act by interfering with the movement of ions through receptor-formed channels. The chemical kinetic experiments, however, give additional information about the action of phencyclidine. They indicate that phencyclidine also increases the rate of receptor inactivation (desensitization) and changes the equilibrium between active and inactive receptor conformations, effects not observed in the presence of cocaine or procaine.
Defective mobilization of Ca2+ by cardiomyocytes can lead to cardiac insufficiency, but the causative mechanisms leading to congestive heart failure (HF) remain unclear. In the present study we performed exhaustive global proteomics surveys of cardiac ventricle isolated from a mouse model of cardiomyopathy overexpressing a phospholamban mutant, R9C (PLN-R9C), and exhibiting impaired Ca2+ handling and death at 24 weeks and compared them with normal control littermates. The relative expression patterns of 6190 high confidence proteins were monitored by shotgun tandem mass spectrometry at 8, 16, and 24 weeks of disease progression. Significant differential abundance of 593 proteins was detected. These proteins mapped to select biological pathways such as endoplasmic reticulum stress response, cytoskeletal remodeling, and apoptosis and included known biomarkers of HF (e.g. brain natriuretic peptide/atrial natriuretic factor and angiotensin-converting enzyme) and other indicators of presymptomatic functional impairment. These altered proteomic profiles were concordant with cognate mRNA patterns recorded in parallel using high density mRNA microarrays, and top candidates were validated by RT-PCR and Western blotting. Mapping of our highest ranked proteins against a human diseased explant and to available data sets indicated that many of these proteins could serve as markers of disease. Indeed we showed that several of these proteins are detectable in mouse and human plasma and display differential abundance in the plasma of diseased mice and affected patients. These results offer a systems-wide perspective of the dynamic maladaptions associated with impaired Ca2+ homeostasis that perturb myocyte function and ultimately converge to cause HF.
Abstract The synthesis of photolabile precursors of neurotransmitters that activate neuronal receptors specific for aspartic acid, glutamic acid, glycine, or γ‐aminobutyric acid is described as outlined in the reaction scheme.
The nicotinic acetylcholine receptor (AChR) controls signal transmission between cells in the nervous system. Abused drugs such as cocaine inhibit this receptor. Transient kinetic investigations indicate that inhibitors decrease the channel-opening equilibrium constant [Hess, G. P. & Grewer, C. (1998) Methods Enzymol. 291, 443–473]. Can compounds be found that compete with inhibitors for their binding site but do not change the channel-opening equilibrium? The systematic evolution of RNA ligands by exponential enrichment methodology and the AChR in Torpedo californica electroplax membranes were used to find RNAs that can displace inhibitors from the receptor. The selection of RNA ligands was carried out in two consecutive steps: ( i ) a gel-shift selection of high-affinity ligands bound to the AChR in the electroplax membrane, and ( ii ) subsequent use of nitrocellulose filters to which both the membrane-bound receptor and RNAs bind strongly, but from which the desired RNA can be displaced from the receptor by a high-affinity AChR inhibitor, phencyclidine. After nine selection rounds, two classes of RNA molecules that bind to the AChR with nanomolar affinities were isolated and sequenced. Both classes of RNA molecules are displaced by phencyclidine and cocaine from their binding site on the AChR. Class I molecules are potent inhibitors of AChR activity in BC 3 H1 muscle cells, as determined by using the whole-cell current-recording technique. Class II molecules, although competing with AChR inhibitors, do not affect receptor activity in this assay; such compounds or derivatives may be useful for alleviating the toxicity experienced by millions of addicts.
