No abstract

In the presence of acetylcholine, the nicotinic acetylcholine receptor undergoes two rapid conformational changes: one in the 1-ms time region, leading to the formation of a transmembrane channel and signal transmission between cells, and the other in the 100-ms time region, leading to an inactive "desensitized" form with altered ligand-binding properties. To determine the properties of the receptor that are relevant for channel opening and signal transmission, we have developed a cell-flow technique that allows measurements to be made with cells prior to receptor desensitization. Here we illustrate the usefulness of the technique. A wide concentration range of both a ligand that controls the opening of receptor channels (carbamoylcholine) and a receptor inhibitor (procaine) was used to measure the dissociation constant of the receptor site controlling channel opening (2.4 X 10-4 M), the channel-opening equilibrium constant (5.5), the inhibition constant for procaine (5.8 x 10-5 M), and the rate coefficients for two desensitization processes of 5 s-' and 0.2 s-'. The cell-flow technique illustrated here is of interest because, by rapid-reaction techniques, it extends the chemical kinetic approach from investigations of reactions in solutions to investigations of many different receptors that exist in membranes of central nervous system cells and whose properties are not well known. EXPERIMENTAL PROCEDURES BC3H1, a nicotinic acetylcholine receptor-containing clonal mammalian cell line (22), was cultured as described by Sine and Taylor (23). The cells were maintained in 35-mm dishes in a differentiating medium containing 0.5% fetal calf serum (GIBCO) as described by Sine and Steinbach (24) for 7 days before use.The whole-cell current recording variant of the patchclamp technique was used as described (12,13). A commercially available amplifier (List L/M-EPC7) suitable for the

Newly synthesized photolabile derivatives of glutamate, caged glutamate, that release free glutamate on a microsecond time scale after a pulse of UV laser light are described. 2-Nitrobenzyl derivatives were attached to the amino or carboxyl groups of glutamate. Substitution with a -COj group at the benzylic carbon accelerates the photolysis reaction when compared to -H and -CH3 substituents. y-O-(aCarboxy-2.nitrobenzyl)glutamate is stable at neutral pH. In 100 mM phosphate buffer at pH 7.0, the compound is photolyzed at 308 nm with a quantum product yield of 0.14.

Recent studies have provided evidence for a role of protein phosphorylation in the regulation of the function of various potassium and calcium channels (for reviews, see refs 1, 2). As these ion channels have not yet been isolated and characterized, it has not been possible to determine whether phosphorylation of the ion channels themselves alters their properties or whether some indirect mechanism is involved. In contrast, the nicotinic acetylcholine receptor, a neurotransmitter-dependent ion channel, has been extensively characterized biochemically and has been shown to be directly phosphorylated. The phosphorylation of this receptor is catalysed by at least three different protein kinases (cyclic AMP-dependent protein kinase, protein kinase C and a tyrosine-specific protein kinase) on seven different phosphorylation sites. However, the functional significance of phosphorylation of the receptor has been unclear. We have now examined the functional effects of phosphorylation of the nicotinic acetylcholine receptor by cAMP-dependent protein kinase. We investigated the ion transport properties of the purified and reconstituted acetylcholine receptor before and after phosphorylation. We report here that phosphorylation of the nicotinic acetylcholine receptor on the gamma- and delta-subunits by cAMP-dependent protein kinase increases the rate of the rapid desensitization of the receptor, a process by which the receptor is inactivated in the presence of acetylcholine (ACh). These results provide the first direct evidence that phosphorylation of an ion channel protein modulates its function and suggest that phosphorylation of postsynaptic receptors in general may play an important role in synaptic plasticity.

The integrated function of the nervous system depends on specific and rapid transmission of signals between its constituent cells. The nicotinic acetylcholine receptor is the best known of a group of membrane-bound proteins responsible for such transmission; for this process to occur, a specific neurotransmitter, in this case acetylcholine, must bind to the receptor, which then forms transmembrane channels through which cations pass. The resulting change in transmembrane voltage determines whether or not a signal is transmitted. The question of how fast this process takes place in any neurotransmitter receptor has remained one of the interesting and most challenging in the field. To answer it, many attempts have been made to evaluate the rate constant for the opening of the acetylcholine receptor channel, but in almost all these studies the rate was measured after the receptor-mediated reaction, which involves the open channel and many intermediate states, had reached a quasi equilibrium. This resulted in a plethora of reported values for the rate constant that differ by a factor of up to 50-fold, even when the measurements were made with the same type of cell. The new approach described here involves the use of single cells of a mammalian cell line (BC3H1), containing muscle-type acetylcholine receptors, and the rapid introduction of neurotransmitter to the cell surface. The rapid delivery was achieved by converting a previously synthesized photolabile precursor of carbamoylcholine to carbamoylcholine, a stable amino-group-containing analogue of acetylcholine, with a single laser pulse and an observed photolysis rate of 7300 s-1.(ABSTRACT TRUNCATED AT 250 WORDS)