PubMed · 5022540
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D C Merrill, C Markland. 1972. Vesical dysfunction induced by the major tranquilizers.. https://doi.org/10.1016/s0022-5347(17)61136-4
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We determined the distances separating five functionally important residues (Gln(10), Lys(27), Trp(29), Arg(33), and Lys(47)) of a three-fingered snake neurotoxin from the reduced disulfide bond alpha(Cys(192)-Cys(193)) located at the alphagamma interface of the Torpedo nicotinic acetylcholine receptor. Each toxin position was substituted individually for a cysteine, which was then linked to a maleimido moiety through three different spacers, varying in length from 10 to 22 A. We estimated the coupling efficiency between the 15 toxin derivatives and the reduced cystine alpha(192-193) by gel densitometry of Coomassie Blue-stained gels. A nearly quantitative coupling was observed between alphaCys(192) and/or alphaCys(193) and all probes introduced at the tip of the first (position 10) and second (position 33) loops of Naja nigricollis alpha-neurotoxin. These data sufficed to locate the reactive thiolate in a "croissant-shaped" volume comprised between the first two loops of the toxin. The volume was further restrained by taking into account the absence or partial coupling of the other derivatives. Altogether, the data suggest that alphaCys(192) and/or alphaCys(193), at the alphagamma interface of a muscular-type acetylcholine receptor, is (are) located in a volume located between 11.5 and 15.5 A from the alpha-carbons at positions 10 and 33 of the toxin, under the tip of the toxin first loop and close to the second one.
Metabolic labeling of a mutant PC12 cell line, A123.7, expressing recombinant rat vesicular acetylcholine transporter (VAChT) with radiolabeled inorganic phosphate was used to demonstrate phosphorylation of the transporter on a serine residue. Mutational analysis was used to demonstrate that serine 480, which is located on the COOH-terminal cytoplasmic tail, is the sole phosphorylation site. Phosphorylation of serine 480 was attributable to the action of protein kinase C. Using a permanently dephosphorylated form of rat VAChT, S480A rVAChT, it was shown that this mutant displays the same kinetics for the transport of acetylcholine and the binding of the inhibitor vesamicol as does the wild type transporter. However, sucrose gradient density centrifugation showed that, unlike wild type VAChT, the S480A mutant did not localize to synaptic vesicles. These results suggest that phosphorylation of serine 480 of VAChT is involved in the trafficking of this transporter.
Dephosphorylation of substrates for cyclic AMP-dependent protein kinase is essential for reversing the effects of this enzyme. It has been proposed that the relevant phosphatase(s) is stimulated by muscarinic cholinergic agonists, thereby accentuating cholinergic antagonism of beta-adrenergic agonists in the heart. To test this hypothesis, dephosphorylation of the three major substrates of cardiac cyclic AMP-dependent protein kinase (phospholamban, troponin-I, and C-protein) was examined. In isolated myocytes, isoproterenol caused concentration-dependent phosphorylation of these three proteins. Simultaneous exposure to acetylcholine with the isoproterenol caused a rightward shift in the concentration-response curve that was similar for protein phosphorylation in myocytes and for the inotropic response of the intact heart. The addition of propranolol after exposure to isoproterenol resulted in protein dephosphorylation, the onset of which was accelerated by acetylcholine. However, acetylcholine did not affect the rate of dephosphorylation for any of the proteins, indicating that phosphatase activity in cardiac muscle is not enhanced by acetylcholine.