MUSCARINE INHIBITS OMEGA-CONOTOXIN-SENSITIVE CALCIUM CHANNELS IN A VOLTAGE-DEPENDENT AND TIME-DEPENDENT MODE IN THE HUMAN NEUROBLASTOMA CELL-LINE SH-SY5Y

1. Calcium channel modulation by muscarine was investigated in culture d human neuroblastoma SH-SY5Y cells using the whole cell variant of th e patch-clamp technique. 2. In SH-SY5Y cells, omega-conotoxin (omega-C gTx)-sensitive, high-voltage-activated Ca2+ current density gradually increased from similar to 1 mu A/cm(2) in undifferentiated cells to 4 mu A/cm(2) after similar to 20 days of application of the differentiat ing agent retinoic acid. 3. In differentiated SH-SY5Y cells, muscarine reversibly decreased high-voltage-activated omega-CgTx-sensitive Ba2 currents in a concentration-dependent way. Maximum inhibition (simila r to 65%) measured at 0 mV was obtained with 30 mu M muscarine and the IC50 was 1 mu M. 4. Current inhibition obtained with 30 mu M muscarin e was suppressed by the specific M2 and M3 antagonists AFDX-116 and 4- diphenylacetoxy-N-methyl-piperidine methiodide (0.3 mu M; 87% suppress ion), but not by the M1 antagonist pirenzepine. 5. Muscarine-induced c urrent suppression was prevented by pretreatment of the cells with per tussis toxin and mimicked by intracellular application of guanosine 5' -[gamma-thio] triphosphate. 6. In several cells, muscarinic inhibition was characterized by a clear slowdown of Ba2+ current activation at l ow test potentials. Both inhibition and slowdown of activation were at tenuated at more positive potentials and could be partially relieved b y strong conditioning depolarizations. 7. These results indicate that muscarinic inhibition of omega-CgTx-sensitive Ca2+ channel current occ urs through activation of specific muscarinic receptors and the modula tory mechanism operates through activation of a guanosine 5'-triphosph ate-binding protein sensitive to pertussis toxin. Our results suggest that a blocking molecule interacts in a voltage-dependent manner with the Ca2+ channel without involvement of intracellular Ca2+ mobilizatio n or activation of protein kinase C or cyclic nucleotide protein kinas es. A simple model describing the reactions involved is proposed.