MUSCARINE INHIBITS w-CONOTOXIN-SENSITIVE CALCIUM CHANNELS IN A VOLTAGE AND
TIME DEPENDENT MODE IN THE HUMAN NEUROBLASTOMA CELL LINE SH-SY5Y.
TOSELLI, Mauro, Paola PERIN and Vanni TAGLIETTI.
Istituto di Fisiologia Generale, Universita' di Pavia Via Forlanini 6, I-
27100, Pavia, Italy.
APStracts 2:0172N, 1995.
SUMMARY AND CONCLUSIONS
1. Calcium channel modulation by muscarine was investigated in cultured human
neuroblastoma SH-SY5Y cells using the whole-cell variant of the patch-clamp
technique. 2. In SH-SY5Y cells w-conotoxin sensitive, high-voltage-activated
Ca 2+ current density gradually increased from about 1[mu]A/cm 2 in
undifferentiated cells to 4 [mu]A/cm 2 after about 20 days of application of
the differentiating agent retinoic acid [Toselli et al., 1991]. 3. In
differentiated SH-SY5Y cells, muscarine reversibly decreased HVA w-CgTx-
sensitive Ba 2+ currents in a concentration-dependent way. Maximum inhibition
(65%) measured at 0 mV was obtained with 30 [mu]M muscarine and the IC 50 was
1[mu]M. 4. Current inhibition obtained with 30 [mu]M muscarine was suppressed
by the specific M2 and M3 antagonists AFDX-116 and 4-DAMP (0.3 [mu]M, 87%
suppression) but not by the M1 antagonist pirenzepine. 5. Muscarine induced
current suppression was prevented by pre-treatment of the cells with pertussis
toxin and mimicked by intracellular application of GTP- y S. 6. In several
cells muscarinic inhibition was characterized by a clear slow down of Ba 2+
current activation at low test potentials. Both inhibition and slow down of
activation were attenuated at more positive potentials, and could be partially
relieved by strong conditioning depolarizations. 7. These results indicate
that muscarinic inhibition of w-CgTx-sensitive Ca 2+ channel current occurs
through activation of specific muscarinic receptors and the modulatory
mechanism operates through activation of a GTP binding protein sensitive to
pertussis toxin. Our results suggest that a blocking molecule interacts in a
voltage dependent manner with the Ca 2+ channel without involvement of
intracellular Ca 2+ mobilization or activation of a protein kinase C or cyclic
nucleotide protein kinases. A simple model describing the reactions involved
is proposed.
Received 29 December 1994; accepted in final form 25 May 1995.
APS Manuscript Number J824-4.
Article publication pending J. Neurophysiol.
ISSN 1080-4757 Copyright 1995 The American Physiological Society.
Published in APStracts on 6 July 1995.