Muscarine inhibits high-threshold calcium currents with two distinct modes in rat embryonic hippocampal neurons.
1. Ca2+ channel modulation by muscarine was investigated in primary cultured embryonic rat hippocampal neurons using the whole-cell variant of the patch-clamp technique. 2. Muscarine produced a reversible and concentration-dependent decrease in the Ba2+ current amplitude. In 65% of neurons sensitive to the agonist, current inhibition was time and voltage dependent, being maximal between -20 and 0 mV and decreasing at depolarizing potentials. In the remaining 35% of neurons, the effects of muscarine were voltage independent, inhibition being constant in a wide potential range between -20 and +80 mV. 3. Different receptors might be involved in the two modes of modulation. Muscarine-induced voltage-dependent inhibition of Ba2+ current was best suppressed by the muscarinic receptor antagonist 4-diphenylacetoxy-N-methyl-piperidine methiodide (81% suppression), while voltage-independent inhibition was best suppressed by AFDX116 (75% suppression). 4. In cells treated with omega-conotoxin (omega-CgTX), the voltage-independent mode of inhibition was strongly prevented, suggesting that the two modulatory mechanisms (voltage dependent and voltage independent) operate on separate classes of high-voltage-activated (HVA) Ca2+ channels. 5. A pertussis toxin-sensitive G-protein is involved in both modes of action of muscarine, since both modes were prevented by pretreatment of the cells with 50 ng ml-1 pertussis toxin. 6. Both modes of modulation were mimicked in different cells by intracellular application of GTP-gamma-S. However, the onset of voltage-independent inhibition was about 5 times slower than that of voltage-dependent inhibition, suggesting involvement of a more complex metabolic pathway for the former mode of channel modulation. 7. Relief of the voltage-dependent inhibition was obtained by depolarizing voltage prepulses and occurred with kinetics that depended on agonist concentration. 8. The voltage-dependent inhibition could be simulated by a kinetic model in which the time course of Ca2+ entry was assumed to be regulated by both the concentration of muscarine and membrane potential.
Toselli
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M
Istituto di Fisiologia Generale, Università di Pavia, Italy.
Taglietti
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D000818:Animals; D001464:Barium / Q000493:pharmacokinetics; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000378:metabolism*; D002478:Cells, Cultured / Q000378:metabolism; D002614:Chelating Agents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004622:Embryo, Mammalian; D005260:Female; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006624:Hippocampus / Q000166:cytology* / Q000196:embryology*; D007700:Kinetics; D008564:Membrane Potentials / Q000502:physiology; D009116:Muscarine / Q000378:metabolism*; D018721:Muscarinic Agonists; D009474:Neurons / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D010455:Peptides / Q000494:pharmacology; D037342:Pertussis Toxin; D011247:Pregnancy; D051381:Rats; D012680:Sensitivity and Specificity; D013997:Time Factors; D010566:Virulence Factors, Bordetella / Q000494:pharmacology; D020866:omega-Conotoxin GVIA
D002121:Calcium Channel Blockers; D015220:Calcium Channels; D002614:Chelating Agents; D018721:Muscarinic Agonists; D010455:Peptides; D010566:Virulence Factors, Bordetella; D001464:Barium; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D004533:Egtazic Acid; D009116:Muscarine; D020866:omega-Conotoxin GVIA; D037342:Pertussis Toxin; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium