MUSCARINE INHIBITS HIGH-THRESHOLD CALCIUM CURRENTS WITH 2 DISTINCT MODES IN RAT EMBRYONIC HIPPOCAMPAL-NEURONS

1. Ca2+ channel modulation by muscarine was investigated in primary cu ltured embryonic rat hippocampal neurons using the whole-cell variant of the patch-clamp technique. 2. Muscarine produced a reversible and c oncentration-dependent decrease in the Ba2+ current amplitude. In 65% of neurons sensitive to the agonist, current inhibition was time and v oltage 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 m ight be involved in the two modes of modulation. Muscarine-induced vol tage-dependent inhibition of Ba2+ current was best suppressed by the m uscarinic receptor antagonist 4-diphenylacetoxy-N-methyl-piperidine me thiodide (81% suppression), while voltage-independent inhibition was b est suppressed by AFDX116 (75% suppression). 4. In cells treated with omega-conotoxin (omega-CgTX), the voltage-independent mode of inhibiti on was strongly prevented, suggesting that the two modulatory mechanis ms (voltage dependent and voltage independent) operate on separate cla sses of high-voltage-activated (HVA) Ca2+ channels. 5. A pertussis tox in-sensitive G-protein is involved in both modes of action of muscarin e, since both modes were prevented by pretreatment of the cells with 5 0 ng ml(-1) pertussis toxin. 6. Both modes of modulation were mimicked in different cells by intracellular application of GTP-gamma-S. Howev er, the onset of voltage-independent inhibition was about 5 times slow er than that of voltage-dependent inhibition, suggesting involvement o f a more complex metabolic pathway for the former mode of channel modu lation. 7. Relief of the voltage-dependent inhibition was obtained by depolarizing voltage prepulses and occurred with kinetics that depende d 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 an d membrane potential.