INTRACELLULAR MECHANISMS OF CALCIUM-CHANNEL MODULATION BY SEROTONIN IN IDENTIFIED HELIX-POMATIA NEURONS

Effects of application of serotonin (5-HT) on voltage-gated calcium cu rrents (I-Ca) Were studied on isolated intracellularly perfused Helix pomatia neurones. Cells were identified in the pedal ganglion in which 5-HT induced reversible potentiation (up to 50%) of the current ampli tude. I-Ca in these cells could also be increased by intracellular int roduction of cAMP (100 mu M) but not cGMP. The effects were not additi ve and could be potentiated by theophylline (5 mM) and IBMX (100-500 m u M). They could be mimicked by forskolin (10-50 mu M) and abolised by tolbutamide (1-5 mM) or protein PK-inhibitor (500 mu g/ml). Methiothe pin (10-50 mu M), a 5-HT1, 2 receptor antagonist, irreversibly inhibit ed this effect, while antagonists of 5-HT2 receptors (cyproheptadine o r ketanserine) as well as 5-HT3 receptor antagonists (ISC 205-930 and cocaine) were without effect. Elevation of intracellular free calcium level ([Ca](i)) by perfusing the cell with Ca-buffered solutions depre ssed the up-regulatory effect in a complicated concentration-dependent way, indicating the involvement of different Ca-binding processes. Ad dition of calmodulin antagonists (50 mu M trifluoperazine or 50 mu M c hlorpromazine), phosphodiesterase inhibitor (IBMX 100-500 mu M) and pr otein phosphatase antagonist (2 mu M okadaic acid) modified the comple x Ca-binding isotherm in a specific way, revealing two tetramolecular binding processes with K-d's of 0.04 and 0.69 mu M respectively. They represent activation of two calmodulin-dependent enzymes: phosphodiest erase which prevents channel phosphorylation by reducing the intracell ular cAMP level and calcineurin which increases its dephosphorylation. Due to this difference in K-d values, changes of [Ca](i) in the physi ological range may serve as a negative feedback mechanism determining the direction, precision and rate of calcium channel modulation by ser otonin.