An Ion Channel Locus for the Protein Kinase C Potentiation of Transmitter Glutamate Release from Guinea Pig Cerebrocortical Synaptosomes

Abstract

The mechanism by which protein kinase C (PKC) activates transmitter release from guinea pig cerebrocortical synaptosomes was investigated by employing parallel fluorescent assays of glutamate release, cytoplasmic free Ca2+, and plasma membrane potential. 4 beta-Phorbol dibutyrate (4 beta-PDBu) enhances the Ca(2+)-dependent, 4-aminopyridine (4AP)-evoked release of glutamate from synaptosomes, the 4AP-evoked elevation of cytoplasmic free Ca2+, and the 4AP-evoked depolarization of the plasma membrane. 4 beta-PDBu itself causes a slow depolarization, which may underlie the small effect of 4 beta-PDBu on spontaneous, KCl-evoked, and Ca(2+)-independent/4AP-evoked glutamate release. Because 4AP (but not KCl) generates spontaneous, tetrodotoxin-sensitive action potentials in synaptosomes, a major locus of presynaptic PKC action is to enhance these action potentials, perhaps by inhibiting delayed rectifier K+ channels.

Presynaptic plasticity: The regulation of Ca2+-dependent transmitter release

“…It is unclear whether similar processes are relevant to the plasticity of the presynaptic nerve terminal. Studies on isolated nerve terminals (Tibbs et al, 1989;Zoltay and Cooper 1990;Barrie et al, 1991) and the squid giant axon (Robitaille and Charlton, 1992) indicate that modulation of presynaptic K + channels may be an important factor in the modulation of the presynaptic input-output relation. Phosphorylation of presynaptic K + channels may prolong presynaptic depolarizations upon incoming action potentials, and may thus lengthen the periods of Ca 2 + influx (presynaptic Ca 2 + channels involved in fast-acting transmitter secretion appear not to inactivate readily, McMahon and Nicholls, 1991) and potentiate transmitter release.…”

“…Phosphorylation of presynaptic K + channels may prolong presynaptic depolarizations upon incoming action potentials, and may thus lengthen the periods of Ca 2 + influx (presynaptic Ca 2 + channels involved in fast-acting transmitter secretion appear not to inactivate readily, McMahon and Nicholls, 1991) and potentiate transmitter release. In fact, this mechanism may be a major pathway in presynaptic plasticity, because presynaptic PKC activation was shown to induce this increased depolarization, Ca 2+ influx and glutamate release (Barrie et al, 1991). In vivo, this PKC stimulation and the proposed lengthening of the depolarization at the nerve terminal may occur through repetitive (high frequency) stimulation, when Ca 2 + levels gradually build up in the presynaptic cytosol or through presynaptic receptor occupation and phospholipase stimulation, possibly in concert with other second messengers (see Section 3 for further discussion).…”

“…3). The activation of PKC attenuates the presynaptic input-output relation by manipulating the repolarization through phosphorylation of K + channels and hereby prolonging Ca :+ channel opening and Ca :+ entrance (see Tibbs et al, 1989;Barrie et al, 1991;Herrero et al, 1992), by regulating the availability of Ca 2+ buffering components through phosphorylation of B-50 and MARCKS-protein (Alexander et al, 1987(Alexander et al, , 1988Graft et al, 1989;McIlroy et al, 1991;Hartwig et al, 1992), by modulating transmitter production through phosphorylation of tyrosine hydroxylase (Woodrow et al, 1992) and probably by modulating recruitment, docking or release of small synaptic vesicles through phosphoryiation of P96 (Robinson 1991, Sim et al, 1991. These mechanisms may also be involved in the maintenance of long term potentiation, since presynaptic PKCactivity was found to correlate with the induction of LTP and increased transmitter release has been implicated in the mechanisms underlying LTP (Bfir et al, 1980(Bfir et al, , 1982Tielen et al, 1983;Lovinger et al, 1985Lovinger et al, , 1986Gianotti et al, 1992).…”

The glutamatergic nerve terminal

“…Ca'+-dependency, effects on spontaneous or evoked release etc.). Recently much of this ambiguity has been resolved by the finding (Banie et al, 1991) that the extent by which phorbol esters stimulate the release of glutamate is greatly enhanced when release is evoked by 4AP rather than by KCl (Fig. 5).…”

Pyridoxine Inhibits Depolarization-Evoked Glutamate Release in Nerve Terminals from Rat Cerebral Cortex: a Possible Neuroprotective Mechanism?

“…Elevated extracellular KCl depolarizes the plasma membrane by shifting the K ϩ equilibrium potential above the threshold potential for activation of voltage-dependent ion channels. Although Na ϩ channels are inactivated under these conditions, VDCCs are activated nonetheless to mediate Ca 2ϩ entry, which supports neurotransmitter release (Barrie et al, 1991). Addition of 15 mM KCl evoked controlled glutamate release of 9.5 Ϯ 0.1 nmol/mg/5 min, which was reduced to 4.9 Ϯ 0.4 nmol/ mg/5 min in the presence of 5 M pyridoxine (n ϭ 7; P Ͻ 0.01) (Fig.…”