Jennifer M. Pocock

Kainic Acid Inhibits the Synaptosomal Plasma Membrane Glutamate Carrier and Allows Glutamate Leakage from the Cytoplasm but Does Not Affect Glutamate Exocytosis

Kainate inhibits the exchange of d‐aspartate into guinea‐pig cerebrocortical synaptosomes. Kainate inhibits the Ca2+‐independent efflux of endogenous glutamate in the presence of a trapping system for the released amino acid but potentiates a Ca2+‐independent net efflux of endogenous and labelled glutamate and aspartate in the ab‐sence of the trap. Dihydrokainate has a similar effect. No discrepancy is seen between the release of endogenous and exogenously accumulated amino acid. These results are consistent with the presence of a slow leak of glutamate or aspartate from the cytoplasm independent of the kainate‐sensitive Na+‐cotransport pathway. In the presence of the trap, glutamate effluxes by both pathways, whereas in the absence of the trap, the Na+‐cotransport pathway opposes the leak. Neither in the presence or absence of the glutamate trap does kainate induce, inhibit, or otherwise affect the Ca2+‐dependent release of endogenous glutamate. The results enable many of the apparent complexities in the presynaptic actions of kainate to be resolved.

Phorbol ester translocation of protein kinase C in guinea-pig synaptosomes and the potentiation of calcium-dependent glutamate release.

The distribution of protein kinase C activity and specific phorbol ester binding sites between soluble and particulate fractions of isolated guinea-pig cerebral cortical synaptosomes is examined following preincubation with phorbol esters. Half-maximal decrease in cytosolic activity requires 10 nM 4 beta-phorbol myristoyl acetate. Specific [3H]phorbol dibutyrate binding sites are translocated from cytoplasmic to particulate fractions in parallel with protein kinase C activity. Depolarization of the plasma membrane by 30 mM KCl does not cause translocation of protein kinase C. 1 microM 4 beta-phorbol myristoyl acetate and 1 microM 4 beta-phorbol didecanoate (but not 1 microM 4 alpha-phorbol didecanoate) enhance the release of glutamate from synaptosomes partially depolarized by 10 mM KCl; however, 4 beta-phorbol myristoyl acetate is ineffective at 20 nM. 1 microM 4 beta-phorbol myristoyl acetate slightly increases the cytosolic free Ca2+ concentration of polarized synaptosomes, but not that following partial depolarization. 4 beta-Phorbol myristoyl acetate causes a concentration-dependent increase in the Ca2+-dependent glutamate release induced by sub-optimal ionomycin concentrations, but is without effect on the release induced by maximal ionomycin. It is concluded that phorbol esters stereospecifically enhance the Ca2+-sensitivity of glutamate release, but that higher concentrations may be required than for protein kinase C translocation in the same preparation. Instead the enhancement may be related to the rapid inactivation of protein kinase C which occurs with phorbol esters.

Flunarizine inhibits both calcium-dependent and -independent release of glutamate from synaptosomes and cultured neurones.

Flunarizine, an established Ca2+ channel antagonist, blocks both exocytotic glutamate release from mammalian cultured cerebellar granule cells and isolated presynaptic nerve endings (synaptosomes) prepared from two distinct areas of the mammalian brain. This blockade of release displays the same flunarizine concentration dependency in synaptosomes in the presence or absence of Ca2+, with total inhibition at a concentration of 10 microM. In cultured neurones, a selective effect on the L-channel-coupled component of the KCl-evoked rise in intracellular Ca2+, [Ca2+]c, can be demonstrated between flunarizine concentrations of 100 nM and 10 microM, while at concentrations above 10 microM, the remaining residual and transient components are affected. In synaptosomes, flunarizine blocks the KCl-evoked elevation in [Ca2+]c in a concentration-dependent manner. Additionally, 10 microM flunarizine directly antagonises ouabain-induced tetrodotoxin (TTX)-sensitive Na+ influx, glutamate, aspartate and GABA release from synaptosomes, whilst inhibiting veratridine-induced Ca(2+)-independent TTX-sensitive Na+ influx and glutamate release at 15 microM and 10 microM in cells and synaptosomes, respectively. In both cultured neurones and synaptosomes, the ability of flunarizine to block both neurotransmitter and cytoplasmic glutamate release is due to a direct antagonism of both voltage dependent Ca2+ channels and tetrodotoxin-sensitive Na+ channels.

A toxin (Aga-GI) from the venom of the spider Agelenopsis aperta inhibits the mammalian presynaptic Ca2+ channel coupled to glutamate exocytosis

Venom of the funnel web spider Agelenopsis aperta was fractionated and screened for activity against the mammalian presynaptic, voltage-dependent Ca2+ channel coupled to glutamate exocytosis. A purified toxin (Aga-GI) from this venom inhibits glutamate exocytosis evoked by elevated potassium or by 4-aminopyridine but is without effect on ionomycin-evoked release. At the same time a partial inhibition of the depolarisation-evoked elevation of cytoplasmic free Ca2+ is seen. The toxin does not inhibit 4-aminopyridine- or potassium-evoked depolarisation, or block Ca(2+)-dependent, potassium-evoked [3H]noradrenaline release. The results indicate that the venom contains a toxin capable of inhibiting the presynaptic voltage-dependent Ca2+ channel coupled to glutamate exocytosis in the mammalian central nervous system. This channel is resistant to block by either omega-conotoxin GVIA or nifedipine. Thus Aga-GI is a novel tool with which to probe this elusive neuronal calcium channel.

The calcium channel coupled to the exocytosis of L-glutamate from cerebellar granule cells is inhibited by the spider toxin, Aga-GI.

The increase in cytosolic calcium, [Ca2+]c, evoked with 50 mM KCl in cerebellar granule cells consists of four components; (1) a rapidly inactivating transient or spike; (2) a nifedipine-sensitive non-inactivating plateau; (3) an Aga-GI (spider toxin) sensitive non-inactivating plateau; (4) a residual non-inactivating plateau insensitive to nifedipine and Aga-GI. None of these components is blocked by synthetic arginine polyamine toxin, spermine, (+)-MK-801 hydrogen maleate, D(-)-2-amino-5-phosphonopentanoic acid or omega-conotoxin-GVIA. The proposed P-type channel antagonist, omega-agatoxin-IVA, has a limited but non-significant effect on the elevated plateau [CA2+]c.L-type Ca2+ channels are located primarily on the soma whereas the component of the plateau which is blocked specifically by Aga-GI is localized primarily on the cell neurites. The latter component is coupled to the exocytosis of endogenous glutamate evoked with 50 mM KCl.

Glutamate exocytosis from cerebellar granule cells: The mechanism of a transition to an L-type Ca2+ channel coupling

When cerebellar granule cells in the presence of 1.3 mM calcium chloride (Ca2+) are depolarized by high potassium chloride (KCl), the release of endogenous glutamate is coupled to a high threshold Ca2+ channel blocked by the spider toxin omega Agatoxin-glutamate-release-inhibitor (Aga-GI) and insensitive to the L-type voltage-dependent Ca2+ channel-inhibitor nifedipine. A prolonged KCl depolarization in the absence of Ca2+ followed by addition of 5 mM Ca2+ results in an enhanced nifedipine-sensitive Ca2+ entry; glutamate exocytosis retains sensitivity to tetanus toxin and bafilomycin A1, is now totally inhibited by nifedipine and shows greatly reduced sensitivity to AGA-GI. Single cell Ca2+ imaging indicates that the L-type channel modulating release is preferentially located at somatic regions rather than neurites. A different pattern of vesicle endocytosis monitored with the fluorescent indicator FM1-43 is seen in response to the two depolarization protocols. Furthermore, vesicles loaded during depolarization with high KCl in the presence of 5 mM Ca2+ extensively exocytose dye in a nifedipine-insensitive manner in response to a second similar stimulation but release little dye in response to stimulus with high KCl in the absence of Ca2+ followed by the addition of 5 mM Ca2+. In contrast, vesicles loaded by stimulating with KCl in the absence of Ca2+ followed by the addition of 5 mM Ca2+ can be released by a second similar stimulus and this release is sensitive to nifedipine. Nifedipine sensitivity is not induced in cerebellar synaptosomes subjected to stimulation with high KCl in the absence of Ca2+ followed by the re-addition of 5 mM Ca2+. The results indicate that different populations of channels and vesicles may be functional during two depolarization protocols.

Phosphorylation of synapsin I and MARCKS in nerve terminals is mediated by Ca2+ entry via an Aga‐GI sensitive Ca2+ channel which is coupled to glutamate exocytosis

Ca2+ entry is a prerequisite for both exocytosis and the phosphorylation of synapsin I and MARCKS proteins in mammalian cerebrocortical synaptosomes. The novel spider toxin Aga‐GI completely blocks KCl‐evoked glutamate exocytosis but only partially inhibits KCl‐evoked cytoplasmic Ca2+ elevations, thus revealing at least two pathways for KCl‐induced Ca2+ entry. Aga‐GI completely attenuates KCl‐induced phosphorylation of synapsin I and MARCKS proteins. We therefore conclude that both exocytosis and the phosphorylation of synapsin I and MARCKS proteins are specifically coupled to Ca2+ entry via a subset of voltage dependent Ca2+ channels at the nerve terminal which are sensitive to Aga‐GI.

The development of Ca2+ channel responses and their coupling to exocytosis in cultured cerebellar granule cells

Using single-cell imaging, we investigated developmental changes in the modulation of KCl-evoked Ca2+ entry by various voltage-dependent Ca2+ channels and the coupling of these channels to exocytosis in cultured cerebellar granule neurons. A component of the KCl-evoked Ca2+ elevation sensitive to nifedipine and localized at cell somata, decreases with culture age. A component blocked by 200 nM omega-Agatoxin-IVA increases with age and whilst localized primarily at the cell somata, also becomes evident at the neurites. The change in activity between nifedipine-sensitive Ca2+ channels and omega-Agatoxin-IVA-sensitive Ca2+ channels occurs at 13 days in vitro at cell somata. A component of Ca2+ entry insensitive to nifedipine and 200 nM omega-Agatoxin-IVA is localized primarily at the neurites and is apparent at all ages. Single-cell imaging of exocytosis using FM1-43 destaining indicates that the residual, but not the nifedipine- or omega-Agatoxin-IVA-sensitive components of Ca2+ entry, modulates exocytosis. However cells cultured for 20-26 days develop a component of Ca2+ entry at the neurites which is sensitive to 200 nM omega-Agatoxin-IVA and omega-Conotoxin-MVIIC and which partially controls release. Immunolocalization studies reveal that binding sites for omega-Conotoxin-GVIA are present throughout development, even though this toxin does not inhibit KCl-evoked [Ca2+]c elevations or exocytosis. 300 nM omega-Agatoxin-IVA labels both somata and, at later developmental stages, neurites, consistent with the functional studies.