Yves Dunant

Zinc inhibits glutamate release via activation of pre‐synaptic KATP channels and reduces ischaemic damage in rat hippocampus

Zinc is concentrated in certain CNS excitatory tracts, especially in hippocampal mossy fibres where it has been suggested to modulate synaptic transmission and plasticity. Using rat mossy fibre synaptosomes depolarized by 4‐aminopyridine, we show here that low zinc concentrations restore the membrane potential and reduce glutamate release. Both effects arose from activation of ATP‐sensitive potassium channels (KATP), since they were mimicked by the KATP opener diazoxide and antagonized by the KATP blocker tolbutamide. Using recombinant channels expressed in COS‐7 cells, we confirmed that micromolar zinc did activate KATP of the type found in hippocampus. We tested the hypothesis that this action of zinc could be beneficial during an ischaemic challenge by using organotypic hippocampal slice cultures. When zinc was applied at micromolar concentrations during a brief anoxic‐hypoglycaemic episode, it significantly attenuated the ensuing neuronal death, whereas chelation of endogenous zinc markedly aggravated cell damage. Protective effect of zinc was mediated through KATP, as was shown by using the opener diazoxide and the blocker tolbutamide. Thus, by activating pre‐synaptic KATP channels, zinc protects neurones from hyper‐excitation, excessive transmitter release and exitotoxicity, and may thus act as an endogenous neuroprotector in conditions such as epilepsy or stroke.

Zinc‐induced changes in ionic currents of clonal rat pancreatic β‐cells: activation of ATP‐sensitive K+ channels

1 The effects of zinc (Zn2+) on excitability and ionic conductances were analysed on RINm5F insulinoma cells under whole‐cell and outside‐out patch‐clamp recording conditions. 2 We found that extracellular application of 10‐20 μM Zn2+ induced a reversible abolition of Ca2+ action potential firing, which was accompanied by an hyperpolarisation of the resting membrane potential. 3 Higher concentrations of Zn2+, in the tens to hundreds micromolar range, induced a reversible reduction of voltage‐gated Ca2+ and, to a lesser extent, K+ currents. Low‐voltage‐activated Ca2+ currents were more sensitive to Zn2+ block than high voltage‐activated Ca2+ currents. 4 The Zn2+‐induced hyperpolarisation arose from a dose‐dependent increase in a voltage‐independent K+ conductance that was pharmacologically identified as an ATP‐sensitive K+ (KATP) conductance. The effect was rapid in onset, readily reversible, voltage independent, and related to intracellular ATP concentration. In the presence of 1 mM intracellular ATP, half‐maximal activation of KATP channels was obtained with extracellular application of 1.7 μM Zn2+. 5 Single channel analysis revealed that extracellular Zn2+ increased the KATP channel open‐state probability with no change in the single channel conductance. 6 Our data support the hypothesis that Zn2+ binding to KATP protein subunits results in an activation of the channels, therefore regulating the resting membrane potential and decreasing the excitability of RINm5F cells. Taken together, our results suggest that Zn2+ can influence insulin secretion in pancreatic β‐cells through a negative feedback loop, involving both KATP and voltage‐gated conductances.

Thiamine and Cholinergic Transmission in the Electric Organ of Torpedo

Abstract: The electric organ of Torpedo marmorata was found to contain as much as 120 ± 24 nmol of thiamine per g of fresh tissue. The vitamin was distributed as nonesterified thiamine (32%), thiamine monophosphate (22%), thiamine diphosphate (8%), and an important proportion of thiamine triphosphate (38%). A high level of thiamine triphosphate was found in synaptosomes isolated from the electric organ. In contrast, the synaptic vesicles did not show any enrichment in thiamine, whereas they contained a marked peak of acetylcholine (ACh) and ATP. Thus thiamine seems to be very abundant in cholinergic nerve terminals; its localization is apparently extravesicular, either in the axoplasm or in association with plasma membrane. When calcium was reduced and magnesium increased in the external medium, the efficiency of transmission was diminished, owing to inhibition of ACh release; in a parallel manner the degree of thiamine phosphorylation was found to increase—this condition is known to modify the repartition of ACh between vesicular and extravesicular compartments. Electrical stimulation, which causes periodic variations of the level of ACh and ATP, also caused significant changes in thiamine esters. In addition, related changes of the vitamin and the transmitter were observed under other conditions, suggesting a functional link between the metabolism of thiamine and that of ACh in cholinergic nerve terminals.

Neurotransmitter release through the V0 sector of V-ATPase

Neurotransmitter release occurs at specialized areas of the nerve terminal membrane, the active zones, where clusters of synaptic vesicles, the neurotransmitter-storing organelles, are observed (Couteaux and PeÂcot-Dechavassine 1974; Harlow et al. 2001). In resting conditions, a population of synaptic vesicles is docked to the active zone membrane, close to voltage-gated calcium channels (Robitaille et al. 1990), within microdomains where, upon stimulation, cytosolic calcium reaches transiently a very high concentration (Llinas et al. 1992). In spite of the high specialization of the active zone structure and high speed of synaptic transmission, proteins involved in docking and fusion of synaptic vesicles are similar to those operating for much slower membrane fusions, from yeast to neurones (Wickner and Haas 2000). In this respect, the role of SNARE complexes for docking synaptic vesicles at the active zones has been well documented (Rothman 1994; Jahn and SuÈdhof 1999). A detailed genetic and pharmacological dissection of yeast homotypic vacuole fusion revealed the existence, after vacuole docking by trans-SNARE complex formation, of a Ca/calmodulin reaction preceeding the ®nal microcystininhibited step of membrane fusion (Wickner and Haas 2000). Recently, Peters et al. (2001) showed that it was the proteolipids of the membrane sector (V0) of V-ATPase which bind to calmodulin and initiate the ®nal step of membrane fusion. The vacuolar-type H-ATPase is indeed composed of a proteolipid membrane sector (V0) and a catalytic sector (V1). The association between V0 and V1 is reversible and participates in the regulation of proton pumping (Nelson and Harvey 1999). Reconstituted V0 proteolipids form a pore that opens in the presence of calcium and calmodulin. During the fusion of two yeast vacuoles, a V0 trans-complex is formed by the apposition of two proteolipid rings, brought into close contact by the SNARE proteins. The V0 trans-complex may therefore form a proteolipid channel spanning the two interacting membranes at the fusion site (Peters et al. 2001). We would like to discuss the relevance of this model for neurotransmitter release. Synaptosomal membranes were shown to contain a proteolipid oligomer that supported a calcium-dependent release of acetylcholine (ACh) when reconstituted in arti®cial membranes (IsraeÈl et al. 1986; see Fig. 1). This oligomer (mediatophore) turned out to be made of the proteolipid c subunit of V-ATPase (Birman et al. 1990). When cells were transfected for this proteolipid, they acquired a Ca-dependent ACh release mechanism that displayed quantal properties (Falk-Vairant et al. 1996; see Fig. 2). Such reconstitution experiments, using liposomes, transfected cells or Xenopus oocytes (Cavalli et al. 1993), showed that a single proteolipid ring not only opens upon calcium action but is suf®cient to let ACh out down its concentration gradient. This was con®rmed by Peters et al. (2001) who measured the release of choline through reconstituted yeast V-ATPase proteolipids, release that required Ca and, in this case, calmodulin. In synapses, the neurotransmitter is pre-concentrated in synaptic vesicles. This process depends on the proton gradient generated by the V-ATPase, and is blocked by N-N 0-dicyclohexylcarbodiimide (DCCD). In contrast, the ef ̄ux of ACh from already loaded synaptic vesicles is not affected by DCCD (Dolezal et al. 1993). This illustrates that ACh and protons follow different routes. Protons bind to a glutamic residue facing the exterior of the proteolipid ring (Harrison et al. 2000) and are translocated during the ATPdriven rotation of this ring (see Nelson and Harvey 1999 for a review on V-ATPases). ACh is most probably released through a pore found in the middle of the proteolipid oligomer by Jones et al. (1995).

Space and time characteristics of transmitter release at the nerve-electroplaque junction of Torpedo.

1. A loose patch electrode was used to stimulate axon terminals and to record evoked electroplaque currents (EPCs) in a limited area of innervated membrane of the electric organ of Torpedo marmorata. Electrophysiological signals were compared to the predictions of a semi‐quantitative model of synaptic transmission which was designed to simulate the release of several packets of neurotransmitter molecules, at the same or at different sites of the synapse, synchronously or with various temporal patterns. 2. The amplitude distribution of EPCs evoked by activation of nerve terminals showed quantal steps. The time to peak of EPCs was in most cases independent of amplitude, but in their decaying phase a positive correlation was seen between half‐decay time and amplitude. Comparison with the model suggested that (i) a dynamic interaction occurred at the end of the EPC between the fields of postsynaptic membrane activated by individual quanta, and (ii) the sites of quantal release in the electric organ are separated from each other by 600‐1000 nm. 3. Spontaneous miniature electroplaque potentials (MEPPs) were recorded externally with the same type of loose patch electrode. The majority (75%) of external MEPPs displayed a homogeneous and rapid time course. This fast MEPP population had a mean time to peak of 0.43 ms, a half‐decay time of 0.45 ms and a time constant of decay of 0.35 ms. 4. Despite homogeneous characteristics of time course, fast MEPPs exhibited a wide amplitude distribution with a main population which could be fitted by a Gaussian curve around 1 mV, and another population of small amplitude. Both the time‐to‐peak and the half‐decay time of fast MEPPs showed a positive correlation with the amplitude from the smallest to the largest events. Acetylcholinesterase was not blocked. 5. In addition to the fast MEPPs, spontaneous signals exhibiting a slow rate of rise, or a slow rate of decay, or both were observed. They occurred at any time during the experiment, independently of the overall frequency. Approximately 15% of the total number of events had a slow rise but their decay phase was nevertheless rapid and could be ascribed to the kinetics of receptors. These slow‐rising MEPPs exhibited a variety of conformations: slow but smooth rise, sudden change of slope and sometimes several bumps or inflexions. Their average amplitude was significantly smaller than that of the main population of fast MEPPs. 6. Composite MEPPs with multiple peaks as well as bursts of small MEPPs were often encountered, even during periods of low frequency.(ABSTRACT TRUNCATED AT 400 WORDS)

Antisense Probes Against Mediatophore Block Transmitter Release in Oocytes Primed with Neuronal mRNAs

Antisense oligodesoxynucleotides were used to determine whether the mediatophore proteolipid is necessary for the Ca2+‐dependent release of the neurotransmitter acetylcholine. Xenopus laevis oocytes were injected with poly(A)+ mRNAs extracted from the electric lobes of Torpedo marmorata. The electric lobes contain an homogeneous population of cholinergic neurons homologous to motoneurons. Addition of antisense probes hybridizing to the mediatophore 15 kDa subunit inhibited the expression of both the mediatophore proteolipid in oocyte membranes and the Ca2+‐dependent acetylcholine release. Expression of other neuronal functions such as synthesis of [14C]acetylcholine from [14C]acetate was not inhibited. Another antisense probe specific for the sequence of a related proteolipid cDNA (the 15 kDa subunit of the chromaffin granule protonophore) was used as a control. It did not hybridize with the Torpedo mediatophore mRNA and, injected in addition to electric lobe mRNAs, it did not inhibit either mediatophore expression or acetylcholine release. We showed in addition that the mRNA primed oocytes did not contain a vesicular pool of acetylcholine. It was concluded (i) that the mediatophore proteolipid is essential for Ca2+‐dependent acetylcholine release and (ii) that the cytosolic pool of neurotransmitter seems to be preferentially used in this system.

Acetylcholine changes underlying transmission of a single nerve impulse in the presence of 4‐aminopyridine in Torpedo

1. Transmission of a single nerve impulse has been investigated at the nerve—electroplaque junction of Torpedo marmorata in the presence of 4‐aminopyridine (4‐AP), a drug which powerfully potentiates evoked transmitter release.

Neurotransmitter release at rapid synapses

The classical concept of the vesicular hypothesis for acetylcholine (ACh) release, one quantum resulting from exocytosis of one vesicle, is becoming more complicated than initially thought. 1) synaptic vesicles do contain ACh, but the cytoplasmic pool of ACh is the first to be used and renewed on stimulation. 2) The vesicles store not only ACh, but also ATP and Ca(2+) and they are critically involved in determining the local Ca(2+) microdomains which trigger and control release. 3) The number of exocytosis pits does increase in the membrane upon nerve stimulation, but in most cases exocytosis happens after the precise time of release, while it is a change affecting intramembrane particles which reflects more faithfully the release kinetics. 4) The SNARE proteins, which dock vesicles close to Ca(2+) channels, are essential for the excitation-release coupling, but quantal release persists when the SNAREs are inactivated or absent. 5) The quantum size is identical at the neuromuscular and nerve-electroplaque junctions, but the volume of a synaptic vesicle is eight times larger in electric organ; at this synapse there is enough ACh in a single vesicle to generate 15-25 large quanta, or 150-200 subquanta. These contradictions may be only apparent and can be resolved if one takes into account that an integral plasmalemmal protein can support the formation of ACh quanta. Such a protein has been isolated, characterised and called mediatophore. Mediatophore has been localised at the active zones of presynaptic nerve terminals. It is able to release ACh with the expected Ca(2+)-dependency and quantal character, as demonstrated using mediatophore-transfected cells and other reconstituted systems. Mediatophore is believed to work like a pore protein, the regulation of which is in turn likely to depend on the SNARE-vesicle docking apparatus.

Thiamine and cholinergic transmission in the electric organ of Torpedo. II. Effects of exogenous thiamine and analogues on acetylcholine release.

Abstract: The electrogenic tissue of Torpedo was found to phosphorylate in vitro external [14C]thiamine by a saturable process. The rate of this metabolisation was increased when acetate, an efficient precursor of acetylcholine (ACh) synthesis in this tissue, was added to the incubation medium, thus increasing the turnover of ACh. Nerve stimulation did not release significant amounts of the previously accumulated [14C]thiamine. Exogenous thiamine modified the size of the electroplaque potential (e.p.p.). At concentrations higher than 10−3 M the nerve‐electroplaque transmission was depressed after a transient increase of the e.p.p. Such a depression was due to a strong decrease of the ACh release. At concentrations equal to or lower than 10−3 M, thiamine affected transmission in a rather complex fashion. ACh release was decreased or increased depending on concentration, time of application, and mode of stimulation. Oxythiamine, a structural antimetabolite of thiamine, affected the transmission in a very characteristic manner at 10−5 M and higher concentrations. The amplitude of the e.p.p. was increased and, more strikingly, its duration was prolonged. These changes were not due to an inhibition of cholinesterase activity but to an enhancement of the evoked release of ACh either on single‐impulse or repetitive stimulation. Another antimetabolite, pyrithiamine, had no effect on the transmission nor on ACh release. From this and our previous work, it is proposed that thiamine is involved, directly or indirectly, in the process of ACh release. The possible mechanisms of this involvement are discussed.

Acetylcholine measured at short time intervals during transmission of nerve impulses in the electric organ of Torpedo

1. The amounts of total acetylcholine (ACh) and ATP, and of vesicle‐bound ACh were measured at short time intervals in the electrogenic tissue of Torpedo marmorata. The aim of this study is to approach with biochemical analysis the speed of electrophysiological phenomena.