Low- and High-Affinity Reactions in Rapid Neurotransmission

Until 1950-1960, most physiologists were reluctant to accept chemical neurotransmission. They believed that chemical reactions were much too slow to account for the speed of synaptic processes. The first breakthrough was to discover the impressive velocity of acetylcholinesterase. Then, nicotinic receptors provided an example of complex ultrarapid reactions: fast activation at a low ligand affinity, then desensitization if the ligand is not rapidly removed. Here, we describe synaptic transmission as a chain of low-affinity rapid reactions, assisted by many slower regulatory processes. For starting quantal acetylcholine release, mediatophores are activated by high Ca2+ concentrations, but they desensitize at a higher affinity if Ca2+ remains present. Several mechanisms concur to the rapid removal of Ca2+ from the submembrane microdomains. Among them, the Ca2+/H+ antiport is a typical low-affinity, high-speed process that certainly contributes to the rapidity of neurotransmissio

Nicotine-induced and depolarisation-induced glutamate release from hippocampus mossy fibre synaptosomes: two distinct mechanisms

Hippocampus mossy fibre terminals activate CA3 pyramidal neurons via two distinct mechanisms, both quantal and glutamatergic: (i) rapid excitatory transmission in response to afferent action potentials and (ii) delayed and prolonged release following nicotinic receptor activation. These processes were analysed here using rat hippocampus mossy fibres synaptosomes. The relationships between synaptosome depolarisation and glutamate release were established in response to high-KCl and gramicidin challenges. Half-maximal release corresponded to a 52 mV depolarisation step. KCl-induced release was accompanied by transient dissipation of the proton gradient across synaptic vesicle membrane. Nicotine elicited a substantial glutamate release from mossy fibre synaptosomes (EC(50) 3.14 microM; V(max) 12.01 +/- 2.1 nmol glutamate/mg protein; Hill's coefficient 0.99). However, nicotine-induced glutamate release was not accompanied by any change in the membrane potential or in the vesicular proton gradient. The effects of acetylcholine (200 microM) were similar to those of nicotine (25 microM). Nicotinic alpha7 receptors were evidenced by immuno-cytochemistry on the mossy fibre synaptosome plasma membrane. Therefore, the same terminals can release glutamate in response to two distinct stimuli: (i) rapid neurotransmission involving depolarisation-induced activation of voltage-gated Ca(2+) channels and (ii) a slower nicotinic activation which does not involve depolarisation or dissipation of the vesicular proton gradient

An invertebrate defense molecule activates membrane conductance in mammalian cells by means of its lectin-like domain

The invertebrate defense molecule Coelomic Cytolytic Factor-1 (CCF-1) and the mammalian cytokine Tumor Necrosis Factor (TNF) share a similar lectin-like domain that, upon interaction with specific sugars, causes lysis of African trypanosomes. In contrast to TNF, CCF-1 does not require an acidification of a lysosomal compartment for this activity. Moreover, we could demonstrate using the whole cell patch clamp technique that both TNF and CCF-1 activate amiloride-sensitive channels in mammalian cells, in a TNF receptor-independent way, but, unlike TNF, CCF-1 does not require acidic conditions for this activity. These data confirm the functional analogies of an invertebrate defense molecule and a mammalian cytokine, based on a similar lectin-like interaction. © 2001 Elsevier Science Ltd. All rights reserved.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

A simple, low-cost and fast Peltier thermoregulation set-up for electrophysiology

Most of the parameters recorded in electrophysiology are strongly temperature dependent. In order to control temperature fluctuations we have built a system that ensures an accurate thermoregulation of the recording chamber. Temperature of physiological preparations can be changed relatively quickly (about 8°C/min) and with a good accuracy (±0.5°C) without inducing thermal oscillations. Contrary to other thermoregulating devices, the temperature regulation is not carried out through the perfused medium but directly at the bottom of the chamber where a 3-cm2 Peltier element has been placed. The element is driven by a dedicated electronic device which controls the amount and the direction of the current flowing across the Peltier thermocouple. All construction details and the appropriate electrical circuits are provided. Using this home-made device, the steady-state chamber temperature could be precisely monitored with a resolution of ±0.1°C in a range of 0–40°C. This set-up was tested in experiments designed to evaluate the temperature dependence of synaptic transmission in the Torpedo nerve–electroplate synapses and of calcium currents recorded from isolated nerve cells. This low-cost method is suitable for a wide range of applications.</p

Membrane interaction of TNF is not sufficient to trigger increase in membrane conductance in mammalian cells

Tumor necrosis factor TNF can trigger increases in membrane conductance of mammalian cells in a receptor-independent manner via its lectin-like domain. A lectin-deficient TNF mutant, lacking this activity, was able to bind to artificial liposomes in a pH-dependent manner, but not to insert into the bilayer, just like wild type TNF. A peptide mimicking the lectin-like domain, which can still trigger increases in membrane currents in cells, failed to interact with liposomes. Thus, the capacity of TNF to trigger increases in membrane conductance in mammalian cells does not correlate with its ability to interact with membranes, suggesting that the cytokine does not form channels itself, but rather interacts with endogenous ion channels or with plasma membrane proteins that are coupled to ion channels

Severe congenital hyperinsulinism caused by a mutation in the Kir6.2 subunit of the adenosine triphosphate-sensitive potassium channel impairing trafficking and function

The ATP-sensitive potassium (K(ATP)) channel, assembled from the inwardly rectifying potassium channel Kir6.2 and the sulfonylurea receptor 1, regulates insulin secretion in beta-cells. A loss of function of K(ATP) channels causes depolarization of beta-cells and congenital hyperinsulinism (CHI), a disease presenting with severe hypoglycemia in the newborn period

Defective tumor necrosis factor-alpha-dependent control of astrocyte glutamate release in a transgenic mouse model of Alzheimer disease.

The cytokine tumor necrosis factor-alpha (TNFalpha) induces Ca2+-dependent glutamate release from astrocytes via the downstream action of prostaglandin (PG) E2. By this process, astrocytes may participate in intercellular communication and neuromodulation. Acute inflammation in vitro, induced by adding reactive microglia to astrocyte cultures, enhances TNFalpha production and amplifies glutamate release, switching the pathway into a neurodamaging cascade (Bezzi, P., Domercq, M., Brambilla, L., Galli, R., Schols, D., De Clercq, E., Vescovi, A., Bagetta, G., Kollias, G., Meldolesi, J., and Volterra, A. (2001) Nat. Neurosci. 4, 702-710). Because glial inflammation is a component of Alzheimer disease (AD) and TNFalpha is overexpressed in AD brains, we investigated possible alterations of the cytokine-dependent pathway in PDAPP mice, a transgenic model of AD. Glutamate release was measured in acute hippocampal and cerebellar slices from mice at early (4-month-old) and late (12-month-old) disease stages in comparison with age-matched controls. Surprisingly, TNFalpha-evoked glutamate release, normal in 4-month-old PDAPP mice, was dramatically reduced in the hippocampus of 12-month-old animals. This defect correlated with the presence of numerous beta-amyloid deposits and hypertrophic astrocytes. In contrast, release was normal in cerebellum, a region devoid of beta-amyloid deposition and astrocytosis. The Ca2+-dependent process by which TNFalpha evokes glutamate release in acute slices is distinct from synaptic release and displays properties identical to those observed in cultured astrocytes, notably PG dependence. However, prostaglandin E2 induced normal glutamate release responses in 12-month-old PDAPP mice, suggesting that the pathology-associated defect involves the TNFalpha-dependent control of secretion rather than the secretory process itself. Reduced expression of DENN/MADD, a mediator of TNFalpha-PG coupling, might account for the defect. Alteration of this neuromodulatory astrocytic pathway is described here for the first time in relation to Alzheimer disease

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

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.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.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.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+.Single channel analysis revealed that extracellular Zn2+ increased the KATP channel open-state probability with no change in the single channel conductance.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

The lectin-like domain of tumor necrosis factor-α increases membrane conductance in microvascular endothelial cells and peritoneal macrophages

Herein, we show that TNF exerts a pH-dependent increase in membrane conductance in primary lung microvascular endothelial cells and peritoneal macrophages. This effect was TNF receptor-independent, since it also occurred in cells isolated from mice deficient in both types of TNF receptors. A TNF mutant in which the three amino acids critical for the lectin-like activity were replaced by an alanine did not show any significant effect on membrane conductance. Moreover, a synthetic 17-amino acid peptide of TNF, which was previously shown to exert lectin-like activity, also increased the ion permeability in these cells. The amiloride sensitivity of the observed activity suggests a binding of TNF to an endogenousion channel rather than channel formation by TNF itself. This may have important implications in mechanisms of TNF-mediated vascular pathology