Benzamil inhibits neuronal and heterologously expressed small conductance Ca2+-activated K+ channels.

Small conductance Ca2+-activated K+ (SK) channels are expressed throughout the soma and dendrites of pyramidal neurons in the neocortex and hippocampal formation, where they participate in the local regulation of membrane excitability and synaptic signals. Through their inter-play with Ca2+ channels, SK channels regulate Ca2+ influx triggered by back-propagating action potentials in dendrites. Inhibition of SK channels affects both the amplitude and duration of Ca2+ transients, but the role of Ca2+ clearance mechanisms and their link to SK channel activity has not been established. Here we report the effect of the Na+/Ca2+ exchanger (NCX) inhibitor benzamil on Ca2+ extrusion and SK channels in the regulation of dendritic Ca2+ signals. Benzamil increased the duration and amplitude of dendritic Ca2+ transients elicited by back-propagating action potentials in hippocampal pyramidal neurons. This data is consistent with previous studies with SK channel blockers and suggests that benzamil inhibits SK channels in addition to the Na+/Ca2+ exchanger. Here we show that indeed both the neuronal SK-mediated IAHP current and the currents mediated by heterologously expressed SK channels were inhibited by benzamil. The inhibition of recombinant SK channels was seen with different K+ concentration gradients, and was stronger at negative voltages. The suppression of SK channels by benzamil is consistent with previous findings on the modulation of Ca2+ signals by SK channels in neurons. We additionally show that benzamil inhibits neuronal voltage-gated calcium currents. The results prompt a careful reassessment of the effects of benzamil on Ca2+ transients in native systems, given the spectrum of ion channels and exchangers this compound targets within a similar range of concentrations.

A role for the Drosophila zinc transporter Zip88E in protecting against dietary zinc toxicity.

Zinc absorption in animals is thought to be regulated in a local, cell autonomous manner with intestinal cells responding to dietary zinc content. The Drosophila zinc transporter Zip88E shows strong sequence similarity to Zips 42C.1, 42C.2 and 89B as well as mammalian Zips 1, 2 and 3, suggesting that it may act in concert with the apically-localised Drosophila zinc uptake transporters to facilitate dietary zinc absorption by importing ions into the midgut enterocytes. However, the functional characterisation of Zip88E presented here indicates that Zip88E may instead play a role in detecting and responding to zinc toxicity. Larvae homozygous for a null Zip88E allele are viable yet display heightened sensitivity to elevated levels of dietary zinc. This decreased zinc tolerance is accompanied by an overall decrease in Metallothionein B transcription throughout the larval midgut. A Zip88E reporter gene is expressed only in the salivary glands, a handful of enteroendocrine cells at the boundary between the anterior and middle midgut regions, and in two parallel strips of sensory cell projections connecting to the larval ventral ganglion. Zip88E expression solely in this restricted subset of cells is sufficient to rescue the Zip88E mutant phenotype. Together, our data suggest that Zip88E may be functioning in a small subset of cells to detect excessive zinc levels and induce a systemic response to reduce dietary zinc absorption and hence protect against toxicity.

A fly's eye view of zinc homeostasis: Novel insights into the genetic control of zinc metabolism from Drosophila.

The core zinc transport machinery is well conserved between invertebrates and mammals, with the vinegar fly Drosophila melanogaster having clear homologues of all major groups of mammalian ZIP and ZNT transport genes. Functional characterization of several of the fly genes has revealed functional conservation between related fly and mammalian zinc transporters in some but not all cases, indicating that Drosophila is a useful model for examining mammalian zinc metabolism. Furthermore, Drosophila research, sometimes quite serendipitously, has provided novel insights into the function of zinc transporters and into zinc-related pathologies, which are highlighted here. Finally, the future research potential of the fly in nutrient metabolism is explored, with reference to emerging experimental technologies.

A role for dZIP89B in Drosophila dietary zinc uptake reveals additional complexity in the zinc absorption process.

Dietary zinc is the principal source of zinc in eukaryotes, with its uptake and distribution controlled by a complex network of numerous membrane-spanning transport proteins. Dietary absorption is achieved by members of the SLC39A (ZIP) gene family, which encode proteins that are generally responsible for the movement of zinc into the cytosol. ZIP4 is thought to be the primary mammalian zinc uptake gene in the small intestine, with mutations in this gene causing the zinc deficiency disease Acrodermatitis enteropathica. In Drosophila, dual knockdown of the major dietary zinc uptake genes dZIP42C.1 (dZIP1) and dZIP42C.2 (dZIP2) results in a severe sensitivity to zinc-deficient media. However, the symptoms associated with ZIP4 loss can be reversed by zinc supplementation and dZIP42C.1 and 2 knockdown has minimal effect under normal dietary conditions, suggesting that additional pathways for zinc absorption exist in both mammals and flies. This study provides evidence that dZIP89B is an ideal candidate for this role in Drosophila, encoding a low-affinity zinc uptake transporter active in the posterior midgut. Flies lacking dZIP89B, while viable and apparently healthy, show indications of low midgut zinc levels, including reduced metallothionein B expression and compensatory up-regulation of dZIP42C.1 and 2. Furthermore dZIP89B mutants display a dramatic resistance to toxic dietary zinc levels which is abrogated by midgut-specific restoration of dZIP89B activity. We postulate that dZIP89B works in concert with the closely related dZIP42C.1 and 2 to ensure optimal zinc absorption under a range of dietary conditions.

Local and systemic effects of targeted zinc redistribution in Drosophila neuronal and gastrointestinal tissues.

While the effects of systemic zinc ion deficiency and toxicity on animal health are well documented, the impacts of localized, tissue-specific disturbances in zinc homeostasis are less well understood. Previously we have identified zinc dyshomeostasis scenarios caused by the targeted manipulation of zinc transport genes in the Drosophila eye. Over expression of the uptake transporter dZIP42C.1 (dZIP1) combined with knockdown of the efflux transporter dZNT63C (dZNT1) causes a zinc toxicity phenotype, as does over expression of dZIP71B or dZNT86D. However, all three genotypes result in different morphologies, responses to dietary zinc, and genetic interactions with the remaining zinc transport genes, indicating that each causes a different redistribution of zinc within affected cells. dZNT86D (eGFP) over expression generates a completely different phenotype, interpreted as a Golgi zinc deficiency. Here we assess the effect of each of these transgenes when targeted to a range of Drosophila tissues. We find that dZIP71B is a particularly potent zinc uptake gene, causing early developmental lethality when targeted to multiple different tissue types. dZNT86D over expression (Golgi-only zinc toxicity) is less deleterious, but causes highly penetrant adult cuticle, sensory bristle and wing expansion defects. The dZIP42C.1 over expression, dZNT63C knockdown combination causes only moderate adult cuticle defects and sensitivity to dietary zinc when expressed in the midgut. The Golgi-only zinc deficiency caused by dZNT86D (eGFP) expression results in mild cuticle defects, highly penetrant wing expansion defects and developmental lethality when targeted to the central nervous system and, uniquely, the fat bodies.

Compartmentalized zinc deficiency and toxicities caused by ZnT and Zip gene over expression result in specific phenotypes in Drosophila.

Movement of zinc ions across cellular membranes is achieved mainly by two families of zinc transport genes encoding multi-transmembrane domain proteins. Members of the Zip family generally transport zinc into the cytosol, either from outside the cell or from the lumen of subcellular organelles such as the endoplasmic reticulum, Golgi, endosomes or storage vacuoles. ZnT proteins move zinc in the opposite direction, resulting in efflux from the cell or uptake into organelles. Zinc homeostasis at both the cellular and systemic level is achieved by the coordinated action of numerous Zip and ZnT proteins, twenty-four in mammals and seventeen in the vinegar fly Drosophila melanogaster. Previously, we have identified a zinc toxicity phenotype in the Drosophila eye, caused by targeted over expression of dZip42C.1 (dZip1) combined with knockdown of dZnT63C (dZnT1). In general, this phenotype was rescued by increased zinc efflux or decreased uptake and was exacerbated by decreased efflux or increased uptake. Now we have identified three additional zinc dyshomeostasis phenotypes caused by over expression of dZnT86D, dZnT86D(eGFP) and dZip71B(FLAG). Genetic and dietary manipulation experiments showed that these three phenotypes all differ both from each other and from our original zinc toxicity phenotype. Based on these data and the approximate subcellular localization of each zinc transport protein, we propose that each phenotype represents a different redistribution of zinc within these cells, resulting in a Golgi zinc toxicity, a Golgi zinc deficiency and a combined Golgi/other organelle zinc toxicity respectively. We are able to group the remaining Drosophila Zip and ZnT genes into several functional categories based on their interaction with the three novel zinc dyshomeostasis phenotypes, allowing the role of each zinc transport protein to be defined in greater detail. This research highlights the differential effects that redistribution of zinc can have within a particular tissue and identifies the Golgi as being particularly sensitive to both excess and insufficient zinc.

In vivo zinc toxicity phenotypes provide a sensitized background that suggests zinc transport activities for most of the Drosophila Zip and ZnT genes.

Members of the ZIP (SLC39A) and ZnT (SLC30A) families of transmembrane domain proteins are predicted to transport the essential transition metal zinc across membranes, regulating cellular zinc content and distribution via uptake and efflux at the outer plasma and organellar membranes. Twenty-four ZIP and ZnT proteins are encoded in mammalian genomes, raising questions of whether all actually transport zinc, whether several function together in the same tissues/cell types, and how the activity of these transporters is coordinated. To address these questions, we have taken advantage of the ability to manipulate several genes simultaneously in targeted cell types in Drosophila. Previously we reported zinc toxicity phenotypes caused by combining overexpression of a zinc uptake gene, dZip42C.1, with suppression of a zinc efflux gene, dZnT63C. Here we show that these phenotypes can be used as a sensitized in vivo system to detect subtle alterations in zinc transport activity that would be buffered in healthy cells. Using two adult tissues, the fly eye and midline (thorax/abdomen), we find that when overexpressed, most of the 17 Drosophila Zip and ZnT genes modify the zinc toxicity phenotypes in a manner consistent with their predicted zinc transport activity. In most cases, we can reconcile that activity with the cellular localization of an enhanced green fluorescent protein tagged version of the protein. Additionally, targeted suppression of each gene by RNA interference reveals several of the fly Zip and ZnT genes are required in the eye, indicating that numerous independent zinc transport genes are acting together in a single tissue.

Small-conductance Ca2+-activated K+ channels modulate action potential-induced Ca2+ transients in hippocampal neurons.

In hippocampal pyramidal neurons, voltage-gated Ca(2+) channels open in response to action potentials. This results in elevations in the intracellular concentration of Ca(2+) that are maximal in the proximal apical dendrites and decrease rapidly with distance from the soma. The control of these action potential-evoked Ca(2+) elevations is critical for the regulation of hippocampal neuronal activity. As part of Ca(2+) signaling microdomains, small-conductance Ca(2+)-activated K(+) (SK) channels have been shown to modulate the amplitude and duration of intracellular Ca(2+) signals by feedback regulation of synaptically activated Ca(2+) sources in small distal dendrites and dendritic spines, thus affecting synaptic plasticity in the hippocampus. In this study, we investigated the effect of the activation of SK channels on Ca(2+) transients specifically induced by action potentials in the proximal processes of hippocampal pyramidal neurons. Our results, obtained by using selective SK channel blockers and enhancers, show that SK channels act in a feedback loop, in which their activation by Ca(2+) entering mainly through L-type voltage-gated Ca(2+) channels leads to a reduction in the subsequent dendritic influx of Ca(2+). This underscores a new role of SK channels in the proximal apical dendrite of hippocampal pyramidal neurons.

Systematic functional characterization of putative zinc transport genes and identification of zinc toxicosis phenotypes in Drosophila melanogaster.

The heavy metal zinc is an essential component of the human diet and is incorporated as a structural component in up to 10% of all mammalian proteins. The physiological importance of zinc homeostasis at the cellular level and the molecular mechanisms involved in this process have become topics of increasing interest in recent years. We have performed a systematic functional characterization of the majority of the predicted Drosophila Zip (zinc/iron regulated transporter-related protein) and ZnT genes, using the Gal4-UAS system to carry out both ubiquitous and targeted over-expression and suppression studies for 13 of the 17 putative zinc transport genes identified to date. We found that six of these 13 genes may be essential for fly viability and that three of the remaining seven demonstrate over-expression phenotypes. Our findings reaffirm the previously proposed function of dZnT63C (CG17723: FBgn005432) as an important zinc efflux protein and indicate that the fly homolog of hZip1, dZip42C.1 (CG9428: FBgn0033096), is a strong zinc importer in Drosophila. By combining over-expression of dZip42C.1 with suppression of dZnT63C we were able to produce easily identifiable zinc toxicosis phenotypes, which can be rescued or worsened by modifying dietary zinc content. Our findings show that a genetically based zinc toxicosis situation can be therapeutically treated or exacerbated by modifications to the diet, providing a sensitized background for future, more detailed studies of Zip/ZnT function.

Fast vesicle replenishment allows indefatigable signalling at the first auditory synapse.

Ribbon-type synapses in inner hair cells of the mammalian cochlea encode the complexity of auditory signals by fast and tonic release through fusion of neurotransmitter-containing vesicles. At any instant, only about 100 vesicles are tethered to the synaptic ribbon, and about 14 of these are docked to the plasma membrane, constituting the readily releasable pool. Although this pool contains about the same number of vesicles as that of conventional synapses, ribbon release sites operate at rates of about two orders of magnitude higher and with submillisecond precision. How these sites replenish their vesicles so efficiently remains unclear. We show here, using two-photon imaging of single release sites in the intact cochlea, that preformed vesicles derived from cytoplasmic vesicle-generating compartments participate in fast release and replenishment. Vesicles were released at a maximal initial rate of 3 per millisecond during a depolarizing pulse, and were replenished at a rate of 1.9 per millisecond. We propose that such rapid resupply of vesicles enables temporally precise and sustained release rates. This may explain how the first auditory synapse can encode with indefatigable precision without having to rely on the slow, local endocytic vesicle cycle.

Pentobarbitone modulates calcium transients in axons and synaptic boutons of hippocampal CA1 neurons.

Although barbiturates, like other general anaesthetics, depress excitatory synaptic transmission in the central nervous system (CNS), the underlying cellular mechanisms remain unresolved. They may increase the likelihood that an action potential will fail to invade every branch of the axonal arbour, thereby decreasing the synaptic drive to the postsynaptic neurons. Alternatively, they may inhibit calcium entry into the presynaptic terminals, thus reducing transmitter release. To resolve these issues, we have used two-photon microscopy to monitor calcium transients evoked by action potentials in axons, axonal varicosities (synaptic boutons) and fine axon collaterals of hippocampal CA1 neurons. Pentobarbitone (75-300 microM) did not block the invasion of the axonal arbour or the synaptic boutons, but it did reduce the amplitude of the calcium transients recorded from the axons in a concentration-dependent manner. At 150 microM, pentobarbitone reduced the transients to 78+/-4% of the control. Pentobarbitone depressed the calcium transients recorded from the synaptic boutons in a concentration-dependent manner. When 150 microM pentobarbitone was applied, the calcium transients recorded from the boutons were 53+/-3% of the control. This concentration of pentobarbitone also reduced the amplitude and frequency of the spontaneous excitatory postsynaptic potentials to 54+/-4 and 42+/-17% of the control, respectively. The local anaesthetic procaine (500 microM) had no significant effect on action potential invasion of axon collaterals, even though it reduced the action potential amplitude by 25%. This data are consistent with the notion that the pentobarbitone-induced depression of presynaptic calcium transients contributes to its depressant effect on excitatory synaptic transmission in the CNS.