Neutralizing aspartate 83 modifies substrate translocation of excitatory amino acid transporter 3 (EAAT3) glutamate transporters

Excitatory amino acid transporters (EAATs) terminate glutamatergic synaptic transmission by removing glutamate from the synaptic cleft into neuronal and glial cells. EAATs are not only secondary active glutamate transporters but also function as anion channels. Gating of EAAT anion channels is tightly coupled to transitions within the glutamate uptake cycle, resulting in Na(+)- and glutamate-dependent anion currents. A point mutation neutralizing a conserved aspartic acid within the intracellular loop close to the end of transmembrane domain 2 was recently shown to modify the substrate dependence of EAAT anion currents. To distinguish whether this mutation affects transitions within the uptake cycle or directly modifies the opening/closing of the anion channel, we used voltage clamp fluorometry. Using three different sites for fluorophore attachment, V120C, M205C, and A430C, we observed time-, voltage-, and substrate-dependent alterations of EAAT3 fluorescence intensities. The voltage and substrate dependence of fluorescence intensities can be described by a 15-state model of the transport cycle in which several states are connected to branching anion channel states. D83A-mediated changes of fluorescence intensities, anion currents, and secondary active transport can be explained by exclusive modifications of substrate translocation rates. In contrast, sole modification of anion channel opening and closing is insufficient to account for all experimental data. We conclude that D83A has direct effects on the glutamate transport cycle and that these effects result in changed anion channel function.     

The secretory granule protein syncollin localizes to HL-60 cells and neutrophils.

The secretory granule protein syncollin was first identified in the exocrine pancreas where a population of the protein is associated with the luminal surface of the zymogen granule membrane. In this study we provide first morphological and biochemical evidence that, in addition to its pancreatic localization, syncollin is also present in neutrophilic granulocytes of rat and human origin. By immunohistological studies, syncollin was detected in neutrophilic granulocytes of the spleen. Furthermore, syncollin is expressed by the promyelocytic HL-60 cells, where it is stored in azurophilic granules and in a vesicular compartment. These findings were confirmed by fractionation experiments and immunoelectron microscopy. Treatment with a phorbol ester triggered the release of syncollin indicating that in HL-60 cells it is a secretory protein that can be mobilized upon stimulation. A putative role for syncollin in host defense is discussed.     

<Emphasis Type="Italic">Tuamotuichthys bispinosus</Emphasis>, a new genus and species from off Tuamotu Islands,South Pacific Ocean (Ophiidiformes,Bythitidae)

A new bythitid genus and species, Tuamotuichthys bispinosus, is described from an adult male (88mm SL), caught by dredge in 1990, off Morane Atoll, Tuamotu Archipelago (23°0843 S, 137°0919 W, 536m). It is characterized by having large scales on head and body, two opercular spines, few gill rakers (total 11, 3 of them long), depressed head, vertically expanded posterior maxilla, and a short predorsal (29% SL), each of which resembles several known genera (Calamopteryx, Cataetyx, Diplacanthopoma, Grammonus, and Lucifuga). The combination of character states, however, does not fit any of the known bythitid genera.     

Functional Properties of the Retinal Glutamate Transporters GLT-1c and EAAT5

In the mammalian retina, glutamate uptake is mediated by members of a family of glutamate transporters known as “excitatory amino acid transporters (EAATs).” Here we cloned and functionally characterized two retinal EAATs from mouse, the GLT-1/EAAT2 splice variant GLT-1c, and EAAT5. EAATs are glutamate transporters and anion-selective ion channels, and we used heterologous expression in mammalian cells, patch-clamp recordings and noise analysis to study and compare glutamate transport and anion channel properties of both EAAT isoforms. We found GLT-1c to be an effective glutamate transporter with high affinity for Na⁺ and glutamate that resembles original GLT-1/EAAT2 in all tested functional aspects. EAAT5 exhibits glutamate transport rates too low to be accurately measured in our experimental system, with significantly lower affinities for Na⁺ and glutamate than GLT-1c. Non-stationary noise analysis demonstrated that GLT-1c and EAAT5 also differ in single-channel current amplitudes of associated anion channels. Unitary current amplitudes of EAAT5 anion channels turned out to be approximately twice as high as single-channel amplitudes of GLT-1c. Moreover, at negative potentials open probabilities of EAAT5 anion channels were much larger than for GLT-1c. Our data illustrate unique functional properties of EAAT5, being a low-affinity and low-capacity glutamate transport system, with an anion channel optimized for anion conduction in the negative voltage range.     

A new species of viviparous brotula genus Pseudogilbia (Ophidiiformes: Dinematichthyidae) from Brazilian reefs,with an updated diagnosis of the genus

In this study, a new species of Pseudogilbia Møller, Schwarzhans & Nielsen 2004 is described based on two male specimens (40–44 mm L_S) from shallow reefs of Bahia, Brazil. Pseudogilbia australis sp. nov. is distinguished from its only congener, Pseudogilbia sanblasensis Møller, Schwarzhans & Nielsen 2004 from Caribbean Panama, by having: two lower preopercular pores (vs. one); dorsal-fin rays 65–67 (vs. 69); anal-fin rays 51–53 (vs. 56); pectoral-fin rays 18 (vs. 20); caudal vertebrae 27–28 (vs. 30); pectoral-fin length 15.0%–15.9% L_S (vs. 14.3); pelvic-fin length 13.5% L_S (vs. 16.4) and a different morphology of the male copulatory organ. Pseudogilbia australis sp. nov. is the only dinematichthyid so far recorded in the South Atlantic. An updated diagnosis for the genus is also provided.     

Noise analysis to study unitary properties of transporter-associated ion channels

Excitatory amino acid transporters (EAATs) do not only mediate secondary-active glutamate uptake but also function as anion channels. We recently used macroscopic current recordings and noise analysis to determine unitary current amplitudes of anion channels associated with a neuronal EAAT isoform, EAAT4. We found that, at symmetrical NO(3)(-), EAAT4 anion channels exhibit a single channel conductance of ~1 pS in the absence as well as in the presence of glutamate. These results indicate that glutamate increases EAAT4 anion currents by modifying exclusively open probabilities, however, leaves unitary current amplitudes unaffected. Noise analysis has been developed for ion channels with a single conductance state and limitations might ensue when using this approach for transporter-associated ion channels. We here performed stochastic simulations of EAAT transporter-associated anion channels and noise analysis of simulated currents to assess the reliability and possible limitations of this technique in studying this special class of ion channels.