Kinetic Properties of Glutamate Receptor Channels in Cultured Embryonic Drosophila Myotubes.

Abstract

Glutamate receptor channels are ubiquitous agonist-gated channels. Pharmacologically they are classified into several subtypes but may have common fundamental channel properties. To build foundation for future molecular biological and genetic studies, we studied kinetics of the glutamate receptor channel in embryonic Drosophila myotubes in culture using the patch clamp technique. There were many brief lasting channel events together with prolonged ones. Brief events were frequently observed in low concentrations whereas the frequency of prolonged events increased with agonist concentrations. Long openings (> 5 ms) were often interrupted by brief closures, most of which lasted less than 100 mu s, thus showing a bursting behavior. At all agonist concentrations, the burst duration was fitted with three exponential components (brief, intermediate and long). The mean duration of the long component increased linearly with the glutamate concentration. The mean closed time and number of brief closures per ms within long bursts were independent of agonist concentration. The mean burst durations of the brief (30-250 mu s) and intermediate component (300-1050 mu s) did not change significantly with agonist concentration. The closing episodes within bursts were rare in the brief and intermediate burst components. The ratio of the fractional areas of the brief or intermediate and long burst components increased linearly with agonist concentration in the log-log plot with a slope of one. These findings suggest that the brief and intermediate components are due to singly-liganded openings and the long component is the result of doubly-liganded openings.

Heterogeneity in synaptic transmission along a Drosophila larval motor axon

“…A genetically-encoded fluorescent Ca 2+ reporter was used to detect postsynaptic Ca 2+ flux through GluRs, which in Drosophila are permeable to Ca 2+ 21 . Versions of the fluorescence resonance energy transfer (FRET)-based reporter Cameleon 22,23 were targeted to postsynaptic sites via fusion to the single-pass transmembrane domain of CD8, as well as to the C-terminal PDZ-interaction domain of the Shaker K + channel 16 .…”

Functional reconstitution ofDrosophila melanogasterNMJ glutamate receptors

“…The expression of fly NMJ iGluRs in Xenopus oocytes, and structural analysis of the GluRIIB ligand binding domain, has uncovered similarities to vertebrate Ca 2+ permeable AMPA and kainate receptors and unique features of Drosophila receptors. The rectification properties of Drosophila larval NMJ iGluRs has not been analyzed in detail before, but biphasic responses were observed previously using either microelectrode or whole-cell recordings (2,44,45), although not for single-channel recording using isolated membrane patches (45,46). This difference mimics the behavior of vertebrate Ca 2+ permeable AMPA and kainate receptors, and is caused by washout of cytoplasmic polyamines in isolated patches (27,28).…”

Rapid feedback regulation of synaptic efficacy during high-frequency activity at theDrosophilalarval neuromuscular junction

“…We therefore expressed LiGluR, our light-gated version of a mammalian kainate receptor, which uses a single cysteine substitution in iGluR6 (L439C) to provide an anchoring site for a maleimide-azobenzene-glutamate (MAG) photoswitch to generate the light-gated receptor (11). iGluR6, one of the closest mammalian homologs to the Drosophila iGluR subunits (19), forms homotetrameric channels that share the Drosophila iGluR property of high permeability to calcium (21,22).…”

“…Inhibition of postsynaptic CaMKII blocked the LiGluR effect, suggesting that calcium enters through the iGluRs and activates CaMKII. Because the experiments were performed under voltage clamp of the muscle (i.e., in absence of activation of voltage-gated calcium channels) and in thapsigargin (depleting the internal calcium stores before the start of the stimulation), the most likely entry route for calcium into the cytoplasm is the calcium-permeable iGluRs themselves (21).…”