A neural gene from Drosophila melanogaster with homology to vertebrate and invertebrate glutamate decarboxylases.

Cross-species hybridization has been used to isolate a second Drosophila gene, with homology to a feline glutamate decarboxylase (Gad) cDNA. The gene differs in sequence, chromosomal location, and spatial expression from the previously reported Drosophila Gad gene, but both encode proteins of 58 kDa. The derived amino acid sequence reveals a typical pyridoxal phosphate binding site and sequence homology consistent with a glutamate decarboxylase function. The protein includes an amino-terminal polyasparagine sequence, and a beta-pleated sheet region, with regularly spaced glutamine and arginine residues, not found in other decarboxylases. Expression in the adult is limited to the neuropil of the first optic ganglion and to regions of the thoracic musculature that may correspond to the location of motor neuron axons. This is consistent with a glial localization for the transcript. There is no overlap with the reported expression of Drosophila Gad. Although the molecular evidence suggests that this gene encodes a pyridoxal phosphate-dependent decarboxylase, glutamate decarboxylase activity associated with this gene could not be demonstrated, and the in vivo substrate is unknown. It is possible that the protein encoded by this gene is novel, not only in sequence and spatial expression, but also in substrate specificity.

Suppression of the membrane defect by divalent cations in the Drosophila mutant shibire.

The single-gene mutant shibire (shits) is temperature-sensitive. It causes reversible paralysis at heat pulses greater than 29 degrees C by blocking synaptic transmission. The synapses of these heat-pulsed flies are depleted in vesicles but contain numerous cisternae. We report that such alterations in synaptic physiology and morphology of heat-pulsed flies can be suppressed by internal perfusion of salines with high concentrations (10-18 mM) of the divalent cations Ca2+ or Mg2+. Synaptic morphology in these perfused flies remains normal even when exposed to nonpermissive temperatures (greater than 29 degrees C); in addition, synaptic transmission maintains a high resistance to failure both in frequency and stimulus duration. We also observed many cisternae in close association with extrajunctional as well as with postsynaptic regions of the sarcolemma in heat-pulsed shits flies not perfused with the increased concentrations of divalent cations. Flies perfused with increased amounts of divalent cations lacked such cisternae in the sarcolemma. The evidence suggests that the divalent cations can mitigate an overall membrane defect expressed by the shits gene, perhaps by influencing lipid-phase transition behavior.

Ion currents in Drosophila flight muscles.

1. The dorsal longitudinal flight muscles of Drosophila melanogaster contain three voltage-activated ion currents, two distinct potassium currents and a calcium current. The currents can be isolated from each other by exploiting the developmental properties of the system and genetic tools, as well as conventional pharmacology.2. The fast transient potassium current (I(A)) is the first channel to appear in the developing muscle membrane. It can be studied in isolation between 60 and 70 hr of pupal development. The channels can be observed to carry both outward and inward currents depending on the external potassium concentration. I(A) is blocked by both tetraethylammonium ion (TEA) and 3- or 4-aminopyridine. The inactivation and recovery properties of I(A) are responsible for a facilitating effect on membrane excitability.3. The delayed outward current (I(K)) develops after maturation of the I(A) system. I(K) can be isolated from I(A) by use of a mutation that removes I(A) from the membrane current response and can be studied before the development of Ca(2+) channels. I(K) shows no inactivation. The channels are more sensitive to blockage by TEA than I(A) channels, but are not substantially blocked by 3- or 4-aminopyridine.4. The calcium current (I(Ca)) is the last of the major currents to develop and must be isolated pharmacologically with potassium-blocking agents. I(Ca) shows inactivation when Ca(2+) is present but not when Ba(2+) is the sole current carrier. When Ca(2+) is the current carrier, the addition of Na(+) or Li(+) retards the inactivation of the net inward current. When the membrane voltage is not clamped, Ba(2+) alone, or Ca(2+) with Na(+) (or Li(+)), produces a plateau response of extended duration.5. The synaptic current (I(J)) evoked by motoneurone stimulation is the fastest and largest of the current systems. It has a reversal potential of approximately -5 mV, indicating roughly equal permeabilities of Na(+) and K(+). During a nerve-driven muscle spike, I(J) is the major inward current, causing a very rapid depolarization away from resting potential. An exceptionally large synaptic current is necessary to rapidly discharge the high membrane capacitance (0.03 muF/cell) in these large (0.05 x 0.1 x 0.8 mm) isopotential cells.