We have performed direct electrophysiological recordings from Drosophila peptidergic synaptic boutons in situ, taking advantage of a mutation, ecdysone, which causes an increase in size of these terminals. Using patch-clamp techniques, we have analyzed voltage-dependent potassium currents at the macroscopic and single-channel level. The synaptic membrane contained at least two distinct voltage-activated potassium currents with different kinetics and voltage sensitivity: an I A -like current with fast activation and inactivation kinetics and voltage-dependent steady-state inactivation; a complex delayed current that includes a slowly inactivating component, resembling the I K described in other preparations; and a noninactivating component. The I A -like current in these peptidergic boutons is not encoded by the gene Shaker, because it is not affected by null mutations at this locus. Rather, synaptic I A has properties similar to those of the Shal-encoded I A . Singlechannel recordings revealed the presence in synaptic membranes of three different potassium channel types (A 2 , K D , K L ), with biophysical properties that could account for the macroscopic currents and resemble those of the Shal, Shab, and Shaw channels described in heterologous expression systems and Drosophila neuronal somata. A 2 channels (6 -9 pS) have brief open times, and like the macroscopic I A they exhibited voltage-dependent steady-state inactivation and a rapidly inactivating ensemble average current profile. K D channels (13-16 pS) had longer open times, activate and inactivate with much slower kinetics, and may account for the slowly inactivating component of the macroscopic current. K L (44 -54 pS) channels produced a noninactivating ensemble average and may contribute to the delayed macroscopic current observed.

The study of regulated vesicle exocytosis, which underlies neurotransmitter and neuropeptide release, has benefited from a convergence of several independent approaches. These include the use of genetically tractable organisms and model preparations that allow a direct characterization of presynaptic ionic currents. Aiming for a comprehensive analysis of release, we had already developed a Drosophila preparation in which electrophysiological recordings from peptidergic terminals are feasible. Here, we report on the characterization of the Ca2+-channel currents present in these terminals. With Ba2+ as the charge carrier, the presynaptic membrane expresses a current type with high-activation threshold and little inactivation. This current is blocked by verapamil and diltiazem at micromolar concentrations, it is relatively insensitive to nifedipine and completely resistant to non-L-type Ca2+-channel antagonists. As a comparison, we also analysed the pharmacology of high-threshold Ba+2 currents on muscle fibres. A high-activation threshold Ca2+-channel current is also present in muscle fibres, albeit with a distinct pharmacological profile. Thus, peptidergic terminals and muscle fibres exhibit different subtypes of voltage-gated Ca2+ channels. The putative role of cysteine string protein (CSP) as a neuronal Ca2+-channel modulator was tested by examining the peptidergic presynaptic current in csp null mutants. We show that CSP is expressed in peptidergic boutons and abolished in the mutant. Direct recordings, under conditions that inhibit calcium influx into glutamatergic terminals, show that Ca2+-currents in peptidergic csp terminals are entirely normal. This result indicates that CSP is not a generic Ca2+-channel modulator and it might perform different functions in fast versus slow forms of release.

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We report the identification and functional characterization of ariadne-1 (ari-1), a novel and vital Drosophila gene required for the correct differentiation of most cell types in the adult organism. Also, we identify a sequence-related gene, ari-2, and the corresponding mouse and human homologues of both genes. All these sequences define a new protein family by the Acid-rich, RING finger, B-box, RING finger, coiled-coil (ARBRCC) motif string. In Drosophila, ari-1 is expressed throughout development in all tissues. The mutant phenotypes are most noticeable in cells that undergo a large and rapid membrane deposition, such as rewiring neurons during metamorphosis, large tubular muscles during adult myogenesis, and photoreceptors. Occasional survivors of null alleles exhibit reduced life span, motor impairments, and short and thin bristles. Single substitutions at key cysteines in each RING finger cause lethality with no survivors and a drastic reduction of rough endoplasmic reticulum that can be observed in the photoreceptors of mosaic eyes. In yeast two-hybrid assays, the protein ARI-1 interacts with a novel ubiquitin-conjugating enzyme, UbcD10, whose sequence is also reported here. The N-terminal RING-finger motif is necessary and sufficient to mediate this interaction. Mouse and fly homologues of both ARI proteins and the Ubc can substitute for each other in the yeast two-hybrid assay, indicating that ARI represents a conserved novel mechanism in development. In addition to ARI homologues, the RBR signature is also found in the Parkinson-disease-related protein Parkin adjacent to an ubiquitin-like domain, suggesting that the study of this mechanism could be relevant for human pathology.