Barry Ganetzky

Molecular analysis of the para locus, a sodium channel gene in Drosophila

Previous behavioral, electrophysiological, pharmacological, and genetic studies of mutations of the para locus in Drosophila melanogaster suggested that these mutations alter the structure or function of sodium channels. To identify the protein encoded by this gene and to elucidate the molecular basis of the mutant phenotypes, genomic DNA from the para locus was cloned. Mutational lesions in nine different para alleles were mapped and found to be distributed over a region of 45 kb. Analysis of cDNAs revealed that the para locus comprises a minimum of 26 exons distributed over more than 60 kb of genomic DNA. The nucleotide sequence of the complementary DNA predicts a protein whose structure and amino acid sequence are extremely similar to those of vertebrate sodium channels. The results support the conclusion that para encodes a functionally predominant class of sodium channels in Drosophila neurons. Furthermore, the para transcript appears to undergo alternative splicing to produce several distinct subtypes of this channel.

Functional role of the β subunit of high conductance calcium-activated potassium channels

Abstract Mammalian high conductance, calcium-activated potassium (maxi-K) channels are composed of two dissimilar subunits, α and β. We have examined the functional contribution of the β subunit to the properties of maxi-K channels expressed heterologously in Xenopus oocytes. Channels from oocytes injected with cRNAs encoding both a and β subunits were much more sensitive to activation by voltage and calcium than channels composed of the α subunit alone, while expression levels, single-channel conductance, and ionic selectivity appeared unaffected. Channels from oocytes expressing both subunits were sensitive to DHS-I, a potent agonist of native maxi-K channels, whereas channels composed of the α subunit alone were insensitive. Thus, α and β subunits together contribute to the functional properties of expressed maxi-K channels. Regulation of coassembly might contribute to the functional diversity noted among members of this family of potassium channels.

Ion Channels and Synaptic Organization: Analysis of the Drosophila Genome

The most obvious conclusion from this survey is that neuronal signaling proteins, for the most part, have been highly conserved throughout evolution. Despite the dramatic differences in complexity and organization of nervous systems and behavioral outputs in worms, flies, and mammals, the key proteins, and presumably the functional mechanisms they govern, are remarkably similar. Apparently, the basic framework for neuronal signaling and synaptic organization was completed early in metazoan evolution, and evolution of much more complicated nervous systems did not entail extensive alteration of this framework. Thus, genomic analysis fully confirms the belief that one can obtain fundamental neurobiological insights from studies of simple model organisms.Still, at this point, it is easier to catalog the information obtained from sequence analysis than to decipher all the functional implications. It is not in any way apparent, for example, why C. elegans, with only 302 neurons, encodes more than 90 K+ channels. Drosophila with a nominally more complex nervous system and behavioral repertoire has a set of only about 30 K+ channel genes. Indeed, most ion channel gene families contain more members in worms than flies. Apparently, increased behavioral complexity does not require an increase in molecular complexity but may depend instead on increases in the number of neurons and the complexity of the wiring diagram. Although somewhat counterintuitive, a greater number of ion channels may be required in worms than flies to provide its simple nervous system with the potential for more extensive signaling capabilities than would otherwise be possible.With the identification in this analysis of new channel subfamilies, it is likely that all the major classes of channel pore-forming subunits have now been described and the complete set of evolutionarily conserved ion channels has been defined. Although this would not have been obvious a priori, there is a striking difference between ion channel pore-forming subunits and auxiliary subunits with respect to evolutionary conservation. With the exception of Ca2+ channels, very few of the accessory subunits identified in mammals have homologous counterparts in worms or flies. The acquisition of novel ion channel subunits that modify the biophysical properties of pore-forming subunits may represent one way of adding additional functional complexity to the basic set of channels.It remains an open question whether the more complex nervous systems of vertebrates, particularly mammals, employ any signaling mechanisms that are fundamentally different from those in invertebrates. If so, the strong conservation of the basic set of signaling proteins argues that these mechanisms either depend on an unknown set of proteins or involve the same components that can be incorporated into novel molecular and cellular networks that generate unique functions and outputs.From the perspective of the genome, it seems safe to predict that elucidation of the fundamental molecular mechanisms of neuronal signaling applicable to all organisms will continue to derive enormous benefit from invertebrate model systems. With future emphasis on understanding the distinctive in vivo functions of the array of proteins now defined only by sequence, the availability of forward and reverse genetic techniques in worms and flies will continue to be of critical importance. The identification of the entire set of ion channels and neuronal signaling proteins opens up a new era in which neurobiologists can work back from the complete gene set toward an understanding of how they function to produce behavior.‡To whom correspondence should be addressed (e-mail: troy@mit.edu).

Synaptic Vesicle Size and Number Are Regulated by a Clathrin Adaptor Protein Required for Endocytosis

Clathrin-mediated endocytosis is thought to involve the activity of the clathrin adaptor protein AP180. However, the role of this protein in endocytosis in vivo remains unknown. Here, we show that a mutation that eliminates an AP180 homolog (LAP) in Drosophila severely impairs the efficiency of synaptic vesicle endocytosis and alters the normal localization of clathrin in nerve terminals. Most importantly, the size of both synaptic vesicles and quanta is significantly increased in lap mutants. These results provide novel insights into the molecular mechanism of endocytosis and reveal a role for AP180 in regulating vesicle size through a clathrin-dependent reassembly process.

Reduced sleep in Drosophila Shaker mutants.

Most of us sleep 7–8 h per night, and if we are deprived of sleep our performance suffers greatly; however, a few do well with just 3–4 h of sleep—a trait that seems to run in families. Determining which genes underlie this phenotype could shed light on the mechanisms and functions of sleep. To do so, we performed mutagenesis in Drosophila melanogaster, because flies also sleep for many hours and, when sleep deprived, show sleep rebound and performance impairments. By screening 9,000 mutant lines, we found minisleep (mns), a line that sleeps for one-third of the wild-type amount. We show that mns flies perform normally in a number of tasks, have preserved sleep homeostasis, but are not impaired by sleep deprivation. We then show that mns flies carry a point mutation in a conserved domain of the Shaker gene. Moreover, after crossing out genetic modifiers accumulated over many generations, other Shaker alleles also become short sleepers and fail to complement the mns phenotype. Finally, we show that short-sleeping Shaker flies have a reduced lifespan. Shaker, which encodes a voltage-dependent potassium channel controlling membrane repolarization and transmitter release, may thus regulate sleep need or efficiency.

International Union of Pharmacology. XLI. Compendium of Voltage-Gated Ion Channels: Potassium Channels

This summary article presents an overview of the molecular relationships among the voltage-gated potassium channels and a standard nomenclature for them, which is derived from the IUPHAR Compendium of Voltage-Gated Ion Channels.1 The complete Compendium, including data tables for each member of the potassium channel family can be found at http://www.iuphar-db.org/iuphar-ic/.

Drosophila Ecdysone Receptor Mutations Reveal Functional Differences among Receptor Isoforms

The steroid hormone ecdysone directs Drosophila metamorphosis via three heterodimeric receptors that differ according to which of three ecdysone receptor isoforms encoded by the EcR gene (EcR-A, EcR-B1, or EcR-B2) is activated by the orphan nuclear receptor USP. We have identified and molecularly mapped two classes of EcR mutations: those specific to EcR-B1 that uncouple metamorphosis, and embryonic-lethal mutations that map to common sequences encoding the DNA- and ligand-binding domains. In the larval salivary gland, loss of EcR-B1 results in loss of activation of ecdysone-induced genes. Comparable transgenic expression of EcR-B1, EcR-B2, and EcR-A in these mutant glands results, respectively, in full, partial, and no repair of that loss.

The maleless protein associates with the X chromosome to regulate dosage compensation in drosophila

The maleless (mle) gene is one of four known regulatory loci required for increased transcription (dosage compensation) of X-linked genes in D. melanogaster males. A predicted mle protein (MLE) contains seven short segments that define a superfamily of known and putative RNA and DNA helicases. MLE, while present in the nuclei of both male and female cells, differs in its association with polytene X chromosomes in the two sexes. MLE is associated with hundreds of discrete sites along the length of the X chromosome in males and not in females. The predominant localization of MLE to the X chromosome in males makes it a strong candidate to be a direct regulator of dosage compensation.

Temperature-Sensitive Paralytic Mutations Demonstrate that Synaptic Exocytosis Requires SNARE Complex Assembly and Disassembly

The neuronal SNARE complex is formed via the interaction of synaptobrevin with syntaxin and SNAP-25. Purified SNARE proteins assemble spontaneously, while disassembly requires the ATPase NSF. Cycles of assembly and disassembly have been proposed to drive lipid bilayer fusion. However, this hypothesis remains to be tested in vivo. We have isolated a Drosophila temperature-sensitive paralytic mutation in syntaxin that rapidly blocks synaptic transmission at nonpermissive temperatures. This paralytic mutation specifically and selectively decreases binding to synaptobrevin and abolishes assembly of the 7S SNARE complex. Temperature-sensitive paralytic mutations in NSF (comatose) also block synaptic transmission, but over a much slower time course and with the accumulation of syntaxin and SNARE complexes on synaptic vesicles. These results provide in vivo evidence that cycles of assembly and disassembly of SNARE complexes drive membrane trafficking at synapses.

Retromer deficiency observed in Alzheimer's disease causes hippocampal dysfunction, neurodegeneration, and Aβ accumulation

Although deficiencies in the retromer sorting pathway have been linked to late-onset Alzheimers disease, whether these deficiencies underlie the disease remains unknown. Here we characterized two genetically modified animal models to test separate but related questions about the effects that retromer deficiency has on the brain. First, testing for cognitive defects, we investigated retromer-deficient mice and found that they develop hippocampal-dependent memory and synaptic dysfunction, which was associated with elevations in endogenous Aβ peptide. Second, testing for neurodegeneration and amyloid deposits, we investigated retromer-deficient flies expressing human wild-type amyloid precursor protein (APP) and human β-site APP-cleaving enzyme (BACE) and found that they develop neuronal loss and human Aβ aggregates. By recapitulating features of the disease, these animal models suggest that retromer deficiency observed in late-onset Alzheimers disease can contribute to disease pathogenesis.