We are interested in characterizing two genes that are required for GABA specific neuronal function. Gamma-aminobutyric acid (GABA) is the primary inhibitory neurotransmitter in vertebrates and invertebrates. Of the 302 neurons in a C. elegans adult hermaphrodite, 26 express GABA. Ablation of these neurons has lead to the identification of three behavioral changes caused by the absence of GABAergic neurons. These include excitatory as well as inhibitory functions for GABAergic neurons in muscle contraction. The DD and VD motor neurons innervate muscles along the body wall and supply inhibitory input during locomotion. In the absence of the DD and VD neurons, the animal hypercontracts its muscles when touched giving it a "shrinker" phenotype. In contrast, the AVL and DVB motor neurons provide excitatory input to the enteric muscles. Animals missing these neurons lack the expulsion step of the defecation cycle and are constipated. Six mutants have been identified that have all or a subset of these phenotypes.
unc-47 and
unc-46 are defective for all GABA functions and are therefore likely to be required for function of the GABAergic neuron rather than their postsynaptic targets. In addition,
unc-47 accumulates high levels of GABA presynaptically suggesting that GABA is not being released from the presynaptic neuron.
unc-47 genetically maps to linkage group III between
stp127 and
unc-50. We have rescued the
unc-47 mutant phenotype with a 5.2 kb genomic fragment containing a single complete 460 amino acid open reading frame. The predicted protein has no sequence homology to any known proteins but does contain eleven putative transmembrane domains. We believe that
unc-47 encodes the GABA vesicular transporter required to pump GABA into synaptic vesicles because it is required for all GABAergic functions, has multiple transmemebrane domains similar to known transport proteins, and mutants accumulate GABA in the presynaptic neuron. Three EMS mutant alleles are being sequenced and a missense mutation has been found in one of them,
n2409, which changes a glycine to an arginine in the middle of transmembrane domain eleven. In addition, we are in the process of making
unc-47::GFP fusions to identify the cellular distribution of
unc-47. Like
unc-47,
unc-46 is required for all GABAergic function, yet
unc-46 mutants fail to accumulate GABA in the presynaptic cell. We believe that
unc-46 encodes a protein required for vesicular transporter function. In support of this an extrachromosomal array of the genomic fragment containing
unc-47 is capable of partially rescuing
unc-46 null mutant animals so that they are no longer constipated and shrink only slightly. This suggests that overexpresion of UNC-47 can partially compensate for a lack of UNC-46. For this reason, it is important to isolate
unc-46 to determine what it encodes and how it interacts with
unc-47.
unc-46 has been genetically mapped to chromosome 5 between lethal genes
let-419 and
let-443, putting it in a physical location that contains > 1Mb of DNA. The
unc-46 mutation will be mapped relative to restriction fragment length polymorphisms (RFLP) in this region as a first step to isolating the gene.