Worm Breeder's Gazette 13(5): 36 (February 1, 1995)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
Department of Biochemistry, UT-Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75235-9038 The M3's are inhibitory motor neurons necessary for the pharynx to repolarize quickly after excitation so that bacteria can be trapped efficiently (WBG 13(4): 72). M3 probably does this by releasing glutamate onto pharyngeal muscle to open glutamate-gated Cl-channels (ibid). We are interested in understanding glutamatergic neurotransmission by taking advantage of mutants that affect M3 function. We know that eat-4 affects the function of M3 and possibly other glutamatergic neurons. We also know that the defect of eat-4 is upstream from pharyngeal muscle and therefore probably in the M3 neuron itself (ibid). Since eat-4 does not appear to affect behavior known to be mediated by neurotransmitters other than glutamate (e.g., GABA, ACh), we suspect that eat-4 may be necessary for either the production or concentration of glutamate in glutamatergic neurons. We previously localized eat-4 to the cosmid ZK512 (WBG 12(5): 65). We have since shown that a 7 kb fragment rescues, and we have rescued eat-4 with a genomic- cDNA fusion. We fused 2.4 kb of genomic sequence from ZK512 in frame to a cDNA yk32h2 that was cloned and partially sequenced by Yoji Kohara. yk32h2 is identical to zk512.6 (found in ACEDB) except in two exons. yk32h2 is nearly full length, but may lack a few hundred bases at the 5' end. A sequence database search of EAT-4 produced several interesting hits, including three putative genes found on LGIII (part of this has been reported by the sequence consortium, in ACEDB). The table below shows percent sequence identity in pairwise comparisons. Members of this putative gene superfamily are found in organisms from mammals to bacteria suggesting an ancient ancestry. EAT-4 is most similar (45% identical) to a neuronal specific sodium-dependent inorganic phosphate cotransporter (BNPI) found in rat brain. BNPI has been shown to have ion transporter activity but its biological function is not clear (Ni et al, PNAS 91: 5607). All members of this superfamily appear to be membrane-associated proteins based on Kyte-Doolittle hydrophobicity analysis. All of the eukaryotic members have very similar hydrophobicity profiles, each having the potential to span the lipid bilayer six times. This similarity at the level of secondary structure further suggests the possibility that they are related. The fact that three of the members are sodium-phosphate transporters (BNPI, and rabbit and human Na-Pi-I) and that all of them seem to be membrane proteins suggests that this family may represent a new class of transporter proteins. We propose to call them the NERD (neural, eat-4 and renal-derived) transporters. eat-4 is probably necessary for all glutamatergic neurotransmission in worms (WBG 13(4): 72). How does a NERD do that? Our first model is that EAT-4 may be a phosphate transporter (like BNPI), necessary for maintaining a high intracellular phosphate concentration in order to stimulate phosphate-activated glutaminase, an enzyme that synthesizes glutamate. (This model was suggested by Robert Edwards.) Alternatively, perhaps EAT-4 is a vesicular glutamate transporter that concentrates glutamate in synaptic vesicles. Table 1: % identity in pairwise comparisons percent identity Eat-4 BNPI K10G9.x C38C10.2 Na-Pi-I C02C2.4 Bac180K Eat-4 100 46 44 32 29 22 26 BNPI 45 100 37 33 31 21 24 K10G9.x 44 38 100 30 29 21 25 C38C10.2 26 28 25 100 26 19 26 Na-Pi-I 24 26 24 26 100 21 20 C02C2.4 22 21 21 23 26 100 23 Bac180K 20 18 19 17 18 24 100 % identity is normalized to the size of protein in each column. BNPI (brain Na+-dependent inorganic phosphate cotransporter I, Ni et al, PNAS 91: 5607). K10G9.x (GENEFINDER predicted CDS in K10G9 and T07A5, we put together the two parts). Na-Pi-I (rabbit renal cortical Na/Pi cotransporter, Werner et al, PNAS 88: 9608). Bac180K (hypothetical ORF found in B. subtilis, Genpept accession D26185). We are grateful to Yoji Kohara for supplying yk32h2 clone.