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Despite many recent studies of G-protein-coupled receptor (GPCR) structures, it is not yet well understood how these receptors activate G proteins. The GPCR assay using baker's yeast, Saccharomyces cerevisiae, is an effective experimental model for the characterization of GPCR-Gα interactions. Here, using the yeast endogenous Gα protein (Gpa1p) as template, we constructed various chimeric Gα proteins with a region that is considered to be necessary for interaction with mammalian receptors. The signaling assay using the yeast pheromone receptor revealed that the chimeric Gα protein harboring 37 gustducin-specific amino acid residues at its C-terminus (GPA1/gust37) maintained functionality in yeast. In contrast, GPA1/gust44, a variant routinely used in mammalian experimental systems, was not functional.

Na 1 -dependent excitatory amino acid transporters (EAATs) normally function to remove extracellular glutamate from brain extracellular space, but EAATs can also increase extracellular glutamate by reversal of uptake. Effects of inhibitors on EAATs can be complex, depending on cell type, whether conditions favor glutamate uptake or uptake reversal and whether the inhibitor itself is a substrate for the transporters. The present study assessed EAAT inhibitors for their ability to inhibit glutamate uptake, act as transporter substrates and block uptake reversal in astrocyte and neuron cultures. L-threo-bhydroxyaspartate (L-TBHA), DL-threo-b-benzyloxyaspartate (DL-TBOA), L-trans-pyrrolidine-2,4-dicarboxylic acid (L-trans2,4-PDC) (1/‐)-cis-4-methy-trans-pyrrolidine-2,4-dicarboxylic acid (cis-4-methy-trans-2,4-PDC) and L-antiendo-3,4-methanopyrrolidine-2,4-dicarboxylic acid (L-antiendo-3,4-MPDC) inhibited L-[ 14 C]glutamate uptake in astrocytes with equilibrium binding constants ranging from 17 mM (DL-TBOA and L-TBHA) ‐4 3mM (cis-4-methy-trans-2,4-PDC). Transportability of inhibitors was assessed in astrocytes and neurons. While L-TBHA, L-trans-2,4-PDC, cis-4-methy-trans-2,4-PDC and L-antiendo3,4-MPDC displayed significant transporter substrate activities in neurons and astrocytes, DL-TBOA was a substrate only in astrocytes. This effect of DL-TBOA was concentrationdependent, leading to complex effects on glutamate uptake reversal. At concentrations low enough to produce minimal DL-TBOA uptake velocity (# 10 mM), DL-TBOA blocked uptake reversal in ATP-depleted astrocytes; this blockade was negated at concentrations that drove substantial DL-TBOA uptake (. 10 mM). These findings indicate that the net effects of EAAT inhibitors can vary with cell type and exposure

Four subtypes of excitatory amino acid transporters (EAAT1-4) have been identified in the mammalian brain. A number of pharmacological agents have been developed to study their intrinsic properties and function. Up to now, blockers were available only for EAAT2, whereas all the inhibitors of glutamate uptake active on the other subtypes were proved to be substrates of the transporters. We synthesized five new derivatives of DL-threo-beta-hydroxyaspartic acid, a well known general substrate of EAATs, and investigated their potential blocking activity on the cloned bovine EAAT1 expressed in the Xenopus oocyte system, by using radiotracer and voltage-clamp techniques. Two of our derivatives proved to be substrates for bovine EAAT1, with reduced electrogenicity compared with their parent compound, and an affinity of 40 and 64 microM. The last three derivatives displayed a blocking activity on bovine EAAT1. The affinity of DL-threo-beta-benzoyloxyaspartate and DL-threo-beta-(1-naphthoyl)oxyaspartate was determined by Schild analysis as 17.2 and 52.1 microM, respectively. These blockers should help in the better understanding of the key intrinsic properties of EAAT1. Moreover, they appear as good candidates for a general blocking activity on EAATs.

We report here the identification of a tachykinin-related peptide (TRP) in crustaceans. Direct MALDI-TOF MS with brain slices of the crayfish Procambarus clarkii indicated the presence of a peptide having an MS number of around 934. Quadrupole orthogonal acceleration time-of-flight (Q-TOF) MS/MS analysis implied the sequence to be APSGFLGMRamide, identical to that of CabTRP Ia, isolated previously from the crab Cancer borealis, and Pev-tachykinin, from the shrimp Penaeus vannamei. The peptide has been shown to be myoactive in the cockroach hindgut, but the structure of its precursor protein had not been elucidated. A cDNA encoding crayfish preproTRP was cloned, revealing that the 225-residue protein contains seven identical copies of the peptide APSGFLGMRamide. This is unique because TRPs identified in other invertebrates were known to exist in several related forms in each species. The conserved structure of TRP in crustaceans was confirmed by cloning preproTRP from the spiny lobster Panulirus interruptus. RT-PCR and Northern blotting analyses suggested that the crayfish preproTRP mRNA is expressed throughout the nervous system, and in situ hybridization studies of the brain revealed that the transcript predominantly localizes to cell clusters 11 (dorsal lateral cells) and 9 (ventral lateral cells).

Achatin-I and fulicin, isolated from the ganglia and atria of the giant land snail Achatina fulica, are a tetrapeptide and pentapeptide containing a d-Phe and d-Asn at position 2, respectively. We succeeded in cloning a cDNA encoding a precursor of achatin-I from the Achatina ganglia, revealing that the d-Phe present in achatin-I is coded by a common l-Phe codon, TTT or TTC. The deduced polypeptide was found to comprise seven repeats of the achatin sequence GFAD and one analog GFGD flanked on both sides by the typical endoproteolytic site KR. Northern blot analysis of transcripts and Southern blot analysis of reverse transcription (RT)-PCR products demonstrated that achatin-I mRNA was localized in the subesophageal ganglia, whereas expression of fulicin mRNA was detected in the atrium as well as in the subesophageal ganglia. Furthermore, localization of the achatin gene transcript in the right and left pedal ganglia compartments was shown by in situ hybridization on sections of subesophageal ganglia, whereas the fulicin transcript was observed in the right and left parietal ganglia. These data suggested that achatin-I plays an essential role in the regulation of the heart as a neurotransmitter or neurohormone through production in the pedal ganglia and transport to the atrium, whereas fulicin serves not only as a neurotransmitter or neurohormone but also as a novel atrial hormone.

The molecular conformation of achatin-II neutral form (H-Gly-Phe-Ala-Asp-OH), an endogenous peptide from the Achatina fulica ganglia, was elucidated by X-ray crystal analysis. The molecule takes an extended β-pleated structure stabilized by 5 intermolecular hydrogen bonds with the antiparallely arranged molecules. This is in contrast with the turn conformation of a neuroactive achatin-I (H-Gly-d-Phe-Ala-Asp-OH) [(1992) FEBS Lett. 276, 95–97]. The conformational comparison of both of the molecules makes clear the structural role which d-Phe residue of achatin-I plays in forming a definite active form.