No abstract

␥-Aminobutyric acid type B (GABA B ) receptors mediate the transmission of slow and prolonged inhibitory signals in the central nervous system. Two splice variants of GABA B receptors, GABA B R1a and GABA B R1b, were recently cloned from a mouse cortical and cerebellar cDNA library. As predicted, these receptors belong to the G protein-coupled receptor superfamily. We have used epitope-tagged versions of GABA B R1a receptors to study the cellular distribution of these proteins in a variety of non-neuronal and neuronal cell types. Here we report that recombinant GABA B receptors fail to reach the cell surface when expressed in heterologous systems and are retained in the endoplasmic reticulum when introduced into COS cells. In addition, we prove that recombinant GABA B receptors are excluded from the cell surface when overexpressed in ganglion neurons and we further demonstrate that they fail to activate in superior cervical ganglion neurons. Together our observations suggest that recombinant GABA B receptors require additional information for functional targeting to the plasma membrane.

GABA(B) receptors are unique among G-protein-coupled receptors (GPCRs) in their requirement for heterodimerization between two homologous subunits, GABA(B1) and GABA(B2), for functional expression. Whereas GABA(B1) is capable of binding receptor agonists and antagonists, the role of each GABA(B) subunit in receptor signaling is unknown. Here we identified amino acid residues within the second intracellular domain of GABA(B2) that are critical for the coupling of GABA(B) receptor heterodimers to their downstream effector systems. Our results provide strong evidence for a functional role of the GABA(B2) subunit in G-protein coupling of the GABA(B) receptor heterodimer. In addition, they provide evidence for a novel "sequential" GPCR signaling mechanism in which ligand binding to one heterodimer subunit can induce signal transduction through the second partner of a heteromeric complex.

B10 cells are an endothelial clonal line derived spontaneously by culture and selection of rat brain microvascular endothelial cells (1), a source of considerable pharmacological potential. The B10 cells respond to extracellular adenine (but not uracil) nucleotides with intracellular Ca 2ϩ mobilization, mediated by a G-protein-coupled P2Y 1 receptor (2). Whereas the known rat P2Y 1 receptor cDNA could be isolated from the B10 cells (2), no transcript for any other then-known P2Y receptor subtype was detectable in them. However, the B10 cells were found to exhibit another second messenger response, namely the inhibition of stimulated adenylyl cyclase, but with a nucleotide agonist specificity very similar to that of the P2Y 1 receptor (2). It was further demonstrated (3) that selective antagonists of the P2Y 1 receptor, such as adenosine 3Ј-phosphate 5Ј-phosphate, were unable to affect that response, although it showed high sensitivity to 2-propylthio-D-␤,␥-difluoromethylene-ATP (AR-C66096), 1 a specific antagonist (4, 5) of the adenylyl cyclase-inhibitory P2Y T or "P2T" receptor for ADP, which was known as an important functional component of blood platelets. That study (3) demonstrated that a second P2Y receptor activity is present in the B10 cell, with some of its functional features in common with the platelet P2Y T receptor.Recently, a cDNA encoding a nucleotide receptor with a novel seven-transmembrane sequence was identified independently by two groups, starting from a human platelet cDNA library or an orphan human DNA sequence, and shown to be distantly but significantly related to the known P2Y receptors (6, 7). This was designated as the P2Y 12 receptor. Its amino acid sequence lies on a previously unrecognized separate branch of the P2Y family (8). It was characterized in both studies to show its adenine nucleotide specificity and its inhibition of forskolin-stimulated adenylyl cyclase, in which it corresponds to the platelet P2Y T receptor (6, 7). That human P2Y 12 receptor cDNA was expressed again in cell line hosts by other groups, confirming the reported series of agonists but with higher potencies in the cAMP decrease (9) and showing their affinities by competition with the binding of radiolabeled 2-MeSADP (9, 10). In the original identification of the human P2Y 12 receptor, a similar cDNA was also derived from a rat platelet library (6), and its RNA was shown to express in Xenopus oocytes, but this receptor was not further characterized.The question, therefore, obviously arises as to whether the cyclase inhibitory receptor for adenine nucleotides of the B10 capillary endothelial cell is in fact the P2Y 12 receptor. However, this cannot necessarily be assumed, because the P2T receptor has commonly been described in the literature as specific to the platelet (11,12). Furthermore, the adenylyl cyclase-inhibitory P2Y receptor activity in the B10 cell differs in an important

The isolation of the peptide inhibitor of M-type K ؉ current, BeKm-1, from the venom of the Central Asian scorpion Buthus eupeus has been described previously (Fillipov A. K., Kozlov, S. A., Pluzhnikov, K. A., Grishin, E. V., and Brown, D. A. (1996) FEBS Lett. 384, 277-280). Here we report the cloning, expression, and selectivity of BeKm-1. A full-length cDNA of 365 nucleotides encoding the precursor of BeKm-1 was isolated using the rapid amplification of cDNA ends polymerase chain reaction technique from mRNA obtained from scorpion telsons. Sequence analysis of the cDNA revealed that the precursor contains a signal peptide of 21 amino acid residues. The mature toxin consists of 36 amino acid residues. BeKm-1 belongs to the family of scorpion venom potassium channel blockers and represents a new subgroup of these toxins. The recombinant BeKm-1 was produced as a Protein A fusion product in the periplasm of Escherichia coli. After cleavage and high performance liquid chromatography purification, recombinant BeKm-1 displayed the same properties as the native toxin. Three BeKm-1 mutants (R27K, F32K, and R27K/F32K) were generated, purified, and characterized. Recombinant wild-type BeKm-1 and the three mutants partly inhibited the native M-like current in NG108-15 at 100 nM. The effect of the recombinant BeKm-1 on different K ؉ channels was also studied. BeKm-1 inhibited hERG1 channels with an IC 50 of 3.3 nM, but had no effect at 100 nM on hEAG, hSK1, rSK2, hIK, hBK, KCNQ1/KCNE1, KCNQ2/KCNQ3, KCNQ4 channels, and minimal effect on rELK1. Thus, BeKm-1 was shown to be a novel specific blocker of hERG1 potassium channels.