We generated mouse mutants with targeted AMPA receptor (AMPAR) GluR-B subunit alleles, functionally expressed at different levels and deficient in Q/R-site editing. All mutant lines had increased AMPAR calcium permeabilities in pyramidal neurons, and one showed elevated macroscopic conductances of these channels. The AMPAR-mediated calcium influx induced NMDA-receptor-independent long-term potentiation (LTP) in hippocampal pyramidal cell connections. Calcium-triggered neuronal death was not observed, but mutants had mild to severe neurological dysfunctions, including epilepsy and deficits in dendritic architecture. The seizure-prone phenotype correlated with an increase in the macroscopic conductance, as independently revealed by the effect of a transgene for a Q/R-site-altered GluR-B subunit. Thus, changes in GluR-B gene expression and Q/R site editing can affect critical architectural and functional aspects of excitatory principal neurons.

Calcium permeability of L-␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors (AMPARs) in excitatory neurons of the mammalian brain is prevented by coassembly of the GluR-B subunit, which carries an arginine (R) residue at a critical site of the channel pore. The codon for this arginine is created by site-selective adenosine deamination of an exonic glutamine (Q) codon at the pre-mRNA level. Thus, central neurons can potentially control the calcium permeability of AMPARs by the level of GluR-B gene expression as well as by the extent of Q͞R-site editing, which in postnatal brain, positions the R codon into >99% of GluR-B mRNA. To study whether the small amount of unedited GluR-B is of functional relevance, we have generated mice carrying GluR-B alleles with an exonic arginine codon. We report that these mutants manifest no obvious deficiencies, indicating that AMPAR-mediated calcium inf lux into central neurons can be solely regulated by the levels of Q͞R site-edited GluR-B relative to other AMPAR subunits. Notably, a targeted GluR-B gene mutant with 30% reduced GluR-B levels had 2-fold higher AMPAR-mediated calcium permeability in hippocampal pyramidal cells with no sign of cytotoxicity. This constitutes proof in vivo that elevated calcium inf lux through AMPARs need not generate pathophysiological consequences.

In this study, a large number of receptor mutants were generated and several N-terminally modified galanin analogues synthesized to refine the previously proposed binding site model for galanin to its GTP-binding-protein-coupled receptor GalRl . In addition to ligand-binding studies, the functionality of mutant receptors was evaluated by assessing their ability to mediate galaninergic inhibition of isoproterenol-stimulated adenylyl cyclase activity. The His264Ala and Phe282Ala receptor mutants, although deficient in binding in the concentration range of galanin used, remain functional albeit 20-fold less efficient than the wild-type receptor in mediating inhibition of stimulated CAMP production by galanin. The His267Ala mutant is, apart from being deficient in galanin binding, also severely impaired in functional coupling. While His264 and Phe282 seem to be important in forming the binding pocket for galanin, His267 might play a role in forming or stabilizing the active conformation of the GalRl receptor rather than directly participating in the formation of the binding pocket for galanin.N-terminal carboxylic acid analogues of galanin have low affinity to wild-type GalRl, but rubstantially increased affinity to the Glu27lLys receptor mutant. This, together with the finding that an alanine substitution of PhellS in TM 111 results in a tenfold decrease in affinity for galanin, suggests that the N-terminus of galanin interacts with PhellS. In contrast to the Phe282AIa mutation in TM VII, a conservative mutation of Phe282 to tyrosine did not alter the affinity for galanin. Thus, the interaction between Tyr9 of galanin and Phe282 is likely to be of an aromatic-aromatic nature.Keywords: galanin ; mutagenesis ; receptor ; binding ; peptide.The neuropeptide galanin plays important roles in physiological functions such as pain signalling, acquisition, feeding, and hormone release (for reviews, see [2, 31). The effects are mediated via 7-transmembrane (7-TM) spanning domain type receptors coupled via inhibitory GTP-binding proteins (GJG,) Structure/activity relationship studies using galanin fragments and analogues show that high-affinity binding as well as agonist action resides in the N-terminal part of galanin, which in its first 14 amino acids is fully conserved in all species studied. Studies of L-Ala substituted analogues of galanin demonstrate that Trp2 and Tyr9 as well as the free N-terminal amino group are the major pharmacophores [S].The ligand-binding sites of GTP-binding-protein-coupled receptors for small ligands such as monoamines [9] and low-molecular-mass, non-peptide antagonists to peptide receptors [ 101 have been delineated in recent years. These small ligands, with molecular masses of less than 500 Da, bind at sites in the TM domains (monoamines) or near the extracellular tops of these helices (non-peptide antagonists) [lo]. Recently, studies on the binding mode of intermediately sized peptides such as substance Neurotoxicology, Stockholm University, S-I 06 91 Stockholm Sweden P and angiotensin I1 h...

Galanin, a neuroendocrine peptide of 29 amino acids, binds to Gi/Go‐coupled receptors to trigger cellular responses. To determine which amino acids of the recently cloned seven‐transmembrane domain‐type human galanin receptor are involved in the high‐affinity binding of the endogenous peptide ligand, we performed a mutagenesis study. Mutation of the His264 or His267 of transmembrane domain VI to alanine, or of Phe282 of transmembrane domain VII to glycine, results in an apparent loss of galanin binding. The substitution of Glu271 to serine in the extracellular loop III of the receptor causes a 12‐fold loss in affinity for galanin. We combined the mutagenesis results with data on the pharmacophores (Trp2, Tyr9) of galanin and with molecular modelling of the receptor using bacteriorhodopsin as a model. Based on these studies, we propose a binding site model for the endogenous peptide ligand in the galanin receptor where the N‐terminus of galanin hydrogen bonds with Glu271 of the receptor, Trp2 of galanin interacts with the Zn2+ sensitive pair of His264 and His267 of transmembrane domain VI, and Tyr9 of galanin interacts with Phe282 of transmembrane domain VII, while the C‐terminus of galanin is pointing towards the N‐terminus of th

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