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The structural features responsible for the high potency and opiate receptor specificity of the opioid peptide dynorphin in the guinea pig ileum myenteric plexus were examined. Successive removal of COOH-terminal amino acids from dynorphin-(1--13) demonstrated important contributions of lysine-13, lysine-11, and arginine-7 to the potency. Removal of the NH2-terminal tyrosine abolished the biologic activity. Several other structural modifications were shown to affect potency: substitution of D-alanine for glycine-2 reduced the potencies of dynorphin-(1--13) amide, -(1--11), and -(1--10); and methyl esterification of the COOH terminus enhanced the potencies of dynorphin-(1--12), -(1--10), -(1--9), -(1--8), and -(1--7). Within the dynorphin sequence, lysine-11 and arginine-7 were found to be important for selectivity of interaction with the dynorphin receptor, which is distinguishable from the mu receptor in this tissue.

Coexpression in Xenopus oocytes of the inwardly rectifying guanine nucleotide binding (G)-protein-gated K channel GIRK1 with a myristoylated modification of the (putative) cytosolic C-terminal tail [GIRK1 aa 183-501 fused in-frame to aa 1-15 of p60src and denoted src+ (183-501)] leads to a high degree of inhibition of the inward G-protein-gated K+ current. The nonmyristoylated segment, src- (183-501), is not active. Although some interference with assembly is not precluded, the evidence indicates that the main mechanism of inhibition is interference with functional activation of the channel by G proteins. In part, the tail functions as a blocking particle similar to a "Shaker ball"; it may also function by competing for the available supply of free G beta gamma liberated by hormone activation of a seven-helix receptor. The non-G-protein-gated weak inward rectifier ROMK1 is less effectively inhibited, and a Shaker K channel was not inhibited. Immunological assays show the presence of a high concentration of src+ (183-501) in the plasma membrane and the absence of any membrane forms for the nonmyristoylated segment.

Opioid receptors on guinea pig brain membranes were alkylated by the naltrexone analogue beta-chlornaltrexamine. Binding of the prototypical mu and kappa ligands, [3H]dihydromorphine and [3H]ethylketocyclazocine, was more readily affected by the reagent than was binding of the delta ligand, 3H-labeled [D-Ala2, D-Leu5]enkephalin. Treatment of membranes with beta-chlornaltrexamine in the presence of dynorphin resulted in significant protection of [3H]ethylketocyclazocine binding sites, without protection of [3H]dihydromorphine or 3H-labeled [D-Ala2, D-Leu5]enkephalin sites. Similarly, [D-Ala2, D-Leu5]enkephalin and sufentanil selectively protected binding sites for 3H-labeled [D-Ala2, D-Leu5]enkephalin and [3H]dihydromorphine, respectively. Scatchard analysis of [3H]ethylketocyclazocine binding to untreated membranes suggested two types of binding site with 40-fold difference in affinities. Membranes treated with beta-chlornaltrexamine in the presence of dynorphin retained about 40% of the high-affinity sites and lost the low-affinity sites. Selective protection of sites with high affinity for dynorphin and ethylketocyclazocine was confirmed in competition binding assays. These results strongly suggest that the three types of opioid receptor are not interconvertible and provide further evidence that the endogenous peptide dynorphin is a highly selective ligand of the kappa opioid receptor.

The murine Ca(2+)-stimulated adenylyl cyclase (type I) (EC 4.6.1.1), which is expressed predominantly in brain, was inactivated by targeted mutagenesis. Ca(2+)-stimulated adenylyl cyclase activity was reduced 40-60% in the hippocampus, neocortex, and cerebellum. Long-term potentiation in the CA1 region of the hippocampus from mutants was perturbed relative to controls. Both the initial slope and maximum extent of changes in synaptic response were reduced. Although mutant mice learned to find a hidden platform in the Morris water task normally, they did not display a preference for the region where the platform had been when it was removed. These results indicate that disruption of the gene for the type I adenylyl cyclase produces changes in behavior and that the cAMP signal transduction pathway may play an important role in synaptic plasticity.

We have measured the opioid receptor reserve in the guinea pig ileum myenteric plexus by means of the site-directed alkylating agent, beta-chlornaltrexamine. Treatment of the tissue with low (less than 10 nM) concentrations of beta-chlornaltrexamine caused a parallel shift of the log concentration-response curves for both normorphine and dynorphin A-(1-13). Analysis of the resulting curves indicated that the Kd values were 1.5 +/- 0.5 X 10(-6) and 10 +/- 4 X 10(-9), respectively. Using the naloxone Ke to distinguish between the mu and kappa receptors in this tissue, we found that the receptor selectivities of normorphine and dynorphin A-(1-13) were unchanged after a maximum parallel shift, thus demonstrating that there are both spare mu and spare kappa receptors present. The spare-receptor fraction for both receptor types was about 90%. In morphine-tolerant preparations (chronic pellet implantation), there was an apparent reduction in the fraction of spare mu receptors without any change in the apparent affinity of normorphine. Reduction in the spare receptor fraction does not necessarily imply reduction in the number of binding sites. We suggest that this reduction in receptor reserve is the basis of opioid tolerance, since the agonist concentration needed to produce a given effect is expected to increase as the receptor reserve decreases.

Activity of several ion channels is controlled by heterotrimeric GTP-binding proteins (G proteins) via a membrane-delimited pathway that does not involve cytoplasmic intermediates. The best studied example is the K+ channel activated by muscarinic agonists in the atrium, which plays a crucial role in regulating the heartbeat. To enable studies of the molecular mechanisms of activation, this channel, denoted KGA, was cloned from a rat atrium cDNA library by functional coupling to coexpressed serotonin type 1A receptors in Xenopus oocytes. KGA displays regions of sequence homology to other inwardly rectifying channels as well as unique regions that may govern G-protein interaction. The expressed KGA channel is activated by serotonin 1A, muscarinic m2, and delta-opioid receptors via G proteins. KGA is activated by guanosine 5'-[gamma-thio]triphosphate in excised patches, confirming activation by a membrane-delimited pathway, and displays a conductance equal to that of the endogenous channel in atrial cells. The hypothesis that similar channels play a role in neuronal inhibition is supported by the cloning of a nearly identical channel (KGB1) from a rat brain cDNA library.

Ca2+ influx controls multiple neuronal functions including neurotransmitter release, protein phosphorylation, gene expression, and synaptic plasticity. Brain L-type Ca2+ channels, which contain either alpha 1C or alpha 1D as their pore-forming subunits, are an important source of calcium entry into neurons. Alpha 1C exists in long and short forms, which are differentially phosphorylated, and C-terminal truncation of alpha 1C increases its activity approximately 4-fold in heterologous expression systems. Although most L-type calcium channels in brain are localized in the cell body and proximal dendrites, alpha 1C subunits in the hippocampus are also present in clusters along the dendrites of neurons. Examination by electron microscopy shows that these clusters of alpha 1C are localized in the postsynaptic membrane of excitatory synapses, which are known to contain glutamate receptors. Activation of N-methyl-D-aspartate (NMDA)-specific glutamate receptors induced the conversion of the long form of alpha 1C into the short form by proteolytic removal of the C terminus. Other classes of Ca2+ channel alpha1 subunits were unaffected. This proteolytic processing reaction required extracellular calcium and was blocked by inhibitors of the calcium-activated protease calpain, indicating that calcium entry through NMDA receptors activated proteolysis of alpha1C by calpain. Purified calpain catalyzed conversion of the long form of immunopurified alpha 1C to the short form in vitro, consistent with the hypothesis that calpain is responsible for processing of alpha 1C in hippocampal neurons. Our results suggest that NMDA receptor-induced processing of the postsynaptic class C L-type Ca2+ channel may persistently increase Ca2+ influx following intense synaptic activity and may influence Ca2+-dependent processes such as protein phosphorylation, synaptic plasticity, and gene expression.