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Protein dimerization provides a mechanism for the modulation of cellular signaling events. In α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors, the rapidly desensitizing, activated state has been correlated with a weakly dimeric, glutamate-binding domain conformation. Allosteric modulators can form bridging interactions that stabilize the dimer interface. While most modulators can only bind to one position with a one modulator per dimer ratio, some thiazide-based modulators can bind to the interface in two symmetrical positions with a two modulator per dimer ratio. Based on small-angle X-ray scattering (SAXS) experiments, dimerization curves for the isolated glutamate-binding domain show that a second modulator binding site produces both an increase in positive cooperativity and a decrease in the EC50 for dimerization. Four body binding equilibrium models that incorporate a second dimer-stabilizing ligand were developed to fit the experimental data. The work illustrates why stoichiometry should be an important consideration during the rational design of dimerizing modulators.

The majority of excitatory neurotransmission in the CNS is mediated by tetrameric AMPA receptors. Channel activation begins with a series of interactions with an agonist that binds to the cleft between the two lobes of the ligand-binding domain of each subunit. Binding leads to a series of conformational transitions, including the closure of the two lobes of the binding domain around the ligand, culminating in ion channel opening. Although a great deal has been learned from crystal structures, determining the molecular details of channel activation, deactivation, and desensitization requires measures of dynamics and stabilities of hydrogen bonds that stabilize cleft closure. The use of hydrogen-deuterium exchange at low pH provides a measure of the variation of stability of specific hydrogen bonds among agonists of different efficacy. Here, we used NMR measurements of hydrogen-deuterium exchange to determine the stability of hydrogen bonds in the GluA2 (AMPA receptor) ligand-binding domain in the presence of several full and partial agonists. The results suggest that the stabilization of hydrogen bonds between the two lobes of the binding domain is weaker for partial than for full agonists, and efficacy is correlated with the stability of these hydrogen bonds. The closure of the lobes around the agonists leads to a destabilization of the hydrogen bonding in another portion of the lobe interface, and removing an electrostatic interaction in Lobe 2 can relieve the strain. These results provide new details of transitions in the binding domain that are associated with channel activation and desensitization.

Ligand-gated ion channels undergo conformational changes that transfer the energy of agonist binding to channel opening. Within ionotropic glutamate receptor (iGluR) subunits, this process is initiated in their bilobate ligand binding domain (LBD) where agonist binding to lobe 1 favors closure of lobe 2 around the agonist and allows formation of interlobe hydrogen bonds. AMPA receptors (GluAs) differ from other iGluRs because glutamate binding causes an aspartate-serine peptide bond in a flexible part of lobe 2 to rotate 180° (flipped conformation), allowing these residues to form cross-cleft H-bonds with tyrosine and glycine in lobe 1. This aspartate also contacts the side chain of a lysine residue in the hydrophobic core of lobe 2 by a salt bridge. We investigated how the peptide flip and electrostatic contact (D655-K660) in GluA3 contribute to receptor function by examining pharmacological and structural properties with an antagonist (CNQX), a partial agonist (kainate), and two full agonists (glutamate and quisqualate) in the wildtype and two mutant receptors. Alanine substitution decreased the agonist potency of GluA3(i)-D655A and GluA3(i)-K660A receptor channels expressed in HEK293 cells and differentially affected agonist binding affinity for isolated LBDs without changing CNQX affinity. Correlations observed in the crystal structures of the mutant LBDs included the loss of the D655-K660 electrostatic contact, agonist-dependent differences in lobe 1 and lobe 2 closure, and unflipped D(A)655-S656 bonds. Glutamate-stimulated activation was slower for both mutants, suggesting that efficient energy transfer of agonist binding within the LBD of AMPA receptors requires an intact tether between the flexible peptide flip domain and the rigid hydrophobic core of lobe 2.

Denatonium, one of the most bitter-tasting substances known, stimulated insulin secretion in clonal HIT-T15 beta-cells and rat pancreatic islets. Stimulation of release began promptly after exposure of the beta-cells to denatonium, reached peak rates after 4-5 min, and then declined to near basal values after 20-30 min. In islets, no effect was observed at 2.8 mmol/;l glucose, whereas a marked stimulation was observed at 8.3 mmol/;l glucose. No stimulation occurred in the absence of extracellular Ca(2+) or in the presence of the Ca(2+)-channel blocker nitrendipine. Stimulated release was inhibited by alpha(2)-adrenergic agonists. Denatonium had no direct effect on voltage-gated calcium channels or on cyclic AMP levels. There was no evidence for the activation of gustducin or transducin in the beta-cell. The results indicate that denatonium stimulates insulin secretion by decreasing KATP channel activity, depolarizing the beta-cell, and increasing Ca(2+) influx. Denatonium did not displace glybenclamide from its binding sites on the sulfonylurea receptor (SUR). Strikingly, it increased glybenclamide binding by decreasing the K(d). It is concluded that denatonium, which interacts with K(+) channels in taste cells, most likely binds to and blocks Kir6.2. A consequence of this is a conformational change in SUR to increase the SUR/glybenclamide binding affinity.

Effects of cocaine and cocaine methiodide were evaluated on the homomeric alpha 7 neuronal nicotinic receptor (nAChR). Whereas cocaine itself is a general nAChR noncompetitive antagonist, we report here the characterization of cocaine methiodide, a novel selective agonist for the alpha 7 subtype of nAChR. Data from (125)I-alpha-bungarotoxin binding assays indicate that cocaine methiodide binds to alpha 7 nAChR with a K(i) value of approximately 200 nM while electrophysiology studies indicate that the addition of a methyl group at the amine moiety of cocaine changes the drug's activity profile from inhibitor to agonist. Cocaine methiodide activates alpha 7 nAChR with an EC(50) value of approximately 50 microM and shows comparable efficacy to ACh in oocyte experiments. While agonist effects are specific for the alpha 7 neuronal nAChR and are not observed with heteromeric neuronal or skeletal muscle nAChR, antagonist effects are present for heteromeric nAChR combinations. Studies of PC12 cells transiently transfected with human alpha 7 cDNA and expressing a variety of functional nicotinic receptor subtypes confirm the specificity of cocaine methiodide agonist effects. Our results indicate that a quaternary structural derivative of cocaine can be used as a specific agonist for the alpha 7 subtype of neuronal nicotinic receptor.

Substance P is known to noncompetitively inhibit activation of muscle and neuronal nicotinic acetylcholine receptors. Neuronal nicotinic receptors formed from different combinations of alpha and beta subunits exhibited differential sensitivity to substance P, with those containing beta-4 subunits having a 25-fold higher affinity than those having beta-2 subunits. To identify the regions and/or amino acid residues of the beta subunit responsible for this difference, chimeric beta subunits were coexpressed with alpha-3 in Xenopus oocytes and the IC50 values for substance P were determined. Amino acid residues between 105 and 109 (beta4 numbering), in the middle of the N-terminal domain, and between 214 and 301, between the extracellular side of M1 and the intracellular side of M3, were identified as major contributors to the apparent affinity of substance P. The affinity of acetylcholine was only affected by residue changes between 105 and 109. Site-directed mutagenesis revealed two amino acids that are important determinants of the affinity of substance P, beta4(V108)/beta2(F106), which is in the middle of the first extracellular domain, and beta4(F255)/beta2(V253), which is within the putative channel lining transmembrane domain M2. However, other residues within these domains must be making subtle but significant contributions, since simultaneous mutation of both these amino acids did not cause complete interconversion of the beta subunit-dependent differences in the receptor affinity for substance P.

Substance P is known to inhibit nicotinic acetylcholine receptors from neuronal tissue, skeletal muscle, and electroplaque. The interaction of substance P with specific combinations of neuronal nicotinic acetylcholine receptor subunits was studied by expressing various combinations of subunits in Xenopus oocytes. The response to acetylcholine was inhibited by substance P with all subunit combinations tested; however, the apparent affinity for substance P varied by 20-30-fold. The affinity seemed to be dependent on the beta subtype expressed (beta 4 or beta 2). This suggests that the beta subunit may contribute, at least partially, to the substance P binding site. In the case of the alpha 7 subtype, which forms a homooligomeric receptor, the apparent affinity for substance P was intermediate between those of the two beta subtypes coexpressed with either alpha 3 or alpha 4. As previously found, the inhibition was noncompetitive. Furthermore, the inhibition was not voltage dependent and, therefore, is unlikely to be due to substance P blocking the channel within the transmembrane portion of the pore.

The binding of [3H]substance P to nicotinic acetylcholine receptor-enriched Torpedo electroplaque membranes was characterized. In the absence of cholinergic agonist, [3H]substance P binding was displaced by unlabeled substance P with an IC50 of 31 +/- 7 microM. In the presence of 10 mM carbamylcholine, displacement reflected two populations of binding sites with IC50 values of 0.93 +/- 0.39 and 30 +/- 5 microM, with the higher affinity component contributing 69 +/- 2% of the inhibition. Equilibrium binding parameters were calculated by transformation of the concentration dependences of inhibition into saturation isotherms. In the absence of agonist, substance P bound with a Kd of 42 +/- 7 microM to 3-4 sites/alpha-bungarotoxin binding site. In the presence of agonist, substance P bound to two sites, a low affinity site not significantly different from that seen in the absence of agonist (Kd = 25 +/- 8 microM, approximately 3 sites/alpha-bungarotoxin site) and a high affinity site with a Kd of 0.55 +/- 0.32 microM (approximately 1 site/2 alpha-bungarotoxin sites, 1 site/receptor). The increase in substance P binding induced by carbamylcholine was blocked by the nicotinic antagonists alpha-bungarotoxin and d-tubocurarine but was not affected by the muscarinic antagonist atropine. The concentration dependence of the carbamylcholine-induced increase had two components, with EC50 values for the agonist of 9.1 +/- 4.2 microM (56 +/- 16% of the increase) and 1.3 +/- 0.5 mM. The structural specificity of agonist-dependent high affinity substance P binding was identical to that seen for inhibition of nicotinic receptor activation and substantially different from that of binding to the G protein-coupled tachykinin receptors. From the time courses of association, it appears that substance P binds preferentially to a transient agonist-induced receptor state. The gamma and delta subunits of the receptor were specifically labeled in an agonist-dependent manner after cross-linking of [3H]substance P to the receptor with the bifunctional cross-linking reagent bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone or after photoaffinity labeling of the receptor with 125I-p-benzoylphenylalanine-substance P. These results demonstrate the existence of a high affinity agonist-induced binding site for substance P on the nicotinic acetylcholine receptor that probably mediates the noncompetitive inhibition by the peptide of receptor activation.

The effect of the neuropeptide substance P on the binding of the cholinergic ligands to the nicotinic acetylcholine receptor of Torpedo electroplaque membranes was examined at a physiological concentration of NaCl (150 mM). Substance P had no effect on the initial rate of 125I-alpha-bungarotoxin binding at concentrations of < 100 microM. The peptide did not bind to the high-affinity local anesthetic site but allosterically modulated [3H]phencyclidine binding, positively in the absence of agonist and negatively in the presence of agonist. Substance P increased the apparent affinity of the cholinergic agonists carbamylcholine and acetylcholine at equilibrium. The effect of substance P on the equilibrium binding of [3H]acetylcholine was examined directly, and the peptide appeared to increase the affinity of the binding of the second molecule of agonist, with no effect on the binding of the first. This indicates that substance P can affect the cooperative interactions between agonist binding sites. Substance P appeared to increase the rate of carbamylcholine-induced desensitization; however, the data are also consistent with an allosteric mechanism that does not involve the desensitized state. To attempt to differentiate between these mechanisms, the rates of recovery were determined after exposure to peptide and/or agonist. The kinetics of recovery are consistent with stabilization of the desensitized state by substance P if the peptide remains bound long enough to allow rapid recovery to the low-affinity state. However, an allosteric modulation of agonist binding that does not involve the desensitized state cannot be ruled out.

The effect of substance P on nicotinic acetylcholine receptor function was examined in Torpedo electroplaque membranes. The peptide inhibited carbamylcholine-stimulated 22Na+ efflux in a concentration-dependent manner. By irreversibly blocking spare receptors with alpha-bungarotoxin, the IC50 for substance P was shown to be less than 3 microM. Inhibition by substance P was slow relative to receptor activation by carbamylcholine, consistent with an enhancement of desensitization or a slow allosteric block.

This report provides direct evidence for a dihydropyridine receptor/calcium channel in the insulin-secreting beta-cell line RINm5F. The receptor/channel can modulate the intracellular Ca++ concentration and the resultant insulin secretion by regulating the influx of extracellular Ca++ through dihydropyridine-sensitive voltage-dependent L-type Ca++ channels. Elevated extracellular K+ or the dihydropyridine Ca++ channel agonist, BAY k 8644 [methyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethyl- phenyl)pyridine-5-carboxylate], stimulated the uptake of 45Ca++, raised [Ca++]i, and increased insulin secretion in a concentration-dependent manner. These actions were inhibited by L-type Ca++ channel blockers including nitrendipine, verapamil and diltiazem. (+)-[3H]PN200-110 bound specifically with high affinity to RINm5F cell membranes (Kd approximately 200 pM). Specific binding was inhibited competitively by dihydropyridines whereas phenylalkylamines inhibited incompletely (+)-[3H]PN200-110 binding, consistent with an allosteric interaction. The benzothiazepine diltiazem had no effect on (+)-[3H]PN200-110 binding in the presence of Ca++, but increased binding allosterically in the absence of Ca++ (in the presence of EGTA). Maximal (+)-[3H]PN200-110 binding required divalent cations, with Mg++, Mn++ and Ba++ essentially as effective as Ca++ in reversing the effects of EGTA, whereas binding was not supported by Cd++ or La . Specific high affinity (+)-[3H]PN200-110 binding was also demonstrated in intact RINm5F cells and shown to be modulated by membrane potential. Depolarization of the cells by raising extracellular K+ from 5 to 80 mM increased the affinity of (+)-[3H]PN200-110 4- to 5-fold (decreased Kd) with no significant effect on the maximum number of binding sites.

Loss of responsiveness of the neuronal-type nicotinic acetylcholine receptor (nAChR) on PC12 cells, a cell line derived from a rat pheochromocytoma, was induced by exposure to carbamylcholine (carbachol). Nicotinic receptor function was assessed by carbachol-induced 22Na+ uptake. We found that, in addition to classically described desensitization, a second process occurs which results in a nonrecoverable loss of nAChR activity. This second process, which we have labeled inactivation, has a slower onset than the classically described desensitization (t1/2 = 14.7 min for inactivation and 0.78 min for desensitization at 1 mM carbachol). Inactivation could not be explained by inadequate washing, a loss of electrochemical driving force, or a loss of cell viability. The onset of inactivation is dependent on the concentration of desensitizing ligand and is blocked by nicotinic antagonists. No recovery of the loss of activity from inactivation was observed even after 2 hr of incubation in recovery buffer. Inactivation does not appear to require formation of a desensitized state since desensitization was reduced in the absence of Ca2+ whereas inactivation was not affected by the absence of Ca2+. The mechanism which underlies inactivation remains to be determined; however, it is possible that inactivation is the first step in nAChR down-regulation and it may also explain previous observations of rapid and prolonged tolerance to the effects of nicotinic agonists.

The nonspecific lipid transfer protein from beef liver was used to modify the phospholipid composition of intact turkey erythrocytes in order to study the dependence of isoproterenol-stimulated adenylate cyclase activity on membrane phospholipid composition. Incorporation of phosphatidylinositol into turkey erythrocytes inhibited isoproterenol-stimulated cyclic AMP accumulation in a linear, concentration-dependent manner. Inhibition was relatively specific for phosphatidylinositol; phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol and phosphatidic acid were from 3 to 7 times less effective as inhibitors of hormone-stimulated cyclase activity. Inhibition by phosphatidylinositol was not reversible when up to 90% of the incorporated phosphatidylinositol was removed, either by incubation with phosphatidylinositol-specific phospholipase C or a second incubation with transfer protein; possibly adenylate cyclase activity depends on a small pool of phosphatidylinositol that is inaccessible to either phospholipase C hydrolysis or removal by lipid transfer protein. Phosphatidylinositol incorporation inhibits adenylate cyclase activity by uncoupling beta-adrenergic receptors from the remainder of the cyclase complex. Phosphatidylinositol incorporation had no effect on stimulation of cAMP accumulation by either cholera toxin or forskolin, indicating that inhibition occurs only at the level of receptor. Phosphodiesterase activity was not altered in phosphatidylinositol-modified cells. Inhibition of cAMP accumulation was not the result of changes in either membrane fluidity or in cAMP transport out of modified turkey erythrocytes. Phosphatidylinositol inhibition of isoproterenol-stimulated cyclase activity may serve as a useful model system for hormone-induced desensitization.

Recent evidence suggests that the molecular interactions of agonists with beta-adrenergic receptors differ from those of antagonists. Most of this evidence has come from studies of agonist inhibition of radiolabeled antagonist binding. We have examined agonist binding directly in rat lung membranes using radiolabeled hydroxybenzylisoproterenol (3H-HBI). Specific binding of 3H-HBI was stereoselective and was inhibited by catecholamines with a potency order characteristic of beta 2-adrenergic receptors. Gpp(NH)p increased the rates of association and dissociation of 3H-HBI from the receptor. In the absence of Gpp(NH)p, Scatchard plots were curvilinear suggesting a complex interaction of the agonist with the receptor. The total number of 3H-HBI binding sites was similar to that of 125I-IHYP binding sites. In the presence of increasing concentrations of Gpp(NH)p, the affinity of 3H-HBI was decreased and Scatchard plots became linear. Sodium chloride mimicked the effect of Gpp(NH)p in lowering the affinity of the receptor for 3H-HBI. Magnesium chloride had the opposite effect in that it promoted high affinity binding. The effect of sodium chloride was largely overcome by the presence of magnesium chloride.

The inhibition of carbamylcholine-stimulated 22Na+ flux by substance P and various peptide analogs was examined in PC12 cells, a line which contains a neuronal-type nicotinic receptor, and BC3H1 cells, a line which contains a muscle-type nicotinic receptor. Substance P produces a noncompetitive inhibition of carbamylcholine-stimulated 22Na+ influx in both cell lines (IC50 = 1.2 microM on PC12 cells and 8.2 microM on BC3H1 cells). The structure-activity relation for substance P analogs was qualitatively similar in both cell lines; however, there were quantitative differences. Substance P was the most potent peptide tested. Analogs of substance P with amino acids removed from the N-terminus resulted in significant decreases in potency, whereas removal of amino acids from the C-terminus resulted in analogs virtually devoid of activity. Compounds purported to be substance P antagonists had actions similar to substance P in reducing carbamylcholine-stimulated 22Na+ flux. The related tachykinins physalaemin and eledoisin had low potencies on both cell lines. These results indicate that the site through which substance P exerts its inhibitory effects on activation of nicotinic receptors is different from the receptors described previously for substance P in more classical systems. In addition, our results indicate that substance P has an effect on both the neuronal-type nicotinic receptor (alpha-bungarotoxin insensitive) expressed on PC12 cells and the muscle-type nicotinic receptor (alpha-bungarotoxin sensitive) expressed on BC3H1 cells.

Whole cell patch-clamp techniques were used to investigate the pharmacological properties of calcium currents in the clonal rat pituitary cell line GH3. Current traces induced by a 100-ms pulse to 0 mV from a holding potential of -80 mV consisted of a component that rapidly inactivated during the pulse and a component that slowly inactivated during the pulse. When the holding potential was reduced to -32 mV, the rapidly inactivating component of the trace disappeared. The dihydropyridine calcium channel blocker nitrendipine affected only the slowly inactivating component of the trace. At a holding potential of -80 mV, nitrendipine blocked the slowly inactivating current with an IC50 of 1 microM. The IC50 for nitrendipine was found to be dependent on the holding potential, decreasing to 10 nM when the holding potential was -32 mV. The dihydropyridine agonist Bay-K 8644, like nitrendipine, affected only the slowly inactivating component. The inorganic blocker Cd2+ blocked both components but the slowly inactivating current was three- to fourfold more sensitive. These results are best explained by the existence of two types of calcium channels in these cells, one sensitive to dihydropyridines and one insensitive to dihydropyridines. These channels appear analogous to the T-type channel (inactivating current) and L-type channel (slowly inactivating current) described in other preparations.

Rats were exposed for one week to either neurotensin (4 micrograms/h), substance P (3.3 micrograms/h), thyrotropin-releasing hormone (5 micrograms/h), or saline administered intracerebroventricularly via mini osmotic-pumps and either haloperidol (2.5 mg/kg i.p., 2 X daily) or vehicle control. The peptide treatments by themselves did not alter [3H]spiroperidol binding in either the nucleus accumbens or the striatum. Neurotensin, however, augmented the increase in [3H]spiroperidol binding caused by the haloperidol treatment in both the nucleus accumbens and striatum.

The actions of three different types of calcium channel blockers on short-circuit current (Isc) in rabbit ileum were studied. These included the phenylalkylamines, verapamil and (l)-desmethoxyverapamil (D888); the dihydropyridines, nifedipine and nitrendipine; and the benzothiazepine, diltiazem. All of the drugs decreased Isc, a change associated with increased Na and Cl absorption. Verapamil and D888 had the largest effects. The dihydropyridine, BAY K 8644, a calcium channel activator, increased Isc and decreased Na and Cl absorption, effects not inhibited by tetrodotoxin. The phenylalkylamines had an additional effect on Isc in the presence of a maximally inhibitory concentration of the dihydropyridines, suggesting the possibility of two distinct calcium channels, one of which is the L-type voltage-activated, dihydropyridine- and phenylalkylamine-sensitive channel, and the other is a channel only sensitive to phenylalkylamines but not to dihydropyridines. [3H]nitrendipine and [3H]D888 binding to an enriched preparation of basolateral membranes from ileal epithelial cells was characterized. Each ligand bound specifically and saturably to an apparently single population of high-affinity sites with [3H]D888 having three times as many binding sites as [3H]nitrendipine. [3H]nitrendipine binding was partially inhibited by verapamil and D888 and was increased by diltiazem; whereas [3H]D888 binding was inhibited completely by verapamil but only partially by nitrendipine and diltiazem. These transport and binding studies suggest the presence of two types of Ca2+ channels in ileal epithelial cells, one of which interacts with the dihydropyridines, the phenylalkylamines, and the benzothiazepines at three different sites and the other channel that only binds the phenylalkylamines.

Regulatory properties of a partially purified Ca2+ -channel preparation from isolated rabbit skeletal muscle triads were examined in proteoliposomes. These properties included (i) inhibition by phenylalkylamine antagonists, such as verapamil, (ii) inhibition by the GTP-binding protein Go in the presence of guanosine 5'-[gamma-thio]triphosphate, and (iii) regulation of phenylalkylamine inhibition as a result of phosphorylation by a polypeptide-dependent protein kinase (PK-P). By selective reconstitution of protein fractions obtained by wheat germ lectin and ion-exchange chromatography, a separation of Ca2+-channel activity (fraction C) from regulatory component(s) (fraction R) responsible for verapamil sensitivity was achieved. Reconstitution of fraction C alone yielded vesicles that exhibited channel-mediated 45Ca2+ uptake that could be directly inhibited by coreconstitution of Go in the presence of guanosine 5'-[gamma-thio]triphosphate. However, the 45Ca2+ uptake obtained with fraction C was not inhibited by verapamil. Coreconstitution of fractions C and R yielded vesicles in which the sensitivity of 45Ca2+ uptake to verapamil was restored. The verapamil sensitivity of this preparation could be inhibited by PK-P. Fraction C, obtained by wheat germ agglutinin-Sepharose chromatography followed by DEAE-Sephacel chromatography, included a 180-kDa protein that was phosphorylated by cAMP-dependent protein kinase (PK-A) but not by PK-P and a 145-kDa protein (180 kDa under nonreducing conditions) that was not phosphorylated by either kinase. Fraction R contained proteins that did not adsorb to wheat germ lectin and included 165-kDa and 55-kDa proteins that were phosphorylated by PK-P but not by PK-A. These results suggest a complex model for Ca2+-channel regulation in skeletal muscle involving a number of distinct, separable protein components.

The interactions of agonists and antagonists with beta-adrenergic receptors appear to be fundamentally different. Antagonists only occupy the receptor while agonists bind to the receptor and induce a specific conformational change. The excellent correlation between the efficacy of full or partial agonists and the magnitude of the changes in the thermodynamic parameters associated with the binding of these compounds suggests that this conformational change is related to the ability of various drugs to stimulate adenylate cyclase. In most instances it is difficult to distinguish an agonist from an antagonist using the in vitro binding of radiolabeled ligands. The thermodynamic analysis described here represents one example of a situation in which agonists and antagonists can be distinguished using in vitro binding techniques. The complexities of agonist interactions with beta-adrenergic receptors are also clearly seen in studies of binding to intact cells and in direct studies using radiolabeled agonists. In the former studies it appeared that there were marked changes in the properties of the receptor that occurred during the 60 min that are required for the binding of IHYP to beta-adrenergic receptors on L6 myoblasts to reach equilibrium. These changes make it difficult to analyze the affinity of the receptor for agonists using intact cells. Finally, the complexities of agonist interactions with the beta-adrenergic receptor are also seen in direct binding studies with radiolabeled agonists. The curvilinear Scatchard plots and the complicated dissociation kinetics attest to this complexity. The importance of the formation of a hypothetical ternary complex between hormone, receptor, and guanine nucleotide-binding regulatory protein is now becoming appreciated. The existence of other components of this system will remain an important subject for future investigation. In any case, it is clear that a full understanding of this system will require the use of not only radiolabeled agonists and antagonists but also of both broken and intact cells.

The dihydropyridine receptor-calcium channel complex, prelabeled with (+)-[3H]PN200-110, was solubilized from rat heart membranes with a detergent mixture of digitonin and Triton X-100. The dissociation of (+)-[3H]PN200-110 was slow enough to permit the hydrodynamic characterization of the complex by means of sucrose gradient sedimentation and gel filtration. The hydrodynamic properties of the complex were determined in several detergents, including Tween 80, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS), and digitonin. S20,w values of 12.5, 15.4, and 21.0 S were obtained in sucrose gradients prepared in Tween 80, CHAPS, and digitonin, respectively. A Stokes radius of 86-87 A was obtained in each of the three detergents. Determination of the partial specific volume of the protein-detergent complex in each case revealed that the differences in S20,w values could be explained by the differences in the properties of the bound detergent species. Partial specific volumes of 0.796, 0.730, and 0.730 ml/g, corresponding to molecular weights of 595,000, 540,000, and 740,000 were obtained for the complex in Tween 80, CHAPS, and digitonin, respectively. This indicated that Tween 80 readily exchanged for the solubilizing mixture of digitonin and Triton X-100, whereas CHAPS did not. Detergent exchange with Tween 80 made it possible to determine the fractional contribution of the receptor protein to the molecular weight of the protein-detergent complex. The molecular weight of the dihydropyridine receptor-calcium channel complex was estimated to be 370,000. The protein-detergent complex was found to have a frictional coefficient of 1.39, consistent with a large transmembrane protein.

We describe the rapid incorporation of the CHAPS solubilized dihydropyridine receptor into phospholipid vesicles. A series of sucrose gradient sedimentation experiments demonstrate that the (+)-[3H]PN200-110-labeled dihydropyridine receptor is associated with lipid vesicles following detergent removal by Extracti-gel chromatography. Solubilization of the receptor results in a loss of (+)-[3H]PN200-110 binding affinity relative to that observed in native membranes; the high affinity binding of (+)-[3H]PN200-110 can be restored upon reincorporation of the receptor into phospholipid vesicles. Similarly, the incorporation of the receptor restores its stability to incubation at 37 degrees C relative to that of the detergent solubilized receptor, thereby mimicking the properties of the membrane bound form of the receptor. The dissociation rate of (+)-[3H]PN200-110 from the reconstituted receptor is shown to be allosterically regulated by verapamil and diltiazem, indicating that the binding sites for these calcium antagonists have been inserted along with the dihydropyridine receptor into phospholipid vesicles. The results presented in this report, thus demonstrate the successful reconstitution of the dihydropyridine receptor into phospholipid vesicles by a variety of criteria. The reconstitution method described here is rapid and efficient, and should now facilitate structure-function studies of this receptor and its interrelationships with other regulatory components of the voltage-sensitive calcium channel system.

Antagonist binding to the beta-adrenergic receptor is largely entropy driven, with only a small enthalpy component. The binding of agonists, on the other hand, is associated with a large decrease in enthalpy which permits a highly unfavourable decrease in entropy. The thermodynamic differences between the binding of agonists and antagonists may provide new insights into the molecular basis for hormone stimulation of adenylate cyclase activity.

The effects of substance P on the functioning of nicotinic acetylcholine receptors in PC12 cells were examined. Carbachol-stimulated 22Na+ uptake was used to assess the functional state of the nicotinic receptor. We found that incubation of the cells with substance P alone caused a loss of receptor function. Receptors recovered from this effect with a t1/2 of 0.94 +/- 0.10 min. Since receptors recovered from carbachol-induced desensitization at a significantly slower rate (t1/2, 1.77 +/- 0.21 min), it was concluded that the two inactive states are not kinetically equivalent. The effects of substance P on carbachol-induced loss of receptor activity were also examined. Substance P had no effect on a component of carbachol-induced loss of activity that was nonrecoverable (inactivation). However, substance P had several effects on the recoverable loss of activity induced by carbachol (desensitization). Substance P caused a shift to the left in the EC50 for carbachol-induced desensitization at equilibrium. If cells were simultaneously incubated with carbachol and substance P7-11, a low-potency analog of substance P, an increase in the rate of formation of a state of the receptor that was kinetically indistinguishable from the state induced by carbachol alone was observed. However, not all inhibition of nicotinic cholinergic function could be explained by an increased rate of formation of a desensitized receptor and it is concluded that substance P causes both enhanced desensitization and block of the nicotinic receptor-linked channel.

An interaction between Ca2+ channel blockers and alpha 2-adrenergic receptors has been demonstrated in rabbit ileum by studying the effect of clonidine on active electrolyte transport, under short-circuited conditions, in the presence and absence of several Ca2+ channel blocking agents. Clonidine, verapamil, diltiazem, cadmium, and nitrendipine all decrease short-circuit current and stimulate NaCl absorption to different extents with clonidine having the largest effect. Exposure to verapamil, diltiazem, and cadmium inhibited the effects of clonidine on transport, whereas nitrendipine had no such effect. Verapamil, diltiazem, and cadmium, but not nitrendipine, also decreased the specific binding of [3H]alpha 2-adrenergic agents to a preparation of ileal basolateral membranes explaining the observed decrease in the transport effects of clonidine. The effective concentrations of the Ca2+ channel blockers that inhibited the effects of clonidine on transport were fairly similar to the concentrations needed to inhibit its specific binding. The displacement of clonidine by calcium channel blockers is ascribed to a nonspecific effect of these agents, although the possibility that their effects are exerted via their binding to the calcium channels is not excluded.

The effect of substance P on the binding of many ligands that interact with the nicotinic acetylcholine receptor was examined in membrane preparations of Torpedo electroplaque and BC3H-1 cells and in solubilized membranes of rat, chick, and goldfish brain. In the absence of carbamylcholine, the affinity of [3H]phencyclidine for the high affinity local anesthetic binding site on Torpedo membranes was increased by substance P with an EC50 of approximately 5 microM. In the presence of carbamylcholine, which itself increases [3H]phencyclidine binding affinity, substance P caused a decrease in the affinity of [3H]phencyclidine. The concentration dependence of the inhibition, however, was inconsistent with a competitive interaction, since the apparent Hill coefficient was significantly less than one. We conclude from these results that substance P does not directly interact with the high affinity local anesthetic binding site on the nicotinic receptor of Torpedo membranes. Substance P also does not appear to interact directly with the agonist binding site since the peptide had no significant effect on [3H]acetylcholine binding to Torpedo membranes. Substance P inhibited [125I]alpha-bungarotoxin binding to both native and Triton X-100 solubilized Torpedo membranes, although the IC50 was 8-fold higher for the solubilized preparation (12 versus 93 microM). We interpret this inhibition in solubilized membranes as evidence that the peptide may interact directly with a binding site on the nicotinic acetylcholine receptor. Substance P also decreased the initial rate of [125I]alpha-bungarotoxin to membranes prepared from BC3H-1 cells (IC50 = 108 microM) and to solubilized membranes from rat, chick, and goldfish brain. In the brain membranes, however, the peptide did not completely inhibit binding; at the highest concentration examined (100 microM), the maximum inhibition observed was 60%. Consistent with the results for [3H]acetylcholine binding to Torpedo membranes, the peptide had no effect on the binding of the cholinergic agonist [3H](-)nicotine to these tissue preparations. These data suggest that substance P may have a general modulatory action on a subclass of nicotinic receptors that include muscle-type, ganglionic-type, and a putative subpopulation of central nervous system receptors.

Detailed kinetic and equilibrium studies of the binding of two radiolabeled 1,4-dihydropyridine calcium antagonists to putative calcium channels in rat brain membranes were performed. (+/-)-[3H]Nitrendipine, a racemic ligand, and (+)-[3H]isopropyl 4-(2,1,3-benzoxadiazol-4-yl)-1, 4-dihydro-2,6-dimethyl-5-methoxycarbonylpyridine-3-carboxylate (PN200-110), a pure isomer, were used and their binding properties were quantitated and compared. Analysis of equilibrium binding revealed a single high affinity component for each radioligand with the same density of binding sites for both ligands. Association rates were determined over a 60-fold range of concentration of each radioligand. For both radioligands, the pseudo-first order association time courses were biphasic with the rate of the faster component dependent on radioligand concentration and the rate of the slower component independent of both the structure of the radioligand and the concentration of the radioligand. Dissociation rates were determined after various times of association. The dissociation of the optically pure radioligand, (+)-[3H]PN200-110, was monophasic at all association times, consistent with a single bound species being present throughout association. However, (+/-)-[3H]nitrendipine dissociation was biphasic after short association times (1-10 min). The biphasic dissociation observed with (+/-)-[3H]nitrendipine is consistent with the two optical isomers binding with approximately the same association rate but having different dissociation rates. These results appear to reflect the existence of two interconvertible binding states of the putative calcium channel in the membrane, one which binds the radioligands with high affinity in a simple bimolecular reaction and one which has no detectable affinity for the ligands. This mechanism of isomerization before ligand binding has been modeled by numerical solution of the differential equations of the scheme providing estimates of the rate constants for each reaction in the scheme.