A rational blueprint for the design of chemically-controlled protein switches.

Small-molecule responsive protein switches are crucial components to control synthetic cellular activities. However, the repertoire of small-molecule protein switches is insufficient for many applications, including those in the translational spaces, where properties such as safety, immunogenicity, drug half-life, and drug side-effects are critical. Here, we present a computational protein design strategy to repurpose drug-inhibited protein-protein interactions as OFF- and ON-switches. The designed binders and drug-receptors form chemically-disruptable heterodimers (CDH) which dissociate in the presence of small molecules. To design ON-switches, we converted the CDHs into a multi-domain architecture which we refer to as activation by inhibitor release switches (AIR) that incorporate a rationally designed drug-insensitive receptor protein. CDHs and AIRs showed excellent performance as drug responsive switches to control combinations of synthetic circuits in mammalian cells. This approach effectively expands the chemical space and logic responses in living cells and provides a blueprint to develop new ON- and OFF-switches.

Selective Pharmacogenetic Activation of Catecholamine Subgroups in the Ventrolateral Medulla Elicits Key Glucoregulatory Responses.

Catecholamine (CA) neurons in the ventrolateral medulla (VLM) contribute importantly to glucoregulation during glucose deficit. However, it is not known which CA neurons elicit different glucoregulatory responses or whether selective activation of CA neurons is sufficient to elicit these responses. Therefore, to selectively activate CA subpopulations, we injected male or female Th-Cre+ transgenic rats with the Cre-dependent DREADD construct, AAV2-DIO-hSyn-hM3D(Gq)-mCherry, at one of four rostrocaudal levels of the VLM: rostral C1 (C1r), middle C1 (C1m), the area of A1 and C1 overlap (A1/C1), and A1. Transfection was highly selective for CA neurons at each site. Systemic injection of the Designer Receptor Exclusively Activated by Designer Drugs (DREADD) receptor agonist, clozapine-N-oxide (CNO), stimulated feeding in rats transfected at C1r, C1m, or A1/C1 but not A1. CNO increased corticosterone secretion in rats transfected at C1m or A1/C1 but not A1. In contrast, CNO did not increase blood glucose or induce c-Fos expression in the spinal cord or adrenal medulla after transfection of any single VLM site but required dual transfection of both C1m and C1r, possibly indicating that CA neurons mediating blood glucose responses are more sparsely distributed in C1r and C1m than those mediating feeding and corticosterone secretion. These results show that selective activation of C1 CA neurons is sufficient to increase feeding, blood glucose levels, and corticosterone secretion and suggest that each of these responses is mediated by CA neurons concentrated at different levels of the C1 cell group.

Tracking the Time-Dependent Role of the Hippocampus in Memory Recall Using DREADDs.

The hippocampus is critical for the storage of new autobiographical experiences as memories. Following an initial encoding stage in the hippocampus, memories undergo a process of systems-level consolidation, which leads to greater stability through time and an increased reliance on neocortical areas for retrieval. The extent to which the retrieval of these consolidated memories still requires the hippocampus is unclear, as both spared and severely degraded remote memory recall have been reported following post-training hippocampal lesions. One difficulty in definitively addressing the role of the hippocampus in remote memory retrieval is the precision with which the entire volume of the hippocampal region can be inactivated. To address this issue, we used Designer Receptors Exclusively Activated by Designer Drugs (DREADDs), a chemical-genetic tool capable of highly specific neuronal manipulation over large volumes of brain tissue. We find that remote (>7 weeks after acquisition), but not recent (1-2 days after acquisition) contextual fear memories can be recalled after injection of the DREADD agonist (CNO) in animals expressing the inhibitory DREADD in the entire hippocampus. Our data demonstrate a time-dependent role of the hippocampus in memory retrieval, supporting the standard model of systems consolidation.

Label-free cell-based dynamic mass redistribution assays.

Label-free cell-based assays offer a powerful approach to drug discovery and compound profiling for endogenously expressed receptors in a variety of cell types, including primary and stem cells. Dynamic mass redistribution (DMR) responses in whole cells following receptor stimulation provide phenotypic activity profiles that are readily amenable to evaluation of compound pharmacology. Protocols are provided in this unit to obtain DMR response profiles in adherent and suspension cells, and then to use known tool compounds to delineate the biology of the underlying signaling pathways from the information-rich kinetic traces that are recorded.

Properties and functions of KATP during mouse perinatal development.

BACKGROUND: Prevailing data suggest that ATP-sensitive potassium channels (K(ATP)) contribute to a surprising resistance to hypoxia in mammalian embryos, thus we aimed to characterize the developmental changes of K(ATP) channels in murine fetal ventricular cardiomyocytes. METHODS: Patch clamp was applied to investigate the functions of K(ATP). RT-PCR, Western blot were used to further characterize the molecular properties of K(ATP) channels. RESULTS: Similar K(ATP) current density was detected in ventricular cardiomyocytes of late development stage (LDS) and early development stage (EDS). Molecular-biological study revealed the upregulation of Kir6.1/SUR2A in membrane and Kir6.2 remained constant during development. Kir6.1, Kir6.2, and SUR1 were detectable in the mitochondria without marked difference between EDS and LDS. Acute hypoxia-ischemia led to cessation of APs in 62.5% of tested EDS cells and no APs cessation was observed in LDS cells. SarcK(ATP) blocker glibenclamide rescued 47% of EDS cells but converted 42.8% of LDS cells to APs cessations under hypoxia-ischemic condition. MitoK(ATP) blocker 5-HD did not significantly influence the response to acute hypoxia-ischemia at either EDS or LDS. In summary, sarcK(ATP) played distinct functional roles under acute hypoxia-ischemic condition in EDS and LDS fetal ventricular cardiomyocytes, with developmental changes in sarcK(ATP) subunits. MitoK(ATP) were not significantly involved in the response of fetal cardiomyocytes to acute hypoxia-ischemia and no developmental changes of K(ATP) subunits were found in mitochondria.

Some cannabinoid receptor ligands and their distomers are direct-acting openers of SUR1 K(ATP) channels.

Here, we examined the chronic effects of two cannabinoid receptor-1 (CB1) inverse agonists, rimonabant and ibipinabant, in hyperinsulinemic Zucker rats to determine their chronic effects on insulinemia. Rimonabant and ibipinabant (10 mg·kg⁻¹·day⁻¹) elicited body weight-independent improvements in insulinemia and glycemia during 10 wk of chronic treatment. To elucidate the mechanism of insulin lowering, acute in vivo and in vitro studies were then performed. Surprisingly, chronic treatment was not required for insulin lowering. In acute in vivo and in vitro studies, the CB1 inverse agonists exhibited acute K channel opener (KCO; e.g., diazoxide and NN414)-like effects on glucose tolerance and glucose-stimulated insulin secretion (GSIS) with approximately fivefold better potency than diazoxide. Followup studies implied that these effects were inconsistent with a CB1-mediated mechanism. Thus effects of several CB1 agonists, inverse agonists, and distomers during GTTs or GSIS studies using perifused rat islets were unpredictable from their known CB1 activities. In vivo rimonabant and ibipinabant caused glucose intolerance in CB1 but not SUR1-KO mice. Electrophysiological studies indicated that, compared with diazoxide, 3 µM rimonabant and ibipinabant are partial agonists for K channel opening. Partial agonism was consistent with data from radioligand binding assays designed to detect SUR1 K(ATP) KCOs where rimonabant and ibipinabant allosterically regulated ³H-glibenclamide-specific binding in the presence of MgATP, as did diazoxide and NN414. Our findings indicate that some CB1 ligands may directly bind and allosterically regulate Kir6.2/SUR1 K(ATP) channels like other KCOs. This mechanism appears to be compatible with and may contribute to their acute and chronic effects on GSIS and insulinemia.

CB1 receptor activation inhibits neuronal and astrocytic intermediary metabolism in the rat hippocampus.

Cannabinoid CB1 receptor (CB1R) activation decreases synaptic GABAergic and glutamatergic transmission and it also controls peripheral metabolism. Here we aimed at testing with ¹³C NMR isotopomer analysis whether CB1Rs could have a local metabolic role in brain areas having high CB1R density, such as the hippocampus. We labelled hippocampal slices with the tracers [2-¹³C]acetate, which is oxidized in glial cells, and [U-¹³C]glucose, which is metabolized both in glia and neurons, to evaluate metabolic compartmentation between glia and neurons. The synthetic CB1R agonist WIN55212-2 (1 µM) significantly decreased the metabolism of both [2-¹³C]acetate (-11.6±2.0%) and [U-¹³C]glucose (-11.2±3.4%) in the tricarboxylic acid cycle that contributes to the glutamate pool. WIN55212-2 also significantly decreased the metabolism of [U-¹³C]glucose (-11.7±4.0%) but not that of [2-¹³C]acetate contributing to the pool of GABA. These effects of WIN55212-2 were prevented by the CB1R antagonist AM251 (500 nM). These results thus suggest that CB1Rs might be present also in hippocampal astrocytes besides their well-known neuronal localization. Indeed, confocal microscopy analysis revealed the presence of specific CB1R immunoreactivity in astrocytes and pericytes throughout the hippocampus. In conclusion, CB1Rs are able to control hippocampal intermediary metabolism in both neuronal and glial compartments, which suggests new alternative mechanisms by which CB1Rs control cell physiology and afford neuroprotection.

[Ontogeny of sulphonylurea-binding regulatory subunits of K(ATP) channels in the pregnant rat myometrium]. / A K(ATP) csatorna szulfonilurea alegységeinek ontogenezise a terhes patkány miometriumban.

K(ATP) channels are composed of sulphonylurea receptors (SURs) and potassium inward rectifiers (Kir(6.x)) that assemble to form a large octameric channel. This study was designed to examine the expression and role of sulphonylurea-binding regulatory subunits 1 [SUR1 (ABCC8)] and 2 [SUR2 (ABCC9)] of the K(ATP) channels in the pregnant rat myometrium with particular regard to the contractility. RT-PCR and Western blot analysis were performed to detect the presence of SUR1 and SUR2. The SUR1 levels were markedly increased in the early stages of pregnancy. The highest level was detected on day 6 of pregnancy, while in the late stages the levels of SUR1 were significantly decreased. The SUR2 level remained unchanged throughout pregnancy. The SUR-non-selective diazoxide and the SUR2-selective pinacidil inhibited oxytocin-induced contractions. Glibenclamide, a K(ATP) channel blocker, antagonized both pinacidil and diazoxide-induced relaxations. It was established that SURs are responsible for pharmacological reactivity of K(ATP) channel openers. We conclude that, both SURs are involved in the K(ATP) channel in the pregnant rat myometrium. It may further be concluded that "pinacidil-like" K(ATP) channel openers may be of therapeutic relevance as tocolytic agents in the future.

Ontogeny of sulfonylurea-binding regulatory subunits of K(ATP) channels in the pregnant rat myometrium.

ATP-sensitive potassium channels (K(ATP) channels) are composed of sulfonylurea receptors (SURs) and potassium inward rectifiers (Kir(6.x)) that assemble to form a large octameric channel. This study was designed to examine the expression and role of sulfonylurea-binding regulatory subunits 1 (SUR1 (ABCC8)) and 2 (SUR2 (ABCC9)) of the K(ATP) channels in the pregnant rat myometrium with particular regard to the contractility. RT-PCR and western blot analyses were performed to detect the presence of SUR1 and SUR2. The SUR1 levels were markedly increased in the early stages of pregnancy. The highest level was detected on day 6 of pregnancy, whereas in the late stages, the levels of SUR1 were significantly decreased. The SUR2 level remained unchanged throughout pregnancy. The SUR non-selective diazoxide and the SUR2-selective pinacidil inhibited oxytocin-induced contractions. Glibenclamide, a K(ATP) channel blocker, antagonized both pinacidil- and diazoxide-induced relaxations. It was established that SURs are responsible for pharmacological reactivity of K(ATP) channel openers. We conclude that both SURs are involved in the K(ATP) channel in the pregnant rat myometrium. It may further be concluded that 'pinacidil-like' K(ATP) channel openers may be of therapeutic relevance as tocolytic agents in the future.

Functional selectivity and biased receptor signaling.

With the emergence of information describing functional selectivity and biased agonists and antagonists has come a lack of confidence in "one size fits all" assays for detection of agonism. Seven-transmembrane receptors are pleiotropic with respect to the signaling protein to which they couple in a cell, and many conformations of the receptor can be formed; this leads to systems where ligands can stabilize unique conformations that go on to selectively activate signaling pathways. Thus, such "biased" ligands can produce cell-specific agonism that may require targeted assays to detect and quantify. It also predicts that ligands can have many different efficacies for the many behaviors that the receptor can exhibit (referred to as "pluridimensional efficacy"), leading to a breakdown in the common classifications of agonist and antagonist. This all poses unique challenges to the pharmacologic nomenclature of drugs, the detection and optimization of new drugs, and the association of phenotypic clinical profiles with pharmacological properties of drugs.

The influence of affinity, efficacy, and slope factor on the estimates obtained by the receptorial responsiveness method (RRM): a computer simulation study.

The receptorial responsiveness method (RRM) was proposed to characterize changes in the concentration of degradable agonists in the microenvironment of their receptors. The characterization is done by providing concentrations of a stable agonist for the same receptor that is equieffective with the change in concentration to be characterized. RRM is based on the analysis of concentration-effect (E/c) curves reflecting the simultaneous action of the degradable and the stable agonist. In the present study, we investigated whether dissimilar affinity and (or) efficacy of the coacting agonists as well as the steepness of the E/c curves influence the reliability of RRM. E/c curves were simulated based on the operational model and then analyzed with RRM. We found that dissimilarity in affinity of the coacting agonists did not affect the accuracy of RRM estimates. In contrast, accuracy of the estimation depended on the magnitude of the concentration to be assessed, the operational slope factor, and the operational efficacy ratio of the coacting agonists. However, our results suggest that proper choice of a stable agonist for a degradable one can help to ensure reliable results, since information about the change in concentration of a degradable agonist is otherwise difficult to obtain.

Effect of asymmetry of concentration-response curves on the results obtained by the receptorial responsiveness method (RRM): an in silico study.

The receptorial responsiveness method (RRM) was proposed to estimate changes in the concentration of an agonist in the microenvironment of its receptor. Usually, this is done by providing the equieffective concentration of another agonist for the same receptor or for a largely overlapping postreceptorial signaling ("test agonist"). The RRM is a special nonlinear regression algorithm to analyze a concentration-response (E/c) curve that represents the simultaneous actions of a single agonist concentration to be estimated and of increasing concentrations of the test agonist. The aim of this study was to explore whether asymmetry of the E/c curve to be analyzed influences the reliability of the RRM. For this purpose, computer simulation was performed by constructing symmetric and asymmetric E/c curves using the operational model of agonism, and then these curves were analyzed with the RRM. To perform the RRM, 2 types of equations were used: one involving the Hill equation, the simplest model of the E/c relationship, and one containing the Richards equation, an advanced model properly handling E/c curve asymmetry. Results of this study indicate that E/c curve asymmetry does not significantly influence the accuracy of the estimates provided by the RRM. Thus, when using the RRM, it is not necessary to replace the Hill equation with the Richards equation to obtain useful estimates. Furthermore, it was found that estimation of a high concentration of a high-efficacy agonist can fail when the RRM is performed with a low-efficacy test agonist in a system characterized by a small operational slope factor.

Diazoxide maintenance of myocyte volume and contractility during stress: evidence for a non-sarcolemmal K(ATP) channel location.

OBJECTIVE: Animal and human myocytes demonstrate significant swelling and reduced contractility during exposure to stress (metabolic inhibition, hyposmotic stress, or hyperkalemic cardioplegia), and these detrimental consequences may be inhibited by the addition of diazoxide (adenosine triphosphate-sensitive potassium channel opener) via an unknown mechanism. Both SUR1 and SUR2A subunits have been localized to the heart, and mouse sarcolemmal adenosine triphosphate-sensitive potassium channels are composed of SUR2A/Kir6.2 subunits in the ventricle and SUR1/Kir6.2 subunits in the atria. This study was performed to localize the mechanism of diazoxide by direct probing of sarcolemmal adenosine triphosphate-sensitive potassium channel current and by genetic deletion of channel subunits. METHODS: Sarcolemmal adenosine triphosphate-sensitive potassium channel current was recorded in isolated wild-type ventricular mouse myocytes during exposure to Tyrode's solution, Tyrode's + 100 µmol/L diazoxide, hyperkalemic cardioplegia, cardioplegia + diazoxide, cardioplegia + 100 µmol/L pinacidil, or metabolic inhibition using whole-cell voltage clamp (N = 7-12 cells per group). Ventricular myocyte volume was measured from SUR1(-/-) and wild-type mice during exposure to control solution, hyperkalemic cardioplegia, or cardioplegia + 100 µmol/L diazoxide (N = 7-10 cells per group). RESULTS: Diazoxide did not increase sarcolemmal adenosine triphosphate-sensitive potassium current in wild-type myocytes, although they demonstrated significant swelling during exposure to cardioplegia that was prevented by diazoxide. SUR1(-/-) myocytes also demonstrated significant swelling during exposure to cardioplegia, but this was not altered by diazoxide. CONCLUSIONS: Diazoxide does not open the ventricular sarcolemmal adenosine triphosphate-sensitive potassium channel but provides volume homeostasis via an SUR1-dependent pathway in mouse ventricular myocytes, supporting a mechanism of action distinct from sarcolemmal adenosine triphosphate-sensitive potassium channel activation.

Activation of SUR2B/Kir6.1 subtype of adenosine triphosphate-sensitive potassium channel improves pressure overload-induced cardiac remodeling via protecting endothelial function.

We sought to explore new strategies targeting SUR2B/Kir6.1, a subtype of adenosine triphosphate (ATP)-sensitive potassium channels (KATP), against pressure overload-induced heart failure. The effects of natakalim, a SUR2B/Kir6.1 selective channel opener, on progression of cardiac remodeling were investigated. Pressure overload-induced heart failure was induced in Wistar rats by abdominal aortic banding. The effects of natakalim (1, 3, and 9 mg·kg⁻¹·d⁻¹ for 10 weeks) on myocardial hypertrophy and heart failure, cardiac histology, vasoactive compounds, and gene expression were assessed. Ten weeks after the onset of pressure overload, natakalim treatment potently inhibited cardiac hypertrophy and prevented heart failure. Natakalim remarkably inhibited the changes of left ventricular hemodynamic parameters and reversed the increase of heart mass index, left ventricular weight index, and lung weight index. Histological examination demonstrated that there was no significant hypertrophy or fibrosis in pressure-overloaded hearts of natakalim-treated rats. Ultrastructural examination of hearts revealed well-organized myofibrils with mitochondria grouped along the periphery of longitudinally oriented fibers in rats from the natakalim group. The content of serum nitric oxide and plasma prostacyclin was increased, whereas that of plasma endothelin-1 and cardiac tissue hydroxyproline and atrial and B-type natriuretic peptide messenger RNA was downregulated in natakalim-treated rats. Natakalim at 0.01-100 µM had no effects on isolated working hearts derived from Wistar rats; however, natakalim had endothelium-dependent vasodilatory effects on the isolated tail artery helical strips precontracted with norepinephrine. These results indicate that natakalim reduces heart failure caused by pressure overloading by activating the SUR2B/Kir6.1 KATP channel subtype and protecting against endothelial dysfunction.

Noble gas xenon is a novel adenosine triphosphate-sensitive potassium channel opener.

BACKGROUND: Adenosine triphosphate-sensitive potassium (KATP) channels in brain are involved in neuroprotective mechanisms. Pharmacologic activation of these channels is seen as beneficial, but clinical exploitation by using classic K channel openers is hampered by their inability to cross the blood-brain barrier. This is different with the inhalational anesthetic xenon, which recently has been suggested to activate KATP channels; it partitions freely into the brain. METHODS: To evaluate the type and mechanism of interaction of xenon with neuronal-type KATP channels, these channels, consisting of Kir6.2 pore-forming subunits and sulfonylurea receptor-1 regulatory subunits, were expressed in HEK293 cells and whole cell, and excised patch-clamp recordings were performed. RESULTS: Xenon, in contrast to classic KATP channel openers, acted directly on the Kir6.2 subunit of the channel. It had no effect on the closely related, adenosine triphosphate (ATP)-regulated Kir1.1 channel and failed to activate an ATP-insensitive mutant version of Kir6.2. Furthermore, concentration-inhibition curves for ATP obtained from inside-out patches in the absence or presence of 80% xenon revealed that xenon reduced the sensitivity of the KATP channel to ATP. This was reflected in an approximately fourfold shift of the concentration causing half-maximal inhibition (IC50) from 26 +/- 4 to 96 +/- 6 microm. CONCLUSIONS: Xenon represents a novel KATP channel opener that increases KATP currents independently of the sulfonylurea receptor-1 subunit by reducing ATP inhibition of the channel. Through this action and by its ability to readily partition across the blood-brain barrier, xenon has considerable potential in clinical settings of neuronal injury, including stroke.

Sulfonylurea receptor 1 mutations that cause opposite insulin secretion defects with chemical chaperone exposure.

The beta-cell ATP-sensitive potassium (K(ATP)) channel composed of sulfonylurea receptor SUR1 and potassium channel Kir6.2 serves a key role in insulin secretion regulation by linking glucose metabolism to cell excitability. Mutations in SUR1 or Kir6.2 that decrease channel function are typically associated with congenital hyperinsulinism, whereas those that increase channel function are associated with neonatal diabetes. Here we report that two hyperinsulinism-associated SUR1 missense mutations, R74W and E128K, surprisingly reduce channel inhibition by intracellular ATP, a gating defect expected to yield the opposite disease phenotype neonatal diabetes. Under normal conditions, both mutant channels showed poor surface expression due to retention in the endoplasmic reticulum, accounting for the loss of channel function phenotype in the congenital hyperinsulinism patients. This trafficking defect, however, could be corrected by treating cells with the oral hypoglycemic drugs sulfonylureas, which we have shown previously to act as small molecule chemical chaperones for K(ATP) channels. The R74W and E128K mutants thus rescued to the cell surface paradoxically exhibited ATP sensitivity 6- and 12-fold lower than wild-type channels, respectively. Further analyses revealed a nucleotide-independent decrease in mutant channel intrinsic open probability, suggesting the mutations may reduce ATP sensitivity by causing functional uncoupling between SUR1 and Kir6.2. In insulin-secreting cells, rescue of both mutant channels to the cell surface led to hyperpolarized membrane potentials and reduced insulin secretion upon glucose stimulation. Our results show that sulfonylureas, as chemical chaperones, can dictate manifestation of the two opposite insulin secretion defects by altering the expression levels of the disease mutants.

Fasting-induced suppression of LH secretion does not require activation of ATP-sensitive potassium channels.

Reproductive hormone secretions are inhibited by fasting and restored by feeding. Metabolic signals mediating these effects include fluctuations in serum glucose, insulin, and leptin. Because ATP-sensitive potassium (K(ATP)) channels mediate glucose sensing and many actions of insulin and leptin in neurons, we assessed their role in suppressing LH secretion during food restriction. Vehicle or a K(ATP) channel blocker, tolbutamide, was infused into the lateral cerebroventricle in ovariectomized mice that were either fed or fasted for 48 h. Tolbutamide infusion resulted in a twofold increase in LH concentrations in both fed and fasted mice compared with both fed and fasted vehicle-treated mice. However, tolbutamide did not reverse the suppression of LH in the majority of fasted animals. In sulfonylurea (SUR)1-null mutant (SUR1(-/-)) mice, which are deficient in K(ATP) channels, and their wild-type (WT) littermates, a 48-h fast was found to reduce serum LH concentrations in both WT and SUR(-/-) mice. The present study demonstrates that 1) blockade of K(ATP) channels elevates LH secretion regardless of energy balance and 2) acute fasting suppresses LH secretion in both SUR1(-/-) and WT mice. These findings support the hypothesis that K(ATP) channels are linked to the regulation of gonadotropin-releasing hormone (GnRH) release but are not obligatory for mediating the effects of fasting on GnRH/LH secretion. Thus it is unlikely that the modulation of K(ATP) channels either as part of the classical glucose-sensing mechanism or as a component of insulin or leptin signaling plays a major role in the suppression of GnRH and LH secretion during food restriction.

5-Fluorotryptamine is a partial agonist at 5-HT3 receptors, and reveals that size and electronegativity at the 5 position of tryptamine are critical for efficient receptor function.

Antagonists, but not agonists, of the 5-HT3 receptor are useful therapeutic agents, and it is possible that partial agonists may also be potentially useful in the clinic. Here we show that 5-fluorotryptamine (5-FT) is a partial agonist at both 5-HT3A and 5-HT3AB receptors with an Rmax (Imax/Imax 5-HT) of 0.64 and 0.45 respectively. It is about 10 fold less potent than 5-HT: EC50=16 and 27 microM, and Ki for displacement of [3H]granisetron binding=0.8 and 1.8 microM for 5-HT3A and 5-HT3AB receptors respectively. We have also explored the potencies and efficacies of tryptamine and a range of 5-substituted tryptamine derivatives. At 5-HT3A receptors tryptamine is a weak (Rmax=0.15), low affinity (EC50=113 microM; Ki=4.8 microM) partial agonist, while 5-chlorotryptamine has a similar affinity to 5-FT (EC50=8.1 microM; Ki=2.7 microM) but is a very weak partial agonist (Rmax=0. 0037). These, and data from 5-methyltryptamine and 5-methoxytryptamine, reveal the importance of size and electronegativity at this location for efficient channel opening.

Receptor theory.

Receptor theory assigns mathematical rules to biological systems in order to quantify drug effects and define what biological systems can and cannot do, leading to the design of experiments that may further modify the model. Drug receptor theory also furnishes the tools for quantifying the activity of drugs in a system-independent manner, essential because drugs are almost always studied in test systems somewhat removed from the therapeutic system for which they are intended. Since biological systems operate at different set points in the body under different conditions, the ability to predict drug effects under a variety of circumstances is important. This unit provides a historical perspective of classical receptor theory and the currently used operational model of drug effects. The mechanism of drug receptor function is also described in terms of the various iterations of the ternary complex model, the two-state theory for ion channels, and a probabilistic model of multiple receptor conformations.

Overview of receptor interactions of agonists and antagonists.

Historically, the earliest methods used to quantitatively measure the fundamental properties of drugs (namely affinity and efficacy) employed isolated tissues, and it is in this realm that the null methods used to define "receptor pharmacology" were described. This unit describes these methods and their use to specifically classify agonists (through potency ratios and determination of relative affinities and efficacies) and antagonists (through analysis of surmountable and insurmountable antagonism) to yield estimates of potency. Different drugs can yield different behaviors in various tissues, so this unit is centered on a flow diagram to indicate the type of analysis appropriate for the behavior observed. For example, some agonists may be full agonists in some tissues and partial agonists in others, while some antagonists may demonstrate surmountable simple competitive antagonism in some tissues and insurmountable non-competitive antagonism in others. Methods exist for determination of affinity and efficacy for all of these behaviors, and these are delineated in this unit.