Luca F. Pisterzi

Oligomeric Size of the M2 Muscarinic Receptor in Live Cells as Determined by Quantitative Fluorescence Resonance Energy Transfer

Fluorescence resonance energy transfer (FRET), measured by fluorescence intensity-based microscopy and fluorescence lifetime imaging, has been used to estimate the size of oligomers formed by the M2 muscarinic cholinergic receptor. The approach is based on the relationship between the apparent FRET efficiency within an oligomer of specified size (n) and the pairwise FRET efficiency between a single donor and a single acceptor (E). The M2 receptor was fused at the N terminus to enhanced green or yellow fluorescent protein and expressed in Chinese hamster ovary cells. Emission spectra were analyzed by spectral deconvolution, and apparent efficiencies were estimated by donor-dequenching and acceptor-sensitized emission at different ratios of enhanced yellow fluorescent protein-M2 receptor to enhanced green fluorescent protein-M2 receptor. The data were interpreted in terms of a model that considers all combinations of donor and acceptor within a specified oligomer to obtain fitted values of E as follows: n = 2, 0.495 ± 0.019; n = 4, 0.202 ± 0.010; n = 6, 0.128 ± 0.006; n = 8, 0.093 ± 0.005. The pairwise FRET efficiency determined independently by fluorescence lifetime imaging was 0.20–0.24, identifying the M2 receptor as a tetramer. The strategy described here yields an explicit estimate of oligomeric size on the basis of fluorescence properties alone. Its broader application could resolve the general question of whether G protein-coupled receptors exist as dimers or larger oligomers. The size of an oligomer has functional implications, and such information can be expected to contribute to an understanding of the signaling process.

Binding of Orthosteric Ligands to the Allosteric Site of the M2 Muscarinic Cholinergic Receptor

The M2 muscarinic receptor has two topographically distinct sites: the orthosteric site and an allosteric site recognized by compounds such as gallamine. It also can exhibit cooperative effects in the binding of orthosteric ligands, presumably to the orthosteric sites within an oligomer. Such effects would be difficult to interpret, however, if those ligands also bound to the allosteric site. Monomers of the hemagglutinin (HA)- and FLAG-tagged human M2 receptor therefore have been purified from coinfected Sf9 cells and examined for any effect of the antagonist N-methyl scopolamine or the agonist oxotremorine-M on the rate at which N-[3H]methyl scopolamine dissociates from the orthosteric site (kobsd). The predominantly monomeric status was confirmed by coimmunoprecipitation and by cross-linking with bis(sulfosuccinimidyl)suberate. Both N-methyl scopolamine and oxotremorine-M acted in a cooperative manner to decrease kobsd by 4.5- and 9.1-fold, respectively; the corresponding estimates of affinity (log KL) are -2.55 ± 0.13 and -2.29 ± 0.14. Gallamine and the allosteric ligand obidoxime decreased kobsd by more than 100-fold (log KL = -4.12 ± 0.04) and by only 1.1-fold (log KL = -1.73 ± 0.91), respectively. Obidoxime reversed the effect of N-methyl scopolamine, oxotremorine-M, and gallamine in a manner that could be described by a model in which all four ligands compete for a common allosteric site. Ligands generally assumed to be exclusively orthosteric therefore can act at the allosteric site of the M2 receptor, albeit at comparatively high concentrations.

Density functional molecular computations on protonated serotonin in the gas phase and various solvent media

Abstract 5-Hydroxytryptamine (serotonin) was geometry optimized at the B3YP/6-31G(d) level of theory to determine the energetically most favourable conformations of the aromatic hydroxyl group and the protonated ethylamine side chain. The hydroxyl group was found to be most stable at anti for all conformations, and the two lowest energy gas phase conformers found were: χ 2 = g + , χ 3 = g − and χ 2 = g − , χ 3 = g + . The protonated amino group was found equally stable at g + , g − and anti . The transition structures linking each gas phase minimum were also computed. Minima found were subjected to solvation calculations in chloroform, DMSO, ethanol and water, which shifted their relative stabilities.

Nursing Workload Associated with Hospital Patient Care

BackgroundNursing costs are poorly estimated in economic studies conducted in hospitals because of a lack of data on nursing times for day-to-day activities relating to patient care.ObjectiveTo determine standard time values for nursing activities for specific indications in a cohort of hospital patients.Study designPatient care hours scores, calculated using the Grace Reynolds Application and Study of PETO (GRASP®) system, were extracted from patient records. A total of 483 patients with five different indications (acute myocardial infarction [MI]: n = 98; diabetes mellitus: n = 93; pneumonia: n = 98; schizophrenia: n = 94; and stroke: n = 100) who were hospitalized in March 2002 at the Sunnybrook Health Sciences Centre, Toronto, Canada were included.Main outcomePatient care hour scores were converted into minutes, and the results were stratified by patient diagnosis and analyzed as to the frequency and duration of each nursing task.ResultsNurses reported that the greatest amount of time was spent providing care for patients with MI, (2.50 ± 2.31 hours of care per patient per hospital stay), followed by those with pneumonia (2.35 ± 2.02 hours), diabetes (1.96 ± 1.31 hours), schizophrenia (1.72 ± 1.95 hours), and stroke (1.70 ± 1.47 hours). The task most frequently undertaken in patients with MI was cardiovascular assessment; in patients with diabetes and pneumonia, it was routine teaching/emotional support; in patients with schizophrenia, it was ongoing assessment; and in patients with stroke, it was administration of medications. Pulmonary artery pressure monitoring consumed the most time in the nursing of patients with MI (110.83 minutes), whereas one-to-one supervision of a patient by a nurse (i.e. 1:1 sitter) consumed the most time in patients with diabetes, pneumonia, schizophrenia, and stroke.ConclusionA standard table of time values for task-specific nursing workload in five indications was created using data obtained from the GRASP® system. Patients with MI required the most nursing care, and those with stroke required the least. This type of data will be important for future studies to determine nursing resource allocation as well as for evaluating the impact of new treatments on the nursing times associated with specific tasks.

Oligomeric Structure of Muscarinic and Adrenergic Receptors in Live Cells

We have determined the oligomeric size and configuration of fluorophore-tagged M1, M2, β1, and β2 receptors in the plasma membrane of Chinese hamster ovary (CHO) cells by examining the distribution of FRET efficiencies measured at the level of single pixels. The receptors were fused at the N-terminus to enhanced green or yellow fluorescent protein, and complementary pairs were co-expressed at different ratios of donor to acceptor (i.e., eGFP2-M1 and eYFP-M1, eGFP2-M2 and eYFP-M2, eGFP2-β1 and eYFP-β1, or eGFP2-β2 and eYFP-β2). Pixel-level emission spectra were recorded from images captured in a single plane; the relative contribution of each fluorophore then was determined by spectral deconvolution and used to calculate the corresponding apparent FRET efficiency. The distributions of efficiencies from 20 cells were analyzed in concert as a sum of Gaussians, with the number of components (n) and the relationships among the means taken as predicted for a dimer (n = 1), a triangular trimer (n = 2), and a tetramer configured as a square (n = 3) and a rhombus (n = 5). Distributions from each of the four receptors required 5 Gaussians, the means of which were related in the manner predicted for a rhombus. The inverse agonists atropine and timolol were without effect on the number of components or the relationship among the means detected for the M2 muscarinic receptor and the β2 adrenergic receptor, respectively. Homo-oligomers of the M1, M2, β1, and β2 receptors therefore appear to be rhombic tetramers in CHO cells. The configuration is unaffected by inverse agonists, at least in the case of the M2 and β2 receptors.