Assessment of Purity, Functionality, Stability, and Lipid Composition of Cyclofos-nAChR-Detergent Complexes from Torpedo californica Using Lipid Matrix and Macroscopic Electrophysiology.

The main objective of the present study was to find detergents that can maintain the functionality and stability of the Torpedo californica nicotinic acetylcholine receptor (Tc-nAChR). We examined the functionality, stability, and purity analysis of affinity-purified Tc-nAChR solubilized in detergents from the Cyclofos (CF) family [cyclofoscholine 4 (CF-4), cyclofoscholine 6 (CF-6), and cyclofloscholine 7 (CF-7)]. The functionality of the CF-Tc-nAChR-detergent complex (DC) was evaluated using the Two Electrode Voltage Clamp (TEVC) method. To assess stability, we used the florescence recovery after photobleaching (FRAP) in Lipidic Cubic Phase (LCP) methodology. We also performed a lipidomic analysis using Ultra-Performance Liquid Chromatography (UPLC) coupled to electrospray ionization mass spectrometry (ESI-MS/MS) to evaluate the lipid composition of the CF-Tc-nAChR-DCs. The CF-4-Tc-nAChR-DC displayed a robust macroscopic current (- 200 ± 60 nA); however, the CF-6-Tc-nAChR-DC and CF-7-Tc-nAChR-DC displayed significant reductions in the macroscopic currents. The CF-6-Tc-nAChR and CF-4-Tc-nAChR displayed higher fractional florescence recovery. Addition of cholesterol produced a mild enhancement of the mobile fraction on the CF-6-Tc-nAChR. The lipidomic analysis revealed that the CF-7-Tc-nAChR-DC displayed substantial delipidation, consistent with the lack of stability and functional response of this complex. Although the CF-6-nAChR-DC complex retained the largest amount of lipids, it showed a loss of six lipid species [SM(d16:1/18:0); PC(18:2/14:1); PC(14:0/18:1); PC(16:0/18:1); PC(20:5/20:4), and PC(20:4/20:5)] that are present in the CF-4-nAChR-DC. Overall, the CF-4-nAChR displayed robust functionality, significant stability, and the best purity among the three CF detergents; therefore, CF-4 is a suitable candidate to prepare Tc-nAChR crystals for structural studies.

Comparison of intraoperative and postoperative pain during canine ovariohysterectomy and ovariectomy.

This study compared physiologic parameters indicating nociception during surgery and pain scores after surgery among dogs undergoing ovariohysterectomy (OHE) and ovariectomy (OVE). Twenty healthy adult female dogs were randomly assigned to either the OHE or the OVE group. Physiologic data collected during surgery included heart rate, respiratory rate, temperature, blood pressure, hemoglobin oxygen saturation, end-tidal CO2 and isoflurane, and vaporizer settings. Postoperative pain was measured using the short form Glasgow Composite Pain Scale, an interactive visual analog scale, and algometry. There were no clinically relevant differences in intraoperative nociception indices between groups. Duration of surgery for OVE was significantly shorter than for OHE (OVE 15.4 minutes, OHE 17.5 minutes, P = 0.04). There was no significant difference between groups in the use of rescue analgesia after surgery, in the average interactive visual analog scale score over the 24-hour postoperative period (P = 0.12), and in algometer readings (P = 0.34).

Comparaison de la douleur peropératoire et postopératoire durant l'ovariohystérectomie et l'ovariectomie canines. Cette étude a comparé les paramètres physiologiques indiquant la nociception durant la chirurgie et la cotation des douleurs après la chirurgie parmi les chiennes subissant une ovariohystérectomie (OHE) et une ovariectomie (OVE). Vingt chiennes adultes en santé ont été réparties au hasard soit au groupe OHE ou au groupe OVE. Les données physiologiques recueillies durant la chirurgie incluaient la fréquence cardiaque, la fréquence respiratoire, la température, la tension artérielle, la saturation en oxygène de l'hémoglobine le PCO2 et l'isoflurane de fin d'expiration ainsi que les réglages du nébuliseur. La douleur postopératoire a été mesurée à l'aide de la forme abrégée de l'échelle de douleur composée de Glasgow, d'une échelle analogue visuelle interactive et de l'algométrie. Il n'y avait pas de différences pertinentes sur le plan clinique dans les indices de nociception peropératoire entre les groupes. La durée de la chirurgie d'OVE était significativement plus courte que celle d'OHE (OVE 15,4 minutes, OHE 17,5 minutes, P = 0,04). Il n'y avait aucune différence significative entre les groupes pour le recours à un analgésique de secours après la chirurgie, dans la note d'échelle visuelle interactive moyenne pendant la période postopératoire de 24 heures (P = 0,12) et dans les lectures de l'algésimètre (P = 0,34).(Traduit par Isabelle Vallières).

Uncovering the lipidic basis for the preparation of functional nicotinic acetylcholine receptor detergent complexes for structural studies.

This study compares the lipid composition, including individual phospholipid molecular species of solubilized nAChR detergent complexes (nAChR-DCs) with those of the bulk lipids from their source, Torpedo californica (Tc) electric tissue. This lipidomic analysis revealed seventy-seven (77) phospholipid species in the Tc tissue. Analysis of affinity-purified nAChR-DCs prepared with C-12 to C-16 phospholipid analog detergents alkylphosphocholine (FC) and lysofoscholine (LFC) demonstrated that nAChR-DCs prepared with FC12, LFC14, and LFC16 contained >60 phospholipids/nAChR, which was more than twice of those prepared with FC14, FC16, and LFC12. Significantly, all the nAChR-DCs lacked ethanolamine and anionic phospholipids, contained only four cholesterol molecules, and a limited number of phospholipid molecular species per nAChR. Upon incorporation into oocytes, FC12 produce significant functionality, whereas LFC14 and LFC16 nAChR-DCs displayed an increased functionality as compared to the crude Tc membrane. All three nAChR-DCs displayed different degrees of alterations in macroscopic activation and desensitization kinetics.