Role of fluidity of membranes on the guanyl nucleotide dependent binding of cholecystokinin-8S to rat brain cortical membranes

The binding of [H-3]cholecystokinin octapeptide (sulphated) ([H-3]CCK-8S), an agonist of the cholecystokinin receptors, to rat. cortical membranes was fast, specific and saturable, with pH optimum at 6.5-7.0. The divalent cations Mg2+ and Ca2+ clearly enhanced [H-3]CCK-8S binding, whereas the monovalent cations Na+ and K+ were inhibitors. Inactivation of the ligand binding ability of these membranes was dependent on the incubation temperature and corresponding tau(1/2) values were 11 days at 4 degrees, 12 hr at 21 degrees, 154 min at 30 degrees and 51 min at 37 degrees, which revealed the apparent activation energy of this process to be 130 +/- 4 kJ/mol. Scatchard analysis of the saturation curves of [H-3]CCK-8S binding was best described by a one site binding model with a K-d = 0.63 +/- 0.18 nM and a maximum binding of 32 +/- 2 fmol/mg protein. The stable GTP analogue guanosin-5'-O-(3-thiotriphosphate) (GTP gamma S) decreased the affinity of [H-3]CCK-8S binding only up to 2-fold without significant influence on maximal binding. Modulation of membrane properties by different detergents revealed that. only in the case of digitonin (0.03-0.04%) did the GTP-dependence of [H-3]CCK-8S binding considerably increase without significant influence on the ligand binding properties in the absence of GTP gamma S. Other detergents studied (sodium cholate, sodium deoxycholate, 3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonate (CHAPS), sucrose monolaurate, series Triton X and Tween) either had little influence on GTP gamma S dependence of [H-3]CCK-8S binding or inactivated the receptor. Parallel studies of fluorescent polarization of diphenylhexatriene (DPH) in rat cortical membranes indicated that digitonin was the only detergent which at low concentrations caused a rapid increase in membrane fluidity and thereafter stabilized it at a certain level. Other detergents studied had only moderate influence on membrane fluidity (CHAPS, cholate, deoxycholate) or caused fast and continuous increase of membrane fluidity (Triton X-100, Tween 80). These data together point to the essential influence of the fluidity of membranes on the regulation of the interactions between G proteins and CCK receptors in rat cortical membranes. Under standard experimental conditions (temperature lower than 30 degrees), the CCK receptor-G protein complex is active for quantitative characterization of the receptors, but the membranes are too rigid for natural communication and regulation. (C) 1998 Elsevier Science Inc.