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 mem branes was fast, specific and saturable, with pH optimum at 6.5-7.0. T he divalent cations Mg2+ and Ca2+ clearly enhanced [H-3]CCK-8S binding , whereas the monovalent cations Na+ and K+ were inhibitors. Inactivat ion of the ligand binding ability of these membranes was dependent on the incubation temperature and corresponding tau(1/2) values were 11 d ays at 4 degrees, 12 hr at 21 degrees, 154 min at 30 degrees and 51 mi n 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-thiotriphos phate) (GTP gamma S) decreased the affinity of [H-3]CCK-8S binding onl y up to 2-fold without significant influence on maximal binding. Modul ation of membrane properties by different detergents revealed that. on ly in the case of digitonin (0.03-0.04%) did the GTP-dependence of [H- 3]CCK-8S binding considerably increase without significant influence o n the ligand binding properties in the absence of GTP gamma S. Other d etergents studied (sodium cholate, sodium deoxycholate, cholamidopropy l)dimethylammonio-1-propanesulfonate (CHAPS), sucrose monolaurate, ser ies Triton X and Tween) either had little influence on GTP gamma S dep endence of [H-3]CCK-8S binding or inactivated the receptor. Parallel s tudies of fluorescent polarization of diphenylhexatriene (DPH) in rat cortical membranes indicated that digitonin was the only detergent whi ch at low concentrations caused a rapid increase in membrane fluidity and thereafter stabilized it at a certain level. Other detergents stud ied had only moderate influence on membrane fluidity (CHAPS, cholate, deoxycholate) or caused fast and continuous increase of membrane fluid ity (Triton X-100, Tween 80). These data together point to the essenti al influence of the fluidity of membranes on the regulation of the int eractions between G proteins and CCK receptors in rat cortical membran es. Under standard experimental conditions (temperature lower than 30 degrees), the CCK receptor-G protein complex is active for quantitativ e characterization of the receptors, but the membranes are too rigid f or natural communication and regulation. (C) 1998 Elsevier Science Inc .