BINDING OF SUBSTANCE-P AGONISTS TO LIPID-MEMBRANES AND TO THE NEUROKININ-1 RECEPTOR

Three new analogues of the neuropeptide substance P (SP) were synthesi zed. The C-terminal message segment was made more hydrophilic in (Arg( 9))SP or more hydrophobic in (Nle(9))SP. In(AcPro(2), Arg(9))SP the ch arge at the N-terminal address segment was reduced, while that of the message segment was increased. The rationale underlying these substitu tions was to correlate the physical-chemical properties of the SP-anal ogues, in particular their lipid-induced conformation and membrane-bin ding affinity, with receptor binding and functional activity. In solut ion, all three analogues exhibited random coil conformations as eviden ced by circular dichroism spectroscopy. Addition of SDS micelles induc ed partially alpha-helical structures. The same structure was also pro duced by negatively charged lipid vesicles for (AcPro(2), Arg(9))SP an d (Arg(9))SP whereas both alpha-helix-like structures and beta-sheet s tructures were observed for SP and (Nle(9))SP. The measurement of the Gibbs adsorption isotherms and monolayer expansion studies provided qu antitative data on the surface area requirement and on the membrane pe netration area of the SP analogues. The thermodynamic parameters for l ipid binding were determined with monolayer expansion constants, K-app , for membranes containing 100% POPG were of the order of 10(3)-10(5) M(-1). The binding was due to electrostatic attraction of the cationic peptides to the negatively charged membrane surface. The intrinsic (h ydrophobic) binding constants, obtained after correcting for electrost atic effects, were much smaller with K-p = 10 +/- 1 M(-1) for (Arg(9)) SP, 9 +/- 1 M(-1) for (AcPro(2), Arg(9))SP, and 39 +/- 3 M(-1) for (Nl e(9))SP. The measurement of the binding affinities to the NK-I recepto r and of the in vitro activities showed that all three peptides behave d as agonists. Their binding affinity to the neurokinin-l receptor dec reased with the size of the side chains at position 9 of the amino aci d sequence but was independent of the cationic charge of the peptides. The fact that even the highly charged (Arg(9))SP has agonistic activi ty provides evidence that the binding epitope at the receptor is in a rather hydrophilic environment. This finding is in agreement with the low hydrophobic binding constants and the weak penetration of the thre e peptides into negatively charged membranes. It argues against a memb rane mediated receptor mechanism and suggests that the agonist approac hes the receptor binding site from the aqueous phase.