Interaction of substance P and its N- and C-terminal fragments with Ca2+: Implications for hormone action

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Abstract
In an attempt to understand the role of Ca2+ on the bioactive conformation of peptide hormones, we have examined the interaction between Ca2+ and the neuropeptide substance P. Using CD spectroscopy to monitor conformational changes caused by Ca2+ binding, we found no significant binding of the cation by substance P in water. However, a substantial conformational change occurred in the hormone on Ca2+ addition in trifluoroethanol or an 80:20 (v/v) mixture of acetonitrile and trifluoroethanol. A biphasic binding of Ca2+ was observed in these solvents with saturation at 2 cations per hormone molecule. Mg2+ caused a relatively smaller conformational change in the hormone. A peptide corresponding to residues 1–7 at the N-terminal fragment of substance P showed a weak nonsaturating binding of Ca2+ in the nonpolar solvents whereas the 7–11 C-terminal fragment peptide displayed a binding indicative of an 1 : 1 Ca2+ / peptide complex. Ca2+ binding by the hormone and the 7–11 fragment was also monitored by changes in fluorescence of the phenylalanyl residues. The results support the conclusion drawn from the CD data about a distinct Ca2+ binding site in the C-terminal part of substance P. The Kd values obtained from fluorescence data were 160 µM for Ca2+ and 1 mM for Mg2+ binding by substance P. The hormone and the two peptide fragments were also tested for their effect on the stability of dimyristoyl lecithin vesicles. Substance P and the N-terminal fragment caused no significant leakage of either fluorescent dyes or K+ trapped in the vesicles. Nor did they cause membrane fusion as monitored by the fluorescence quenching method. In contrast, the C-terminal 7–11 fragment caused considerable leakage of vesicular contents and promoted membrane fusion. When tested for their ability to translocate Ca2+ across the lipid bilayer in dimyristoyl lecithin vesicles containing the Ca2+ indicator Arsenazo III, both substance P and the 7–11 fragment showed significant Ca2+ transport while the 1–7 fragment showed negligible transport. The apparent transport of the 7–11 fragment is attributable to its effect on the integrity of the bilayer membrane. These data distinguish the N- and C-terminal domains of substance P in terms of their lipid-dependent interaction with Ca2+. In light of the requirement of extracellular Ca2+ for substance P action, the data are also suggestive of the possibility of the involvement of Ca2+ in the bioactive conformation of this hormone and in its interaction with the receptor. © 1992 John Wiley & Sons, Inc.