Ž . Brain Research 821 1999 294–308 Research report Structure–activity relationships of the Helix neuropeptide, SEPYLRFamide, and its N-terminally modified analogues on identified Helix lucorum neurones A.S. Pivovarov a , R. Sharma b , R.J. Walker b, ) a Department of Higher NerÕous ActiÕity, Biological Faculty, Moscow LomonosoÕ State UniÕersity, Moscow 119899, Russian Federation b School of Biological Sciences, UniÕersity of Southampton, Bassett Crescent East, Southampton S016 7PX, England, UK Accepted 22 December 1998 Abstract Metabotropic and ionotropic effects evoked by the endogenous Helix heptapeptide, SEPYLRFamide, and four analogues, i.e., where Ž . the amino acid sequences at the N-terminal EPYLRFamide, SEGYLRFamide, SRPYLRFamide and SKPYLRFamide were modified, were compared on identified Helix lucorum LPa2, LPa3, RPa3, RPa2 neurones using two electrode voltage clamp and current clamp Ž . techniques. All peptides bath application reduce reversibly the inward current to local ionophoretic application of acetylcholine onto the neurone soma with an order of potency: EPYLRFamide sSEGYLRFamide sSRPYLRFamide )SEPYLRFamide )SKPYLRFamide. The reductions of the acetylcholine-induced inward current evoked by SEPYLRFamide and its analogues at concentrations of 0.01–10 mM are not accompanied by a change of amplitude of the leak inward current caused by constant negative shift of a holding potential. At concentration of 50 mM all peptides increase reversibly the resting membrane conductance to an equal degree. Local application under pressure of SEPYLRFamide and its analogues onto the soma of neurones evoke hyperpolarizations with similar values. These results indicate that the N-terminal three amino acids of the peptide molecule are not responsible for the degree of ionotropic effect on the neurones studied. In contrast the amino acid sequence at the N-terminal modifies the degree of the modulatory effects of the Ž 1 2 3 . YLRFamide-related analogues. Changes at the SEPYLRFamide N-terminal Ser -Glu -Pro intensify the inhibitory action of the 1 Ž 1 2 . 3 analogues as compared with effect evoked by the endogenous peptide, that is, removal of Ser Glu -Pro , replacement of Pro with 3 Ž 1 2 3 . 2 2 Ž 1 2 3 . 2 2 Ž 1 2 3 . Gly Ser -Glu -Gly , replacement of Glu with Arg Ser -Arg -Pro . Replacement of Glu with Lys Ser -Lys -Pro reduces the modulatory potency. It is concluded that ionotropic and metabotropic effects of these YLRFamide-related peptides may occur at different membrane binding sites. q 1999 Elsevier Science B.V. All rights reserved. Keywords: SEPYLRFamide; EPYLRFamide; SEGYLRFamide; SRPYLRFamide; SKPYLRFamide; Acetylcholine; Identified neurones; Helix lucorum 1. Introduction Heptapeptide, Ser 1 -Glu 2 -Pro 3 -Tyr 4 -Leu 5 -Arg 6 -Phe 7 - Ž . NH SEPYLRFamide , a FMRFamide-related peptide, 2 w x was identified from the land snail, Helix aspersa 18 . The structure of SEPYLRFamide is similar to the endogenous FMRFamide-related heptapeptide SKPYMRFamide which was extracted from CNS of the freshwater snail, Lymnaea wx stagnalis 5 . These two peptides are different with respect ) Corresponding author. Fax: q44-01703-594319; E-mail: physiol@soton.ac.uk to amino acids in 2 and 5 positions: Lymnaea heptapeptide contains Lys 2 instead of Glu 2 and Met 5 instead of Leu 5 . The biological activity of SKPYMRFamide has been investigated on identified neurones of H. aspersa, Helix w x lucorum and L. stagnalis 11,15,16 . SKPYMRFamide acts as an inhibitory neuromodulator of cholinergic trans- mission in Helix parietal ganglia and has direct excitatory effects on L. stagnalis neurones but inhibitory effects on H. lucorum neurones. SKPYMRFamide achieves its modu- latory action at the postsynaptic level via inhibition of Ž . w x acetylcholine ACh receptors 15,16 . This peptide re- versibly decreases excitatory responses evoked by local ionophoretic application of ACh onto the soma of identi- w x fied F1, F2, F4, F5r6 neurones in H. aspersa 15 and w x LPa2, LPa3, RPa3 neurones in H. lucorum 16 . SKPYM- 0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. Ž . PII: S0006-8993 99 01097-5