Structure–activity relationships of the Helix neuropeptide, SEPYLRFamide, and its N-terminally modified analogues on identified Helix lucorum neurones

Metabotropic and ionotropic effects evoked by the endogenous Helix heptapeptide, SEPYLRFamide, and four analogues, i.e., where 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 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=SEGYLRFamide=SRPYLRFamide>SEPYLRFamide>SKPYLRFamide. The reductions of the acetylcholine-induced inward current evoked by SEPYLRFamide and its analogues at concentrations of 0.01–10 μM 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 μM 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 YLRFamide-related analogues. Changes at the SEPYLRFamide N-terminal (Ser 1-Glu 2-Pro 3) intensify the inhibitory action of the analogues as compared with effect evoked by the endogenous peptide, that is, removal of Ser 1 (Glu 1-Pro 2), replacement of Pro 3 with Gly 3 (Ser 1-Glu 2-Gly 3), replacement of Glu 2 with Arg 2 (Ser 1-Arg 2-Pro 3). Replacement of Glu 2 with Lys 2 (Ser 1-Lys 2-Pro 3) reduces the modulatory potency. It is concluded that ionotropic and metabotropic effects of these YLRFamide-related peptides may occur at different membrane binding sites.