Structure‐activity relationship of the neurotransmitter alpha‐bag cell peptide on Aplysia LUQ neurons: Implications regarding its inactivation in the extracellular space

Alpha‐bag cell peptide [α‐BCP (Ala‐Pro‐Arg‐Leu‐Arg‐Phe‐Tyr‐Ser‐Leu)] is a neurotransmitter that mediates bag cell‐induced inhibition of left‐upper‐quadrant (LUQ) neurons L2, L3, L4, and L6 in the abdominal ganglion of Aplysia. Our recent biochemical studies have shown that α‐BCP[1–9] is cleaved into α‐BCP[1–2], [3–9], [1–5], [6–9], and [7–9] by a combination of three distinct peptidase activities located within the extracellular spaces of the CNS: A diaminopeptidase‐IV (DAP‐IV)‐like enzyme cleaves α‐BCP[1–9] at the 2–3 peptide bond; a neutral metalloendopeptidase (NEP)‐like enzyme cleaves either α‐BCP[1–9] or α‐BCP[3–9] at the 5–6 bond; an aminopeptidase M‐II (APM‐II)‐like enzyme cleaves α‐BCP[6–9] at the 6–7 bond, but cleaves neither α‐BCP[1–9], nor the other ganglionic peptidase products. To further understand the manner in which α‐BCP is inactivated after release, that is loses its electro‐physiological activity, we studied its structure‐activity relationship by recording intracellularly from LUQ neurons in isolated abdominal ganglia that were arterially perfused with peptides dissolved in artificial sea water. The effects of α‐BCP[1–9] and 15 of its fragments ([1–8], [1–7], [1–6], [1–5], [2–9], [3–9], [3–8], [6–9], [7–9], [8–9], [6–7], [6–8], [1–2], Phe, Tyr) indicated that the sequence Phe6‐Tyr7 was both necessary and sufficient to produce LUQ inhibitory activity. The combined results of our electrophysiological and biochemical studies strongly suggest that α‐BCP[1–9] is inactivated by the serial actions of the NEP‐like and APM‐II‐like peptidases; that is, the NEP‐like enzyme yields an electro‐physiologically active product, α‐BCP[6–9], that is cleaved by the APM‐II‐like enzyme to yield inactive α‐BCP[7–9]. Furthermore, because α‐BCP[6–9] is more active than α‐BCP[1–9], cleavage by the NEP‐like enzyme potentiates α‐BCP's activity.