Brain Research, 533 (1990) 137-140 Elsevier 137 BRES 24365 Microcystin-LR, a potent protein phosphatase inhibitor, prolongs the serotonin- and cAMP-induced currents in sensory neurons of Aplysia californica Mitsuyuki Ichinose 1, Shogo Endo 1, Stuart D. Critz 1, Shirish Shenolikar 2 and John H. Byrne 1 Departments of JNeurobiology and Anatomy, and 2Pharmacology, University of Texas Medical School, Houston, TX 77225 (U.S.A.) (Accepted 31 July 1990) Key words: Aplysia; Microcystin-LR;Serotonin; cAMP; Protein phosphatase; Phosphatase inhibitor; S-channel Microcystin-LR(MCYST-LR), a hepatotoxin produced by cyanobacteria, inhibited purified protein phosphatases (PrPs) from rabbit skeletal muscle and the enzymes from Aplysia with an ICs0 of approximately 10 -1° M. MCYST-LR also prolonged both serotonin- (5-HT) and cyclic adenosine monophosphate-induced inward currents in sensory neurons of Aplysia. These results, which are consistent with inhibition of Aplysia PrPs, indicate that MCYST-LR may be a useful probe to elucidate the function of PrPs in neural tissues. The function of certain membrane channels can be modulated by phosphorylation in response to physiolog- ical stimuli. In Aplysia sensory neurons, for example, serotonin (5-HT) stimulates a cAMP-dependent protein kinase leading to increased protein phosphorylation and closure of the S-K + channel 1'21'22. This represents an extensively studied model of learning and memory TM. Little is known about mechanisms regulating dept~os- phorylation and reversal of the serotonergic response. Recently, we demonstrated the presence of two protein phosphatases, termed PrP-1 and PrP-2A, in neuronal tissues from Aplysia that are biochemically similar to their mammalian counterparts 8-w. We also showed that okadaic acid (OA), a potent inhibitor of these PrPs 2, prolonged the responses to 5-HT and 8-bromo-cAMP in voltage-clamped sensory neurons ~4. Recent studies have shown that MCYST-LR, a hepa- totoxic cyclic heptapeptide from Microcystis aeruginosa H" 23, activated phosphorylase a in the absence of changes in intracellular calcium or cAMP in intact hepatocytes ~8. Subsequently, MCYST-LR was shown to be a potent inhibitor of mammalian phosphorylase a phosphatases 16, suggesting that MCYST-LR-induced decrease in hepatic PrP activity accounted for the activation of phosphorylase a, Here, we report that MCYST-LR is also an inhibitor of Aplysia PrPs, being more potent than OA. Initially, we confirmed the biochemical effects of MCYST-LR on vertebrate PrPs. PrP-1 and PrP-2A were purified from rabbit skeletal muscle according to the method of DeGuzman and Lee 7. PrP-2B was a gift of Dr. Randall L. Kincaid, Section of Immunology, Laboratory of Physiologic and Pharmacologic Studies, NIAAA. Activity of these PrPs was determined using either 32p-labelled phosphorylase a or p-nitrophenyl phosphate (PNPP, Sigma) as substrate (see legend Fig. 1). A dose-dependent inhibition of phosphorylase a phospha- tase activity in the presence of MCYST-LR (Calbiochem) was observed (Fig. 1). Both PrP-1 and PrP-2A were potently inhibited with an identical ICs0 of approximately 10-1° M. Complete inhibition of phosphorylase a dephos- phorylation was observed at concentrations greater than 10 .-8 M. Thus, MCYST-LR was 10-100 times more potent than OA as an inhibitor of PrP-1 and PrP- 2A 2'5A6'2°. With PNPP as substrate, MCYST-LR (10-6 M) inhibited PrP-2B activity by 60% but had no effect on alkaline phosphatase activity. The effect of MCYST-LR on Aplysia PrPs was assayed by the dephosphorylation of phosphorylase a, using either tissue extracts (containing both prpol and PrP-2A) or salt-extracts of particulate fractions from abdominal ganglion, containing primarily PrP-18'1° (Fig. 2). These tissue fractions were prepared as described in the legend for Fig. 2. The addition of MCYST-LR resulted in a dose-dependent inhibition of phosphorylase a dephos- phorylation, with an ICs0 of approximately 10-1° M, with both Aplysia extracts (Fig. 2). The inhibition of salt- extracted PrP from the particulate fraction of abdominal ganglion resembled that seen with purified PrP-1 from Correspondence: J.H. Byrne, Department of Neurobiologyand Anatomy, Universityof Texas Medical School, P.O. Box 20708, Houston, TX 77225, U.S.A. 0006-8993/90/$03.50 ~ 1990 Elsevier Science Publishers B.V. (Biomedical Division)