Prorocentrolide-A from Cultured Prorocentrum lima Dinoflagellates Collected in Japan Blocks Sub-Types of Nicotinic Acetylcholine Receptors

Abstract

Prorocentrolides are members of the cyclic imine phycotoxins family. Their chemical structure includes a 26-membered carbo-macrocycle and a 28-membered macrocyclic lactone arranged around a hexahydroisoquinoline that incorporates the characteristic cyclic imine group. Six prorocentrolides are already known. However, their mode of action remains undetermined. The aim of the present work was to explore whether prorocentrolide A acts on nicotinic acetylcholine receptors (nAChRs), using competition-binding assays and electrophysiological techniques. Prorocentrolide-A displaced [¹²⁵I]α-bungarotoxin binding to Torpedo membranes, expressing the muscle-type (α1₂β1γδ) nAChR, and in HEK-293 cells, expressing the chimeric chick neuronal α7-5HT₃ nAChR. Functional studies revealed that prorocentrolide-A had no agonist action on nAChRs, but inhibited ACh-induced currents in Xenopus oocytes that had incorporated the muscle-type α1₂β1γδ nAChR to their membranes, or that expressed the human α7 nAChR, as revealed by voltage-clamp recordings. Molecular docking calculations showed the absence of the characteristic hydrogen bond between the iminium group of prorocentrolide-A and the backbone carbonyl group of Trp147 in the receptor, explaining its weaker affinity as compared to all other cyclic imine toxins. In conclusion, this is the first study to show that prorocentrolide-A acts on both muscle and neuronal nAChRs, but with higher affinity on the muscle-type nAChR.

Keywords: Xenopus oocytes; binding assays; cyclic imine toxins; dinoflagellate toxin; molecular docking; nicotinic acetylcholine receptors; nicotinic currents; prorocentrolides.

Figure 1

General chemical structure of prorocentrolides and analogues. The nature of substituents R¹ to R⁸ (colored in red) is detailed in Table 1. The cyclic imine group is colored in orange.

Figure 2

Effect of prorocentrolide-A on the human α7 nAChR expressed in Xenopus oocytes. (A) Typical inward nicotinic currents evoked by ACh (350 μM) applied for 3 s and recorded at −60 mV holding membrane potential. The blue tracings above the current traces denote the perfusion of ACh. The red tracing (above the current trace) denotes the perfusion of 2.5 μM prorocentrolide-A. Note that no current was evoked by the perfusion of the prorocentrolide alone, which indicates that it has no direct agonist action on the α7-receptor, while when applied together with ACh (red and blue tracing, MIX, 3rd current trace from left) a block of the peak inward current was observed. The washout of the prorocentrolide from the medium by the fast perfusion system allowed a partial recovery of the ACh evoked current (4th and 5th current tracings) as compared to the control currents (first two tracings). (B) Concentration-dependent inhibition of ACh-elicited nicotinic currents by prorocentrolide-A in oocytes expressing the human α7 nAChR. Peak amplitudes of ACh-evoked currents (mean ± SEM), recorded at −60 mV in the presence of the prorocentrolide were normalized to control currents, and fitted to the Hill equation (nH = 0.93). The concentration of ACh used was the EC₅₀ determined.

Figure 3

Prorocentrolide-A and α-cobratoxin displaced, in a concentration-dependent manner, the specific [¹²⁵I]α–BTX binding to HEK-293 cells, expressing the chimeric chick neuronal α7-5HT₃ nAChR (A) and to Torpedo membranes expressing the muscle-type α1₂β1γδ nAChR; and (B) each point in the curves represents the mean ± SEM of three different experiments performed in duplicate.

Figure 4

Docking conformations of prorocentrolide-A at the subunit interface of muscle-type (left) and neuronal α7 nAChR (right). Residues within 4 Å from the ligand are shown. Loop C is hidden for more clarity.

Figure 5

LC-MS chromatogram of the sample used in the experiments here reported. Note the relative abundance of prorocentrolide-A. The small peak component revealed at 9.55 min corresponds to the 9,51-dihydroprorocentrolide.