Marine Macrocyclic Imines, Pinnatoxins A and G: Structural Determinants and Functional Properties to Distinguish Neuronal α7 from Muscle α1(2)βγδ nAChRs

Abstract

Pinnatoxins are macrocyclic imine phycotoxins associated with algal blooms and shellfish toxicity. Functional analysis of pinnatoxin A and pinnatoxin G by binding and voltage-clamp electrophysiology on membrane-embedded neuronal α7, α4β2, α3β2, and muscle-type α12βγδ nicotinic acetylcholine receptors (nAChRs) reveals high-affinity binding and potent antagonism for the α7 and α12βγδ subtypes. The toxins also bind to the nAChR surrogate, acetylcholine-binding protein (AChBP), with low Kd values reflecting slow dissociation. Crystal structures of pinnatoxin-AChBP complexes (1.9-2.2 Å resolution) show the multiple anchoring points of the hydrophobic portion, the cyclic imine, and the substituted bis-spiroketal and cyclohexene ring systems of the pinnatoxins that dictate tight binding between the opposing loops C and F at the receptor subunit interface, as observed for the 13-desmethyl-spirolide C and gymnodimine A congeners. Uniquely, however, the bulky bridged EF-ketal ring specific to the pinnatoxins extends radially from the interfacial-binding pocket to interact with the sequence-variable loop F and govern nAChR subtype selectivity and central neurotoxicity.

Fig. 1. Chemical structures of the pinnatoxin variants and the open-ring PnTx-A AK derivative

The 7-membered cyclic imines common to other spirolides are highlighted with a grey background while the substructures that distinguish the pinnatoxins from other spirolides are boxed. Locations of group substitutions (R¹ to R⁴) that differ amongst the various pinnatoxins are indicated. PnTx-A: R¹ = CO₂, R² = OH, R³ = R⁴ = H; PnTx-G: R¹ = CH=CH₂/O-acyl esters (C14 to C24 acyl chain with variable numbers of double bonds), R² = OH, R³ = R⁴ = H; PnTx-B/C: R¹ = NH₂CO₂, R² = OH, R³ = R⁴ = H; PnTx-D: R¹ = CO(CH₂)₂CO₂, R² = H, R³ = OH, R⁴ = CH₃; PnTx-E: R¹ = COH(CH₂)₂CO₂, R² = H, R³ = OH, R⁴ = CH₃; PnTx-F: R¹ = C₄H₆O₂ (butyrolactone), R² = H, R³ = OH, R⁴ = CH₃.

Fig. 2. Inhibition of ACh-evoked currents recorded from human α7 nAChR transiently expressed in oocytes

The amplitudes of the ACh-induced currents recorded in the presence of PnTx-A (filled circles) and of PnTx-G (empty circles) (mean ± SEM; 3–4 oocytes per concentration) were normalized to control currents and are expressed as relative current values. The holding membrane potential during recordings was −60 mV.

Fig. 3. Overall and close-up views of the pentameric PnTx-AChBP complexes, and structural comparisons

(A) The pentamer in the PnTx-A complex is viewed from the “membrane” side (left), and in a radial perspective towards one subunit interface with the apical side at top and the membrane side at bottom (center). The main and side chains at the (+) and (−) faces of the exemplified interface are displayed in yellow and cyan, respectively. The position and orientation of bound PnTx-A and PnTx-G are identical. Bound PnTx-A (pink bonds and molecular surface, red oxygens, blue nitrogen) and PnTx-G (light blue bonds and molecular surface, red oxygens, blue nitrogen) are perfectly ordered, as assessed by the quality of the 1.9Å and 2.2 Å resolution 2Fo-Fc electron density maps contoured at 1.2σ (blue) (right). (B) Close-up views of bound PnTx-A (left) and PnTx-G (right) in the aromatic nest at the subunit interface, showing details of the cyclic imine environment (radial perspective). The Cys190-Cys191 disulfide bridge embedded at the tip of loop C is displayed in brown and loop F is in green. The 5,6-bicycloketal ring (i.e., ring EF in Fig. 1) is highlighted in blue. Side chains (in yellow and cyan) and solvent molecules (red dots) that interact specifically with bound PnTx-A/G are shown and labeled. The dashed lines denote hydrogen bonds. Note the distinctive interaction networks of the R¹ substituents at the membrane side of the interface. (C) Overlays of A-AChBP as bound to PnTx-A with L-AChBP (PDB code 1UW6) (left) and the α7/A-AChBP chimera mutant III (PDB code 3SH1) (right). Residues in the ligand-binding pockets of L-AChBP and α7 that differ from those in A-AChBP are colored in orange for the (+) face and light blue for the (-) face and labeled with italics. Other color codes as in panel B.

Fig. 4. Surface complementarity of bound PnTx-A and PnTx-G with the A-AChBP subunit interface and comparison with the SPX, GYM and MLA complexes

(A) The molecular surface buried by bound PnTx-A (pink toxin and surface) and PnTx-G (light blue toxin) at the (+) (yellow) and (−) (blue) faces of the A-AChBP subunit interface, is viewed radially from the pentamer outer periphery (transparent brown loop C, green loop F). (B–D) Superimposition of PnTx-A (pink toxin) with SPX (orange toxin and surface), GYM (green toxin and surface) and MLA (magenta toxin and surface) as bound to A-AChBP. Compared with the accommodation modes of SPX and GYM within the ligand-binding pocket, the additional contact points with loop F residues mediated by the 5,6-bicycloketal ring (blue) found in PnTx-A are evident. In contrast, the N-phenyl succinimide moiety of MLA extends to the bottom of the binding pocket.