Structural Determinants Mediating Tertiapin Block of Neuronal Kir3.2 Channels

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Tertiapin (TPN) is a 21 amino acid venom peptide from Apis mellifera that inhibits certain members of the inward rectifier potassium (Kir) channel family at a nanomolar affinity with limited specificity. Structure-based computational simulations predict that TPN behaves as a pore blocker; however, the molecular determinants mediating block of neuronal Kir3 channels have been inconclusive and unvalidated. Here, using molecular docking and molecular dynamics (MD) simulations with ‘potential of mean force’ (PMF) calculations, we investigated the energetically most favored interaction of TPN with several Kir3.x channel structures. The resulting binding model for Kir3.2-TPN complexes was then tested by targeted mutagenesis of the predicted contact sites, and their impact on the functional channel block was measured electrophysiologically. Together, our findings indicate that a high-affinity TPN block of Kir3.2 channels involves a pore-inserting lysine side chain requiring (1) hydrophobic interactions at a phenylalanine ring surrounding the channel pore and (2) electrostatic interactions with two adjacent Kir3.2 turret regions. Together, these interactions collectively stabilize high-affinity toxin binding to the Kir3.2 outer vestibule, which orients the ε-amino group of TPN-K21 to occupy the outermost K+ binding site of the selectivity filter. The structural determinants for the TPN block described here also revealed a favored subunit arrangement for assembled Kir3.x heteromeric channels, in addition to a multimodal binding capacity of TPN variants consistent with the functional dyad model for polybasic peptide pore blockers. These novel findings will aid efforts in re-engineering the TPN pharmacophore to develop peptide variants having unique and distinct Kir channel blocking properties.

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Biochemistry, v. 59, issue 7, p. 836-850