Many plants of the Ericaceae family, Rhododendron, Pieris, Agarista and Kalmia, contain diterpene grayanotoxins. Consumption of grayanotoxin containing leaves, flowers or secondary products as honey may result in intoxication specifically characterized by dizziness, hypotension and atrial-ventricular block. Symptoms are caused by an inability to inactivate neural sodium ion channels resulting in continuous increased vagal tone. Grayanotoxin containing products are currently sold online, which may pose an increasing risk. In humans, intoxication is rarely lethal, in contrast to cattle and pet poisoning cases. Scientific evidence for the medicinal properties of grayanotoxin containing preparations, such as honey or herbal preparation in use in folk medicine, is scarce, and such use may even be harmful.

Human ether-à-go-go related gene (hERG) 1 channels conduct the rapid delayed rectifier K⁺current (I_Kr) and are essential for the repolarization of the cardiac action potential. hERG1 inhibition by structurally diverse drugs may lead to life threatening arrhythmia. Putative binding determinants of hERG1 channel blockers include T623, S624 and V625 on the pore helix and residues G648, Y652 and F656, located on segment S6. We and others have previously hypothesized that additional binding determinants may be located on helix S5, which is in close contact with the S6 segments. In order to test this hypothesis, we performed a detailed investigation combining ionic current measurements with two-microelectrode voltage clamp and molecular modeling techniques. We identified a novel aromatic high affinity binding determinant for blockers located in helix S5, F557, which is equally potent as Y652. Modeling supports a direct interaction with the outer pore helix.

Inward rectifier potassium (Kir) channel activity is controlled by plasma membrane lipids. Phosphatidylinositol-4,5-bisphosphate (PIP₂) binding to a primary site is required for opening of classic inward rectifier Kir2.1 and Kir2.2 channels, but interaction of bulk anionic phospholipid (PL⁻) with a distinct second site is required for high PIP₂ sensitivity. Here we show that introduction of a lipid-partitioning tryptophan at the second site (K62W) generates high PIP₂ sensitivity, even in the absence of PL⁻. Furthermore, high-resolution x-ray crystal structures of Kir2.2[K62W], with or without added PIP₂ (2.8- and 2.0-Å resolution, respectively), reveal tight tethering of the C-terminal domain (CTD) to the transmembrane domain (TMD) in each condition. Our results suggest a refined model for phospholipid gating in which PL⁻ binding at the second site pulls the CTD toward the membrane, inducing the formation of the high-affinity primary PIP₂ site and explaining the positive allostery between PL⁻ binding and PIP₂ sensitivity.

In excitable cells, K_IR2.x ion-channel-carried inward rectifier current (I_K1) is thought to set the negative and stable resting membrane potential, and contributes to action potential repolarization. Loss- or gain-of-function mutations correlate with cardiac arrhythmias and pathological remodelling affects normal K_IR2.x protein levels. No specific I_K1 inhibitor is currently available for in vivo use, which severely hampers studies on the precise role of I_K1 in normal cardiac physiology and pathophysiology. The diamine antiprotozoal drug pentamidine (P) acutely inhibits I_K1 by plugging the cytoplasmic pore region of the channel. We aim to develop more efficient and specific I_K1 inhibitors based on the P structure. We analysed seven pentamidine analogues (PA-1 to PA-7) for I_K1 blocking potency at 200 nM using inside-out patches from K_IR2.1 expressing HEK-293 cells. PA-6 showed the highest potency and was tested further. PA-6 blocked K_IR2.x currents of human and mouse with low IC₅₀ values (12–15 nM). Modelling indicated that PA-6 had less electrostatic but more lipophilic interactions with the cytoplasmic channel pore than P, resulting in a higher channel affinity for PA-6 (ΔG −44.1 kJ/Mol) than for P (ΔG −31.7 kJ/Mol). The involvement of acidic amino acid residues E224 and E299 in drug-channel interaction was confirmed experimentally. PA-6 did not affect I_Nav1.5, I_Ca-L, I_Kv4.3, I_Kv11.1, and I_Kv7.1/minK currents at 200 nM. PA-6 inhibited the inward (50 nM 40%; 100 nM 59%; 200 nM 77%) and outward (50 nM 40%; 100 nM 76%; 200 nM 100%) components of I_K1 in isolated canine adult-ventricular cardiomyocytes (CMs). PA-6 prolonged action potential duration of CMs by 8 (n = 9), 26 (n = 5), and 34% (n = 11) at 50, 100, and 200 nM, respectively. Unlike P, PA-6 had no effect on K_IR2.1 channel expression at concentrations from 0.1 to 3 μM. However, PA-6 at 10 μM increased K_IR2.1 expression levels. Also, PA-6 did not affect the maturation of hERG, except when applied at 10 μM. PA-6 has higher efficiency and specificity to K_IR2.x-mediated current than P, lengthens action potential duration, and does not affect channel trafficking at concentrations relevant for complete I_K1 block.

Ibogaine is a psychoactive indole alkaloid. Its use as an antiaddictive agent has been accompanied by QT prolongation and cardiac arrhythmias, which are most likely caused by human ether a go-go-related gene (hERG) potassium channel inhibition. Therefore, we studied in detail the interaction of ibogaine with hERG channels heterologously expressed in mammalian kidney tsA-201 cells. Currents through hERG channels were blocked regardless of whether ibogaine was applied via the extracellular or intracellular solution. The extent of inhibition was determined by the relative pH values. Block occurred during activation of the channels and was not observed for resting channels. With increasing depolarizations, ibogaine block grew and developed faster. Steady-state activation and inactivation of the channel were shifted to more negative potentials. Deactivation was slowed, whereas inactivation was accelerated. Mutations in the binding site reported for other hERG channel blockers (Y652A and F656A) reduced the potency of ibogaine, whereas an inactivation-deficient double mutant (G628C/S631C) was as sensitive as wild-type channels. Molecular drug docking indicated binding within the inner cavity of the channel independently of the protonation of ibogaine. Experimental current traces were fit to a kinetic model of hERG channel gating, revealing preferential binding of ibogaine to the open and inactivated state. Taken together, these findings show that ibogaine blocks hERG channels from the cytosolic side either in its charged form alone or in company with its uncharged form and alters the currents by changing the relative contribution of channel states over time.