Tertiapin

It is well known that vagal nerve tone plays a crucial role in atrial fibrillation (AF). Acetylcholine-activated potassium current (IKACh) mediates much of the cardiac response to vagal nerve stimulation (VNS), but the contribution of IKACh to AF remains unknown. We investigated the role of the IKACh channel in canine AF models using tertiapin, a selective IKACh blocker. Tertiapin (4-41 nmol kg(-1), i.v.) terminated AF in the canine VNS-induced and aconitine-induced AF models. The muscarinic M-receptor antagonist AF-DX-116 terminated AF in these models, but the adenosine receptor antagonist DPCPX had no effect. Thus it is likely that IKACh activation via the M-receptor has a crucial role in both models. Tertiapin (12 nmol kg(-1), i.v.) preferentially prolonged the atrial effective refractory period (ERP) but not the ventricular ERP under the VNS condition. This peptide (4-41 nmol kg(-1), i.v.) did not affect PQ, QRS and corrected QT intervals in isoflurane-anaesthetised dogs. These results suggest that a selective IKACh blocker is effective in canine AF models without affecting ventricular repolarisation, and might be useful for the treatment of patients with AF.

Venoms are comprised of diverse mixtures of proteins, peptides, and small molecules. Identifying individual venom components and their target(s) with mechanism of action is now attainable to understand comprehensively the effectiveness of venom cocktails and how they collectively function in the defense and predation of an organism. Here, structure-based computational methods were used with bioinformatics tools to screen and identify potential biological targets of tertiapin (TPN), a venom peptide fromApis mellifera(European honey bee). The small hive beetle (Aethina tumida(A. tumida)) is a natural predator of the honey bee colony and was found to possess multiple inwardly rectifying K+(Kir) channel subunit genes from a genomic BLAST search analysis. Structure-based virtual screening of homology modelledA. tumidaKir (atKir) channels found TPN to interact with a docking profile and interface "footprint" equivalent to known TPN-sensitive mammalian Kir channels. The results support the hypothesis thatatKir channels, and perhaps other insect Kir channels, are natural biological targets of TPN that help defend the bee colony from infestations by blocking K+transport viaatKir channels. From these in silico findings, this hypothesis can now be subsequently tested in vitro by validatingatKir channel block as well as in vivo TPN toxicity towardsA. tumida. This study highlights the utility and potential benefits of screening in virtual space for venom peptide interactions and their biological targets, which otherwise would not be feasible.

Tertiapin (TPN), a short peptide isolated from the venom of the honey bee, is a potent and selective blocker of the inward rectifier K+(Kir) channel Kir3.2. Here we examine in atomic detail the binding mode of TPN to Kir3.2 using molecular dynamics, and deduce the key residue in Kir3.2 responsible for TPN selectivity. The binding of TPN to Kir3.2 is stable when the side chain of either Lys16 (TPNK16-Kir3.2) or Lys17 (TPNK17-Kir3.2) of the toxin protrudes into the channel pore. However, the binding affinity calculated from only TPNK17-Kir3.2 and not TPNK16-Kir3.2 is consistent with experiment, suggesting that Lys17 is the most plausible pore-blocking residue. The alanine mutation of Kir3.2-Glu127, which is not present in TPN-resistant channels, reduces the inhibitory ability of TPN by over 50 fold in TPNK17-Kir3.2, indicating that Kir3.2-Glu127 is important for the selectivity of TPN.

1. In an isolated right atrial preparation, an increase in right atrial pressure (RAP) produces an increase in atrial rate. This rate response is larger and occurs faster when there is background vagal or muscarinic stimulation. 2. We hypothesized that in the latter situation, an increase in RAP antagonizes the effect of muscarinic stimulation through stretch inactivation of the mechanosensitive muscarinic potassium current I(K,ACh). 3. In two groups of bath-mounted right atria isolated from male Wistar rats (control n = 12; 300 nmol/L tertiapin-Q treated (to block I(K,ACh)) n = 10), we examined the change in atrial rate when RAP was raised from 2 to 8 mmHg; oxotremorine-M (oxo-M; from 10 to 500 nmol/L) was added to incrementally activate muscarinic receptors. 4. In both control and tertiapin-Q-treated groups, oxo-M reduced atrial rate, but its effect was less ( approximately 40-50%) in the latter group (P < 0.001). In control preparations, responses to an increase in RAP became progressively larger and quicker as the concentration of oxo-M was increased, whereas in tertiapin-Q treated preparations oxo-M did not affect either the amplitude or the speed of the response (P < 0.0001 for both). 5. The results support the hypothesis that atrial stretch antagonizes muscarinic slowing by its effect on I(K,ACh). We suggest that through this mechanism, parasympathetic control of heart rate may be modulated continuously by RAP.

Red blood cells (RBCs) are the most abundant cells in the human blood that have been extensively studied under morphology, ultrastructure, biochemical and molecular functions. Therefore, RBCs are excellent cell models in the study of biologically active compounds like drugs and toxins on the structure and function of the cell membrane. The aim of the present study was to explore erythrocyte ghost's proteome to identify changes occurring under the influence of three bee venom peptides-melittin, tertiapin, and apamin. We conducted preliminary experiments on the erythrocyte ghosts incubated with these peptides at their non-hemolytic concentrations. Such preparations were analyzed using liquid chromatography coupled with tandem mass spectrometry. It was found that when higher concentrations of melittin and apamin were used, fewer proteins were identified. Moreover, the results clearly indicated that apamin demonstrates the greatest influence on the RBCs ghosts proteome. Interestingly, the data also suggest that tertiapin exerted a stabilizing effect on the erythrocyte membrane. The experiments carried out show the great potential of proteomic research in the projects focused on the toxin's properties as membrane active agents. However, to determine the specificity of the effect of selected bee venom peptides on the erythrocyte ghosts, further proteomic research should be focused on the quantitative analysis.

Sinus node (SAN) dysfunction (SND) manifests as low heart rate (HR) and is often accompanied by atrial tachycardia or atrioventricular (AV) block. The only currently available therapy for chronic SND is the implantation of an electronic pacemaker. Because of the growing burden of SND in the population, new pharmacological therapies of chronic SND and heart block are desirable. We developed a collection of genetically modified mouse strains recapitulating human primary SND associated with different degrees of AV block. These mice were generated with genetic ablation of L-type Cav1.3 (Cav1.3-/-), T-type Cav3.1 (Cav3.1-/-), or both (Cav1.3-/-/Cav3.1-/-). We also studied mice haplo-insufficient for the Na+channel Nav1.5 (Nav1.5+/) and mice in which the cAMP-dependent regulation of hyperpolarization-activated f-(HCN4) channels has been abolished (HCN4-CNBD). We analysed, by telemetric ECG recording, whether pharmacological inhibition of the G-protein-activated K+current (IKACh) by the peptide tertiapin-Q could improve HR and AV conduction in these mouse strains. Tertiapin-Q significantly improved the HR of Cav1.3-/-(19%), Cav1.3-/-/Cav3.1-/-(23%) and HCN4-CNBD (14%) mice. Tertiapin-Q also improved cardiac conduction of Nav1.5+/-mice by 24%. Our data suggest that the development of pharmacological IKAChinhibitors for the management of SND and conduction disease is a viable approach.

Renal outer medullary K+ channel, ROMK (Kir1.1, kcnj1) is expressed in the kidney and brain, but its role in the central nervous system remains unknown. Recent studies suggested an involvement of the ROMK channel in mental diseases. Tertiapin (TPN) is a European honey bee venom peptide and is reported to selectively block the ROMK channel. Here, we have chemically synthesized a series of mutated TPN peptides, including TPN-I8R and -M13Q (TPN-RQ), reported previously, and examined their blocking activity on the ROMK channel. Among 71 peptides tested, TPN-RQ was found to block the ROMK channel most effectively. Whole-cell patch-clamp recordings showed the essential roles of two disulfide bonds and the circular structure for the blockade activity. To examine the central role, we injected TPN-RQ intracerebroventricularly and examined the effects on depression- and anxiety-like behaviors in mice. TPN-RQ showed an antidepressive effect in tail-suspension and forced swim tests. The injection of TPN-RQ also enhanced the anxiety-like behavior in the elevated plus-maze and light/dark box tests and impaired spontaneous motor activities in balance beam and wheel running tests. Administration of TPM-RQ suppressed the anti-c-Fos immunoreactivity in the lateral septum, without affecting immunoreactivity in antidepressant-related nuclei, e.g. the dorsal raphe nucleus and locus coeruleus. TPN-RQ may exert its antidepressive effects through a different mechanism from current drugs.

Tertiapin, a short peptide from honey bee venom, has been reported to specifically block the inwardly rectifying K(+) (Kir) channels, including G protein-coupled inwardly rectifying potassium channel (GIRK) 1+GIRK4 heteromultimers and ROMK1 homomultimers. In the present study, the effects of a stable and functionally similar derivative of tertiapin, tertiapin-Q, were examined on recombinant human voltage-dependent Ca(2+)-activated large conductance K(+) channel (BK or MaxiK; alpha-subunit or hSlo1 homomultimers) and mouse inwardly rectifying GIRK1+GIRK2 (i.e., Kir3.1 and Kir3.2) heteromultimeric K(+) channels expressed in Xenopus oocytes and in cultured newborn mouse dorsal root ganglion (DRG) neurons. In two-electrode voltage-clamped oocytes, tertiapin-Q (1-100 nM) inhibited BK-type K(+) channels in a use- and concentration-dependent manner. We also confirmed the inhibition of recombinant GIRK1+GIRK2 heteromultimers by tertiapin-Q, which had no effect on endogenous depolarization- and hyperpolarization-activated currents sensitive to extracellular divalent cations (Ca(2+), Mg(2+), Zn(2+), and Ba(2+)) in defolliculated oocytes. In voltage-clamped DRG neurons, tertiapin-Q voltage- and use-dependently inhibited outwardly rectifying K(+) currents, but Cs(+)-blocked hyperpolarization-activated inward currents including I(H) were insensitive to tertiapin-Q, baclofen, barium, and zinc, suggesting absence of functional GIRK channels in the newborn. Under current-clamp conditions, tertiapin-Q blocked the action potential after hyperpolarization (AHP) and increased action potential duration in DRG neurons. Taken together, these results demonstrate that the blocking actions of tertiapin-Q are not specific to Kir channels and that the blockade of recombinant BK channels and native neuronal AHP currents is use-dependent. Inhibition of specific types of Kir and voltage-dependent Ca(2+)-activated K(+) channels by tertiapin-Q at nanomolar range via different mechanisms may have implications in pain physiology and therapy.

Tertiapin is a 21-residue peptide isolated from honey bee venoms. A recent study indicated that tertiapin is a potent blocker of certain types of inwardly rectifying K(+) (Kir) channels (). We examined the effect of tertiapin on ion channel currents in rabbit cardiac myocytes using the patch-clamp technique. In the whole-cell configuration, tertiapin fully inhibited acetylcholine (1 microM)-induced muscarinic K(+) (K(ACh)) channel currents in atrial myocytes with the half-maximum inhibitory concentration of approximately 8 nM through approximately 1:1 stoichiometry. The potency of tertiapin in inhibiting K(ACh) channels was not significantly different at -40 and -100 mV. Tertiapin also inhibited the K(ACh) channel preactivated by intracellular guanosine 5'-O-(3-thiotriphosphate), a nonhydrolyzable GTP analog. A constitutively active Kir channel, the I(K1) channel, was at least 100 times less sensitive to tertiapin. Another Kir channel in cardiac myocytes, the ATP-sensitive K(+) channel, was virtually insensitive to tertiapin (1 microM). The voltage-dependent K(+) and the L-type Ca(2+) channels were not affected by tertiapin (1 microM). At the single-channel level, tertiapin inhibited the K(ACh) channel from the outside of the membrane by reducing the NP(o) (N is the number of functional channels, and the P(o) is the open probability of each channel) without affecting the single-channel conductance or fast kinetics. Therefore, tertiapin potently and selectively blocks the K(ACh) channel in cardiac myocytes in a receptor- and voltage-independent manner. Tertiapin is a novel pharmacological tool to identify the functional role of the K(ACh) channel in the parasympathetic regulation of the heart beat.

Tertiapin (TPN) is a 21 amino acid venom peptide fromApis melliferathat 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.