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Tertiapin has been isolated from the venom of the Honeybee Apis mellifera. Tertiapin-Q is an oxidation-resistant mutant of the wild-type tertiapin where Methionine 13 has been replaced by a Glutamine. Tertiapin-Q blocks the inwardly rectifying Kir1.1 (ROMK1) and Kir3.1/3.4 (GIRK1/GIRK4 also known as IKACh) potassium channels with Kd values of around 2 nM and 8 nM respectively.Tertiapin-Q also inhibits calcium-activated large conductance BK potassium channels (KCa1.1) in a concentration and voltage-dependent manner (IC50 ~ 5 nM), in addition to inhibiting Kir3.1/3.2 (GIRK1/GIRK2) heteromultimer potassium channels with a Kd close to 270 nM. Tertiapin-Q can prevent dose-dependent acetylcholine(ACh)-induced atrioventricular blocks in mammalian hearts, as KCNJ3/KCNJ5 channels (also named I(KACh)), are activated by ACh found in mammalian myocytes.
AA sequence: Ala-Leu-Cys3-Asn-Cys5-Asn-Arg-Ile-Ile-Ile-Pro-His-Gln-Cys14-Trp-Lys-Lys-Cys18-Gly-Lys-Lys-NH2
Disulfide bonds: Cys3-Cys14 and Cys5-Cys18
Length (aa): 21
Formula: C106H175N35O24S4
Molecular Weight: 2452 Da
Appearance: White lyophilized solid
Solubility: water and saline buffer
CAS number: [252198-49-5]
Source: Synthetic
Purity rate: > 97 %
Inward rectifier potassium channels (Kir) play critical roles in cell physiology. Despite representing the simplest tetrameric potassium channel structures, the pharmacology of this channel family remains largely undeveloped. In this respect, tertiapin (TPN), a 21 amino acid peptide isolated from bee venom, has been reported to inhibit Kir1.1 and Kir3.1/3.4 channels with high affinity by binding to the M1-M2 linker region of these channels. The features of the peptide-channel interaction have been explored electrophysiologically, & these studies have identified ways by which to alter the composition of the peptide without affecting its biological activity. In the present study, the TPN derivative, TPN-Y1/K12/Q13, has been synthesized & radiolabeled to high specific activity with (125)I. TPN-Y1/K12/Q13 & mono-iodo-TPN-Y1/K12/Q13 ([(127)I]TPN-Y1/K12/Q13) inhibit with high affinity rat but not human Kir1.1 channels stably expressed in HEK293 cells. [(125)I]TPN-Y1/K12/Q13 binds in a saturable, time-dependent, & reversible manner to HEK293 cells expressing rat Kir1.1, as well as to membranes derived from these cells, & the pharmacology of the binding reaction is consistent with peptide binding to Kir1.1 channels. Studies using chimeric channels indicate that the differences in TPN sensitivity between rat & human Kir1.1 channels are due to the presence of two nonconserved residues within the M1-M2 linker region. When these results are taken together, they demonstrate that [(125)I]TPN-Y1/K12/Q13 represents the first high specific activity radioligand for studying rat Kir1.1 channels & suggest its utility for identifying other Kir channel modulators.
Felix, J. P.,et al. (2006) Characterization of Kir1.1 channels with the use of a radiolabeled derivative of tertiapin,Biochemistry. PMID: 16906771
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 & ROMK1 homomultimers. In the present study, the effects of a stable & functionally similar derivative of tertiapin, tertiapin-Q, were examined on recombinant human voltage-dependent Ca(2+)-activated largeconductance K(+) channel (BK or MaxiK; alpha-subunit or hSlo1 homomultimers) & mouse inwardly rectifying GIRK1+GIRK2 (i.e., Kir3.1 & Kir3.2) heteromultimeric K(+) channels expressed in Xenopus oocytes & 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- & concentration-dependent manner. We also confirmed the inhibition of recombinant GIRK1+GIRK2 heteromultimers by tertiapin-Q, which had no effect on endogenous depolarization- & hyperpolarization-activated currents sensitive to extracellular divalent cations (Ca(2+), Mg(2+), Zn(2+), & Ba(2+)) in defolliculated oocytes. In voltage-clamped DRG neurons, tertiapin-Q voltage- & use-dependently inhibited outwardly rectifying K(+) currents, but Cs(+)-blocked hyperpolarization-activated inward currents including I(H) were insensitive to tertiapin-Q, baclofen, barium, & zinc, suggesting absence of functional GIRK channels in the newborn. Under current-clamp conditions, tertiapin-Q blocked the action potential after hyperpolarization (AHP) & 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 & that the blockade ofrecombinant BK channels & native neuronal AHP currents is use-dependent. Inhibition of specific types of Kir & voltage-dependent Ca(2+)-activated K(+) channels by tertiapin-Q at nanomolar range via different mechanisms may have implications in pain physiology & therapy.
Kanjhan, R., C et al. (2005) Tertiapin-Q blocks recombinant and native large conductance K+ channels in a use-dependent manner, J Pharmacol Exp Ther. PMID: 15947038
Tertiapin-Q (TPN(Q)), a honey bee toxin derivative, inhibits inward-rectifier K(+) channels by binding to their external vestibule. In the present study we found that TPN(Q) inhibition of the channels is profoundly affected by extracellular pH. This pH dependence mainly reflects titration of histidine residue 12 in TPN(Q) by extracellular protons, since it largely vanishes when the histidine residue is replaced with alanine. Not surprisingly, this alanine derivative of TPN(Q) binds to the channel with much lower affinity. Quantitative thermodynamic cycle analysis shows that deprotonation of the histidine residue reduces the TPN(Q)-ROMK1 binding energy by 1.6 kcal/mol. To eliminate pH sensitivity but retain high affinity, we derivatized TPN(Q) by replacing histidine 12 with lysine. This derivative-denoted tertiapin-KQ (TPN(KQ))-not only is practically insensitive to extracellular pH but also binds to the channel with even higher affinity than TPN(Q) at extracellular pH 7.6.
Ramu, Y., et al. (2001) Titration of tertiapin-Q inhibition of ROMK1 channels by extracellular protons, Biochemistry. PMID: 11297426
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 atrialmyocytes 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 & -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(+) & the L-type Ca(2+) channels were not affected by tertiapin (1 microM). At the single-channel level, tertiapin inhibited theK(ACh) channel from the outside of the membrane by reducing the NP(o) (N is the number of functional channels, & the P(o) is the open probability of each channel) without affecting the single-channel conductance or fast kinetics. Therefore, tertiapin potently & selectively blocks the K(ACh) channel in cardiac myocytes in a receptor- & 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.
Kitamura, H., et al. (2000) Tertiapin potently and selectively blocks muscarinic K(+) channels in rabbit cardiac myocytes, J Pharmacol Exp Ther. PMID: 10734170
Tertiapin-Q (TPN(Q)) is a derivative of honey bee toxin tertiapin (TPN) whose methionine residue is replaced with a glutamine residue. TPN(Q) inhibits the ROMK1 & GIRK1/4 inward-rectifier K(+) channels with affinities very similar to TPN. However, unlike native TPN, TPN(Q) is nonoxidizable by air. The stability of TPN(Q) allows us to investigate how it interacts with the targeted channels. We found that the interaction between TPN(Q) & the ROMK1 channel is a bimolecular reaction, i.e., one TPN(Q) molecule binds to one channel. The interaction surface in TPN(Q) is primarily formed by its alpha helix rather than the beta sheets with which scorpion toxins form their interaction surface. The mutagenesis studies on both the channel & TPN(Q) together strongly suggest that to block the K(+) pore TPN(Q) plugs its alpha helix into the vestibule of the K(+) pore, while leaving the extended structural portion sticking out of the vestibule into the extracellular media.
Jin, W., et al. (1999) Mechanisms of inward-rectifier K+ channel inhibition by tertiapin-Q, Biochemistry. PMID: 10572004
Tertiapin (TPN), a small protein derived from honey bee venom, inhibits the GIRK1/4 & ROMK1 channels with nanomolar affinities. Methionine residue 13 in TPN interacts with residue F148 in the channel, located just outside of the narrow region of the ROMK1 pore. The methionine residue in TPN can be oxidized by air, which significantly hinders TPN binding to the channels. To overcome the reduction in TPN affinity due to oxidation of M13, we replaced M13 in TPN with fourteen different residues. Out of the fourteen derivatives, only the one in which M13 was replaced by glutamine, TPNQ, binds to the channel with a Ki value very similar to that of native TPN. Since TPNQ is stable & functionally resembles native TPN, it will be a very useful molecular probe for studying the inward-rectifier K+ channels.
Jin, W., and Lu, Z. (1999) Synthesis of a stable form of tertiapin: a high-affinity inhibitor for inward-rectifier K+ channels, Biochemistry. PMID: 10572003
