Tertiapin-Q

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.

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.

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.