Titration of tertiapin-Q inhibition of ROMK1 channels by extracellular protons.

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.