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

Tertiapin-Q (TPNQ), a honey bee toxin derivative, inhibits inward-rectifier K+ channels by binding to their external vestibule. In the present study w e found that TPNQ inhibition of the channels is profoundly affected by extr acellular pH. This pH dependence mainly reflects titration of histidine res idue 12 in TPNQ by extracellular protons, since it largely vanishes when th e histidine residue is replaced with alanine. Not surprisingly, this alanin e derivative of TPNQ binds to the channel with much lower affinity. Quantit ative thermodynamic cycle analysis shows that deprotonation of the histidin e residue reduces the TPNQ-ROMK1 binding energy by 1.6 kcal/mol. To elimina te pH sensitivity but retain high affinity, we derivatized TPNQ by replacin g histidine 12 with lysine. This derivative-denoted tertiapin-KQ (TPNKQ)-no t only is practically insensitive to extracellular pH but also binds to the channel with even higher affinity than TPNQ at extracellular pH 7.6.