Kir channels serve diverse and important roles throughout the human body and malfunctions of these channels are implicated in various channelopathies. Specific inhibitors for different subtypes of Kir channels are not available. However, Tertiapin-Q (TPNQ), a polypeptide isolated from honey bee venom, differentially inhibits certain subtypes of Kir channels with nanomolar affinity: ROMK1 (Kir1.1) and GIRK1/GIRK4 (Kir3.1/Kir3.4). Modification of TPNQ to increase selectivity for target channels bears great therapeutic potential. The in silico studies based on TPNQ-docked channel models, ROMK1_IRK2 (Kir1.1_Kir2.2) and GIRK2 (Kir3.2), predicted specific paired residue interactions and were experimentally validated here. In ROMK1 E123A mutant, the TPNQ sensitivity was decreased by ~2-fold while GIRK2 E127A mutant reduced the TPNQ sensitivity by greater than 10-fold. Also, we could observe the additional effect, ~ 18 fold, of GIRK1 subunits, ~1.7 fold, and E127A mutation, ~10 fold, on the TPNQ sensitivity in the heteromeric mutant channel, GIRK1/GIRK2 E152D_E127A as compared with the homomeric GIRK2 E152D. Finally, we introduced the Kir3.2 E152D mutant as a good representative of wild-type behavior particularly for the TPNQ study. Overall, this type of structure-function studies suggests an efficient and cost effective way toward design and development of specific Kir channel blockers by targeting on specific paired interactions between TPNQ and the Kir channels.