Inward rectifier potassium (Kir) channels comprise transmembrane and cytoplasmic domains. Various cytoplasmic regulators associate with the cytoplasmic domain to control the gate at the transmembrane domain. Despite increasing knowledge about its structure, it remains unclear how conformational change in the cytoplasmic domain couples to the gate. To approach this question, we focused on 2 spatially adjacent residues, Glu236 and Met313, of G protein-gated Kir channel subunit Kir3.2, whose side chains face the cytoplasmic pore when it is in a closed state. These 2 residues reside on adjacent β-strands which form a β-sheet. Since the cytoplasmic pore appeared to expand its inner diameter during gating, it could be speculated that effect of mutations reflects conformational changes surrounding these residues. We introduced mutations into either of residues, expressed them with m2-muscarinic receptor in Xenopus oocytes and measured the acetylcholine (ACh)-dependent potassium currents. Mutants at Glu236 tended to shift dose-response curves to the rightward direction. On the other hand, mutations at Met313 tended to render the concentration-dependent curve with shallow slope. These results suggest that, even though 2 β-strands lie adjacent in the closed state, both strands shift differently during gating. One of the Glu236 mutants showed the mal-conduction and the constitutive activity. In crystal structure of the mutant, the residue introduced contacts with main chains of the adjacent subunits. These results strongly suggest that the Kir channels change the inner diameter of the cytoplasmic pore during gating, and the strands differently contribute to pore expansion to control of Kir channel gating.