The　inwardly　rectifying　potassium　channel　Kir3.2,　a　member　of　the　inward　rectifier　potassium　(Kir)　channel　family,　exerts　important　biological　functions　through　transporting　potassium　ions　outside　of　the　cell,　during　which　a　large-scale　synergistic　movement　occurs　among　its　different　domains.　Currently,　it　is　not　fully　understood　how　the　binding　of　the　ligand　to　the　Kir3.2　channel　leads　to　the　structural　changes　and　which　key　residues　are　responsible　for　the　channel　gating　and　allosteric　dynamics.　Here,　we　construct　the　Gaussian　network　model　(GNM)　of　the　Kir3.2　channel　with　the　secondary　structure　and　covalent　interaction　information　considered　(sscGNM),　which　shows　a　better　performance　in　reproducing　the　channel’s　flexibility　compared　with　the　traditional　GNM.　In　addition,　the　sscANM-based　perturbation　method　is　used　to　simulate　the　channel’s　conformational　transition　caused　by　the　activator　PIP2’s　binding.　By　applying　certain　forces　to　the　PIP2　binding　pocket,　the　coarse-grained　calculations　generate　the　similar　conformational　changes　to　the　experimental　observation,　suggesting　that　the　topology　structure　as　well　as　PIP2　binding　are　crucial　to　the　allosteric　activation　of　the　Kir3.2　channel.　We　also　utilize　the　sscGNM-based　thermodynamic　cycle　method　developed　by　us　to　identify　the　key　residues　whose　mutations　significantly　alter　the　channel’s　binding　free　energy　with　PIP2.　We　identify　not　only　the　residues　important　for　the　specific　binding　but　also　the　ones　critical　for　the　allosteric　transition　coupled　with　PIP2　binding.　This　study　is　helpful　for　understanding　the　working　mechanism　of　Kir3.2　channels　and　can　provide　important　information　for　related　drug　design.　©　2024　American　Chemical　Society