Regulation of inwardly-rectifying potassium (Kir) channels by intracellular ligands couples cell membrane excitability to important signaling cascades and metabolic pathways. Particularly, the ATP-sensitive potassium (KATP) channels, highly expressed in diverse excitable tissues, are the keystone components of glucose-stimulated insulin secretion in pancreatic β-cells. KATP channels are constituted by a canonical pore-forming transmembrane domain (TMD) and a large cytoplasmic domain (CTD) where ATP binds and inhibits the channel activity. A non-covalent interface domain in Kir channel structures is speculated as a mediator of coupling between the cytoplasmic (‘ligand-sensing’) and transmembrane (‘gating’) domains. The work described in this thesis aims to investigate the molecular mechanism that link ligand binding to the channel gating. A significant barrier is that many functionally important channel motifs are highly sensitive to mutagenesis, resulting in a loss-of-function (LOF) phenotype. We have developed a novel ‘forced gating’ approach by substituting a glutamate in the hydrophobic Kir channel bundle crossing (F168E), generating channels that open upon alkalization, likely due to mutual repulsion of the introduced glutamate side chain. This ‘forced gating’ approach is implemented in mutagenic scans of the Kir channel domain interface comprising numerous motifs, including the transverse ‘slide’ helix, the C-linker, the βC-βD loop, and the G-loop. Without exception, expression on the ‘forced gating’ background rescued all loss-of-function interfacial mutants in alkaline pH, and enabled functional characterization of all interfacial mutants on generation of conductive channels and ATP-dependent gating. Our findings highlight a small subset of ‘anchor residues’ at the Kir6.2 TMD-CTD interface, including D58 and T61 in the ‘slide helix’, R177 in the C-linker, T294 in the G-loop, and D204 in the βC-βD loop, which are required for both formation of conductive channels, and appropriate transmission of ligand binding to the channel gating. Disruption of these residues uncouples the TMD and CTD, causing loss-of-function phenotype combined with profound ligand insensitivity. Additionally, in contrast to a traditional ‘allosteric rescue’ approach (Kir6.2[C166S]) with an elevated intrinsic channel open probability, the ‘forced gating’ approach has a more general application due to its efficiency in rescuing all loss-of-function interfacial mutants without inherently perturbing ATP sensitivity in Kir6.2 channel.