Voltage-gated K⁺ channels are tetramer, and four α (main) subunits form a single K⁺ channel. Each α subunit possesses a single voltage sensor domain consisting of four transmembrane segments (S1-S4) and contributes voltage-dependent gating. The fourth transmembrane segment (S4) is a central part of voltage-sensing. Membrane depolarization induces upward movement of the S4 segments, which leads to the channel opening. Most of the voltage-gated K⁺ channels have auxiliary subunits. These subunits could modulate gating properties and even change the affinity of agonist/antagonist. Therefore, it is essential to understand the structures of the macromolecular complex and how the auxiliary subunits modify the voltage-dependent gating. KCNQ1 channel is one of the most well-studied ion channels for gating modulation because its auxiliary subunit KCNE drastically changes the gating properties. For example, KCNE3 makes the KCNQ1 channel constitutively open. Recent cryo-EM structures clearly show KCNE3 interacts with the voltage sensor domain via the S1 segment of KCNQ1. By introducing mutations on putative interaction sites, we identified that the tight interaction between the S1 segment and KCNE3 is required to stabilize the channel's open state. This KCNQ1-KCNE3 complex could be an excellent model to study how auxiliary subunits modulate the gating behavior of voltage-gated ion channels.