KCNQ1 and KCNE1 co-assembly generates the I_KS K⁺ current, which is crucial to the cardiac action potential repolarization. Mutations in their corresponding genes cause long QT syndrome (LQT) and atrial fibrillation. The A-kinase anchor protein, yotiao (also known as AKAP9), brings the I_KS channel complex together with signaling proteins to achieve regulation upon β1-adrenergic stimulation. Recently, we have shown that KCNQ1 helix C interacts with the KCNE1 distal C-terminus. We postulated that this interface is crucial for I_KS channel modulation. Here, we examined the yet unknown molecular mechanisms of LQT mutations located at this intracellular intersubunit interface. All LQT mutations disrupted the internal KCNQ1–KCNE1 intersubunit interaction. LQT mutants in KCNQ1 helix C led to a decreased current density and a depolarizing shift of channel activation, mainly arising from impaired phosphatidylinositol-4,5-bisphosphate (PIP₂) modulation. In the KCNE1 distal C-terminus, the LQT mutation P127T suppressed yotiao-dependent cAMP-mediated upregulation of the I_KS current, which was caused by reduced KCNQ1 phosphorylation at S27. Thus, KCNQ1 helix C is important for channel modulation by PIP₂, whereas the KCNE1 distal C-terminus appears essential for the regulation of I_KS by yotiao-mediated PKA phosphorylation.