KCNQ1 channels play vital roles in cardiovascular, gastric and other systems. The conductance and dynamics of KCNQ1 could be modulated differently by different single transmembrane helical auxiliary proteins (such as KCNE1, KCNE2 and others). Using combinational methods of electrophysiology, immunofluorescence, solution NMR and related backbone flexibility analysis, we examined detail KCNQ1 function modulations by different regions of KCNE1 or KCNE2. In the chimera experiments, decreased surface expression and consequent low activities of KCNQ1 were observed in the presence of KCNE2 N-terminus. The transmembrane domains (TMDs) of KCNE1 and KCNE2 were illustrated to associate with the KCNQ1 channel in different modes: Ile64 in KCNE2-TMD interacting with Phe340 and Phe275 in KCNQ1, while two pairs of interacting residues (Phe340-Thr58 and Ala244-Tyr65) in the KCNQ1/KCNE1 complex. The KCNE1 C-terminus could modulate gating property of KCNQ1, whereas KCNE2 C-terminus had only minimal influences on KCNQ1. All of the results demonstrated different KCNQ1 function modulations by different regions of the two auxiliary proteins.