KCNQ1 is a voltage-gated potassium channel α subunit. KCNQ1 forms a tetramer and gives rise to K+ conductance in many tissues such as heart, inner ear, kidney, intestine and pancreas. Biophysical properties of KCNQ1 are dynamically modulated by the presence of KCNE proteins. KCNE1, for example, increases KCNQ1 current amplitude, slows down the activation and deactivation kinetics and greatly shifts the conductance-voltage (G-V) curve in the positive (depolarizing) direction. This KCNQ1-KCNE1 complex underlies the slowly-activating K+ current called IKs in the heart, and regulates the excitability of cardiac myocytes. KCNE3, co-expressed with KCNQ1 in the intestine, makes KCNQ1 channel constitutively-active. Therefore, the subunit organization of KCNQ1-KCNE complex is essential for the ion channel properties and the physiological functions of KCNQ1. One of the fundamental questions for KCNQ1 channel is the stoichiometry of KCNQ1-KCNE complex. Previous works indicate the stoichiometry of KCNQ1 and KCNE1 is fixed at 4:2; two KCNE1 proteins bind to a tetrameric KCNQ1 channel. We recently applied the subunit counting method using single GFP molecule imaging to address this question (Nakajo et al. 2010). KCNE1-GFP fusion protein was co-expressed with KCNQ1 channels in Xenopus oocytes. Using a total internal reflection microscope, a single bleaching event from a single GFP molecule tagged to KCNE1 protein was observed as a step-wise reduction of fluorescence. The number of KCNE1 proteins in a single ion channel complex can be estimated by the number of bleaching events from a single optical spot. By counting the number of KCNE1 proteins, we found that some of the KCNQ1 ion channel complexes contain four KCNE1 proteins. This result indicates that “4:4” channel (four KCNQ1 subunits with four KCNE1 subunits) does exist. Interestingly, the number of KCNE1 proteins in KCNQ1 channel complex was not fixed but variable. More KCNE1 proteins are expressed, more “4:4” channels are produced. Because “4:4” channel showed slower activation kinetics compared to “4:2” channel, an increase in KCNE1 expression may elevate the excitability of cardiac myocytes. Our results imply that the dynamic interaction between KCNQ1 and KCNE proteins may occur on the plasmamembrane. If that is the case, KCNQ1 channels expressed on the plasmamembrane can be directly regulated by the association and dissociation of KCNE proteins. To investigate this possibility, we examined electrophysiological properties of series of tandem constructs of KCNQ1 and KCNE1 with various linker lengths. If the stoichiometry of KCNQ1-KCNE1 complex is flexible and dynamically changeable on the plasmamembrane, KCNQ1 channel properties would depend on the linker length between KCNE1 and KCNQ1. KCNE1-KCNQ1 constructs (producing “4:4” channel) showed a linker-length dependent change; as linker length increased, G-V curves were shifted to hyperpolarizing direction. On the other hand, KCNE1-KCNQ1-KCNQ1 constructs (producing “4:2” channel) showed no apparent linker-length dependent change in the G-V curves. These results suggest that 1st and 2nd KCNE1 proteins firmly bind to KCNQ1 channel while 3rd and 4th KCNE1 proteins may bind loosely. 3rd and 4th KCNE1 might dynamically regulate “4:2” channel by association/dissociation on the plasmamembrane. Further experiments are needed to prove the dynamic interaction between KCNQ1 and KCNE proteins on the plasmamembrane. It is also important to understand how KCNE proteins regulate KCNQ1 channels. Growing evidences from different groups including us indicate that KCNE proteins give influence on the movement of the voltage-sensing domain (VSD; S1-S4 segments) of KCNQ1 channel. By using cysteine-modifying MTS reagents, we showed that the accessibility of S4 segment was significantly changed by the presence of KCNE proteins (Nakajo and Kubo, 2007). As longer depolarization was required to be modified by MTSES, we concluded that the VSD of KCNQ1 was stabilized in the “down” state. On the other hand, KCNE3 stabilized the VSD in the “up” state and that is the reason why KCNE3 makes KCNQ1 a constitutively-open channel. By comparing the amino acid sequence of human KCNQ1 to that of Ci-KCNQ1 (KCNQ1 ortholog from marine chordate Ciona Intestinalis), we successfully identified S1 segment of KCNQ1 as a binding site for KCNE3 protein (Nakajo et al. 2011). KCNE1 also interacts with the VSD and modifies its movement. Because KCNQ1-KCNE channel complex can form different stoichiometries, it is important to know how each VSD is modified by KCNE proteins towards further understanding the modulation of KCNQ1 channel by KCNE proteins.