biostudies-literatureTedeschi GNIDCD NIH HHSNational Institutes of Health, National Institute of General MedicineNational Institutes of HealthNIGMS NIH HHS10719https://www.ebi.ac.uk/biostudies/studies/S-EPMC8140153biostudies-literatureUnknown11(1)Voltage-gated potassium (Kv) channels are a family of membrane proteins that facilitate K⁺ ion diffusion across the plasma membrane, regulating both resting and action potentials. Kv channels comprise four pore-forming α subunits, each with a voltage sensing domain, and they are regulated by interaction with β subunits such as those belonging to the KCNE family. Here we conducted a comprehensive biophysical characterization of stoichiometry and protein diffusion across the plasma membrane of the epithelial KCNQ1-KCNE2 complex, combining total internal reflection fluorescence (TIRF) microscopy and a series of complementary Fluorescence Fluctuation Spectroscopy (FFS) techniques. Using this approach, we found that KCNQ1-KCNE2 has a predominant 4:4 stoichiometry, while non-bound KCNScientific reportsFluorescence Fluctuation Spectroscopy enables quantification of potassium channel subunit dynamics and stoichiometry.PMC8140153P41 GM103540R21 DC015736R21DC0157368P41-GM103540Papanikolaou MAbbott GWDigman MAScipioni LTedeschi GfalseFluorescence Fluctuation Spectroscopy enables quantification of potassium channel subunit dynamics and stoichiometry.Voltage-gated potassium (Kv) channels are a family of membrane proteins that facilitate K⁺ ion diffusion across the plasma membrane, regulating both resting and action potentials. Kv channels comprise four pore-forming α subunits, each with a voltage sensing domain, and they are regulated by interaction with β subunits such as those belonging to the KCNE family. Here we conducted a comprehensive biophysical characterization of stoichiometry and protein diffusion across the plasma membrane of the epithelial KCNQ1-KCNE2 complex, combining total internal reflection fluorescence (TIRF) microscopy and a series of complementary Fluorescence Fluctuation Spectroscopy (FFS) techniques. Using this approach, we found that KCNQ1-KCNE2 has a predominant 4:4 stoichiometry, while non-bound KCN2021-01-01T00:00:00Z2021 May2025-04-04T07:22:36.611Z2025-04-04T07:22:36.611ZS-EPMC81401533402117710.1038/s41598-021-90002-2