Single Molecule Fluorescence Imaging Reveals the Stoichiometry of BKγ1 Subunit in Living HEK293 Cell Expression...

Large conductance Ca²⁺-activated K⁺ (BK_Ca) channels are ubiquitously expressed in plasma membrane of both excitable and non-excitable cells and possess significant physiological functions. A tetrameric complex of α subunit (BKα) forms a functional pore of BK_Ca channel. The properties of BK_Ca channel, such as voltage-dependence, Ca²⁺ sensitivity and pharmacological responses, are extensively modulated by co-expressing accessory β subunits (BKβ), which can associate with BKα in one to one manner. Although the functional significance of newly identified γ subunits (BKγ) has been revealed, the stoichiometry between BKα and BKγ1 remains unclear. In the present study, we utilized a single molecule fluorescence imaging with a total internal reflection fluorescence (TIRF) microscope to directly count the number of green fluorescent protein (GFP)-tagged BKγ1 (BKγ1-GFP) within a single BK_Ca channel complex in HEK293 cell expression system. BKγ1-GFP significantly enhanced the BK channel activity even when the intracellular Ca²⁺ concentration was kept lower, i.e., 10 nM, than the physiological resting level. BKγ1-GFP stably formed molecular complexes with BKα-mCherry in the plasma membrane. Counting of GFP bleaching steps revealed that a BK_Ca channel can contain up to four BKγ1 per channel at the maximum. These results suggest that BKγ1 forms a BK_Ca channel complex with BKα in a 1 : 1 stoichiometry in a human cell line.