Molecular assembly and dynamics of fluorescent protein-tagged single K Ca1.1 channel in expression system and vascular smooth muscle cells

摘要

The large-conductance Ca 2+-activated K + (K Ca1.1, BK) channel has pivotal roles in the regulation of vascular tone. To clarify the molecular dynamics of BK channels and their functionally coupled protein on the membrane surface, we examined single-molecule imaging of fluorescent-labeled BK subunits in the plasma membrane using total internal reflection fluorescence (TIRF) microscopy. The dynamic mobility of yellow fluorescent protein (YFP)-tagged BK?? subunit (BK??-YFP) expressed in human embryo kidney 293 (HEK) cells was detected in TIRF regions at the level of individual channels and their clusters on the plasma membrane with a diffusion coefficient of 6.7 ?? 10 3 nm 2/s. When BK??-YFP was coexpressed with cyan fluorescent protein (CFP)-tagged BK??1 subunit (BK??1 -CFP) in HEK cells, the mobility was reduced by ~50%. Fluorescent image analyses suggest that green fluorescent protein (GFP)-tagged BK?? subunit (BK??-GFP) expressed in vascular smooth muscle cells (VSMCs), at low density, preferentially formed a heterotetrameric molecular assembly with native BK?? subunits, rather than homotetrameric BK?? -GFP. Movement of BK??-YFP in VSMCs (0.29 ?? 10 3 nm 2/s) was far more restricted than BK??-YFP/BK??1-CFP in HEK cells (2.5 ?? 10 3 nm 2/s). Actin disruption by pretreatment with cytochalasin D in VSMCs appeared to increase the mobile behavior of BK??-YFP, which was then significantly reduced by addition of jasplakinolide. Most BK??-YFP colocalized with caveolin 1 (Cav1)-CFP in VSMCs, but unexpectedly not frequently in HEK cells. Fluorescence resonance energy transfer analyses showed the direct interaction between BK??-YFP and Cav1-CFP, particularly in VSMCs. These results, obtained by single molecule imaging in living cells, indicate that the dynamics of BK?? molecules on the membrane surface are strongly restricted or regulated by its auxiliary ??-subunit, cytoskeleton, and direct interaction with Cav1 in VSMCs. ? 2012 the American Physiological Society.