Molecular assembly and dynamics of fluorescent protein-tagged single K_Ca1.1 channel in expression system and vascular smooth muscle cells

Abstract

The large-conductance Ca(2+)-activated K(+) (K(Ca)1.1, 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 BKα subunit (BKα-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 BKα-YFP was coexpressed with cyan fluorescent protein (CFP)-tagged BKβ1 subunit (BKβ1-CFP) in HEK cells, the mobility was reduced by ∼50%. Fluorescent image analyses suggest that green fluorescent protein (GFP)-tagged BKα subunit (BKα-GFP) expressed in vascular smooth muscle cells (VSMCs), at low density, preferentially formed a heterotetrameric molecular assembly with native BKα subunits, rather than homotetrameric BKα-GFP. Movement of BKα-YFP in VSMCs (0.29 × 10(3) nm(2)/s) was far more restricted than BKα-YFP/BKβ1-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 BKα-YFP, which was then significantly reduced by addition of jasplakinolide. Most BKα-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 BKα-YFP and Cav1-CFP, particularly in VSMCs. These results, obtained by single molecule imaging in living cells, indicate that the dynamics of BKα molecules on the membrane surface are strongly restricted or regulated by its auxiliary β-subunit, cytoskeleton, and direct interaction with Cav1 in VSMCs.

Modulation of TMEM16A-Channel Activity as Ca2+ Activated Cl− Conductance via the Interaction With Actin Cytoskeleton in Murine Portal Vein

“…Actin disruption attenuates CFTR Cl − current and activates BK Ca current and ClC-2 current (10,13,14). In addition, dynamic mobilization of BK Ca channel is restricted by actin filaments (5). In this study, we found that the activity of TMEM16A channel is also modified by the actin cytoskeleton.…”

Caveolin-1 Facilitates the Direct Coupling between Large Conductance Ca2+-activated K+ (BKCa) and Cav1.2 Ca2+ Channels and Their Clustering to Regulate Membrane Excitability in Vascular Myocytes

“…Under these conditions, BK Ca current density measured in aortic myocytes expressing BK␣-YFP was comparable with that measured in myocytes expressing yellow fluorescent proteins (YFP) alone. This meant that in our system artifacts by overexpression of fluorescent protein-labeled ion channels were minimized due to their relatively low expression levels (30).…”

“…Single-molecule Imaging-Single-molecule imaging was performed using a TIRF imaging system with an objective lens (CFI Plan Apo TIRF 60ϫ/1.45 or CFI Apo TIRF 100ϫ/1.49, oil immersion; Nikon, Tokyo, Japan) as described previously (30). Data were collected with an EM-CCD camera and analyzed by AQUACOSMOS software (Hamamatsu Photonics, Hamamatsu, Japan).…”

“…The fluorescence of YFP was photobleached using a mercury lamp (100 W, C-SHG1; Nikon) and a G-2A filter cube (Ex510 -560/DM575/BA590; Nikon) for 2.5 min. FRET efficiency (E FRET ) was calculated as the percentage increase in CFP emission after YFP photobleaching, as described previously (30).…”

Enhancement of Ca2+Influx and Ciliary Beating by Membrane Hyperpolarization due to ATP-Sensitive K+Channel Opening in Mouse Airway Epithelial Cells

“…A whole-cell patch clamp was applied to a single ciliary cell with a patch pipette using a CEZ-2400 amplifier (Nihon Kohden, Tokyo, Japan) as previously described (Imaizumi et al, 1989;Yamamura et al, 2012). The membrane currents and voltage signals were stored and analyzed using a Digidata 1440A and a pCLAMP 10.2 (Axon Instruments, Foster City, CA).…”