Research ArticleVASCULAR BIOLOGY

Membrane depolarization activates BK channels through ROCK-mediated β1 subunit surface trafficking to limit vasoconstriction

See allHide authors and affiliations

Science Signaling  09 May 2017:
Vol. 10, Issue 478, eaah5417
DOI: 10.1126/scisignal.aah5417
  • Fig. 1 Depolarization stimulates surface trafficking of BK β1 subunits in arterial myocytes.

    (A) Representative Western blots illustrating surface (S) and intracellular (I) BKα and β1 proteins in rat cerebral arteries obtained using arterial surface biotinylation. (B) Mean data, n = 6 to 8 animals for each group; *P < 0.05 compared to control, #P < 0.05 compared to SNP, P < 0.05 compared to 30 K+, and δP < 0.05 compared to 60 K+ alone. All stimuli were applied for 10 min unless stated otherwise. (C) Immunofluorescence images of BKα and β1 and the calculated normalized FRET (N-FRET) under each condition in arterial myocytes. Scale bars, 10 μm. (D) Mean data for BKα and β1 immunoFRET, n = 6 images for each group obtained from four different animals; *P < 0.05 compared to control and #P < 0.05 compared to 60 K+.

  • Fig. 2 Rab11A knockdown inhibits depolarization-induced surface trafficking of β1 subunits in arterial myocytes.

    (A) Representative Western blots illustrating surface and intracellular β1 protein in cerebral arteries transfected with scrambled or rab11A shRNA and modulation by 60 K+ (10 min). (B) Mean data, n = 6 animals for each group; *P < 0.05 compared to scrambled (scrm) control and #P < 0.05 compared to scrambled 60 K+. (C) Immunofluorescence and immunoFRET images of BKα and β1 in arterial myocytes. Scale bars, 10 μm. (D) Mean data, n = 6 images for each group obtained from four different animals; *P < 0.05 compared to scrambled shRNA. (E) Representative Western blot of β1 protein in depolarized control arteries and arteries expressing dominant-negative (DN) rab11A. (F) Mean data, n = 6 animals for each group; *P < 0.05 compared to control and #P < 0.05 compared to 60 K+.

  • Fig. 3 Extracellular Ca2+ removal and CaV1.2 channel and ROCK inhibitors attenuate depolarization-induced β1 surface trafficking in arteries.

    (A) Representative Western blots illustrating surface and intracellular BKα and β1 protein using arterial surface biotinylation. (B) Mean data from biotinylation experiments showing effect of extracellular Ca2+ removal and different antagonists on depolarization-induced changes in the surface abundance of β1 protein, n = 6 animals for each group; *P < 0.05 compared to 6 K+ and #P < 0.05 compared to 60 K+. (C) Representative Western blots illustrating surface and intracellular BKα and β1 protein from arteries treated with scrambled siRNA or CaV1.2 siRNA. (D) Mean data, n = 5 animals for each group. (E) Representative Western blots illustrating surface and intracellular BKα and β1 protein from arteries treated with scrambled siRNA or ROCK1 or ROCK2 siRNA. (F) Mean data, n = 6 animals for each group; *P < 0.05 compared to scrambled control and #P < 0.05 compared to 60 K+.

  • Fig. 4 Depolarization-induced ROCK activation increases BK channel apparent Ca2+-sensitivity in arterial myocytes.

    (A) Representative Western blots illustrating total protein from arteries under control conditions, treated with 60 K+ or 60 K+ + HA1100 (10 μM). (B) Mean data, n = 6 animals for each group; *P < 0.05 compared to control and #P < 0.05 compared to 60 K+. (C) Representative traces of single BK channel activity and activation by lithocholate in the same patches pulled from either control myocytes (6 K+) or myocytes exposed to 30 K+ or 30 K+ + HA1100 (10 μM). Traces are shown from experiments performed with 10 μM free intracellular Ca2+ concentration [Ca2+]i. (D) Mean data, n = 6 recordings for each data point; *P < 0.05 compared to 6 K+ and #P < 0.05 compared to 30 K+. (E) Mean data illustrating lithocholate activation of BK channels with 10 μM free [Ca2+]i, n = 6 recordings for each data set obtained from eight different animals; *P < 0.05 compared to 6 K+ control, #P < 0.05 compared to respective control under the same condition, and δP < 0.05 compared to 30 K+ + lithocholate.

  • Fig. 5 Rab11A knockdown inhibits BK channels in pressurized arteries.

    (A) Original diameter recordings illustrating constriction in response to iberiotoxin, dilation in response to NS1619, and dilation in response to lithocholate in either control (red trace) or rab11A knockdown (blue trace) arteries. (B) Mean data, n = 6 animals for each group; *P < 0.05 compared to control. (C) Mean data illustrating dilation to lithocholate, n = 6 animals for each group; *P < 0.05 compared to control.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/10/478/eaah5417/DC1

    Fig. S1. Effect of different antagonists on depolarization- and SNP-induced changes in the surface abundance of BKα and β1 protein.

    Fig. S2. Depolarization-induced surface trafficking of β1 subunits in human cerebral arteries.

    Fig. S3. Overexpression of β1 subunits in HEK293 cells and colocalization analysis of BKα-β1 FRET with WGA.

    Fig. S4. Effect of Rab11A shRNA or Rab11A dominant-negative mutant on Rab11A abundance and surface BKα protein.

    Fig. S5. Regulation of surface BKα protein by antagonists and CaV1.2 and BKα proteins by CaV1.2 siRNA.

    Fig. S6. Effect of ROCK 1 or ROCK2 knockdown and HA1100 on respective protein abundance.

    Fig. S7. Rab11A knockdown does not alter vasoconstriction by membrane depolarization or myogenic tone.

  • Supplementary Materials for:

    Membrane depolarization activates BK channels through ROCK-mediated β1 subunit surface trafficking to limit vasoconstriction

    M. Dennis Leo, Xue Zhai, Padmapriya Muralidharan, Korah P. Kuruvilla, Simon Bulley, Frederick A. Boop, Jonathan H. Jaggar*

    *Corresponding author. Email: jjaggar{at}uthsc.edu

    This PDF file includes:

    • Fig. S1. Effect of different antagonists on depolarization- and SNP-induced changes in the surface abundance of BKα and β1 protein.
    • Fig. S2. Depolarization-induced surface trafficking of β1 subunits in human cerebral arteries.
    • Fig. S3. Overexpression of β1 subunits in HEK293 cells and colocalization analysis of BKα-β1 FRET with WGA.
    • Fig. S4. Effect of Rab11A shRNA or Rab11A dominant-negative mutant on Rab11A abundance and surface BKα protein.
    • Fig. S5. Regulation of surface BKα protein by antagonists and CaV1.2 and BKα proteins by CaV1.2 siRNA.
    • Fig. S6. Effect of ROCK 1 or ROCK2 knockdown and HA1100 on respective protein abundance.
    • Fig. S7. Rab11A knockdown does not alter vasoconstriction by membrane depolarization or myogenic tone.

    [Download PDF]

    Technical Details

    Format: Adobe Acrobat PDF

    Size: 914 KB


    Citation: M. D. Leo, X. Zhai, P. Muralidharan, K. P. Kuruvilla, S. Bulley, F. A. Boop, J. H. Jaggar, Membrane depolarization activates BK channels through ROCK-mediated β1 subunit surface trafficking to limit vasoconstriction. Sci. Signal. 10, eaah5417 (2017).

    © 2017 American Association for the Advancement of Science

Navigate This Article