Effects of LDL on BK channel current in BK-HEK 293 cells.

<p><b>A</b>. Voltage-dependent BK current recorded with voltage protocol as shown in the inset in cells treated with LDL (75 µg/mL) or LDL plus MβCD treatment. <b>B</b>. <i>I-V</i> relationships of BK current in control, LDL or LDL plus MβCD treatment (n = 25, *<i>P</i><0.05; **<i>P</i><0.01 vs. control). <b>C</b>. Percentage values compared to control of BK current at +60 mV in control (n = 25), with LDL (n = 32, **<i>P</i><0.01 vs. control), and LDL plus MβCD treatment (n = 10, ^##<i>P</i><0.01 vs. LDL alone). <b>D</b>. Activation variables calculated with <i>I-V</i> relationships of the channel current in panel <b>B</b> were fitted to a Boltzmann function. <b>E</b>. Western immunoblots showing KCa1.1 protein and the auxiliary β1-subunit protein in cells treated with LDL or LDL plus MβCD. <b>F</b>. Mean percentage values of KCa1.1 protein and the auxiliary β1-subunit protein in cells with LDL or LDL plus MβCD (n = 6-9, **<i>P</i><0.01 vs. control, ^#<i>P</i><0.05 vs. LDL alone).</p

Effect of cholesterol on KCa1.1 protein in cultured human coronary artery smooth muscle cells.

<p>A. BK current was recorded in a representative human coronary artery smooth muscle cell with the voltage protocol shown in the inset, and the current was inhibited by 1 µM paxilline. B. BK current was recorded in cells treated without/with MβCD or MβCD-cholesterol. C. Mean values of BK current at +80 mV in cells treated with MβCD (n = 5) or MβCD-cholesterol (n = 5, **<i>P</i><0.01 vs. control, n = 6). D. Western immunoblots showing KCa1.1 protein in human coronary artery smooth muscle cells treated with cholesterol depletion (MβCD), cholesterol enrichment (MβCD-cholesterol), LDL, or LDL plus MβCD. E. Mean percentage values of BK protein in cells with different treatments (<i>n</i> = 4–6, **<i>P</i><0.01 vs. control, ^#<i>P</i><0.05 vs. LDL alone).</p

Effects of silencing KCa1.1 or Kir2.1 on cell migration in human cardiac c-kit⁺ progenitor cells.

<p><b>(A)</b>: Images of human cardiac c-kit⁺ progenitor cells with wound-healing migration assay in confluent cells transfected with control siRNA, KCa1.1 siRNA or Kir2.1 siRNA (40 nM for each group). <b>(B)</b>: Images of migrated human cardiac c-kit⁺ progenitor cells to the lower membrane in cells transfected with control siRNA, KCa1.1 siRNA or Kir2.1 siRNA (40 nM for each group). <b>(C)</b>: Mean values of ratio of migrated cells in cells transfected with control siRNA, KCa1.1 siRNA or Kir2.1 siRNA (40 nM for each group, n = 5 for each group, *<i>P</i><0.05, **<i>P</i><0.01 vs. control siRNA).</p

Cholesterol regulation and channel protein degradation in BK-HEK 293 cells with co-expressing β1-subunit.

<p><b>A</b>. BK channel current recorded with the voltage protocol (inset) in cells treated with bafilomycin A1, lactacystin, MβCD-cholesterol, MβCD-cholesterol plus bafilomycin A1, or MβCD-cholesterol plus lactacystin. <b>B</b>. BK channel current at +60 mV in cells treated with bafilomycin A1 (n = 16, <i>P</i> = NS vs. control), lactacystin (n = 15, <i>P</i> = NS vs. control), MβCD-cholesterol (MβCD-chol, n = 19, **<i>P</i><0.01 vs. control), MβCD-cholesterol plus bafilomycin A1 (n = 14, ^##<i>P</i><0.01 vs. MβCD-cholesterol), or MβCD-cholesterol plus lactacystin (n = 18, ^##<i>P</i><0.01 vs. MβCD-cholesterol). <b>C</b>. Western immunoblots showing KCa1.1 protein in cells treated with bafilomycin A1, lactacystin, MβCD-cholesterol, MβCD-cholesterol plus bafilomycin A1, or MβCD-cholesterol plus lactacystin. <b>D</b>. Mean percentage values of KCa1.1 protein in cells treated with bafilomycin A1, lactacystin, MβCD-cholesterol, MβCD-cholesterol plus bafilomycin A1, or MβCD-cholesterol plus lactacystin (n = 6, **<i>P</i><0.01 vs. control; ^##<i>P</i><0.01 vs. MβCD-cholesterol).</p

Cholesterol effect on KCa1.1 current.

<p><b>A</b>. Voltage-dependent KCa1.1 current recorded in hKCa1.1-HEK 293 cells with the voltage protocol (<i>inset</i>) in cells treated with cholesterol depletion (MβCD) or cholesterol enrichment (MβCD-cholesterol). <b>B</b>. <i>I-V</i> relationships of hKCa1.1 current in control (n = 19), cholesterol depletion (n = 16, *<i>P</i><0.05 or **<i>P</i><0.01 vs. control at 0 to +60 mV) or cholesterol-enrichment (n = 11, <i>P</i> = NS vs. control). <b>C</b>. Percentage values of hKCa1.1 current at +60 mV in control (n = 16), cholesterol depletion (n = 19, **<i>P</i><0.01 vs. control) or cholesterol enrichment (n = 11, ^##<i>P</i><0.01 vs. MβCD alone). <b>D</b>. Single channel activity of hKCa1.1 channels recorded in cell-attached mode at +60 mV in cells treated with 5 mM MβCD or MβCD-cholesterol. The labels C, O₁ and O₂ indicate the close level, 1^st and 2^nd opening levels of the channel. <b>E</b>. Histograms of open probability of hKCa1.1 single channels in cells treated without (control) or with cholesterol depletion (MβCD). <b>F</b>. Mean values of open probability of hKCa1.1 channels in control (n = 8), MβCD (n = 6), and MβCD-cholesterol (n = 6, *<i>P</i><0.05 vs. control). <b>G</b>. Conductance of hKCa1.1 channels in control, MβCD, and MβCD-cholesterol (n = 6–8, <i>P</i> = NS vs. control).</p

Single channel activity of BK channels.

<p><b>A</b>. Single channel activity of BK channels recorded with cell-attached mode at +60 mV in cells treated with 5 mM MβCD or MβCD-cholesterol. The labels C, O₁ and O₂ indicate the close level, 1^st and 2^nd opening levels of the channel. <b>B</b>. Histograms of open probability of BK single channels in cells treated without (control) or with cholesterol enrichment (MβCD-cholesterol). <b>C</b>. Mean values of open probability of BK channels in control (n = 7), MβCD (n = 6), and MβCD-cholesterol (n = 7, **<i>P</i><0.01 vs. control). <b>D</b>. Conductance of BK channels in control, MβCD, and MβCD-cholesterol (n = 6–7, <i>P</i> = NS vs. control).</p

Protein expression of BK channels in BK-HEK 293 cells with co-expressing auxiliary β1-subunit.

<p><b>A</b>. Western immunoblots showing KCa1.1 protein and the auxiliary β1-subunit protein in cells treated with cholesterol depletion (MβCD) or cholesterol enrichment (MβCD-cholesterol). <b>B</b>. Mean percentage values of KCa1.1 protein and the β1-subunit protein in cells with different treatments (n = 6–9, **<i>P</i><0.01 vs. control). <b>C</b>. Immunocytochemistry showing membrane expression of KCa1.1 protein in cells treated with cholesterol depletion (MβCD) or cholesterol enrichment (MβCD-cholesterol). Cholesterol enrichment induced a reduction of membrane expression of BK channels.</p

Inhibition of membrane current by ion channel blockers in human cardiac c-kit⁺ progenitor cells.

<p><b>(A)</b>: BK_Ca and I_Na.TTX were co-expressed in a human cardiac c-kit⁺ progenitor cell, and inhibited respectively by 1 μM paxilline and 30 nM TTX. <b>(B)</b>: I_Kir and BK_Ca were co-expressed in a typical human cardiac c-kit⁺ progenitor cell, and inhibited respectively by 500 μM Ba²⁺ and 1 μM paxilline.</p

Effects of specific siRNA molecules on expression of ion channel genes and proteins in human cardiac c-kit⁺ progenitor cells.

<p><b>(A)</b>: PCR images and relative level of KCa1.1 mRNA in cells treated with lipofectamine 2000 (Lipo), control siRNA, or KCa1.1 siRNA. <b>(B)</b>: Western blots and relative level of KCa1.1 protein in cell treated with lipofectamine 2000, control siRNA, or KCa1.1 siRNA. <b>(C)</b>: PCR images and relative level of Kir2.1 mRNA in cells treated with lipofectamine 2000, control siRNA, or Kir2.1 siRNA. <b>(D)</b>: Western blots and relative level of Kir2.1 protein in cell treated with lipofectamine 2000, control siRNA, or Kir2.1 siRNA (n = 6 for each group, **<i>P</i><0.01 vs. control siRNA).</p

Effects of silencing KCa1.1 or Kir2.1 on cell migration in human cardiac c-kit⁺ progenitor cells.

<p><b>(A)</b>: Images of human cardiac c-kit⁺ progenitor cells with wound-healing migration assay in confluent cells transfected with control siRNA, KCa1.1 siRNA or Kir2.1 siRNA (40 nM for each group). <b>(B)</b>: Images of migrated human cardiac c-kit⁺ progenitor cells to the lower membrane in cells transfected with control siRNA, KCa1.1 siRNA or Kir2.1 siRNA (40 nM for each group). <b>(C)</b>: Mean values of ratio of migrated cells in cells transfected with control siRNA, KCa1.1 siRNA or Kir2.1 siRNA (40 nM for each group, n = 5 for each group, *<i>P</i><0.05, **<i>P</i><0.01 vs. control siRNA).</p