Cholesterol activates BK channels by increasing KCNMB1 protein levels in the plasmalemma

Figures

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  Figure 1. In vitro CLR enrichment increases smooth muscle CLR in rat MCA and activates BK channels.A, original snapshots of filipin-stained segments of rat MCAs following a 20 min-long CLR enrichment with 0.625 mM CLR. Within each row, panels reflect (from left to right): filipin staining (blue), plasmalemmal marker wheat germ agglutinin (red), smooth muscle layer image in visible light (gray), and an overlay of all three frames (multicolor). B, scattered data showing increase in filipin-associated fluorescence following a 20 min-long enrichment of rat MCA segments with 0.625 mM CLR. p = 0.0087 by two-tailed Mann–Whitney test. C, BK recordings from an I/O patch excised from rat MCA myocyte with naïve CLR content (black, number of channels N = 4) or CLR-enriched for 20 min prior to patch excision (blue, N = 4). Free [Ca2+] in the bath solution was set at 30 μM. Here and in all other figures, Po: open probability of a single channel. D, Boltzmann fitting of NPo/NPomax-V data from individual patches from rat MCA myocytes with naïve CLR (black) and following a 20-min-long enrichment with CLR prior to patch excision (blue). Averaged data for each group are depicted by thick black and blue…

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  Figure 2. High-CLR diet in vivo increases smooth muscle CLR in rat MCA and activates BK channels.A, original snapshots of filipin-stained segments of rat MCAs harvested after 29 weeks from placing 25 day-old rats on high-CLR diet (2% CLR). Red arrows point at the plasmalemma of individual myocytes. B, scattered data showing increase in filipin-associated fluorescence following enrichment of rat MCA segments with CLR in the course of high-CLR diet. p = 0.0027 by one-tailed Mann–Whitney test. C, BK recordings from an I/O patch excised from rat MCA myocyte from rats on control chow (black, number of channels N = 2) or on high-CLR chow offered for 29–32 weeks (blue, N = 6). Free [Ca2+] in the bath solution were set at 30 μM. D, Boltzmann fitting of NPo/NPomax-V data from individual patches from rat MCA myocytes in control chow-fed (black) and CLR chow-fed (blue) groups. Averaged data for each group are depicted by thick black and blue curves, respectively. E, scattered data showing decrease in Vhalf following in vivo accumulation of within rat MCA smooth muscle. p = 0.0095 by two-tailed Mann–Whitney test.

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  Figure 3. Loss of cellular environment results in BK channel inhibition by CLR.A, BK recordings from an I/O patch from rat MCA myocyte excised following a 5 min-long enrichment of myocyte with CLR in vitro (black, number of channels N = 3) or excised and then enriched with CLR for 5 min (blue, N = 7). Free [Ca2+] in the bath solution was set at 30 μM. B, Boltzmann fitting of NPo/NPomax-V data from individual patches from rat MCA myocytes when patches are excised after (black) and before (blue) 5-min-long enrichment with CLR. Averaged data for each group are depicted by thick black and blue curves, respectively. C, scattered data showing increase in Vhalf following application of CLR-enriching media to excised patches as opposed to CLR enrichment of myocytes prior to patch excision. p = 0.0260 by two-tailed Mann–Whitney test.

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  Figure 4. Block of cellular trafficking switches BK channel activation by CLR enrichment to inhibition.A, BK recordings from an I/O patch from rat MCA myocyte excised following a 60 min-long incubation of myocyte in 10 μM brefeldin-A (BFA) without (black, number of channels N = 18) or with addition of CLR-enriching solution for 20 min at minute 40 of incubation (red, N = 17). Free [Ca2+] in the bath solution was set at 30 μM. B, Boltzmann fitting of NPo/NPomax-V data from individual patches from rat MCA myocytes subjected to BFA block of cellular trafficking without (black) and with subsequent addition of CLR-enriching solution (red). Averaged data for each group are depicted by thick black and red curves, respectively. C, scattered data showing increase in Vhalf when CLR enrichment of rat MCA myocytes was performed in the presence of BFA as opposed to BFA treatment alone prior to patch excision. p = 0.0286 by two-tailed Mann–Whitney test.

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  Figure 5. CLR enrichment in vitro increases plasmalemmal abundance of KCNMB1 protein, but not BK channel alpha subunits.A, representative Western blot images showing that 20-min-long incubation of rat MCA segments in CLR-enriching solution results in upregulation of KCNMB1 cell surface (S) but not intracellular fraction (I). The CLR-driven upregulation of surface KCNMB1 is blunted by 10 μM BFA. In contrast, distribution of BK channel alpha subunits between S and I fractions remains unchanged by either treatment. B, scattered data showing surface KCNMB1 as percentage of total KCNMB1 protein level in control, after CLR enrichment, and after CLR enrichment in presence of 10 μM BFA. p = 0.0005 by one-way ANOVA with Tukey posttest; ∗∗∗p < 0.001, ∗∗p < 0.01. C, scattered data presented as a ratio of surface KCNMB1 over surface BK alpha subunit protein. p = 0.0007 by one-way ANOVA with Tukey post-test; ∗∗∗p < 0.001, ∗∗p < 0.01.

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  Figure 6. In vitro CLR enrichment of myocytes from basilar (BA) and coronary (CA) arteries that were reported to have the highest amounts of KCNMB1 protein in plasmalemma fails to activate BK currents.A, BK recordings from an I/O patch from rat BA (top records, purple) and CA (bottom records, orange) myocyte excised following a 20 min-long incubation of myocytes in bath (left recordings) of CLR-enriching solution (right recordings). Free [Ca2+] in the bath solution was set at 30 μM. BA bath record is from a patch with N = 8; BA CLR record is from a path with N = 7. CA bath record is from a patch with N = 6; CA CLR record is from a patch with N = 2. B, Boltzmann fitting of NPo/NPomax-V data from individual patches from rat BA and CA myocytes susbjected to either a 20 min-long incubation in bath or CLR-enriching solution prior to excision. Averaged data for each group are depicted by thick purple and orange curves, respectively. C, scattered data showing lack of decrease in Vhalf when CLR enrichment was performed on myocytes from BA or CA that are naturally high in KCNMB1 plasmalemmal fraction.

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  Figure 7. In vitro CLR enrichment of myocytes from MCA of rats on high-CLR diet inhibits BK channels.A, BK recordings from MCA of rat subjected to high-CLR diet for 29 weeks and to a 20 min-long CLR enrichment in vitro prior to patch excision. Free [Ca2+] in the bath solution was set at 30 μM, number of channels in the patch N = 13. B, Boltzmann fitting of NPo/NPomax-V data from individual myocyte membrane patches of rats on high-CLR chow without in vitro manipulation with CLR level (blue) and following a 20-min-long CLR enrichment in vitro prior to patch excision (red). Averaged data for each group are depicted by thick blue and red curves, respectively. C, scattered data showing increase in Vhalf when in vitro enrichment with CLR was performed on myocytes from rats receiving high-CLR chow when compared with rats on high-CLR chow without in vitro manipulations with CLR level. p = 0.0286 by two-tailed Mann–Whitney test.

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  Figure 8. In vitro CLR enrichment of myocytes from BA and MCA reveals BK lack of effect and activation of BK currents, respectively, when recordings are performed at low calcium level.A, BK recordings from an I/O patch from rat BA (top records, purple) and MCA (bottom records, black and blue) myocyte excised following a 20 min-long incubation of myocytes in bath (left recordings) of CLR-enriching solution (right recordings). Free [Ca2+] in the bath solution was set at 3 μM. BA Bath record is from a patch with N = 8; BA CLR record is from a patch with N = 6. MCA bath record is from a patch with N = 8; MCA CLR record is from a patch with N = 6. B, Boltzmann fitting of NPo/NPomax-V data from individual patches from rat BA and MCA myocytes subjected to either 20-min-long incubation in bath or CLR-enriching solution prior to excision. Averaged data for each group are depicted by thick purple (BA) and black and blue (MCA) curves, respectively. C, scattered data showing lack of decrease in Vhalf when CLR enrichment was performed on myocytes from BA, while enrichment of MCA myocytes still rendered a decrease in Vhalf. p = 0.0159 by two-tailed Mann–Whitney test.

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  Figure 9. CLR enrichment fails to activate BK channels in MCA myocyte patches from KCNMB1 KO mice.A, BK recordings from an I/O patch from MCA of C57BL/6 (wild type, wt, top traces) and KCNMB1-/- (KO, bottom traces) mouse myocytes. Traces in black and gray depict BK channel activity in patches with naïve CLR level, while traces in blue and red depict BK channel activity in patches from myocytes that were subjected to a 1 h-long enrichment with CLR in vitro prior to patch excision. Free [Ca2+] in the bath solution was set at 30 μM. Wt bath record is from a patch with N = 6; wt CLR record is from a patch with N = 5; KCNMB1-/- bath record is from a patch with N = 4; KCNMB1-/- CLR record is from a patch with N = 4. B, Boltzmann fitting of NPo/NPomax-V data from individual patches from MCA myocytes of wt and KCNMB1-/- mouse with naïve CLR levels (black and gray) or from myocytes subjected to a 1 h-long incubation in CLR-enriching solution prior to patch excision (blue and red). Averaged data for each group are depicted by thick black (wt, naïve CLR), gray (KCNMB1-/-, naïve CLR), blue (wt, CLR enrichment), and red (KCNMB1-/-,CLR enrichment) curves. C, scattered data showing lack of decrease in…