AKAP5 complex facilitates purinergic modulation of vascular L-type Ca2+ channel CaV1.2

Figures

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  Fig. 1. ht31 prevents glucose/NF546-induced cAMP in human cells. a Average ICUE3-PM FRET trace (mean = solid line; SEM = shade) in response to application of 10 μM ht31 and after 1 μM forskolin in human arterial myocytes. Time courses of the average ICUE3-PM FRET traces (mean = solid line; SEM = shade) in human arterial myocytes in response to b 15 mM D-glucose (D-glu) and c 500 nM NF546 and after 1 μM forskolin in control (CT) and ht31-treated cells. d Plot of maximum FRET responses for human male/female arterial myocytes exposed to the ht31 peptide alone (n = 17 cells/2 humans), 15 mM D-glu (n = 19 cells/3 humans for −ht31 and n = 36 cells/3 humans for +ht31) and NF546 (n = 18 cells/3 humans for −ht31 and n = 14 cells/3 humans for +ht31) in the absence and presence of forskolin. *P < 0.05 with Kruskal–Wallis one-way ANOVA with Dunn’s multiple comparisons. The single asterisks highlight significant differences between all datasets in the absence of forskolin. The double asterisks indicate a statistical difference within the same experimental group in the absence and presence forskolin. P = 0.0002 for 15 mM D-glu-ht31 and ht31-ht31 + 15 mM D-glu + forskolin. P = 0.0002 for NF546-NF546 +…

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  Fig. 2. AKAP5 is necessary for glucose/NF546-induced cAMP synthesis. Averaged ICUE3-PM FRET traces (mean = solid line; SEM = shade) and plot of maximum FRET ratio for wild-type (WT) and AKAP5−/− male arterial myocytes in response to a, b 20 mM D-glucose (D-glu; n = 21 cells/6 WT mice; n = 13 cells/6 AKAP5−/− mice), c, d 500 nM NF546 (n = 23 cells/6 WT mice; n = 18 cells/6 AKAP5−/− mice), and e, f 20 mM D-glu + NF546 (n = 22 cells/6 WT mice; n = 13 cells/6 AKAP5−/− mice) and 1 µM forskolin. *P < 0.05 with two-tailed Mann–Whitney test. The single asterisks highlight significant differences between datasets in the absence of forskolin. The double asterisks indicate a statistical difference within the same experimental group in the absence and presence forskolin. All significant P values are <0.0001. Data represent mean ± SEM. Source data are provided as Source data file.

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  Fig. 3. AKAP5/AC5 mediate IBa potentiation and vasoconstriction upon glucose/NF546. Representative a diameter recordings, b summary percentage myogenic tone, and c change in myogenic tone from wild-type (WT; n = 9 arteries/5 mice) and AKAP5−/− (n = 7 arteries/6 mice) cerebral arteries in 10 and 20 mM D-glucose (D-glu). *P < 0.05 with two-tailed Wilcoxon test for b (P = 0.0039 for WT 10 mM D-glu-20 mM D-glu) and two-tailed Mann–Whitney test for c (P = 0.0012 for WT-AKAP5−/−). d Averaged ICUE3-PM traces (mean = solid line; SEM = shade) and e maximum FRET ratio for WT and AC5−/− arterial myocytes upon 500 nM NF546 (n = 23 cells/6 WT mice; n = 18 cells/5 AC5−/− mice), 20 mM D-glu + 500 nM NF546 (n = 22 cells/6 WT mice; n = 15 cells/5 AC5−/− mice) and after 1 µM forskolin. *P < 0.05 with two-tailed Mann–Whitney test. Single asterisks highlight significant differences between datasets in the absence of forskolin. Double asterisks indicate statistical differences within the same experimental group before and after forskolin. All significant P values are <0.0001. f Exemplary IBa traces and g amalgamated current density from WT (n = 7 cells/5 mice), AKAP5−/− (n = 7 cells/5 mice) and AC5−/− (n = 7…

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  Fig. 4. Glucose/NF546 effects on myogenic tone/blood flow require AKAP5. a Exemplary open cranial window images of wild-type (WT; n = 18 arteries/3 mice) and AKAP5−/− (n = 21 arteries/3 mice) middle cerebral arteries and branches exposed to 10 mM D-glucose (D-glu), 20 mM D-glu + NF546, and 0 Ca2+ + 1 μM nifedipine (nif). b Summary myogenic tone data. *P < 0.05 with Friedman one-way ANOVA with Dunn’s multiple comparisons. Significance was compared to the 10 mM D-glu group. P < 0.0001 for WT 10 mM D-glu-20 mM D-glu and P = 0.0004 for WT 10 mM D-glu-20 mM D-glu + NF546. c Representative pseudo-colored blood flow images of cerebral pial arteries through a cranial window exposed to 10 mM D-glu + 10 mM mannitol, 20 mM D-glu, and 0 Ca2+ + 1 μM nif in WT and AKAP5−/− mice. d Summary blood flow data in WT (n = 18 arteries/5 mice) and AKAP5−/− (n = 12 arteries/3 mice) mice. *P < 0.05 with Friedman one-way ANOVA with Dunn’s multiple comparisons. Significance was compared to the +10 mM mannitol group. P = 0.0007 for WT mannitol-20 mM D-glu and P < 0.0001 for WT mannitol-20 mM D-glu + NF546. The yellow ovals highlight regions that were used for analysis. e Representative open cranial window images of…

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  Fig. 5. AKAP5 closely associates with P2Y11-like, AC5, and CaV1.2. Representative super-resolution Airyscan intensity projection images and line intensity profile of arterial myocytes triple-labeled for either a, b AKAP5 (cyan), P2Y11 (yellow), and CaV1.2 (magenta) or c, d AKAP5 (cyan), AC5 (yellow), and CaV1.2 (magenta). Merged images are on the right side panels. The magnified insets are from regions highlighted by the white squares in the AKAP5 images. Insets’ scale bars = 2 μm. Line intensity profiles obtained from the regions below the dotted lines in the magnified insets in (a) and (c). Similar results were observed in six different cells from two independent preparations per condition. Representative super-resolution GSD reconstruction maps of arterial myocytes labeled for e AKAP5 and P2Y11, f AKAP5 and AC5, or g AKAP5 and CaV1.2 (scale bar = 2 µm). Two magnified areas obtained from the region highlighted by the yellow boxes in the main figure are shown (scale bar = 200 nm). These magnified panels are intended to provide a clearer view of cluster size and distribution in two areas of a cell. Enlarged merged images (left) and associated x–y fluorescence intensity profile (right)…

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  Fig. 6. AKAP5 fosters the P2Y11, AC5, PKA, and CaV1.2 nanocomplex. a Exemplary merged fluorescence proximity ligation assay (PLA; red)/nucleus (blue; left panels) and differential interference contrast (right panels) images of wild-type (WT) and AKAP5−/− cerebral arterial myocytes co-labeled for P2Y11 + AKAP5, AC5 + AKAP5, and P2Y11 + AC5. b Quantification of proximity ligation assay (PLA) fluorescent puncta per cell area (puncta/µm2) for WT and AKAP5−/− cerebral arterial myocytes labeled for P2Y11 + CaV1.2 (n = 27 cells/3 WT mice; n = 26 cells/3 AKAP5−/− mice), P2Y11 + PKAcat (n = 27 cells/3 WT mice; n = 26 cells/3 AKAP5−/− mice), P2Y11 + AKAP5 (n = 28 cells/3 WT mice; n = 15 cells/3 AKAP5−/− mice), CaV1.2 + AKAP5 (n = 27 cells/3 WT mice; n = 24 cells/3 AKAP5−/− mice), AC5 + AKAP5 (n = 29 cells/3 WT mice; n = 18 cells/3 AKAP5−/− mice), P2Y11 + AC5 (n = 22 cells/3 WT mice; n = 27 cells/3 AKAP5−/− mice), CaV1.2 + PKARIIα (n = 18 cells/3 WT mice; n = 17 cells/3 AKAP5−/− mice), CaV1.2 + AC5 (n = 28 cells/3 WT mice; n = 20 cells/3 AKAP5−/− mice), and PKARIIα + AC5 (n = 36 cells/3 WT mice; n = 22 cells/3 AKAP5−/− mice). *P < 0.05 with two-tailed Mann–Whitney test. Comparison between WT and…

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  Fig. 7. PDE inhibition restores cAMP signal but not vasoconstriction in AKAP5−/− tissue. a Time courses of the average ICUE3-PM traces (mean = solid line; SEM = shade) and b summary data of wild-type (WT; n = 22 cells/3 mice) and AKAP5−/− (n = 12 cells/3 mice) arterial myocytes pretreated with the broad phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methylxanthine (IBMX; 100 μM) in response to 20 mM D-glucose (D-glu) + NF546 and after application of forskolin. *P < 0.05 with two-tailed Mann–Whitney test. The single asterisks highlight significant differences between datasets in the absence of forskolin. The double asterisks indicate a statistical difference within the same experimental group in the absence and presence forskolin. All significant P values are <0.0001. c Exemplary diameter recordings and d summary myogenic tone data from WT (n = 6 arteries/4 mice) and AKAP5−/− (n = 6 arteries/3 mice) cerebral arteries pretreated with IBMX (100 μM) before and after application of 20 mM D-glu and subsequent NF546. Scales = 10 μm (vertical) and 5 min (horizontal). Initial diameters shown on the traces left side. *P < 0.05 with Friedman one-way ANOVA with Dunn’s multiple comparisons.…