β-Adrenergic control of sarcolemmal Ca V 1.2 abundance by small GTPase Rab proteins

Figures

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  Fig. 1.. Internal pools of presynthesized CaV1.2 channels reside on endosomes. (A) Two-color Airyscan superresolution images of control and 100 nM ISO-stimulated AMVMs immunostained to examine distributions of CaV1.2 and EEA1+ early endosomes. Binary colocalization maps (Bottom) display pixels in which CaV1.2 and endosomal expression precisely overlapped. (B) Histograms showing percent colocalization between EEA1 and CaV1.2 in male and female AMVMs, in control (male: n = 3, n = 15; female n = 3, n = 13) and ISO-stimulated conditions (male: n = 3, n = 14; female n = 3, n = 15). (C) Immunostaining of CaV1.2 and Rab11+ recycling endosomes and (D) accompanying histogram summarizing results from control (male: n = 4, n = 15; female n = 3, n = 14) and ISO conditions (male: n = 3, n = 15; female n = 3, n = 11). (E) Immunostaining of CaV1.2 and Rab7+ LEs and lysosomes. (F) Histogram summarizing results from control (males: n = 3, n = 15, females: n = 3, n = 14) and ISO-stimulated AMVMs (males: n = 3, n = 15, females: n = 3, n = 11). Error bars indicate SEM. Two-way ANOVA **P < 0.01; *P < 0.05. Pictured are male AMVMs; for females see SI Appendix, Fig. S1.

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  Fig. 2.. Rab4a and Rab11a regulate an ISO-stimulated boost in CaV1.2 recycling. (A) TIRF images of Cav1.2-tRFP distribution in the PM of tsA-201 cells before (Left) and after 100 nM ISO (Right; n = 18). (B) Time course and kinetics (solid red line) of the fold-change in CaV1.2-tRFP intensity in the TIRF footprint before, during, and after ISO stimulation. (C) Histogram summarizing CaV1.2 channel cluster density (number/μm2) in the TIRF footprint in control and ISO-stimulated conditions. (D–F) Same layout format in tsA-201 cells coexpressing constitutively active (GTP-locked) Rab4a (n = 10). (G–I) Same layout format in tsA-201 cells coexpressing dominant-negative (GDP-locked) Rab4a (n = 12). (J–L) Same layout format in tsA-201 cells coexpressing dominant-negative (GDP-locked) Rab11a (n = 8). (M) Calculation and resultant theoretical time-course and kinetics of Rab11a-dependent ISO-stimulated recycling. Data that passed a normality test (C and L) were analyzed with a paired t test, others (F and I) were analyzed with a nonparametric Wilcoxon test. ***P < 0.001; **P < 0.01; *P < 0.05.

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  Fig. 3.. Actin and MT polymerization are essential for ISO-stimulated CaV1.2 recycling. Two-color Airyscan images of fixed AMVMs immunostained to examine the relative localization of CaV1.2 and (A) α-tubulin, or (B) phalloidin-stained actin. (C) The distribution of α-tubulin in untreated (Top) and nocodazole-treated AMVMs (Bottom). (D) Airyscan images of CaV1.2 and EEA1 distribution in nocodazole-treated control (n = 3, n = 12; Left) and ISO-stimulated (n = 3, n = 10; Right) AMVMs. (Bottom) Binary colocalization maps display pixels in which CaV1.2 and EEA1 expression precisely overlapped. (E) Histogram summarizing percent colocalization of EEA1 with CaV1.2 in nocodazole-treated cells. (F) Actin distribution in untreated (Top) and lat-A–treated cells (Bottom). (G) Airyscan images and binary colocalization maps of CaV1.2 and EEA1 distribution in lat-A–treated control (n = 3, n = 12; Left) and ISO-stimulated (n = 3, n = 12; Right) AMVMs. (H) Histogram showing percent colocalization of EEA1 with CaV1.2 in lat-A–treated AMVMs. (I) Airyscan images and binary colocalization maps of AMVMs treated with both lat-A and nocodazole under control (n = 3, n = 12) and ISO stimulated conditions (n = 3, n…

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  Fig. 4.. Dynamic imaging to unmask the mobile channel population. (A) TIRF images of GFP fluorescence emission from Cavβ2a-paGFP transduced AMVMs before (Top) and after 100 nM ISO (Bottom). Images illustrating inserted, endocytosed, static, and merged channel populations are shown to the right. (Bottom) Time course of the changes in Cavβ2a-paGFP intensity in ROIs (i–iii) indicated by yellow circles on TIRF images (n = 5, n = 7). Same format for cells pretreated with (B) nocodazole (n = 5, n = 7), (C) lat-A (n = 3, n = 6), or (D) both cytoskeleton disruptors (n = 3, n = 6). (E–H) Histograms summarizing statistics for these experiments analyzed with Kruskal–Wallis tests. **P < 0.01; *P < 0.05.

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  Fig. 5.. βAR-mediated ICa enhancement is blunted by reducing dynamic channel insertion. (A) Whole-cell currents elicited from a representative AMVM during a 300-ms depolarization step from −40 mV to 0 mV before (control: black) and during application of 100 nM ISO (blue). (B) I-V plot summarizing the results from n = 7 cells (from n = 7 animals) subjected to test potentials ranging from −50 mV to +100 mV. Currents were normalized to cell capacitance to generate current density. (C) Voltage-dependence of the normalized conductance (G/Gmax) before and during ISO application, fit with Boltzmann functions. (D–F) Whole-cell currents (D), I-V (E), and G/Gmax plots (F) from AMVMs pretreated for 2 h with 10 μM nocodazole (n = 4, n = 7). (G, H) The same for cells treated with 5 μM lat-A (n = 5, n = 5). (J–L) Results from cells treated with a combination of both (n = 3, n = 6).

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  Fig. 6.. βAR-stimulated CaV1.2 superclustering and enhanced sarcolemmal expression requires an intact MT cytoskeleton. Superresolution GSD localization maps of control (Left) and 100 nm ISO‐stimulated (Right), fixed, AMVMs immunostained to examine CaV1.2 channel distribution under (A) untreated (control: n = 4, n = 16; ISO: n = 4, n = 17), (B) nocodazole-treated (control: n = 3, n = 8; ISO: (n = 3, n = 15), (C) lat-A–treated (control: n = 3, n = 8; ISO: (n = 3, n = 9), and (D) combo-treated (control: n = 2, n = 10; ISO: (n = 3, n = 13) conditions. Maps were pseudocolored “cyan hot” and received a one-pixel median filter for display purposes. Yellow (control) and blue (ISO) boxes indicate the location of the zoomed‐in regions displayed in the center. (E and F) Aligned dot plots showing mean CaV1.2 channel cluster areas and normalized total integrated density in each condition. Two-way ANOVA. ***P < 0.001; **P < 0.01; *P < 0.05.