Nanoscale coupling of junctophilin-2 and ryanodine receptors regulates vascular smooth muscle cell contractility

Abstract

Junctophilin proteins maintain close contacts between the endoplasmic/sarcoplasmic reticulum (ER/SR) and the plasma membrane in many types of cells, as typified by junctophilin-2 (JPH2), which is necessary for the formation of the cardiac dyad. Here, we report that JPH2 is the most abundant junctophilin isotype in native smooth muscle cells (SMCs) isolated from cerebral arteries and that acute knockdown diminishes the area of sites of interaction between the SR and plasma membrane. Superresolution microscopy revealed nanometer-scale colocalization of JPH2 clusters with type 2 ryanodine receptor (RyR2) clusters near the cell surface. Knockdown of JPH2 had no effect on the frequency, amplitude, or kinetics of spontaneous Ca²⁺ sparks generated by transient release of Ca²⁺ from the SR through RyR2s, but it did nearly abolish Ca²⁺ spark-activated, large-conductance, Ca²⁺-activated K⁺ (BK) channel currents. We also found that JPH2 knockdown was associated with hypercontractility of intact cerebral arteries. We conclude that JPH2 maintains functional coupling between RyR2s and BK channels and is critically important for cerebral arterial function.

Keywords: Ca2+ signaling; cerebral arteries; electrophysiology; ion channels; super-resolution microscopy.

Fig. 1.

JPH2 maintains the proximity of the plasma membrane and SR in cerebral artery SMCs. (A) Representative end-point RT-PCR analysis for expression of Jph1, Jph2, Jph3, and Jph4 in RNA samples isolated from whole cerebral arteries (CA), and in SMCs isolated from cerebral arteries, whole brain, and skeletal muscle (SkM; n = 3 independent experiments). β-Actin (Actb) was used as a positive control. (B) Relative expression levels of Jph1 and Jph2 mRNAs from isolated SMCs, normalized to Actb expression (n = 3; *P < 0.05). (C) Representative Wes protein analysis of whole cerebral artery (CA) and heart lysates probed with an anti-JPH2 antibody. (D) Deconvolved confocal slice of isolated SMCs from cerebral arteries treated with control or Jph2-targeting morpholinos and stained with dyes that specifically target the plasma membrane (PM; red) or SR (green). Merged images show the effects of morpholino treatment on close interactions of the PM and SR at the periphery of the cell in respective insets. (Scale bars: full images, 10 µm; Insets, 1 µm.) (E) Representative surface analysis of z-stack reconstructions of SMCs isolated from cerebral arteries treated with control or Jph2-targeting morpholinos and labeled with dyes staining the PM (red) or SR (green). Colocalization surface representations (Coloc; yellow) were generated from voxels that were positive for both channels. (Scale bar, 10 µm.) (Insets) Magnified views of colocalizing surfaces. (Scale bar, 1 µm.) (F) Summary data showing mean PM and SR volumes, the number of PM–SR colocalizing sites per cell, and the mean volume of individual PM–SR colocalizing sites in SMCs isolated from cerebral arteries treated with control or Jph2-targeting morpholinos (n = 634 to 817 individual colocalization sites in n = 12 to 13 cells per group from 3 animals; *P < 0.05).

Fig. 2.

Nanometer-scale colocalization of JPH2 and RyR2 at the plasma membrane of cerebral artery SMCs. (A) Superresolution localization maps for a native cerebral artery SMC immunolabeled for JPH2 (green) and RyR2 (red) imaged using GSDIM in epifluorescence illumination mode. Merged images and colocalized protein clusters identified by object-based analysis (OBA) are also shown. (Scale bar, 1 µm.) The middle row shows an expanded view of the white rectangle from the top merged panel. (Scale bar, 0.5 µm.) The bottom row is a further expanded view of an interacting cluster (white box). (Scale bar, 0.2 µm.) Maps are representative of n = 9 cells from 3 animals. (B) Superresolution localization maps for a native cerebral artery SMCs immunolabeled for JPH2 (green) and RyR2 (red) imaged using GSDIM in TIRF illumination mode. (Scale bar, 1 µm.) The bottom row shows an expanded view of clusters interacting in the white square from the merged panel. (Scale bar, 0.2 µm.) Maps are representative of n = 7 cells from 3 animals.

Fig. 3.

Jph2 knockdown has no effect on Ca²⁺ spark frequency, amplitude, or kinetics. (A) Representative confocal Ca²⁺ images of pressurized (60 mmHg), Fluo-4-AM–loaded cerebral arteries treated with control or Jph2-targeting morpholinos. Colored boxes show selected ROIs where Ca²⁺ sparks occurred. (Scale bar, 10 µm.) (B) Representative changes in fractional fluorescence (F/F₀) as a function of time for ROIs in A. The trace color corresponds to the color of the respective ROI box. (C) Summary data showing the Ca²⁺ spark frequency (in Hertz) normalized to surface area (in Hertz per 100 square micrometers) in cerebral arteries (n = 5 to 6 cerebral arteries/group from 4 animals), as well as the amplitude (F/F₀), half-duration [half-time (t_1/2), in seconds], rise time (t_1/2, in seconds), and decay time (t_1/2, in seconds) of individual Ca²⁺ spark events recorded from each group (n = 616 events for control, n = 601 events for Jph2-targeted). There were no significant differences.

Fig. 4.

JPH2 is required for functional coupling of RyR2 and BK. (A) Representative recordings of STOCs in SMCs isolated from cerebral arteries treated with control or Jph2-targeting morpholinos recorded over a range of membrane potentials (−60 to 0 mV) using perforated patch-clamp electrophysiology. Summary data show the frequency (in Hertz) of STOCs as a function of membrane potential (n = 5 to 6 cells per group from 3 animals; *P < 0.05). (B) Representative BK (paxilline-sensitive) currents recorded from SMCs isolated from cerebral arteries treated with control or Jph2-targeting morpholinos using conventional whole-cell patch-clamp electrophysiology. BK currents were recorded over a series of command voltage steps (−100 to +100 mV). Summary of whole-cell current data (n = 8 cells/group from 4 animals). There were no significant differences.

Fig. 5.

Jph2 knockdown causes vascular hypercontractility. (A) Representative traces showing changes in luminal diameter over a range of intraluminal pressures (5 to 140 mmHg) for cerebral arteries treated with control (black) or Jph2-targeting (blue) morpholinos. The passive response to changes in intraluminal pressure for both arteries is indicated by the gray trace. (B) Summary data for myogenic tone as a function of intraluminal pressure for both groups (n = 6 arteries per group from 4 to 5 animals; *P < 0.05). (C) Representative traces showing changes in luminal diameter over a range of intraluminal pressures (5 to 140 mmHg) for mesenteric arteries treated with control (black) or Jph2-targeting (blue) morpholinos. The passive response to changes in intraluminal pressure for both groups is indicated by the gray trace. (D) Summary data for myogenic tone as a function of intraluminal pressure for both groups (n = 6 arteries per group from 4 to 5 animals; *P < 0.05). (E) Representative traces showing changes in luminal diameter over a range of intraluminal pressures (5 to 140 mmHg) for cerebral arteries treated with control morpholinos (black) or cerebral arteries treated with control morpholinos in the presence of tetracaine (10 µM; red). The passive response to changes in intraluminal pressure for both arteries is indicated by the gray trace. (F) Summary data for myogenic tone as a function of intraluminal pressure for both groups (n = 6 arteries per group from 4 to 5 animals; *P < 0.05).