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.
Highlights
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
We found that Jph2 is the most abundant junctophilin isotype in native smooth muscle cells (SMCs) obtained from cerebral resistance arteries, and that this structural protein is necessary for site-specific juxtaposition of the SR and plasma membrane at the periphery of these cells
These data suggest that Jph1 and Jph2 are present in SMCs, and that other Jph isotypes may be expressed in other cell types present in whole arteries, such as fibroblasts, pericytes, and/or endothelial cells
Summary
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. To investigate the impact of JPH2 knockdown on spontaneous Ca2+ spark activity, we used high-speed (∼50 fps) spinning-disk confocal microscopy to image Ca2+ signals in pressurized (60 mmHg), intact cerebral arteries treated with Jph2-targeting or control morpholinos and loaded with the Ca2+ indicator dye Fluo-4-AM (Fig. 3 and Movies S3 and S4).
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