Junctophilin‐2 Supports Functional Coupling Between Type 2 Ryanodine Receptors and BK Channels in Vascular Smooth Muscle Cells
Authors: Yamasaki Evan , Pritchard Harry A.T. , Pires Paulo W. , Earley Scott 
Published: June 2021

Junctophilin‐2 (JPH2) is a structural protein that tethers the sarcoplasmic reticulum (SR) to the plasma membrane (PM) within the cardiac dyad. JPH2 is vital for efficient excitation‐contraction coupling in cardiomyocytes and is critically important for maintaining the structure of Ca2+ microdomains that enable crosstalk between ion channels on the PM and SR. Contractile regulation of vascular smooth muscle cells (SMCs) also relies on close interactions between the PM and SR, but little is known about the molecular architecture of these peripheral coupling sites. This study tested the hypothesis that JPH2 is critically important for maintaining interactions between the SR and PM and associated signaling pathways in SMCs. Using RT‐PCR, we found that mRNA encoding JPH2 was present in pools of native, contractile SMCs isolated from enzymatically dispersed cerebral pial arteries and enriched using fluorescence‐activated cell sorting. The other JPH family members (JPH1, JPH3, and JPH4) were not detected in this assay. JPH2 protein expression was confirmed using the Wes capillary electrophoresis‐based immunoassay. Super‐resolution microscopy showed significant co‐localization of JPH2 and type 2 ryanodine receptors (RyR2) in immunolabeled contractile SMCs compared to randomized controls. In SMCs from cerebral arteries, RyR2 on the SR are functionally coupled with Ca2+‐activated K+ (BK) channels on the PM to form a negative feedback system that limits the magnitude and duration of vasoconstriction. Selective pharmacological inhibitors of JPH2 are not available. Therefore, we employed a molecular interference approach using selective morpholinos to knockdown JPH2 expression in isolated cerebral pial arteries. JPH2 protein levels were reduced by ~50% compared with scrambled sequence controls. Perforated patch‐clamp electrophysiology was carried over a range of membrane potentials to record spontaneous transient outward currents (STOCs), which represent the activation of clusters of BK channels by Ca2+ released from the SR through RyR2. These studies found that STOCs were essentially absent from SMCs isolated from vessels treated with JPH2‐targeted morpholinos, but STOC frequency and amplitude in SMCs treated with scrambled control morpholinos were voltage dependent and did not differ from untreated controls. To complement our knockdown strategy, we designed a novel inhibitory peptide that is homologous to the Membrane Occupation and Recognition Nexus (MORN) domain of JPH2. This domain is necessary for JPH2 to interact with the PM. Cells were loaded with either the MORN domain peptide or a scrambled control through the patch pipette and STOCs were recorded using the conventional whole cell patch‐clamp method. These experiments showed that STOC frequency was significantly reduced in SMCs loaded with the MORN domain peptide compared to scrambled controls. This study is the first to show that JPH2 is present in contractile arterial SMCs and is critically important for maintaining functional coupling between RyR2 and BK channels.

Support or Funding Information

Support: R01HL091095 (to SE) and AHA15POST2472002 (to PWP).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.