Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, C091

Oral Communications

Junctophilin-2 Supports Functional Coupling Between Type 2 Ryanodine Receptors and BK Channels in Vascular Smooth Muscle Cells

H. A. Pritchard1, E. Yamasaki2, P. W. Pires2, M. Nelson3,1, A. S. Greenstein1, S. Earley2

1. Division of Cardiovascular Sciences, University of Manchester, Manchester, United Kingdom. 2. Department of Pharmacology, Center for Cardiovascular Research, University of Nevada, Reno School of Medicine, Reno, Nevada, United States. 3. Department of Pharmacology, University of Vermont, Burlington, Vermont, United States.

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 critical for maintaining interactions between the SR and PM and associated signaling pathways in SMCs. C57/Bl6 mice were euthanized according to approved protocols by the Institutional Animal Care and Use Committee of the University of Nevada, Reno, and the UK Home Office Guidance on the Operation of the Animals (Scientific Procedures) Act 1986. We found that mRNA encoding JPH2 was present in native, contractile SMCs purified from enzymatically dispersed cerebral pial arteries and enriched using fluorescence-activated cell sorting. 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. In cerebral arteries SMC, RyR2 on the SR are functionally coupled with Ca2+-activated K+ (BK) channels on the PM, a negative feedback system that buffers vasoconstriction. We employed a molecular interference approach using selective morpholinos to knockdown JPH2 expression in isolated cerebral pial arteries, due to the lack of selective pharmacology. JPH2 protein levels were reduced by ~50% compared with scrambled sequence controls. Perforated patch-clamp electrophysiology was performed 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 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, necessary for JPH2 to interact with the PM. Conventional whole cell patch clamp method was employed. These experiments showed that STOC frequency was significantly reduced in SMCs loaded with the MORN domain peptide compared to a scrambled control. This study is the first to show the presence of JHP2 in contractile arterial SMCs and it role in maintaining functional coupling between RyR2 and BK channels.

Where applicable, experiments conform with Society ethical requirements