Interaction between CaV2.1 and Junctophilin3/4 depends on the II-III loop of CaV2.1 and on the α-helical region of Junctophilin3/4

Neuronal junctophilins (JPH3 and JPH4) form junctions between the endoplasmic reticulum (ER) and plasma membrane (PM) through their C-terminal transmembrane (TM) domain, which is embedded in the ER membrane, and N-terminal domain, which binds to the PM. JPHs also recruit and slow the inactivation of the voltage-gated Ca²⁺ channel Ca_V2.1. Here, we identified the domains responsible for Ca_V2.1-JPH interactions by co-expressing the isolated GFP-tagged Ca_V2.1 cytoplasmic domains with mCherry-tagged JPH3/4 in tsA201 cells. Among the Ca_V2.1 domains, only the II-III loop colocalized with JPH3 and JPH4 as well as with the TM-truncated JPH3-ΔTM and JPH4-ΔTM constructs, which cannot form ER-PM junctions. Further fragmentation of the II-III loop showed that both JPH-ΔTM constructs colocalized with the proximal half of the loop containing the synprint domain, known to bind presynaptic proteins, but only JPH4-ΔTM colocalized with the distal half and only JPH4 slowed the inactivation of a Ca_V2.1 construct lacking most of the synprint region. JPH colocalization with the II-III loop persisted when JPH divergent and TM domains were deleted but was lost when the α-helical domain was also removed. Swapping the α-helical domains between JPH3 and JPH4 led to a corresponding exchange in their ability to interact with the II-III loop distal segment. Thus, the α-helical domain appears necessary for JPH binding to the synprint-containing II-III loop half and for the differential binding of JPH3 and JPH4 to the loop distal half. Furthermore, the binding of JPH α-helical domain to the Ca_V2.1 II-III loop is essential for slowing Ca_V2.1 inactivation.