Contraction of striated muscle is triggered by a massive release of calcium from the sarcoplasmic reticulum (SR) into the cytoplasm. This intracellular calcium release is initiated by membrane depolarization, which is sensed by voltage-gated calcium channels CaV1.1 (in skeletal muscle) and CaV1.2 (in cardiac muscle) in the plasma membrane (PM), which in turn activate the calcium-releasing channel ryanodine receptor (RyR) embedded in the SR membrane. This cross-communication between channels in the PM and in the SR happens at specialized regions, the SR-PM junctions, where these two compartments come in close proximity. Junctophilin1 and Junctophilin2 are responsible for the formation and stabilization of SR-PM junctions in striated muscle and actively participate in the recruitment of the two essential players in intracellular calcium release, CaV and RyR.
JPH1 knock-out mice die within 24 h after birth due to suckling defects leading to undernourishment. The suckling defect is likely due to muscle weakness since the neuronal suckling reflexes are normal in knock-out mice . Functional studies on isolated hindlimb muscle showed abnormal twitch tension and a greater dependency on extracellular calcium in KO mice muscles, suggesting that a significant fraction of RyR1s in the junctional SR are not directly coupled with the CaV1.1 channels in the T-tubules and therefore operate via calcium-induced calcium release. Nonetheless, knock-out (KO) mice are still relatively mobile and show skeletal muscle-type EC coupling to a certain degree, indicating that JPH2 can support voltage-induced Ca2+ release in the absence of JPH1. From a structural point of view, although no major disorganization of the fiber is noticed at the light microscopy level, evident alterations are noticeable at the ultrastructural level . In particular, the skeletal muscle of wt and JPH1 KO mice show a similar development in the embryonic stages until shortly after birth. At this age, wt muscle experiences a significant increase in JPH1 expression, which is temporally correlated with the transition from immature SR-PM junctions, mainly organized in dyads at this stage, into fully formed triads. This transition is absent in JPH1 KO muscle , suggesting that JPH2 is important in forming the dyads, while JPH1 has a crucial role in the conversion from dyads to triads in the fully mature skeletal muscle. The knocking down of junctophilins using sh-RNA, leads to the impairment of store-operated Ca2+ entry (SOCE), altered intracellular calcium release and intracellular calcium stores  and to a reduction in RyR1 and CaV1.1 co-clustering associated with a decrease in CaV1.1 membrane expression  both in myotubes and muscle fibers. In both these studies, a shRNA against both JPH1 and JPH2 was used; hence, it was impossible to distinguish each isoform’s relative contribution to the resulting phenotype.
Remarkable advancements in protein folding prediction were recently achieved by the artificial intelligence software Alphafold2 . Alphafold2 is a giant leap forward in the reliability of protein folding prediction compared to similar existing software , and it has already been used to predict the structure of nearly the entire human proteome. Based on Aplhafold2 prediction models, junctophilin 1 and 2 show a similar 3D structure, also shared by the neuronal isoforms JPH3 and JPH4 (UniProt protein ID: Q9HDC5, Q9BR39, Q8WXH2, Q96JJ6 for human JPH1, JPH2, JPH3 and JPH4, respectively). The structure of the most ordered domains of junctophilin, specifically the MORN repeats, the α-helical region and the transmembrane domain, are predicted with high confidence by Alphafold2. In contrast, the joining and divergent domains are likely disordered, at least in the isolated protein, and the structure cannot be predicted with reasonable confidence. According to the prediction model (Figure 3), the MORN domains are arranged in an extended “halfpipe” configuration, with the α-helical domain lying on the convex side of this half-pipe (Figure 3B,C) and establishing interactions with charged residues in the MORN domains (see the zoomed-in region in Figure 3C for an example).
Figure 3. Structure of the MORNs and α-helical domains of human junctophilin1 and junctophilin2.(A) Schematic representation of junctophilin domain as shown in Figure 2; the solid red lines indicate the regions for which the structure is predicted with high fidelity by Alphafold2 and illustrated in (B,C). (B,C) predicted structures of the MORNs- -helical domains of junctophilin1 (B) and junctophilin2 (C). The α-helical domain (in red) lies on the convex side of the MORN domains half-pipe structure (in blue) in both junctophilin1 and junctophilin2. β-sheet hairpins forming MORN domains I and VIII (green parentheses) and the position of the N-terminal end (N-t) and C-terminal end (C-t) of the joining domain (in pink), which is absent in this representation, are indicated in (B). The inset in (C) shows some of the residues that form the hydrogen bonds that stabilize the association between the MORN domains and the α-helical domain of junctophilin2.
The structure obtained using Alphafold2 is substantially different from what was previously predicted using RaptorX software by Gross and colleagues . In the structure described by Gross et al., the α-helical domain extends beyond the MORN domains without interacting with them at all. However, Alphafold2 software is considered to be more accurate than most (if not all) of the currently existing structure-predicting software, especially for proteins for which no homologous structures exist , and the reciprocal arrangement of JPH2 MORN motifs and α-helical domain predicted by Alphafold2 agrees with data from Li and collaborators  based on the crystal structure of the protein MORN4. MORN4 contains a series of MORN motifs arranged in a half-pipe configuration followed by a brief α-helical region. The helical region stabilizes the MORN domains by lying over part of the convex side of the half-pipe. The structure solved by Li and colleagues is in many ways very similar to the sequence predicted by Alphafold2 for JPHs.
Furthermore, in MORN 4, the concave side of the MORN half-pipe structure, containing most of the conserved residues that define the MORN domain, engages in the binding with the α-helical region of myosin3a. It is conceivable that the concave side of the junctophilin MORN motifs could also participate in protein–protein interactions with components of the EC coupling machinery. The particular arrangement of the α-helical domain with respect to the MORN motifs predicted by Alphafold2 and suggested by the observations of Li and colleagues challenges the classic view of the α-helical domain as the spacer that spans most of the junctional gap (see schematic representation in Figure 2) and points to the divergent domain as the region that most likely fulfills this role.
This entry is adapted from 10.3390/biom12010109