Molecular evolution of the junctophilin gene family

Abstract

Junctophilins (JPHs) are members of a junctional membrane complex protein family important for the physical approximation of plasmalemmal and sarcoplasmic/endoplasmic reticulum membranes. As such, JPHs facilitate signal transduction in excitable cells between plasmalemmal voltage-gated calcium channels and intracellular calcium release channels. To determine the molecular evolution of the JPH gene family, we performed a phylogenetic analysis of over 60 JPH genes from over 40 species and compared conservation across species and different isoforms. We found that JPHs are evolutionary highly conserved, in particular the membrane occupation and recognition nexus motifs found in all species. Our data suggest that an ancestral form of JPH arose at the latest in a common metazoan ancestor and that in vertebrates four isoforms arose, probably following two rounds of whole genome duplications. By combining multiple prediction techniques with sequence alignments, we also postulate the presence of new important functional regions and candidate sites for posttranslational modifications. The increasing number of available sequences yields significant insight into the molecular evolution of JPHs. Our analysis is consistent with the emerging concept that JPHs serve dual important functions in excitable cells: structural assembly of junctional membrane complexes and regulation of intracellular calcium signaling pathways.

Fig. 1.

Schematic representation of junctophilin (JPH). Model figure showing a JPH2 protein (red) spanning the junctional membrane domain between the plasma membrane (PM) and endo/sarcoplasmic reticulum (ER/SR). Functional domains within JPH are indicated. MORN, Membrane Occupation and Recognition Nexus; TT, transverse tubule; LTTC, L-type Ca²⁺ channel; RyR2, ryanodine receptor type 2. Mutations identified in patients with hypertrophic cardiomyopathy are indicated in green (7) and yellow (13).

Fig. 2.

JPH phylogeny tree. Phylogeny tree of 64 JPH isoforms representing 41 species generated using the maximum likelihood method, with the LG substitution model and BIONJ initial tree. Branch support is calculated using the approximate likelihood ratio test and shown for each branch out of 100. Isoforms are listed by the Latin name. The branch length for Hydra magnipapillata was truncated for clarity; the actual length is shown. *The first 150 amino acids Gallus gallus JPH1 were removed (see methods).

Fig. 3.

Conservation and secondary structure of JPH domains. Alignment of all 64 isoforms of JPH, showing location of reported domains, conservation, and gaps in alignment. Note the conserved region between the MORN motifs. TM, transmembrane.

Fig. 4.

Conservation of MORN motifs. Sequence logo from all 8 MORN motifs of all 64 isoforms combined, resulting in a total of 512 MORN motifs averaged. The extra amino acid (at position 7) in Drosophila and Hydra MORN motif 4 was excluded (see text). Hydrophobic residues are shown in black, hydrophilic in blue, and neutral in green (8).

Fig. 5.

Predicted secondary structure of human JPH isoforms. Blue, alpha helix; purple, random coil; red, extended strand.

Fig. 6.

Alignment of published JPH2 mutations associated with cardiomyopathy in patients. Four mutations in JPH2 associated with heart disease have been published. The mutation is highlighted by a black box, and the alignment of the mutated site is shown by the gray shading. S101R, Y141H, and S165F from Ref. ; G505S from Ref. .