Sequence conservation of JPH1, JPH3, and JPH4 with JPH2 and comparison of the ability of the four JPH isoforms to recruit Ca_V1.1 into ER–PM junctions. (A) Schematic representation of the domain architecture of JPHs, including the MORN domains, responsible for the association with the PM and the C-terminal TM domain that embeds these proteins in the ER membrane. The numbers beneath the schematic indicate the percentage of identical residues for each domain between JPH1 and JPH2 (green), JPH3 and JPH2 (red), and JPH4 and JPH2 (purple). A detailed sequence alignment is illustrated in Fig. 5. (B) Mid-level confocal optical sections of tsA201 cells transfected with cDNAs for YFP-Ca_V1.1 (green), β1a, and Stac3. A substantial amount of the YFP fluorescence appears to be ER associated in the cell interior, and for the cells illustrated in the left and center panels, there is no clear association of YFP with the cell surface. In other cells (right panel), there was a fairly continuous band of YFP-Ca_V1.1 closely associated with the cell surface. (C) Mid-level optical sections of tsA201 cells transfected with cDNAs for YFP-Ca_V1.1 (green) and mCherry (mCh)-tagged JPH1, JPH2, JPH3, and JPH4 (red). cDNAs for β1a and Stac3 were also transfected. Colocalization, visible as yellow areas in the rightmost merged images, is primarily found in discrete spots (some indicated by arrowheads) at the cell’s periphery, presumptive ER–PM junctions. The total number of cells analyzed for each condition was no JPH, 14 from one transfection; JPH1, 16 from one transfection; JPH2, 36 from two transfections; JPH3, 37 from two transfections; and JPH4, 17 from one transfection. Scale bars, 5 μm.