Interactions of Junctophilins and STIM1 with ER Calcium-Releasing Channels

Abstract

Junctions between the endoplasmic reticulum (ER) and plasma membrane (PM) are found in many cell types, where they mediate local Ca2+ signaling by recruiting a variety of ER and PM channels to the two sides of these junctions. Stim1 and Junctophilins (JPs) are among the major proteins responsible for forming junctions. Stim1 organizes mostly transient junctions in response to ER Ca2+ depletion and is essential for store-operated Ca2+ entry. Junctophilins 1 and 2 coordinate the organization of the calcium releasing units in striated muscle, but the role of the neuronal junctophilins, JP3 and JP4, is less understood. Because studies on double knock-out mice indicated the importance of JP3/JP4 for brain function, we are using expression in tsA201 cells to compare JP3, JP4 and Stim1. Previously, we showed that JP3 and JP4 were both able to recruit the PM channels CaV1.2, CaV2.1 and CaV2.2 to junctions, and that JP3, but not JP4, could recruit the ER channels RyR1 and RyR2. Here we show that RyR3 is recruited to junctions by JP3, and also by JP4 (less efficiently than JP3). RyR1 and RyR3 constructs lacking ER transmembrane domains co-localized with JP3, indicative of an interaction which likely contributes to the ability of JP3 to recruit the full-length proteins to junctions. By contrast, RyR3 lacking transmembrane domains did not co-localize with JP4. Co-localization also did not occur between IP3R1 and either JPs. On the other hand, constitutively active Stim1D76A displayed moderate co-localization with IP3R1 but none with the RyRs. These results suggest that neuronal ER-PM junctions formed by Stim1, JP3 and JP4 likely harbor different sets of channels and that the JP3/JP4 ratio in junctophilin-formed junctions might modulate the composition of such microdomains with possible functional implications.