In skeletal muscle, depolarization of the plasma membrane (PM) causes conformational changes of the calcium channel CaV1.1, which then activate RYR1 to release calcium from the sarcoplasmic reticulum (SR). Because it does not require extracellular calcium entry, this process is termed voltage-induced calcium release. In skeletal muscle, junctophilins (JPH) 1 and 2 are responsible for forming the SR–PM junctions at which voltage-induced calcium release occurs; structurally similar junctions with different molecular constituents are formed in neurons by JPH3 and JPH4. Studies on mice models demonstrated that JPH1 knockout mice can still perform voltage-induced calcium release, although the complementary approach to verify whether JPH1 alone also supports this release is not easily practicable due to the embryonic lethality of JPH2 knockout mice. In a previous work, we showed that voltage-induced calcium release could be recapitulated in HEK293-derived cells transfected with cDNAs for JPH2 and CaV1.1, β1a, Stac3, and RYR1. Here, we used this reconstitutional approach to test whether JPH1 and the more distantly related JPH3 and JPH4 can also support voltage-induced calcium release in HEK293-derived cells. Our data show that all the four isoforms colocalize with CaV1.1 at ER–PM junctions and that JPH1, JPH2, and JPH3, but not JPH4, cause colocalization of RYR1 with CaV1.1 at the junctions. To test for function, potassium depolarization was applied to cells in which WT CaV1.1 was replaced with the calcium impermeant mutant CaV1.1(N617D) to eliminate extracellular calcium entry. Calcium transients were observed in cells expressing JPH1, JPH2, and JPH3, indicating that these isoforms support voltage-induced calcium release, but not in cells expressing JPH4. Thus, the JPHs seem to act primarily to (1) form ER–PM junctions and (2) recruit the required set of signaling proteins to these junctions; voltage-induced calcium release can be supported by any JPH isoform fulfilling both of these functions.

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