Neuronal Junctophilin 3 Can Replace Muscle Junctophilin 2 in Voltage-induced Calcium Release

Abstract

We recently showed that skeletal-muscle, voltage-induced calcium release could be recapitulated in tsA201 cells by expression of 5 muscle junctional proteins: CaV1.1, CaVβ1a, Stac3, RyR1 and Junctophilin-2 (JP2). In tsA201 cells, JP2 induced junctions at which CaV1.1, CaVβ1a, Stac3 accumulated on the PM side, and RyR1 accumulated on the ER side. To determine whether isoform-specific properties of JP2 are required for voltage-gated Ca2+ release, we have now replaced JP2 with the neuronal Junctophilin-3 (JP3). We found that JP3 is more effective at recruiting RyR1 to junctions than JP2, either when the JP's are expressed alone or when CaV1.1, CaVβ1a and Stac3 are also present, which increased RyR1 recruitment for both junctophilins. Strikingly, a truncated, cytoplasmically diffusible RyR1 (RyR11:4300), lacking the ER transmembrane domain, strongly co-localizes with JP3 alone, but very poorly with JP2 alone. CaV1.1, CaVβ1a and Stac3 co-expression confers on JP2 the ability to recruit RyR11:4300 and further increases the recruitment of RyR11:4300 by JP3. We next tested whether JP3 could not only recruit the other junctional proteins but functionally replace JP2. Freeze-fracture electron microscopy of cells expressing JP3 and the other four proteins revealed the presence of CaV1.1 arranged in tetrads, indicating mechanical links between CaV1.1 and RyR1. Moreover, depolarization elicited Ca2+ transients not requiring extracellular Ca2+ entry (as in skeletal muscle). However, the transients for JP3 were slightly smaller and slower than those for JP2. Our results indicate that the major role of JP2 in striated muscle excitation-contraction coupling is to form and stabilize ER-PM junctions and to recruit the necessary proteins to these junctions. Voltage-dependent Ca2+ release does not require the presence of a muscle Junctophilin, because such release still occurs, albeit less efficiently, with the neuronal JP3. Supported by NIH-AR070298.