Interactions between Neuronal Junctophilins and Voltage Gated Ion Channels

Junctions between the endoplasmic reticulum (ER) and plasma membrane (PM) occur in diverse cell types. In many of these junctions, calcium efflux from the ER occurs via ryanodine receptors or IP3 receptors, triggered by voltage-gated ion channels or ligand receptors in the PM. Junctophilins (JPs) have been identified as proteins that cause formation of ER-PM junctions in muscle (JP1, JP2) and neurons (JP3, JP4). Knockout of JP1 or JP2 is perinatal or embryonic lethal. Single knockout of neuronal JP produces only a mild (JP3) or no (JP4) phenotype; knockout of both causes hippocampal abnormalities, irregular hindlimb reflexes and impaired memory. Here, we have examined the ability of neuronal junctophilins to cause voltage-gated channels to localize within junctional domains of the PM by co-expressing, in tsA201 cells, voltage-gated channels and JP3 or JP4 tagged with different fluorescent proteins. In the absence of JPs, the L-type channel CaV1.2 had a relatively uniform distribution, while P/Q-type (CaV2.1), N-type (CaV2.2) and T-type (CaV3.1) channels were present in the surface at a low density making them difficult to visualize. Both JP3 and JP4 caused CaV1.2, CaV2.1 and CaV2.2 to cluster at sites co-localized with the junctophilin. Such clusters were not observed for CaV3.1. KV2.1 clustered without junctophilin at sites previously shown to represent PM junctions with the ER. When co-expressed, JP4 co-localized with the KV2.1 clusters, whereas JP3 did not, raising the possibility that the two junctophilins have distinct roles. Overall our results show that junctophilins are not just structural proteins necessary for ER-PM formation, but that they actively recruit neuronal channels to spatially restricted PM domains. Supported by: NIH grants AR070298 to KGB and GM109888 to MM Tamkun (who provided GFP-KV2.1). HM Colecraft provided CaV3.1-YFP.