Role of Neuronal Junctophilins in Recruitment and Modulation of Voltage-Gated Calcium Channels in PM-ER Junctions

Abstract

Junctions between the plasma membrane (PM) and the endoplasmic reticulum (ER), which have been observed in many cell types, are subcellular compartments in which multiple channels, both in the PM (ligand or voltage-gated channels) and in the ER (ryanodine receptors and IP3 receptors) cross-talk with one other and regulate local calcium dynamics. Junctophilins (JPs) were among the first identified proteins to be involved in the formation and stabilization of PM-ER junctions, principally in muscle and neurons. While the crucial role in cardiac and skeletal muscle contractility of the two muscle isoforms JP1 and JP2 is demonstrated by the severity of the KO phenotypes (perinatal and embryonic lethality respectively), the roles of the neuronal junctophilins (JP3 and JP4) appear to be more subtle and less well understood. While knock-outs for JP3 or JP4 exhibit only mild or no phenotype, double knock-outs show motor discoordination, impaired motor learning and impaired memory, suggesting multiple roles of JPs in the nervous system. Here we tried to clarify how voltage-gated calcium channels localize and function in neuronal PM-ER junctions by co-expressing some of these channels with the two JPs, in tsA201 cells and using confocal microscopy and electrophysiology to assess their interaction. Our results show that JP3 and JP4 selectively recruited L-type, P/Q-type and N-type, but not T-type, channels to PM-ER junctions and that JP3, and to a greater extent JP4, substantially slowed inactivation of P/Q and N-type channels. A similar effect on channel activity was observed using JPs in which deletion of the C-terminal, ER-anchoring transmembrane domain disrupts their ability to form clusters. This indicates that channel activity is regulated by direct interaction with JPs and not as a result of channel clustering in the PM-ER junction.