Significance: Voltage-gated calcium channels are essential for diverse cellular functions. For example, Ca _V 1.1 channels trigger skeletal muscle contraction and Ca _V 1.2 channels regulate neural gene expression in response to neuronal activity. Thus, it is important to understand the cellular mechanisms that regulate delivery of these channels to the plasma membrane and that govern calcium movements via the membrane-inserted channels. Here we show that the cellular adapter protein “Stac3” participates in both processes. Specifically, Stac3 binds to both Ca _V 1.1 and Ca _V 1.2. This binding is essential for efficient delivery of Ca _V 1.1 to the plasma membrane, but not for Ca _V 1.2. However, binding of Stac3, or the related protein Stac2, to Ca _V 1.2 causes a dramatic slowing of inactivation, thereby increasing calcium entry via Ca _V 1.2.

Significance: Almost all cell types rely on calcium signals to maintain homeostasis and trigger specific cell responses. In eukaryotic cells, store-operated Ca ²⁺ entry (SOCE) is one of the mechanisms used to ensure control over cytosolic Ca ²⁺ signaling and internal Ca ²⁺ stores. Mutations in either Orai1 or stromal interaction molecule 1 (STIM1) lead to lethal severe combined immune deficiencies. Using a morphological approach with transmission and freeze–fracture electron microscopy, we describe STIM1-Orai1 interactions and visualize the distribution of individual Orai1 channels on the cell surface. This approach confirms STIM1-Orai1 interaction at specialized endoplasmic reticulum (ER)–plasma membrane junctions following ER depletion and provides new insight on STIM1-Orai1 stoichiometry.

Stac protein (named for its SH3- and cysteine-rich domains) was first identified in brain 20 years ago and is currently known to have three isoforms. Stac2, Stac1, and Stac3 transcripts are found at high, modest, and very low levels, respectively, in the cerebellum and forebrain, but their neuronal functions have been little investigated. Here, we tested the effects of Stac proteins on neuronal, high-voltage-activated Ca²⁺channels. Overexpression of the three Stac isoforms eliminated Ca²⁺-dependent inactivation (CDI) ofl-type current in rat neonatal hippocampal neurons (sex unknown), but not CDI of non-l-type current. Using heterologous expression in tsA201 cells (together with β and α₂-δ₁auxiliary subunits), we found that CDI for Ca_V1.2 and Ca_V1.3 (the predominant, neuronall-type Ca²⁺channels) was suppressed by all three Stac isoforms, whereas CDI for the P/Q channel, Ca_V2.1, was not. For Ca_V1.2, the inhibition of CDI by the Stac proteins appeared to involve their direct interaction with the channel's C terminus. Within the Stac proteins, a weakly conserved segment containing ∼100 residues and linking the structurally conserved PKC C1 and SH3_1 domains was sufficient to fully suppress CDI. The presence of CDI forl-type current in control neonatal neurons raised the possibility that endogenous Stac levels are low in these neurons and Western blotting indicated that the expression of Stac2 was substantially increased in adult forebrain and cerebellum compared with neonate. Together, our results indicate that one likely function of neuronal Stac proteins is to tune Ca²⁺entry via neuronall-type channels.SIGNIFICANCE STATEMENTStac protein, first identified 20 years ago in brain, has recently been found to be essential for proper trafficking and function of the skeletal musclel-type Ca2+ channel and is the site of mutations causing a severe, inherited human myopathy. In neurons, however, functions for Stac protein have remained unexplored. Here, we report that one likely function of neuronal Stac proteins is tuning Ca2+ entry vial-type, but not that via non-l-type, Ca2+ channels. Moreover, there is a large postnatal increase in protein levels of the major neuronal isoform (Stac2) in forebrain and cerebellum, which could provide developmental regulation ofl-type channel Ca2+ signaling in these brain regions.

Calsequestrin (CASQ) is the major component of the sarcoplasmic reticulum (SR) lumen in skeletal and cardiac muscles. This calcium-binding protein localizes to the junctional SR (jSR) cisternae, where it is responsible for the storage of large amounts of Ca²⁺, whereas it is usually absent, at least in its polymerized form, in the free SR. The retention of CASQ inside the jSR is due partly to its association with other jSR proteins, such as junctin and triadin, and partly to its ability to polymerize, in a high Ca²⁺ environment, into an intricate gel that holds the protein in place. In this work, we shed some light on the still poorly described in situ structure of polymerized CASQ using detailed EM images from thin sections, with and without tilting, and from deep-etched rotary-shadowed replicas. The latter directly illustrate the fundamental network nature of polymerized CASQ, revealing repeated nodal points connecting short segments of the linear polymer.

Junctions between the endoplasmic reticulum and plasma membrane that are induced by the neuronal junctophilins are of demonstrated importance, but their molecular architecture is still poorly understood and challenging to address in neurons. This is due to the small size of the junctions and the multiple isoforms of candidate junctional proteins in different brain areas. Using colocalization of tagged proteins expressed in tsA201 cells, and electrophysiology, we compared the interactions of JPH3 and JPH4 with different calcium channels. We found that JPH3 and JPH4 caused junctional accumulation of all the tested high-voltage-activated Ca_V isoforms, but not a low-voltage-activated Ca_V. Also, JPH3 and JPH4 noticeably modify Ca_V2.1 and Ca_V2.2 inactivation rate. RyR3 moderately colocalized at junctions with JPH4, whereas RyR1 and RyR2 did not. By contrast, RyR1 and RyR3 strongly colocalized with JPH3, and RyR2 moderately. Likely contributing to this difference, JPH3 binds to cytoplasmic domain constructs of RyR1 and RyR3, but not of RyR2.