Phillip J. Dittmer

Learning and memory require coordinated structural and functional plasticity at neuronal glutamatergic synapses located on dendritic spines. Here, we investigated how the endoplasmic reticulum (ER) controls postsynaptic Ca ²⁺ signaling and long-term potentiation of dendritic spine size, i.e., sLTP that accompanies functional strengthening of glutamatergic synaptic transmission. In most ER-containing (ER+) spines, high-frequency optical glutamate uncaging (HFGU) induced long-lasting sLTP that was accompanied by a persistent increase in spine ER content downstream of a signaling cascade engaged by N-methyl-D-aspartate receptors (NMDARs), L-type Ca ²⁺ channels (LTCCs), and Orai1 channels, the latter being activated by stromal interaction molecule 1 (STIM1) in response to ER Ca ²⁺ release. In contrast, HFGU stimulation of ER-lacking (ER−) spines expressed only transient sLTP and exhibited weaker Ca ²⁺ signals noticeably lacking Orai1 and ER contributions. Consistent with spine ER regulating structural metaplasticity, delivery of a second stimulus to ER− spines induced ER recruitment along with persistent sLTP, whereas ER+ spines showed no additional increases in size or ER content in response to sequential stimulation. Surprisingly, the physical interaction between STIM1 and Orai1 induced by ER Ca ²⁺ release, but not the resulting Ca ²⁺ entry through Orai1 channels, proved necessary for the persistent increases in both spine size and ER content required for expression of long-lasting late sLTP.

In neurons, regulation of activity-dependent transcription by the nuclear factor of activated T-cells (NFAT) depends upon Ca²⁺influx through voltage-gated L-type calcium channels (LTCC) and NFAT translocation to the nucleus following its dephosphorylation by the Ca²⁺-dependent phosphatase calcineurin (CaN). CaN is recruited to the channel by A-kinase anchoring protein (AKAP) 79/150, which binds to the LTCC C-terminus via a modified leucine-zipper (LZ) interaction. Here we sought to gain new insights into how LTCCs and signaling to NFAT are regulated by this LZ interaction. RNA interference–mediated knockdown of endogenous AKAP150 and replacement with human AKAP79 lacking its C-terminal LZ domain resulted in loss of depolarization-stimulated NFAT signaling in rat hippocampal neurons. However, the LZ mutation had little impact on the AKAP–LTCC interaction or LTCC function, as measured by Förster resonance energy transfer, Ca²⁺imaging, and electrophysiological recordings. AKAP79 and NFAT coimmunoprecipitated when coexpressed in heterologous cells, and the LZ mutation disrupted this association. Critically, measurements of NFAT mobility in neurons employing fluorescence recovery after photobleaching and fluorescence correlation spectroscopy provided further evidence for an AKAP79 LZ interaction with NFAT. These findings suggest that the AKAP79/150 LZ motif functions to recruit NFAT to the LTCC signaling complex to promote its activation by AKAP-anchored calcineurin.

Significance: At synaptic contacts between neurons, memory is supported by long-lasting potentiation of synaptic transmission and structural modification of the postsynaptic element, the dendritic spine. Such modification of function and structure occurs in response to strong synaptic stimulation. Here we show that this kind of stimulation also rapidly modifies Ca ²⁺ signaling in a narrow zone of dendrite centered at the dendrite’s junction with its stimulated spine. This zone is ±10–20 µm in extent along the dendrite, and we show that its length can be increased by activation of neuromodulatory β adrenergic receptors and protein kinase A signaling. β-adrenergic tuning of the length of this zone regulates the range along the dendrite over which crosstalk between stimulated spines can occur.

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.