William A. Sather

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.

Excitation-driven entry of Ca²⁺through L-type voltage-gated Ca²⁺channels controls gene expression in neurons and a variety of fundamental activities in other kinds of excitable cells. The probability of opening of Ca_V1.2 L-type channels is subject to pronounced enhancement by cAMP-dependent protein kinase (PKA), which is scaffolded to Ca_V1.2 channels by A-kinase anchoring proteins (AKAPs). Ca_V1.2 channels also undergo negative autoregulation via Ca²⁺-dependent inactivation (CDI), which strongly limits Ca²⁺entry. An abundance of evidence indicates that CDI relies upon binding of Ca²⁺/calmodulin (CaM) to an isoleucine–glutamine motif in the carboxy tail of Ca_V1.2 L-type channels, a molecular mechanism seemingly unrelated to phosphorylation-mediated channel enhancement. But our work reveals, in cultured hippocampal neurons and a heterologous expression system, that the Ca²⁺/CaM-activated phosphatase calcineurin (CaN) is scaffolded to Ca_V1.2 channels by the neuronal anchoring protein AKAP79/150, and that overexpression of an AKAP79/150 mutant incapable of binding CaN (ΔPIX; CaN-binding PXIXIT motif deleted) impedes CDI. Interventions that suppress CaN activity—mutation in its catalytic site, antagonism with cyclosporine A or FK506, or intracellular perfusion with a peptide mimicking the sequence of the phosphatase's autoinhibitory domain—interfere with normal CDI. In cultured hippocampal neurons from a ΔPIX knock-in mouse, CDI is absent. Results of experiments with the adenylyl cyclase stimulator forskolin and with the PKA inhibitor PKI suggest that Ca²⁺/CaM-activated CaN promotes CDI by reversing channel enhancement effectuated by kinases such as PKA. Hence, our investigation of AKAP79/150-anchored CaN reconciles the CaM-based model of CDI with an earlier, seemingly contradictory model based on dephosphorylation signaling.