Zn 2+ plays essential roles in biology, and cells have adopted exquisite mechanisms for regulating steady-state Zn 2+ levels. Although much is known about total Zn 2+ in cells, very little is known about its subcellular distribution. Yet defining the location of Zn 2+ and how it changes with signaling events is essential for elucidating how cells regulate this essential ion. Here we create fluorescent sensors genetically targeted to the endoplasmic reticulum (ER) and Golgi to monitor steady-state Zn 2+ levels as well as flux of Zn 2+ into and out of these organelles. These studies reveal that ER and Golgi contain a concentration of free Zn 2+ that is 100 times lower than the cytosol. Both organelles take up Zn 2+ when cytosolic levels are elevated, suggesting that the ER and Golgi can sequester elevated cytosolic Zn 2+ and thus have the potential to play a role in influencing Zn 2+ toxicity. ER Zn 2+ homeostasis is perturbed by small molecule antagonists of Ca 2+ homeostasis and ER Zn 2+ is released upon elevation of cytosolic Ca 2+ pointing to potential exchange of these two ions across the ER. This study provides direct evidence that Ca 2+ signaling can influence Zn 2+ homeostasis and vice versa, that Zn 2+ dynamics may modulate Ca 2+ signaling.

Transition metals are essential enzyme cofactors that are required for a wide range of cellular processes. Paradoxically, whereas metal ions are essential for numerous cellular processes, they are also toxic. Therefore cells must tightly regulate metal accumulation, transport, distribution, and export. Improved tools to interrogate metal ion availability and spatial distribution within living cells would greatly advance our understanding of cellular metal homeostasis. In this work, we present genetically encoded sensors for Zn 2؉ based on the principle of fluorescence resonance energy transfer. We also develop methodology to calibrate the probes within the cellular environment. To identify both sources of and sinks for Zn 2؉ , these sensors are genetically targeted to specific locations within the cell, including cytosol, plasma membrane, and mitochondria. Localized probes reveal that mitochondria contain an elevated pool of Zn 2؉ under resting conditions that can be released into the cytosol upon glutamate stimulation of hippocampal neurons. We also observed that Zn 2؉ is taken up into mitochondria following glutamate/Zn 2؉ treatment and that there is heterogeneity in both the magnitude and kinetics of the response. Our results suggest that mitochondria serve as a source of and a sink for Zn 2؉ signals under different cellular conditions.Although mammalian cells are known to concentrate transition metals, it is now well established that under resting conditions, "free" (e.g. unbound) metals are maintained at extremely low levels. Estimates of the total Zn 2ϩ concentration in mammalian cells typically range from 100 to 500 M (1); yet free Zn 2ϩ concentrations are tightly buffered by proteins such as metallothionein to maintain cytosolic Zn 2ϩ concentrations in the picomolar to nanomolar range (2-5). However, there is emerging evidence that this static picture is dramatically altered by different cellular conditions, such as redox perturbations caused by oxidative stress (6, 7) and cellular signals such as nitric oxide (8 as mitochondrial function (7, 9, 10). Elucidation of the sources and dynamics of these Zn 2ϩ signals would greatly advance our understanding of the interplay between metal regulation and cellular function.There has been a huge effort in the past few years to develop sensitive and selective fluorescent probes to monitor Zn 2ϩ in biological systems. The majority of this work has focused on the generation of small molecule fluorescent indicators (reviewed by Que et al. (11)). Yet there are also examples of sensors based partially on Zn 2ϩ -binding proteins, such as carbonic anhydrase (12) and metallothionein (13), and peptide scaffolds (14). Although many of these sensors have begun to provide insight into Zn 2ϩ concentrations within cells, one limitation is that it is challenging to explicitly target them to subdomains within the cell. Localized probes are necessary to generate a complete picture of cellular Zn 2ϩ homeostasis in mammalian cells. For this reason, sensors that are genetically encode...

SUMMARY Potentiation of synaptic strength relies on postsynaptic Ca2+ signals, modification of dendritic spine structure and changes in gene expression. One Ca2+ signaling pathway supporting these processes routes through L-type Ca2+ channels (LTCC), whose activity is subject to tuning by multiple mechanisms. Here we show in hippocampal neurons that LTCC inhibition by the endoplasmic reticulum (ER) Ca2+ sensor, stromal interaction molecule 1 (STIM1), is engaged by the neurotransmitter glutamate, resulting in regulation of spine ER structure and nuclear signaling by the NFATc3 transcription factor. In this mechanism, depolarization by glutamate activates LTCC Ca2+ influx, releases Ca2+ from the ER and consequently drives STIM1 aggregation and an inhibitory interaction with LTCCs that increases spine ER content but decreases NFATc3 nuclear translocation. These findings of negative feedback control of LTCC signaling by STIM1 reveal interplay between Ca2+ influx and release from stores that controls both postsynaptic structural plasticity and downstream nuclear signaling.

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 CaV1.2 L-type channels is subject to pronounced enhancement by cAMP-dependent protein kinase (PKA), which is scaffolded to CaV1.2 channels by A-kinase anchoring proteins (AKAPs). CaV1.2 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 IQ motif in the carboxy tail of CaV1.2 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 CaV1.2 channels by the neuronal anchoring protein AKAP79/150 and that over-expression of an AKAP79/150 mutant incapable of binding CaN (ΔPIX) 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.

SUMMARY Long-term information storage in the brain requires continual modification of the neuronal transcriptome. Synaptic inputs located hundreds of micrometers from the nucleus can regulate gene transcription, requiring high-fidelity, long-range signaling from synapses in dendrites to the nucleus in the cell soma. Here, we describe a synapse-to-nucleus signaling mechanism for the activity-dependent transcription factor NFAT. NMDA receptors activated on distal dendrites were found to initiate L-type Ca 2+ channel (LTCC) spikes that quickly propagated the length of the dendrite to the soma. Surprisingly, LTCC propagation did not require voltage-gated Na + channels or back-propagating action potentials. NFAT nuclear recruitment and transcriptional activation only occurred when LTCC spikes invaded the somatic compartment, and the degree of NFAT activation correlated with the number of somatic LTCC Ca 2+ spikes. Together, these data support a model for synapse to nucleus communication where NFAT integrates somatic LTCC Ca 2+ spikes to alter transcription during periods of heightened neuronal activity.