Insertion of the voltage-sensitive domain into circularly permuted red fluorescent protein as a design for genetically encoded voltage sensor.

Visualization of electrical activity in living cells represents an important challenge in context of basic neurophysiological studies. Here we report a new voltage sensitive fluorescent indicator which response could be detected by fluorescence monitoring in a single red channel. To the best of our knowledge, this is the first fluorescent protein-based voltage sensor which uses insertion-into-circular permutant topology to provide an efficient interaction between sensitive and reporter domains. Its fluorescent core originates from red fluorescent protein (FP) FusionRed, which has optimal spectral characteristics to be used in whole body imaging techniques. Indicators using the same domain topology could become a new perspective for the FP-based voltage sensors that are traditionally based on Förster resonance energy transfer (FRET).

Ca2+ dependent surface trafficking of norepinephrine transporters depends on threonine 30 and Ca2+ calmodulin kinases.

The antidepressant-sensitive norepinephrine (NE) transporter (NET) inactivates NE released during central and peripheral neuronal activity by transport into presynaptic cells. Altered NE clearance due to dysfunction of NET has been associated with the development of mental illness and cardiovascular diseases. NET activity in vivo is influenced by stress, neuronal activity, hormones and drugs. We investigated the mechanisms of Ca2+ regulation of NET and found that Ca2+ influenced both Vmax and Km for NE transport into cortical synaptosomes. Changes in extracellular Ca2+ triggered rapid and bidirectional surface trafficking of NET expressed in cultured cells. Deletion of residues 28-47 in the NET NH2-terminus abolished the Ca2+ effect on surface trafficking. Mutagenesis studies identified Thr30 in this region as the essential residue for both Ca2+- dependent phosphorylation and trafficking of NET. Depolarization of excitable cells increased surface NET in a Thr30 dependent manner. A proteomic analysis, RNA interference, and pharmacological inhibition supported roles of CaMKI and CaMKII in Ca2+-modulated NE transport and NET trafficking. Depolarization of primary noradrenergic neurons in culture with elevated K+ increased NET surface expression in a process that required external Ca2+ and depended on CaMK activity. Hippocampal NE clearance in vivo was also stimulated by depolarization, and inhibitors of CaMK signaling prevented this stimulation. In summary, Ca2+ signaling influenced surface trafficking of NET through a CaMK-dependent mechanism requiring Thr30.

Imaging Membrane Potential with Two Types of Genetically Encoded Fluorescent Voltage Sensors.

Genetically encoded voltage indicators (GEVIs) have improved to the point where they are beginning to be useful for in vivo recordings. While the ultimate goal is to image neuronal activity in vivo, one must be able to image activity of a single cell to ensure successful in vivo preparations. This procedure will describe how to image membrane potential in a single cell to provide a foundation to eventually image in vivo. Here we describe methods for imaging GEVIs consisting of a voltage-sensing domain fused to either a single fluorescent protein (FP) or two fluorescent proteins capable of Förster resonance energy transfer (FRET) in vitro. Using an image splitter enables the projection of images created by two different wavelengths onto the same charge-coupled device (CCD) camera simultaneously. The image splitter positions a second filter cube in the light path. This second filter cube consists of a dichroic and two emission filters to separate the donor and acceptor fluorescent wavelengths depending on the FPs of the GEVI. This setup enables the simultaneous recording of both the acceptor and donor fluorescent partners while the membrane potential is manipulated via whole cell patch clamp configuration. When using a GEVI consisting of a single FP, the second filter cube can be removed allowing the mirrors in the image splitter to project a single image onto the CCD camera.

Developing Fast Fluorescent Protein Voltage Sensors by Optimizing FRET Interactions.

FRET (Förster Resonance Energy Transfer)-based protein voltage sensors can be useful for monitoring neuronal activity in vivo because the ratio of signals between the donor and acceptor pair reduces common sources of noise such as heart beat artifacts. We improved the performance of FRET based genetically encoded Fluorescent Protein (FP) voltage sensors by optimizing the location of donor and acceptor FPs flanking the voltage sensitive domain of the Ciona intestinalis voltage sensitive phosphatase. First, we created 39 different "Nabi1" constructs by positioning the donor FP, UKG, at 8 different locations downstream of the voltage-sensing domain and the acceptor FP, mKO, at 6 positions upstream. Several of these combinations resulted in large voltage dependent signals and relatively fast kinetics. Nabi1 probes responded with signal size up to 11% ΔF/F for a 100 mV depolarization and fast response time constants both for signal activation (~2 ms) and signal decay (~3 ms). We improved expression in neuronal cells by replacing the mKO and UKG FRET pair with Clover (donor FP) and mRuby2 (acceptor FP) to create Nabi2 probes. Nabi2 probes also had large signals and relatively fast time constants in HEK293 cells. In primary neuronal culture, a Nabi2 probe was able to differentiate individual action potentials at 45 Hz.

Genetically Encoded Protein Sensors of Membrane Potential.

Organic voltage-sensitive dyes offer very high spatial and temporal resolution for imaging neuronal function. However these dyes suffer from the drawbacks of non-specificity of cell staining and low accessibility of the dye to some cell types. Further progress in imaging activity is expected from the development of genetically encoded fluorescent sensors of membrane potential. Cell type specificity of expression of these fluorescent protein (FP) voltage sensors can be obtained via several different mechanisms. One is cell type specificity of infection by individual virus subtypes. A second mechanism is specificity of promoter expression in individual cell types. A third, depends on the offspring of transgenic animals with cell type specific expression of cre recombinase mated with an animal that has the DNA for the FP voltage sensor in all of its cells but its expression is dependent on the recombinase activity. Challenges remain. First, the response time constants of many of the new FP voltage sensors are slower (2-10 ms) than those of organic dyes. This results in a relatively small fractional fluorescence change, ΔF/F, for action potentials. Second, the largest signal presently available is only ~40% for a 100 mV depolarization and many of the new probes have signals that are substantially smaller. Large signals are especially important when attempting to detect fast events because the shorter measurement interval results in a relatively small number of detected photons and therefore a relatively large shot noise (see Chap. 1). Another kind of challenge has occurred when attempts were made to transition from one species to another or from one cell type to another or from cell culture to in vivo measurements.Several laboratories have recently described a number of novel FP voltage sensors. Here we attempt to critically review the current status of these developments in terms of signal size, time course, and in vivo function.

Monitoring brain activity with protein voltage and calcium sensors.

Understanding the roles of different cell types in the behaviors generated by neural circuits requires protein indicators that report neural activity with high spatio-temporal resolution. Genetically encoded fluorescent protein (FP) voltage sensors, which optically report the electrical activity in distinct cell populations, are, in principle, ideal candidates. Here we demonstrate that the FP voltage sensor ArcLight reports odor-evoked electrical activity in the in vivo mammalian olfactory bulb in single trials using both wide-field and 2-photon imaging. ArcLight resolved fast odorant-responses in individual glomeruli, and distributed odorant responses across a population of glomeruli. Comparisons between ArcLight and the protein calcium sensors GCaMP3 and GCaMP6f revealed that ArcLight had faster temporal kinetics that more clearly distinguished activity elicited by individual odorant inspirations. In contrast, the signals from both GCaMPs were a saturating integral of activity that returned relatively slowly to the baseline. ArcLight enables optical electrophysiology of mammalian neuronal population activity in vivo.

Calcium-dependent interactions of the human norepinephrine transporter with syntaxin 1A.

The norepinephrine (NE) transporter (NET) terminates noradrenergic signaling by clearing released NE at synapses. The activity of NET can be rapidly regulated by depolarization and receptor activation via Ca2+ and kinase/phosphatase-linked pathways. The SNARE protein syntaxin 1A (SYN1A) interacts with NET and influences transporter surface trafficking and catalytic activity. In this study, we establish a link between changes in intracellular Ca2+ and SYN1A/NET interactions. SYN1A influenced NE transport only in the presence of Ca2+ in brain cortical synaptosomes. Although NET/SYN1A associations were sensitive to manipulations of Ca2+ in CHO cells, in vitro binding experiments using purified NET and SYN1A fusion proteins demonstrated a lack of direct Ca2+ sensitivity. Disruption of NET/SYN1A interaction abolished inhibition of NE transport by phorbol ester (PMA) to activate protein kinase C (PKC), but had no effect on transport inhibition by the Ca2+ calmodulin kinase (CaMK) inhibitor KN93. Furthermore, PMA enhanced Ca2+-dependent modulation of NE transport in synaptosomes. Our data reveal roles for SYN1A in the Ca2+-dependent regulation of NET, likely reliant on regulation by PKC signaling, but independent of CaMK.

Amphetamine induces a calcium/calmodulin-dependent protein kinase II-dependent reduction in norepinephrine transporter surface expression linked to changes in syntaxin 1A/transporter complexes.

Norepinephrine (NE) transporters (NETs) are high-affinity transport proteins that mediate the synaptic clearance of NE after vesicular release. NETs represent a major therapeutic target for antidepressants and are targets of multiple psychostimulants including amphetamine (AMPH) and cocaine. Recently, we demonstrated that syntaxin 1A (SYN1A) regulates NET surface expression and, through binding to the transporter's NH(2) terminus, regulates transporter catalytic function. AMPH induces NE efflux and may also regulate transporter trafficking. We monitored NET distribution and function in catecholaminergic cell lines (CAD) stably transfected with either full-length human NET (CAD-hNET) or with an hNET N-terminal deletion (CAD-hNETDelta(28-47) cells). In hNET-CAD cells, AMPH causes a slow and small reduction of surface hNET with a modest increase in hNET/SYN1A associations at the plasma membrane. In contrast, in CAD-hNETDelta(28-47) cells, AMPH induces a rapid and substantial reduction in surface hNETDelta(28-47) accompanied by a large increase in plasma membrane hNETDelta(28-47)/SYN1A complexes. We also found that AMPH in CAD-hNETDelta(28-47) cells induces a robust increase in cytosolic Ca2+ and concomitant activation of calcium/calmodulin-dependent protein kinase II (CaMKII). Inhibition of either the increase in intracellular Ca2+ or CaMKII activity blocks AMPH-stimulated hNETDelta(28-47) trafficking and the formation of hNETDelta(28-47)/SYN1A complexes. Here, we demonstrate that AMPH stimulation of CAMKII stabilizes an hNET/SYN1A complex. This hNET/SYN1A complex rapidly redistributes, upon AMPH treatment, when mechanisms supported by the transporter's NH2 terminus are eliminated.

Proteomic analysis of human norepinephrine transporter complexes reveals associations with protein phosphatase 2A anchoring subunit and 14-3-3 proteins.

The norepinephrine transporter (NET) terminates noradrenergic signals by clearing released NE at synapses. NET regulation by receptors and intracellular signaling pathways is supported by a growing list of associated proteins including syntaxin1A, protein phosphatase 2A (PP2A) catalytic subunit (PP2A-C), PICK1, and Hic-5. In the present study, we sought evidence for additional partnerships by mass spectrometry-based analysis of proteins co-immunoprecipitated with human NET (hNET) stably expressed in a mouse noradrenergic neuroblastoma cell line. Our initial proteomic analyses reveal multiple peptides derived from hNET, peptides arising from the mouse PP2A anchoring subunit (PP2A-Ar) and peptides derived from 14-3-3 proteins. We verified physical association of NET with PP2A-Ar via co-immunoprecipitation studies using mouse vas deferens extracts and with 14-3-3 via a fusion pull-down approach, implicating specifically the hNET NH2-terminus for interactions. The transporter complexes described likely support mechanisms regulating transporter activity, localization, and trafficking.

Cell surface trafficking of the antidepressant-sensitive norepinephrine transporter revealed with an ectodomain antibody.

The antidepressant-sensitive L-norepinephrine (NE) transporter (NET;SLC6A2) is a critical determinant of neurotransmitter inactivation following NE release at synapses. Although regulated trafficking of NET has been documented in transfected cells, a lack of reagents suitable for reporting native NET surface exposition has limited validation of this concept in neurons. In the current report, we document the utility of a novel antibody (43408) directed at conserved sequences in the NET second extracellular loop. Using human NET (hNET) stably transfected cells, we document loss of NET surface expression following acute (30 min) phorbol ester treatments. In superior cervical ganglion (SCG) cultures, NET surface expression is prominent on varicosities defined by FM1-43 labeling of living neurons or synaptophysin labeling of fixed preparations. Moreover, NET surface density can be rapidly augmented by brief depolarization (5 min, 40 mM K(+)). Similarly, in brainstem cultures, we demonstrate an increase in NET surface labeling following either depolarization or angiotensin II stimulation. These findings provide the first evidence for regulated trafficking of NET in neurons and support the suggestion that activity-dependent NET trafficking may provide additional modulatory capacity for noradrenergic signaling.

A regulated interaction of syntaxin 1A with the antidepressant-sensitive norepinephrine transporter establishes catecholamine clearance capacity.

Norepinephrine (NE) transporters (NETs) terminate noradrenergic synaptic transmission and represent a major therapeutic target for antidepressant medications. NETs and related transporters are under intrinsic regulation by receptor and kinase-linked pathways, and clarification of these pathways may suggest candidates for the development of novel therapeutic approaches. Syntaxin 1A, a presynaptic soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein, interacts with NET and modulates NET intrinsic activity. NETs colocalize with and bind to syntaxin 1A in both native preparations and heterologous systems. Protein kinase C activation disrupts surface NET/syntaxin 1A interactions and downregulates NET activity in a syntaxin-dependent manner. Syntaxin 1A binds the NH(2) terminal domain of NET, and a deletion of this domain both eliminates NET/syntaxin 1A associations and prevents phorbol ester-triggered NET downregulation. Whereas syntaxin 1A supports the surface trafficking of NET proteins, its direct interaction with NET limits transporter catalytic function. These two contradictory roles of syntaxin 1A on NET appear to be linked and reveal a dynamic cycle of interactions that allow for the coordinated control between NE release and reuptake.