Cardiac Kir2.1 and NaV1.5 Channels Traffic Together to the Sarcolemma to Control Excitability.

RATIONALE: In cardiomyocytes, NaV1.5 and Kir2.1 channels interact dynamically as part of membrane bound macromolecular complexes. OBJECTIVE: The objective of this study was to test whether NaV1.5 and Kir2.1 preassemble during early forward trafficking and travel together to common membrane microdomains. METHODS AND RESULTS: In patch-clamp experiments, coexpression of trafficking-deficient mutants Kir2.1Δ314-315 or Kir2.1R44A/R46A with wild-type (WT) NaV1.5WT in heterologous cells reduced inward sodium current compared with NaV1.5WT alone or coexpressed with Kir2.1WT. In cell surface biotinylation experiments, expression of Kir2.1Δ314-315 reduced NaV1.5 channel surface expression. Glycosylation analysis suggested that NaV1.5WT and Kir2.1WT channels associate early in their biosynthetic pathway, and fluorescence recovery after photobleaching experiments demonstrated that coexpression with Kir2.1 increased cytoplasmic mobility of NaV1.5WT, and vice versa, whereas coexpression with Kir2.1Δ314-315 reduced mobility of both channels. Viral gene transfer of Kir2.1Δ314-315 in adult rat ventricular myocytes and human induced pluripotent stem cell-derived cardiomyocytes reduced inward rectifier potassium current and inward sodium current, maximum diastolic potential and action potential depolarization rate, and increased action potential duration. On immunostaining, the AP1 (adaptor protein complex 1) colocalized with NaV1.5WT and Kir2.1WT within areas corresponding to t-tubules and intercalated discs. Like Kir2.1WT, NaV1.5WT coimmunoprecipitated with AP1. Site-directed mutagenesis revealed that NaV1.5WT channels interact with AP1 through the NaV1.5Y1810 residue, suggesting that, like for Kir2.1WT, AP1 can mark NaV1.5 channels for incorporation into clathrin-coated vesicles at the trans-Golgi. Silencing the AP1 ϒ-adaptin subunit in human induced pluripotent stem cell-derived cardiomyocytes reduced inward rectifier potassium current, inward sodium current, and maximum diastolic potential and impaired rate-dependent action potential duration adaptation. CONCLUSIONS: The NaV1.5-Kir2.1 macromolecular complex pre-assembles early in the forward trafficking pathway. Therefore, disruption of Kir2.1 trafficking in cardiomyocytes affects trafficking of NaV1.5, which may have important implications in the mechanisms of arrhythmias in inheritable cardiac diseases.

Neurogranin, a link between calcium/calmodulin and protein kinase C signaling in synaptic plasticity.

Neurogranin (Ng) (also named RC3, p17 or BICKS) is a small protein originally identified in rat brain and abundantly expressed in several telencephalic areas, such as the cerebral cortex, hippocampus, amygdala, and striatum. In neurons, it is found concentrated at dendritic spines where it participates in synaptic signaling events through the regulation of calmodulin (CaM) availability. Ng features an IQ motif that mediates its interaction with CaM and phosphatidic acid (PA) and that is phosphorylated by protein kinase C (PKC) at serine 36 (Ser36). Ser36-phosphorylated Ng is unable to bind either CaM or PA. Ng knockout mice display an apparently normal phenotype; however, they show severe deficits in spatial and emotional learning and a decrease in LTP induction, mostly due to the attenuation of the signaling that depends on calcium/CaM kinase II (CaMKII), PKC, and protein kinase A (PKA) activation. The present review is an update on the most relevant information about Ng expression, localization, interactions, and modifications as well as on its role in synaptic plasticity.

Activity-dependent translocation of neurogranin to neuronal nuclei.

Long-term changes of synaptic plasticity depend on protein synthesis and transcription. Ng (neurogranin) is a small protein concentrated at dendrites and spines of forebrain neurons, involved in synaptic plasticity through the regulation of CaM (calmodulin)-mediated signalling. Ng presents a central IQ motif that mediates its binding to CaM and PA (phosphatidic acid) and that can be phosphorylated by PKC (protein kinase C). In the present manuscript, we report that Ng displays a strong nuclear localization when expressed in cell lines and hippocampal neurons, either alone or fused to GFP (green fluorescent protein; GFP-Ng). Furthermore, using subcellular fractionation and immunocytochemical techniques, we were able to localize endogenous Ng in the nuclei of rat forebrain neurons. Nuclear localization of Ng depends on its IQ motif and is reduced by binding to cytoplasmic CaM. Also, PKC stimulation induces a transient nuclear translocation of Ng in acute hippocampal slices. A similar translocation is observed in the neurons of the cerebral cortex and hippocampus after the induction of generalized seizures in adult rats. In summary, the results of the present study show that a fraction of rat brain Ng is localized in the neuronal nuclei and that synaptic activity regulates its translocation from the cytoplasm. The possible involvement of Ng in the regulation of intranuclear Ca2+/CaM-dependent signalling and gene expression is discussed.

Mechanisms of endoplasmic-reticulum export of glycine transporter-1 (GLYT1).

The GLYT1 (glycine transporter-1) regulates both glycinergic and glutamatergic neurotransmission by controlling the reuptake of glycine at synapses. Trafficking to the cell surface of GLYT1 is critical for its function. In the present paper, by using mutational analysis of the GLYT1 C-terminal domain, we identified the evolutionarily conserved motif R(575)L(576)(X(8))D(585) as being necessary for ER (endoplasmic reticulum) export. This is probably due to its capacity to bind Sec24D, a component of the COPII (coatomer coat protein II) complex. This ER export motif was active when introduced into the related GLYT2 transporter but not in the unrelated VSVG (vesicular-stomatitis virus glycoprotein)-GLYT1 protein in which this motif was mutated but was not transported to the plasma membrane, although this effect was rescued by co-expressing these mutants with wild-type GLYT1. This behaviour suggests that GLYT1 might form oligomers along the trafficking pathway. Cross-linking assays performed in rat brain synaptosomes and FRET (fluorescence resonance energy transfer) microscopy in living cells confirmed the existence of GLYT1 oligomers. In summary, we have identified a motif involved in the ER exit of GLYT1 and, in analysing the influence of this motif, we have found evidence that oligomerization is important for the trafficking of GLYT1 to the cell surface. Because this motif is conserved in the NSS (sodium- and chloride-dependent neurotransmitter transporter) family, it is possible that this finding could be extrapolated to other related transporters.

Neurogranin Expression Is Regulated by Synaptic Activity and Promotes Synaptogenesis in Cultured Hippocampal Neurons.

Neurogranin (Ng) is a calmodulin (CaM)-binding protein that is phosphorylated by protein kinase C (PKC) and is highly enriched in the dendrites and spines of telencephalic neurons. It is proposed to be involved in regulating CaM availability in the post-synaptic environment to modulate the efficiency of excitatory synaptic transmission. There is a close relationship between Ng and cognitive performance; its expression peaks in the forebrain coinciding with maximum synaptogenic activity, and it is reduced in several conditions of impaired cognition. We studied the expression of Ng in cultured hippocampal neurons and found that both protein and mRNA levels were about 10% of that found in the adult hippocampus. Long-term blockade of NMDA receptors substantially decreased Ng expression. On the other hand, treatments that enhanced synaptic activity such as long-term bicuculline treatment or co-culture with glial cells or cholesterol increased Ng expression. Chemical long-term potentiation (cLTP) induced an initial drop of Ng, with a minimum after 15 min followed by a slow recovery during the next 2-4 h. This effect was most evident in the synaptosome-enriched fraction, thus suggesting local synthesis in dendrites. Lentiviral expression of Ng led to increased density of both excitatory and inhibitory synapses in the second and third weeks of culture. These results indicate that Ng expression is regulated by synaptic activity and that Ng promotes the synaptogenesis process. Given its relationship with cognitive function, we propose targeting of Ng expression as a promising strategy to prevent or alleviate the cognitive deficits associated with aging and neuropathological conditions.

Neurogranin binds to phosphatidic acid and associates to cellular membranes.

Neurogranin (Ng) is a 78-amino-acid-long protein concentrated at dendritic spines of forebrain neurons that is involved in synaptic plasticity through the regulation of CaM (calmodulin)-mediated signalling. Ng features a central IQ motif that mediates binding to CaM and is phosphorylated by PKC (protein kinase C). We have analysed the subcellular distribution of Ng and found that it associates to cellular membranes in rat brain. In vitro binding assays revealed that Ng selectively binds to PA (phosphatidic acid) and that this interaction is prevented by CaM and PKC phosphorylation. Using the peptide Ng-(29-47) and a mutant with an internal deletion (Ng-IQless), we have shown that Ng binding to PA and to cellular membranes is mediated by its IQ motif. Ng expressed in NIH-3T3 cells accumulates at peripheral regions of the plasma membrane and localizes at intracellular vesicles that can be clearly visualized following saponin permeabilization. This distribution was affected by PLD (phospholipase D) and PIP5K (phosphatidylinositol 4-phosphate 5-kinase) overexpression. Based on these results, we propose that Ng binding to PA may be involved in Ng accumulation at dendritic spines and that Ng could modulate PA signalling in the postsynaptic environment.

Brugada syndrome trafficking-defective Nav1.5 channels can trap cardiac Kir2.1/2.2 channels.

Cardiac Nav1.5 and Kir2.1-2.3 channels generate Na (INa) and inward rectifier K (IK1) currents, respectively. The functional INa and IK1 interplay is reinforced by the positive and reciprocal modulation between Nav15 and Kir2.1/2.2 channels to strengthen the control of ventricular excitability. Loss-of-function mutations in the SCN5A gene, which encodes Nav1.5 channels, underlie several inherited arrhythmogenic syndromes, including Brugada syndrome (BrS). We investigated whether the presence of BrS-associated mutations alters IK1 density concomitantly with INa density. Results obtained using mouse models of SCN5A haploinsufficiency, and the overexpression of native and mutated Nav1.5 channels in expression systems - rat ventricular cardiomyocytes and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) - demonstrated that endoplasmic reticulum (ER) trafficking-defective Nav1.5 channels significantly decreased IK1, since they did not positively modulate Kir2.1/2.2 channels. Moreover, Golgi trafficking-defective Nav1.5 mutants produced a dominant negative effect on Kir2.1/2.2 and thus an additional IK1 reduction. Moreover, ER trafficking-defective Nav1.5 channels can be partially rescued by Kir2.1/2.2 channels through an unconventional secretory route that involves Golgi reassembly stacking proteins (GRASPs). Therefore, cardiac excitability would be greatly affected in subjects harboring Nav1.5 mutations with Golgi trafficking defects, since these mutants can concomitantly trap Kir2.1/2.2 channels, thus unexpectedly decreasing IK1 in addition to INa.

Kir2.1-Nav1.5 Channel Complexes Are Differently Regulated than Kir2.1 and Nav1.5 Channels Alone.

Cardiac Kir2.1 and Nav1.5 channels generate the inward rectifier K+ (IK1) and the Na+ (INa) currents, respectively. There is a mutual interplay between the ventricular INa and IK1 densities, because Nav1.5 and Kir2.1 channels exhibit positive reciprocal modulation. Here we compared some of the biological properties of Nav1.5 and Kir2.1 channels when they are expressed together or separately to get further insights regarding their putative interaction. First we demonstrated by proximity ligation assays (PLAs) that in the membrane of ventricular myocytes Nav1.5 and Kir2.1 proteins are in close proximity to each other (<40 nm apart). Furthermore, intracellular dialysis with anti-Nav1.5 and anti-Kir2.1 antibodies suggested that these channels form complexes. Patch-clamp experiments in heterologous transfection systems demonstrated that the inhibition of the Ca2+/calmodulin-dependent protein kinase II (CaMKII) decreased the INa and the IK1 generated by Nav1.5 and Kir2.1 channels when they were coexpressed, but not the IK1 generated by Kir2.1 channels alone, suggesting that complexes, but not Kir2.1 channels, are a substrate of CaMKII. Furthermore, inhibition of CaMKII precluded the interaction between Nav1.5 and Kir2.1 channels. Inhibition of 14-3-3 proteins did not modify the INa and IK1 densities generated by each channel separately, whereas it decreased the INa and IK1 generated when they were coexpressed. However, inhibition of 14-3-3 proteins did not abolish the Nav1.5-Kir2.1 interaction. Inhibition of dynamin-dependent endocytosis reduced the internalization of Kir2.1 but not of Nav1.5 or Kir2.1-Nav1.5 complexes. Inhibition of cytoskeleton-dependent vesicular trafficking via the dynein/dynactin motor increased the IK1, but reduced the INa, thus suggesting that the dynein/dynactin motor is preferentially involved in the backward and forward traffic of Kir2.1 and Nav1.5, respectively. Conversely, the dynein/dynactin motor participated in the forward movement of Kir2.1-Nav1.5 complexes. Ubiquitination by Nedd4-2 ubiquitin-protein ligase promoted the Nav1.5 degradation by the proteasome, but not that of Kir2.1 channels. Importantly, the Kir2.1-Nav1.5 complexes were degraded following this route as demonstrated by the overexpression of Nedd4-2 and the inhibition of the proteasome with MG132. These results suggested that Kir2.1 and Nav1.5 channels closely interact with each other leading to the formation of a pool of complexed channels whose biology is similar to that of the Nav1.5 channels.

Activity dependent internalization of the glutamate transporter GLT-1 mediated by ß-arrestin 1 and ubiquitination.

GLT-1 is the main glutamate transporter in the brain and undergoes trafficking processes that control its concentration on the cell surface thereby shaping glutamatergic neurotransmission. We have investigated how the traffic of GLT-1 is regulated by transporter activity. We report that internalization of GLT-1 from the cell surface is accelerated by transportable substrates like glutamate or aspartate, as well as by the transportable inhibitor L-trans-2,4-PDC, but not by the non-substrate inhibitor WAY 213613 in primary mixed cultures and in transiently transfected HEK293 cells. Analysis of the mechanism of endocytosis in HEK293 cells revealed that glutamate promoted the association with the transporter of the adaptor protein ß-arrestin and the ubiquitin ligase Nedd4-2. The addition of glutamate is accompanied by an increase in the transporter ubiquitination, and the internalization is suppressed by an ubiquitination inhibitor (PYR41), and in a mutant defective in C-terminal lysines. The glutamate triggered endocytosis was also suppressed by siRNA for ß-arrestin. This regulatory mechanism might be relevant in controlling the amount of transporter on the cell surface in conditions such as ischemia or traumatic brain injury, where extracellular concentrations of glutamate are persistently elevated.

Visualization of phosphatidic acid fluctuations in the plasma membrane of living cells.

We developed genetically-encoded fluorescent sensors based on Förster Resonance Energy Transfer to monitor phosphatidic acid (PA) fluctuations in the plasma membrane using Spo20 as PA-binding motif. Basal PA levels and phospholipase D activity varied in different cell types. In addition, stimuli that activate PA phosphatases, leading to lower PA levels, increased lamellipodia and filopodia formation. Lower PA levels were observed in the leading edge than in the trailing edge of migrating HeLa cells. In MSC80 and OLN93 cells, which are stable cell lines derived from Schwann cells and oligodendrocytes, respectively, a higher ratio of diacylglycerol to PA levels was demonstrated in the membrane processes involved in myelination, compared to the cell body. We propose that the PA sensors reported here are valuable tools to unveil the role of PA in a variety of intracellular signaling pathways.

Age-associated decrease of SIRT1 expression in rat hippocampus: prevention by late onset caloric restriction.

We have studied the effect of aging and late onset caloric restriction (CR) on the expression of SIRT1 in hippocampus and cerebral cortex of the rat. Quantitative analysis showed that there is a significant reduction of SIRT1 protein levels in hippocampus with aging. Late onset, moderate CR prevented the deleterious effect of aging on SIRT1 content. Examination of SIRT1 immunoreactivity in coronal sections from hippocampus supported these results, and confirmed that old animals are able to respond to the beneficial effects of CR by regulating SIRT1 protein expression. Differences in the amounts of SIRT1 transcripts among animal groups were not found, which suggest that post-transcriptional mechanisms could be involved in the effects of aging and CR on SIRT1 expression.

Setting up and running an advanced light microscopy and imaging facility.

During the last twenty years, interest in light microscopy and imaging techniques has grown in various fields, such as molecular and cellular biology, developmental biology, and neurobiology. In addition, the number of scientific articles and journals using these techniques is rapidly increasing. Nowadays, most research institutions require sophisticated microscopy systems to cover their investigation demands. In general, such instruments are too expensive and complex to be purchased and managed by a single laboratory or research group, so they have to be shared with other groups and supervised by specialized personnel. This is the reason why microscopy and imaging facilities are becoming so important at research institutions nowadays. In this unit, we have gathered and presented a number of issues and considerations from our own experience that we hope will be helpful when planning or setting up a new facility.