Biosci Rep (2006) 26:399–412 DOI 10.1007/s10540-006-9029-5 ORIGINAL PAPER
SK3 Trafficking in Hippocampal Cells: The Role of Different Molecular Domains Ilaria Decimo Æ Renza Roncarati Æ Silvia Grasso Æ Marcel Clemens Æ Christian Chiamulera Æ Guido Fumagalli
Published online: 24 October 2006 Springer Science+Business Media, LLC 2006
Abstract The regulative steps that control trafficking of ion channels are fundamental determinants of their qualitative and quantitative expression on the cell membrane. In this work the trafficking of the small conductance calcium-activated potassium channel, SK3 was studied in neurons in order to identify relevant molecular domains involved in this process. Hippocampal cell cultures were transfected with fusion proteins of green fluorescent protein (GFP) and different SK3 subunit truncations. The differential distribution of the mutants was analyzed by confocal microscopy and compared to the localization of the control fusion protein with full length SK3. The transport of chimeric proteins was quantified from fluorescence images by developing a morphometric analytical method. We found that the full length SK3 was distributed in cell body, axon and dendrites, whereas the deleted forms GFPD578–736 (deletion of the entire C-terminal domain), GFPDCaMBD (deletion of the calmodulin-binding site) and GFPDN (deletion of the N-terminal domain) were not transported into cell processes but accumulated in the cell body. The GFPD640–736 (deletion of the distal C-terminal domain) showed a distribution similar to control. The quantification and statistical analysis confirmed the differences in distribution across the three groups. In conclusion, the current work provides evidence for a fundamental role of the N-terminal domain and the calmodulin binding domain in SK3 trafficking in neurons. Keywords SK3 Æ Small conductance calcium activated potassium channel Æ Hippocampal neurons Æ Trafficking
I. Decimo (&) Æ S. Grasso Æ M. Clemens Æ C. Chiamulera Æ G. Fumagalli Department of Medicine and Public Health, University of Verona, P.le L. Scuro 10, 37100 Verona, Italy e-mail:
[email protected] R. Roncarati Siena Biotech S.p.A. Discovery Research, Via Fiorentina 1, 53100 Siena, Italy
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Introduction The family of the small conductance Ca2+ activated potassium channels (SK channels) comprises three members, SK1, SK2, and SK3 (Kohler et al. 1996), which are all expressed in the central nervous system (CNS) (Stocker and Pederzani 2000; Tacconi et al. 2001; Sailer et al. 2002, 2004). They share the tetrameric structure with each subunit containing six transmembrane regions and intracellular N- and C-terminals. SK channels are voltage independent and activated by an increase in intracellular Ca2+ concentration (Xia et al. 1998); Ca2+ sensitivity is mediated by calmodulin, which is constitutively bound to a 92-amino acid domain embedded in the C-terminal region (Keen et al. 1999). The major role of SK channels in the CNS is to contribute to the afterhyperpolarization that follows an action potential and to regulate membrane excitability and firing properties of CNS neurons (Lancaster and Adams 1986; Sah 1996; Sah and Davies 2000; Hosseini et al. 2001; Sah and Faber 2002; Bond et al. 2004; Villalobos et al. 2004). The SK channels are functionally relevant for several integrated functions of the CNS, including sleep-waking cycle (Gandolfo et al. 1996), long-term potentiation (Blank et al. 2003), synaptic plasticity and memory encoding (Stackman et al. 2002; Tzounopoulos and Stackman 2003; Faber et al. 2005). The importance of SK channels in the regulation of neuronal excitability is also underlined by their direct involvement in some CNS pathologies such as cerebellar ataxia (Shakkottai et al. 2004). Furthermore, a mutation of the SK3 channel, acting as dominant negative regulator of SK function, have been detected in patients suffering from schizophrenia (Miller et al. 2001; Tomita et al. 2003). Subcellular localization is an important determinant of the physiological significance and role of ion channels. In neuronal cells, immunolabeling and functional studies with the SK-specific ligand apamin showed that SK channels exhibit dendritic localization (Stackman et al. 2002; Womack and Khodakhah 2003; Faber et al. 2005). Recent immunofluorescence studies suggested a presynaptic localization in motor neurons and in hippocampal neurons (Roncarati et al. 2001; Obermair et al. 2003). The subcellular localization of membrane proteins in neurons is the result of the integration of cellular functions involved in protein sorting and trafficking. Many ion channels carry specific trafficking motifs that regulate their exit from the endoplasmic reticulum (ER) to the trans-Golgi network (TGN), from where they are directed to discrete plasma membrane domains assuming a highly compartmentalized distribution (Deutsch 2003). Little is known about the molecular regulation of SK channel transport. Recent experimental evidence underlines the importance of the calmodulin binding domain (CaMBD) for cell surface expression of SK channels (Joiner et al. 2001; Lee et al. 2003). Previously, we transfected non-neuronal cells with full length or truncated mutants SK3 channels and identified three different domains of the SK3 channel subunit necessary for surface expression (Roncarati et al. 2005). In particular, we showed that the N-terminal domain and CaMBD were necessary for efficient channel transport from the ER to the cell membrane and that the distal C-terminal region was necessary for the post-Golgi transport of SK3. In the present investigation we extend our study on the subcellular distribution and determinants for intracellular trafficking to neuronal hippocampal cells in culture. Five different constructs encoding fusion proteins of GFP and SK3 either wild type (w.t.) or deleted were transfected into rat hippocampal cells in culture and their differential distribution analyzed by confocal microscopy. The results presented
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here demonstrate that the N-terminal and CaMBD domains are necessary for SK3 channel transport along dendrites and axons.
Materials and Methods Cell Cultures Rat primary cultures of dissociated hippocampal neurons were prepared from Sprague– Dawely E18–E19 pups, as described by Banker and Cowan (1977). Briefly, hippocampi were dissociated by trypsin treatment and trituration and plated on poly-D-lysine- and laminin-coated coverslips (19 mm of diameter) at a density of 75,000 cells/well. Neurons were cultured in Neuron Chow (Neurobasal Medium (Gibco 21103-049) 2% B27 supplements, 500 lM glutamine, 12.5 lM glutamate) in a humidified 5% CO2 incubator at 37C. Half volume of the medium was changed after 4 days with Neuron Chow without glutamate. Cells were transfected with various plasmids, 6 days after plating. Construct Preparations All the plasmid constructs were prepared as previously described (Roncarati et al. 2005). Briefly human SK3 cDNA (accession number AJ251016) and C-terminal and N-terminal deletion mutants were inserted into plasmid pEGFPC1 obtaining the construct described in Fig. 1.
Fig. 1 Schematic drawing of GFP-tagged SK3 and its mutated constructs. Transmembrane regions are indicated as boxes S1–S6. Deletions are defined by the first and last a.a. deleted; in the GFPDCaMBD a.a. 579–638 were removed; in GFPDN the a.a. from 1 to 271 were removed
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Transfections For transfections of hippocampal cells a calcium-phosphate method was used. Briefly, for two wells of a 12-well plate 10 lg of Quiagen-kit purified DNA, 10 ll of 2.5 M CaCl2, and sterile water up to 100 ll were mixed in a tube. 2· HEBS solution (50 mM Hepes, 1.5 mM Na2HPO4, 10 mM KCl, 280 mM NaCl, 12 mM glucose, pH 7.05) was added and the mixture was incubated at room temperature for 30 min. Cell plated on cover slips were pre-equilibrated in DMEM at 37C/5% CO2. Then 100 ll of the mixture was dispensed dropwise in each well and incubated at 37C/5% CO2 for 10 min, or until fine sand-like precipitates were visible at the optical microscope. Cells were washed with DMEM and placed back in their original conditioned medium. Cells were transfected after 6 days in vitro (DIV) and were used for immunofluorescence at 10 DIV. Immunofluorescence and Confocal Microscopy Hippocampal neurons grown on cover slips were fixed for 20 min with 4% paraformaldehyde and 4% sucrose in PBS buffer and subsequently rinsed in PBS. Neurons were permeabilized for 30 min in PBS containing 0.2% bovine serum albumin and 0.2% Triton X-100 (PBS/BSA/TRITON). Neurons were incubated with primary antibodies in PBS/BSA/TRITON for 90 min at room temperature. After rinsing in PBS/BSA/ TRITON, secondary antibodies were applied for 1 h. After final washing steps in PBS, preparations were mounted on anti-bleaching 1,4-diazabicyclo[2.2.2]octane (Sigma) in PBS containing 50% glycerol. Slides were observed by using a Zeiss LSM 510 confocal microscope equipped with argon (488 nm) and helium/neon (543 nm) excitation lasers. For 488 nm excitation, emission was selected with a 510–530 nm bandpass filter, whereas for 546 nm excitation emission was selected using a 560 nm longpass filter. Primary Antibodies The rabbit polyclonal antibody specific for SK3 (Alomone Labs) was used at 1:1,000 dilution; the mouse monoclonal anti-MAP2 antibody (Roche) was used at 1:200 dilution. For the staining of the Golgi apparatus, a monoclonal antibody directed against the GM130 Golgi protein (BD Biosciences; 1:200 dilution) was utilized whereas for the staining of endoplasmic reticulum the mouse anti-KDEL monoclonal antibody was employed (Stressgen Biotechnologies; 1:50 dilution). Secondary Antibodies Cy3-conjugated goat anti-rabbit antibody (Amersham, 1:1,000) and Cy3-conjugated goat anti-mouse (Amersham, 1:1,000) were used. Staining of lysosomes in living neurons was performed by incubation with 100 nM Lysotracker (Molecular Probes) for 30 min at 37C. Image Quantification The transfected hippocampal cells were imaged with a Zeiss LSM 510 confocal microscope at a magnification of 40·. Since the concentration of GFP was higher in the cell bodies than in the neuronal processes, fluorescence signal in neuronal processes was
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detected only by saturating the signal intensity of the cell body. Therefore two images for each cell were acquired, one in which detector gain and offset was optimized for detection of soma details without saturating pixels and a second optimized for fluorescence detection in dendrites. The pinhole was fixed at 145 lm. These parameters were unchanged for all acquisitions. Quantification of images was carried out using ‘‘Scion Image’’ software (http:// www.scioncorp.com). For each cell, the image in which the cell body was not saturated was used to calculate the area of the cell body. The signal fluorescence in the neuronal processes for each cell was quantified according to the following procedure. First, the area of the cell body as defined in the unsaturated cell body image was removed from the image in which the cell body was saturated. In the resulting image an estimate was then made of the background level, (b), and its standard deviation, (r). A threshold was then applied to the image to exclude all pixels below b + 4r thus effectively removing background pixels form the image. The total number of pixels and their mean intensity value above this threshold were calculated. The following formula was applied to obtain a normalized measure of the fluorescence signal in neuronal processes: Total pixels number Pixels mean intensity value : Cell body signal area
Statistics Fluorescence signal values (as calculated by the above described formula) were analyzed for normal distribution for each construct group (Graphpad Prism, V.4.02). Overall comparison between groups was analyzed with non-parametric one-way ANOVA (Kruskal–Wallis test) followed by post-hoc Dunn’s test for multiple comparisons.
Results Subcellular Distribution of GFP–SK3 in Neurons Rat primary cultures of dissociated hippocampal neurons were transfected 1 week after plating. The subcellular distribution of the chimeric protein was analyzed by fluorescence confocal microscopy 4 days after transfection. The transfection efficiency was about 0.1%. To determine the nature of the neurites of the hippocampal cells, we used antibodies directed against the microtubule associated protein 2 (MAP2), a specific marker of dendrites. Axons were identified as MAP2-negative long and fine processes. The GFP–SK3 protein was observed in cell bodies, dendrites and axons (Fig. 2). In particular GFP–SK3 signal appeared diffuse in the soma but excluded from the nucleus; along neurites, GFP–SK3 signal was often punctated suggesting that the fluorescent tagged channel accumulated in vesicular structures. To verify that the GFP fluorescent signal reflected channel distribution, we immunostained transfected neurons with an anti-SK3 antibody which showed a complete co-localization with GFP fluorescence (Fig. 2). When untagged SK3 was used to transfect neurons, anti-SK3 immunofluorescence had the same subcellular distribution
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Fig. 2 Expression and localization of GFP–SK3 fusion protein in cultured hippocampal neurons. In the upper row representative confocal images of neurons transfected with GFP–SK3 and stained with antiMAP2 antibody (A, red) are shown. In B, one neuron expressing the GFP–SK3 protein is visible; the green fluorescent signal is present in the soma and processes. In C, images A and B are superimposed; the yellow color indicates coincidence of fluorescent signals. The arrow points to the axon of the transfected cell which is devoid of MAP2 immunoreactivity (red) and shows presence of GFP–SK3 (green). In the lower row, the GFP green signal (E) of a GFP–SK3 expressing cell is compared to the distribution of the red immunolabeling signal obtained with an anti-SK3 antibody (D). The two images are superimposed in F where yellow indicates coincidence of the two fluorescent signals. Scale bar 50 lm
as GFP–SK3 (data not show). Finally, hippocampal neurons were transfected with GFP alone. In this case, the fluorescent signal was evenly distributed in the cells and their neurites and was also seen in the nucleus (not shown). These results confirm that the GFP fluorescence reflects channel distribution in the transfected cells and that the GFP tag did not interfere with assembly and trafficking of the channel. The Role of the C-terminal and N-terminal Domains in Neuronal Targeting of SK3 In order to identify the presence of axon–dendrite targeting domains in the C-terminal region of SK3, we analysed the neuronal distribution of three different mutants truncated in the C-terminus (Fig. 1). First, the effects of the complete deletion of the Cterminal domain (GFPD578–736; deletion from amino acid 578, at the end of the sixth transmembrane domain, to the last amino acid 736) were investigated. GFPD578–736 was localized in the soma only and was excluded from both dendrites and axons (Fig. 3). This subcellular distribution suggested that targeting to axons and dendrites requires the presence of a targeting domain contained in the C-terminal domain. To better define the role of this region two C-terminal partially truncated mutants were used: GFPD640–736 and GFPDCaMBD. In the GFPD640–736 construct, the deletion involves the C-terminal region, distally from the CaMBD. The GFPDCaMBD mutant lacks the calmodulin binding domain (from a.a. 579 to 638) and retains the distal part of the C-terminal region. The GFPD640–736 mutant was observed in soma,
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Fig. 3 Expression and localization of GFP–SK3 C-terminal and N-terminal mutant proteins in cultured hippocampal neurons. Representative confocal images of hippocampal neurons transfected with GFPD578–736 (A–C), GFPD640–736 (D–F), GFPDCaMBD (G–I) and GFPDN (J–L) are presented. The left column (A, D, G, J) shows MAP2 immunostaining (red). The middle column (B, E, H, K) shows the GFP–SK3 green fluorescent signal of the corresponding neurons. The right column presents the merged images (C, F, I, L) where the yellow color indicates co-localization between the mutant proteins and MAP2. Scale bar is 50 lm
dendrites and axons (Fig. 3) with a pattern of subcellular distribution that was indistinguishable from that of the GFP–SK3 w.t. protein. These data indicate that the distal C-terminal domain has no role in defining SK3 channel distribution in hippocampal neurons. In neurons transfected with the GFPDCaMBD mutant, the fluorescent protein accumulated in form of small clusters in the soma and the proximal dendrites, but was excluded from the distal portions of dendrites and axons (Fig. 3). Thus, it seems the CaMBD is necessary for dendritic and axonal targeting of SK3 channels. The potential role of the SK3 N-terminal region in neuronal trafficking was investigated by transfecting hippocampal cells with a GFP-tagged N-terminal truncated construct (GFPDN, Fig. 1). The GFPDN signal was seen in the soma, excluded from the nucleus, and was not detected in dendrites and axons (Fig. 3). This suggests that the N-terminal domain is also necessary for neuronal SK3 trafficking. To determine whether the accumulation in the soma of the mutant channels (GFPD578–736, GFPDCaMBD, GFPDN), reflected ER retention, the ER and the Golgi
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apparatus were immunolocalized in the same cells (Fig. 4). We observed mainly co-localization with the ER and occasionally a partial co-localization between the transfected protein and the Golgi marker. No co-localization was found with a marker of lysosomes. These results indicate that accumulation in the soma of these deleted subunits is mainly caused by a block in the ER and that regions of both the SK3 N-terminal and Cteminal domains are necessary for progression downstream along the secretory pathway. Quantitative Data Analysis Confirm the Role of N-terminal and C-terminal Domains in SK3 Trafficking The qualitative results describing the subcellular distribution of the GFP–SK3 protein and its deletion mutants were supported by quantitative morphometric analysis of fluorescence distribution in transfected neurons. For each analyzed neuron we quantified the fluorescence signal area and intensity in the neurites as described in ‘‘Materials and methods’’. The values obtained were normalized to the cell body area in order to account for cell size variability. Since data were not normally distributed (D’Agostino & Pearson omnibus normality test, K2 = 35.65, 34.37, 43.36, 13.04 and 13.41 values, respectively, for GFP–SK3, GFPD640–736, GFPD578–736, GFPDN and GFPDCaMBD constructs), the arbitrary median values obtained for each construct fluorescence were compared by non-parametric one-way ANOVA (Kruskal–Wallis test) for testing differences in distribution. Median values of fluorescence distribution are shown in Fig. 5 for each construct. Overall ANOVA analysis showed a significant construct effect (n = 5 groups, Kruskal–Wallis statistics = 72.85, P < 0.001). Post-hoc Dunn’s Test showed that GFP–SK3 fluorescence distribution is statistically different from the distribution of GFPD578–736 (P < 0.001), GFPDCaMBD (P < 0.001), GFPDN (P < 0.001) but not from GFPD640–736 (P > 0.05). Similarly, GFPD640–736 values result significantly different from the distribution of GFPD578–736 (P < 0.01), GFPDCaMBD (P < 0.05), GFPDN (P < 0.001). Finally, distribution of fluorescence of constructs GFPD578–736, GFPDCaMBD and GFPDN are not significantly different from each other (NS, Dunn’s Test). Taken together these data identify two groups which are significantly different: GFPD578–736, GFPDCaMBD and GFPDN display a fluorescence signal distribution in neurites that is statistically lower than GFP–SK3 and GFPD640–736. This confirms the qualitative observations suggesting an essential role of both the N- and C-terminal domains for distribution of SK channels in axons and dendrites.
Discussion Subcellular Distribution of SK3 Channel The aim of this work was to identify molecular domains of SK3 channels relevant for its trafficking and subcellular distribution in neurons. In a previous study we characterized the subcellular distribution of the GFP–SK3 protein in PC12 cells. These cells express endogenous SK3 channel (Terstappen et al. 1999); when exposed to NGF, they form long neurites that lack the biochemical and functional differentiation that distinguishes axons from dendrites in neurons in vivo. Therefore PC12 cells are not a useful model when the goal is to study intracellular trafficking in neuronal systems.
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Fig. 4 Intracellular localization of SK3 deletion mutants GFPD578–736, GFPDCaMBD and GFPDN. Representative confocal images of transfected neurons (a) GFPD578–736 (b) GFPDCaMBD and (c) GFPDN stained with anti-KDEL antibody to visualize the ER (red), anti-GM130 antibody to visualize Golgi apparatus (red) and lysotracker to visualize lysosomes (red). In merged, yellow represents regions of overlap between the marker (red) and the indicated construct (green). Scale bar is 20 lm
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Fig. 4 continued
Hippocampal neurons were selected because they are a well established experimental model system in vitro where neurons reach the morphological and functional differentiation seen in vivo (Bartlett and Banker 1984a, b; Dotti et al. 1988). These cells have been extensively used for studying the mechanisms governing the subcellular distribution and the intracellular trafficking of membrane proteins in neurons (Sampo
Median values of fluorescence in neuronal processes
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*** = p
