Cysteine string protein (CSP) is an insulin secretory granule-associated protein regulating beta -cell exocytosis
1998; Springer Nature; Volume: 17; Issue: 17 Linguagem: Inglês
10.1093/emboj/17.17.5048
ISSN1460-2075
AutoresHilary F. Brown, Olof Larsson, Robert Bränström, Shao Nian Yang, Barbara Leibiger, Ingo B. Leibiger, Gabriel Fried, Tilo Moede, Jude T. Deeney, G. R. Brown, Gunilla Jacobsson, Christopher J. Rhodes, Janice E.A. Braun, Richard H. Scheller, Barbara E. Corkey, Per‐Olof Berggren, Björn Meister,
Tópico(s)Lipid Membrane Structure and Behavior
ResumoArticle1 September 1998free access Cysteine string protein (CSP) is an insulin secretory granule-associated protein regulating β-cell exocytosis Hilary Brown Hilary Brown Department of Neuroscience, The Berzelius Laboratory, Karolinska Institute, Stockholm, Sweden The Rolf Luft Center for Diabetes Research, Department of Molecular Medicine, Karolinska Institute, Stockholm, Sweden Search for more papers by this author Olof Larsson Olof Larsson The Rolf Luft Center for Diabetes Research, Department of Molecular Medicine, Karolinska Institute, Stockholm, Sweden Search for more papers by this author Robert Bränström Robert Bränström The Rolf Luft Center for Diabetes Research, Department of Molecular Medicine, Karolinska Institute, Stockholm, Sweden Search for more papers by this author Shao-Nian Yang Shao-Nian Yang The Rolf Luft Center for Diabetes Research, Department of Molecular Medicine, Karolinska Institute, Stockholm, Sweden Search for more papers by this author Barbara Leibiger Barbara Leibiger The Rolf Luft Center for Diabetes Research, Department of Molecular Medicine, Karolinska Institute, Stockholm, Sweden Search for more papers by this author Ingo Leibiger Ingo Leibiger The Rolf Luft Center for Diabetes Research, Department of Molecular Medicine, Karolinska Institute, Stockholm, Sweden Search for more papers by this author Gabriel Fried Gabriel Fried Department of Women and Child Health, Karolinska Hospital, Stockholm, Sweden Search for more papers by this author Tilo Moede Tilo Moede The Rolf Luft Center for Diabetes Research, Department of Molecular Medicine, Karolinska Institute, Stockholm, Sweden Search for more papers by this author Jude T. Deeney Jude T. Deeney The Rolf Luft Center for Diabetes Research, Department of Molecular Medicine, Karolinska Institute, Stockholm, Sweden Search for more papers by this author Graham R. Brown Graham R. Brown The Rolf Luft Center for Diabetes Research, Department of Molecular Medicine, Karolinska Institute, Stockholm, Sweden Search for more papers by this author Gunilla Jacobsson Gunilla Jacobsson Department of Neuroscience, The Berzelius Laboratory, Karolinska Institute, Stockholm, Sweden Search for more papers by this author Christopher J. Rhodes Christopher J. Rhodes Center for Diabetes Research, Departments of Internal Medicine and Pharmacology, University of Texas South Western Medical Center, Dallas, TX, 75235 USA Search for more papers by this author Janice E.A. Braun Janice E.A. Braun Department of Molecular and Cellular Physiology, Howard Hughes Medical Institute, Beckman Center, Stanford University, Stanford, CA, 94305 USA Search for more papers by this author Richard H. Scheller Richard H. Scheller Department of Molecular and Cellular Physiology, Howard Hughes Medical Institute, Beckman Center, Stanford University, Stanford, CA, 94305 USA Search for more papers by this author Barbara E. Corkey Barbara E. Corkey Diabetes and Metabolism Unit, Evans Department of Medicine, Boston University School of Medicine, Boston, MA, 02118 USA Search for more papers by this author Per-Olof Berggren Per-Olof Berggren The Rolf Luft Center for Diabetes Research, Department of Molecular Medicine, Karolinska Institute, Stockholm, Sweden Search for more papers by this author Björn Meister Corresponding Author Björn Meister Department of Neuroscience, The Berzelius Laboratory, Karolinska Institute, Stockholm, Sweden Search for more papers by this author Hilary Brown Hilary Brown Department of Neuroscience, The Berzelius Laboratory, Karolinska Institute, Stockholm, Sweden The Rolf Luft Center for Diabetes Research, Department of Molecular Medicine, Karolinska Institute, Stockholm, Sweden Search for more papers by this author Olof Larsson Olof Larsson The Rolf Luft Center for Diabetes Research, Department of Molecular Medicine, Karolinska Institute, Stockholm, Sweden Search for more papers by this author Robert Bränström Robert Bränström The Rolf Luft Center for Diabetes Research, Department of Molecular Medicine, Karolinska Institute, Stockholm, Sweden Search for more papers by this author Shao-Nian Yang Shao-Nian Yang The Rolf Luft Center for Diabetes Research, Department of Molecular Medicine, Karolinska Institute, Stockholm, Sweden Search for more papers by this author Barbara Leibiger Barbara Leibiger The Rolf Luft Center for Diabetes Research, Department of Molecular Medicine, Karolinska Institute, Stockholm, Sweden Search for more papers by this author Ingo Leibiger Ingo Leibiger The Rolf Luft Center for Diabetes Research, Department of Molecular Medicine, Karolinska Institute, Stockholm, Sweden Search for more papers by this author Gabriel Fried Gabriel Fried Department of Women and Child Health, Karolinska Hospital, Stockholm, Sweden Search for more papers by this author Tilo Moede Tilo Moede The Rolf Luft Center for Diabetes Research, Department of Molecular Medicine, Karolinska Institute, Stockholm, Sweden Search for more papers by this author Jude T. Deeney Jude T. Deeney The Rolf Luft Center for Diabetes Research, Department of Molecular Medicine, Karolinska Institute, Stockholm, Sweden Search for more papers by this author Graham R. Brown Graham R. Brown The Rolf Luft Center for Diabetes Research, Department of Molecular Medicine, Karolinska Institute, Stockholm, Sweden Search for more papers by this author Gunilla Jacobsson Gunilla Jacobsson Department of Neuroscience, The Berzelius Laboratory, Karolinska Institute, Stockholm, Sweden Search for more papers by this author Christopher J. Rhodes Christopher J. Rhodes Center for Diabetes Research, Departments of Internal Medicine and Pharmacology, University of Texas South Western Medical Center, Dallas, TX, 75235 USA Search for more papers by this author Janice E.A. Braun Janice E.A. Braun Department of Molecular and Cellular Physiology, Howard Hughes Medical Institute, Beckman Center, Stanford University, Stanford, CA, 94305 USA Search for more papers by this author Richard H. Scheller Richard H. Scheller Department of Molecular and Cellular Physiology, Howard Hughes Medical Institute, Beckman Center, Stanford University, Stanford, CA, 94305 USA Search for more papers by this author Barbara E. Corkey Barbara E. Corkey Diabetes and Metabolism Unit, Evans Department of Medicine, Boston University School of Medicine, Boston, MA, 02118 USA Search for more papers by this author Per-Olof Berggren Per-Olof Berggren The Rolf Luft Center for Diabetes Research, Department of Molecular Medicine, Karolinska Institute, Stockholm, Sweden Search for more papers by this author Björn Meister Corresponding Author Björn Meister Department of Neuroscience, The Berzelius Laboratory, Karolinska Institute, Stockholm, Sweden Search for more papers by this author Author Information Hilary Brown1,2, Olof Larsson2, Robert Bränström2, Shao-Nian Yang2, Barbara Leibiger2, Ingo Leibiger2, Gabriel Fried3, Tilo Moede2, Jude T. Deeney2, Graham R. Brown2, Gunilla Jacobsson1, Christopher J. Rhodes4, Janice E.A. Braun5, Richard H. Scheller5, Barbara E. Corkey6, Per-Olof Berggren2 and Björn Meister 1 1Department of Neuroscience, The Berzelius Laboratory, Karolinska Institute, Stockholm, Sweden 2The Rolf Luft Center for Diabetes Research, Department of Molecular Medicine, Karolinska Institute, Stockholm, Sweden 3Department of Women and Child Health, Karolinska Hospital, Stockholm, Sweden 4Center for Diabetes Research, Departments of Internal Medicine and Pharmacology, University of Texas South Western Medical Center, Dallas, TX, 75235 USA 5Department of Molecular and Cellular Physiology, Howard Hughes Medical Institute, Beckman Center, Stanford University, Stanford, CA, 94305 USA 6Diabetes and Metabolism Unit, Evans Department of Medicine, Boston University School of Medicine, Boston, MA, 02118 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (1998)17:5048-5058https://doi.org/10.1093/emboj/17.17.5048 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Cysteine string proteins (CSPs) are novel synaptic vesicle-associated protein components characterized by an N-terminal J-domain and a central palmitoylated string of cysteine residues. The cellular localization and functional role of CSP was studied in pancreatic endocrine cells. In situ hybridization and RT–PCR analysis demonstrated CSP mRNA expression in insulin-producing cells. CSP1 mRNA was present in pancreatic islets; both CSP1 and CSP2 mRNAs were seen in insulin-secreting cell lines. Punctate CSP-like immunoreactivity (CSP-LI) was demonstrated in most islets of Langerhans cells, acinar cells and nerve fibers of the rat pancreas. Ultrastructural analysis showed CSP-LI in close association with membranes of secretory granules of cells in the endo- and exocrine pancreas. Subcellular fractionation of insulinoma cells showed CSP1 (34/36 kDa) in granular fractions; the membrane and cytosol fractions contained predominantly CSP2 (27 kDa). The fractions also contained proteins of 72 and 70 kDa, presumably CSP dimers. CSP1 overexpression in INS-1 cells or intracellular administration of CSP antibodies into mouse ob/ob β-cells did not affect voltage-dependent Ca2+-channel activity. Amperometric measurements showed a significant decrease in insulin exocytosis in individual INS-1 cells after CSP1 overexpression. We conclude that CSP is associated with insulin secretory granules and that CSP participates in the molecular regulation of insulin exocytosis by mechanisms not involving changes in the activity of voltage-gated Ca2+-channels. Introduction Cysteine string proteins (CSPs) are highly conserved proteins associated with synaptic vesicles (Zinsmaier et al., 1990; see Braun and Scheller, 1995; Buchner and Gundersen, 1997). The proteins were originally discovered as synapse-associated antigens in the nervous system of Drosophila and, independently, in Torpedo as candidate functional components of presynaptic voltage-sensitive N-type Ca2+-channels, expressed ectopically in frog oocytes (Gundersen and Umbach, 1992). CSPs are characterized by a central multiple palmitoylated string of cysteine residues and by an N-terminal J-domain, the latter being a 70 amino acid region of homology shared by bacterial DnaJ, a signature domain present in all members of the DnaJ molecular chaperone/heat shock protein (Hsp) family (Caplan et al., 1993; Braun and Scheller, 1995; see Buchner and Gundersen, 1997). CSPs exist in at least two isoforms, CSP1 and CSP2, generated by alternate RNA splicing (Zinsmaier et al., 1990; Chamberlain and Burgoyne, 1996). CSP2 represents a truncated version of CSP1 lacking the C-terminal 31 amino acid sequence. Several lines of evidence show that CSPs are associated with rat brain synaptic vesicle membranes (Mastrogiacomo and Gundersen, 1995). A general role for CSPs in trafficking events in both neuronal and non-neuronal cells has been implicated since CSP has been identified in membranes of pancreatic zymogen granules (Braun and Scheller, 1995), secretory granules of neurosecretory neurons in the neurohypophysis (Pupier et al., 1997) and in a chromaffin granule-enriched fraction (Kohan et al., 1995; Chamberlain et al., 1996). Detection of CSP mRNA in all human tissues examined suggests a widespread expression of the csp gene (Coppola and Gundersen, 1996). Functionally, CSPs have been demonstrated to be regulatory components of synaptic exocytosis, since deletion of the csp gene in Drosophila generates a phenotype characterized by premature death and temperaturesensitive inhibition of synaptic transmission, resulting in reversible paralysis at elevated temperatures (Zinsmaier et al., 1994). However, the precise role of CSPs in synaptic vesicle exocytosis remains largely unknown. Apart from a suggested functional interaction of CSPs with presynaptic voltage-sensitive N-type Ca2+-channels (Gundersen and Umbach, 1992; Mastrogiacomo et al., 1994a), it has been proposed that CSPs operate as molecular chaperones, e.g. assisting in folding or the conformational change of proteins participating in membrane trafficking (Caplan et al., 1993; Zinsmaier et al., 1994; Bohen et al., 1995; see Buchner and Gundersen, 1997). The cysteine-rich string domain in CSPs has led to another hypothetical model for the functional role of CSPs. The cysteine residues of CSPs have been shown to possess a high degree of post-translational fatty acid acylation. This compact acylation conveys a hydrophobic domain to the protein, which also contains highly polar N- and C-termini, and as a consequence CSP has been proposed to be uniquely suited to catalyze membrane fusion (Gundersen et al., 1995). Stimulus–secretion coupling in the pancreatic β-cell involves complex interaction between different signal transduction pathways (see Efendic et al., 1991; Berggren and Larsson, 1994). Glucose metabolism results in an increase in the cellular ATP:ADP ratio, closure of KATP-channels, depolarization of the plasma membrane and opening of voltage-gated L-type Ca2+-channels. The subsequent Ca2+-influx is a major signal for initiating insulin secretion in the pancreatic β-cell (Berggren and Larsson, 1994). Several proteins that regulate vesicular docking and fusion events, originally characterized in the nervous system, have recently been identified in the endocrine pancreas (Jacobsson et al., 1994), and roles for several of these proteins in the secretion of insulin have been revealed (Jacobsson et al., 1994; Martin et al., 1995, 1996; Regazzi et al., 1995, 1996; Sadoul et al., 1995; Kirali-Bourri et al., 1996; Nagamatsu et al., 1996, 1997; Wheeler et al., 1996; Lang et al., 1997; Mizuta et al., 1997). With the suggestion that CSP may be a modulator of Ca2+-channel activity and Ca2+-influx being the key event in the initiation of insulin secretion, we have attempted to identify the presence of CSP in pancreatic β-cells and to determine its role in insulin secretion. We have investigated the cellular localization of CSP in the pancreas at the mRNA and protein levels by using in situ hybridization, RT–PCR, RNase protection assay, immunofluorescence histochemistry combined with confocal laser microscopy, immunoelectron microscopy, immunoblotting and subcellular fractionation. The possible functional role of CSP in the β-cell has been evaluated by studies of voltage-gated Ca2+-channel activity and insulin secretion in individual cells using patch–clamp and amperometric techniques. Results CSP mRNA in insulin-producing cells In situ hybridization of sections of cultured RINm5F cells as well as rat brain, each with two probes complementary to two different regions of CSP mRNA, showed a strong hybridization signal (data not shown). Hybridization of sections from RINm5F cells with radiolabeled CSP probe in the presence of an excess (100×) of cold probe did not result in any hybridization signal (data not shown). Slides that were emulsion-dipped and hematoxylin-eosin counter-stained, showed silver grains overlying the cytoplasm of individual RINm5F cells (data not shown). Two isoforms of CSP have so far been described, CSP1 and CSP2. The mRNA of the latter isoform has a 72 bp insertion, which contains a stop codon and leads to a shorter 3.3 kDa protein (Chamberlain and Burgoyne, 1996). In order to study which of the isoforms is expressed in the pancreatic islets and in the insulin-producing cell line INS-1, we performed RT–PCR analysis employing primers that flank the insertion region. As shown in Figure 1A, INS-1 cells express both isoforms (lane 2) of CSP, whereas pancreatic islets contain only CSP1 (lanes 3 and 4). Therefore, we selected the CSP1 isoform for overexpression studies. Figure 1.(A) Analysis of CSP mRNA isoforms by RT–PCR in rat cerebellum (lane 1), INS-1 cells (lane 2), rat pancreatic islets (lane 3) and ob/ob-mouse pancreatic islets (lane 4). CSP1 isoform corresponds to the 217 bp PCR product and CSP2 isoform to the 289 bp fragment. Lane M shows DNA length marker [pBluescriptII KS(+) digested with HpaII]. (B) Expression of CSP protein detected by immunoblotting using an antiserum to recombinant CSP in rat islets, islets from ob/ob mice, INS-1 cells and rat brain. Download figure Download PowerPoint CSP-immunoreactivity in rat pancreatic islets, INS-1 and mouse β-cells CSP protein was demonstrated by Western blotting in homogenates from rat pancreatic islets, ob/ob mouse β-cells and INS-1 cells (Figure 1B). Incubation of cryostat sections with an antiserum generated to CSP peptide or an antiserum generated to recombinant CSP protein gave identical labeling patterns in both pancreas and brain. In the pancreas, there was strong CSP-like immunoreactivity (CSP-LI) in many cells of the islets of Langerhans and also in nerve fibers and terminals present around blood vessels and extending into the islets (Figure 2A and B). Confocal laser microscopy revealed that CSP-LI was punctate within the cytoplasm of endocrine cells (Figure 2B). The exocrine pancreas demonstrated slightly weaker, punctate CSP-LI in the apical region of acinar cells (Figure 2C). Figure 2.(A–C) Immunofluorescence micrographs of sections of rat pancreas obtained via confocal laser microscopy after incubation with CSP peptide antiserum. Strong CSP-LI is present in the cytoplasm of cells in the islets of Langerhans and also in some nerve fibers and terminals within the islets (solid arrows) (A). At higher magnification (B), it can be seen that the immunofluorescence within the endocrine cells is punctate. Also note weak, punctate CSP-LI in the acinar cells of the exocrine pancreas (open arrow in A). Higher magnification of cells in the exocrine pancreas shows punctate CSP-LI accumulated in the apical region of the acinar cells and in the lumen (C). Bars = 100 μm. Download figure Download PowerPoint Incubation with non-immune serum gave no fluorescence in the rat pancreas (data not shown). Preabsorption of the CSP peptide antiserum with the corresponding immunogen (10−6 M) gave no immunostaining in exocrine or endocrine cells. Direct double-labeling immunofluorescence histochemistry of rat pancreatic islets demonstrated CSP-LI in insulin- (cf. Figure 3A with B), glucagon- (cf. Figure 3C with D) and somatostatin- (cf. Figure 3E with F) containing cells. Figure 3.Immunofluorescence micrographs of sections of rat pancreas obtained via confocal laser microscopy after direct double-labeling combining antiserum to CSP (A, C and E) with antiserum to insulin (B), glucagon (D) and somatostatin (F). Punctate CSP-LI is present in insulin-, glucagon- and somatostatin-containing cells (see arrows). Bar = 100 μm. Download figure Download PowerPoint Punctate CSP-LI was also demonstrated in cultured RINm5F cells (data not shown), INS-1 cells (Figure 4A) and in cells from ob/ob mouse β-cells (Figure 4B). Double-labeling of such cells showed extensive, but not complete, co-localization with insulin (Figure 4C). Figure 4.Immunofluorescence photographs obtained via confocal laser microscopy after incubation of INS-1 cells (A) and ob/ob mouse β-cells (B) with CSP antiserum and after double-labeling with insulin antiserum (C). Download figure Download PowerPoint Ultrastructural localization of CSP Immunoelectron microscopy, using an antiserum to recombinant CSP, revealed gold particles localized to the membrane area of secretory vesicles in virtually all endocrine cells of the islets of Langerhans (Figure 5A and B). Insulin was detected in several secretory vesicles. Acinar cells of the exocrine pancreas showed gold particles localized to the membrane area of the zymogen granules (Figure 5C). Localization of gold particles to other organelles of the exocrine pancreas was not apparent. Figure 5.Immunoelectron microscopy of sections from the endocrine (A and B) and exocrine (C) pancreas after incubation with an antiserum to recombinant CSP. The endocrine pancreas shows gold particles located to the membrane area of secretory vesicles (SV) [arrows in (A)]. (B) Enlargement of rectangle in (A). Insulin (INS) can be seen in some SV. The exocrine pancreas shows gold particles located to the membrane area of zymogen granules (ZG). ER, endoplasmic reticulum; M, mitochondrion; N, nucleus. Bars in (A) and (C) = 200 nm and bar in (B) = 100 nm. Download figure Download PowerPoint CSP proteins in subcellular fractions and tissue/cell homogenates The presence and subcellular localization of CSP protein were also examined in pancreas by subcellular fractionation and immunoblotting using antiserum to recombinant CSP. Subcellular fractions of insulinoma cells showed an enrichment of 34 and 36 kDa proteins in the granule fraction with a weak signal at 27 kDa (Figure 6). The membrane fraction showed an enrichment of a 27 kDa protein with a very weak signal at 36 and 34 kDa (Figure 6). The cytosol fraction demonstrated a 27 kDa protein (Figure 6). Homogenates of rat pancreas and liver showed a 36 kDa protein together with a very weakly stained 27 kDa protein (data not shown). Rat brain contained a strong protein band at 34 kDa and a weak band at 27 kDa (Figure 6). Higher molecular weight proteins that reacted with our antiserum to recombinant CSP were also detected. Proteins of 72/70 kDa were identified in all preparations except in brain where the molecular weight was 68 kDa (Figure 6). Figure 6.Expression of CSP protein detected by immunoblotting using an antiserum to recombinant CSP after subcellular fractionation of rat insulinoma tissue and rat brain. An equal amount of protein has been added to the gel. Proteins corresponding to the expected sizes of 72, 68, 36, 34 and 27 kDa are indicated by arrows. Download figure Download PowerPoint Effect of CSP overexpression and CSP antibodies on voltage-dependent Ca2+-channels The possible effects of CSP on voltage-dependent Ca2+-channel activity was tested using the whole-cell configuration of the patch–clamp technique. Overexpression of rat CSP1 in INS-1 cells led to an ∼400-fold increase in CSP mRNA amounts (Figure 7A) and a 2.6-fold increase in CSP protein levels (Figure 7B). Immunohistochemical analysis of CSP1 overexpressing cells compared with mock-transfected cells did not reveal any obvious differences in either CSP distribution or co-localization with insulin (Figure 7C). Figure 7.(A) RNase protection analysis of CSP and β-actin mRNA in CSP1-overexpressing INS-1 cells (lane 1) and mock-transfected cells (lane 2). (B) Western blot analysis of CSP protein levels in CSP-overexpressing cells (CSP) and mock-transfected cells (Mock). Equal amounts of protein have been used for gel electrophoresis. (C) Co-localization of CSP- and insulin-immunoreactivity in CSP1-overexpressing cells (CSP) [(a) and (c)] and mock-transfected cells (Mock) [(b) and (d)]. Bar = 10 μm. Overexpression of CSP1 results in a 400-fold increase in CSP1 mRNA and a 2.6-fold increase in CSP protein levels. The distribution of the overexpressed CSP protein and co-localization with insulin is similar to that in mock-transfected cells. Download figure Download PowerPoint To analyze the effect of CSP1 overexpression on the function of voltage-dependent Ca2+-channels at the single-cell level, we co-expressed CSP1 and green fluorescent protein (GFP) in INS-1 cells. Following identification of the positively transfected cells via GFP fluorescence (Figure 8A), the whole-cell configuration was established and membrane currents were recorded during voltage steps to membrane potentials between −60 and +50 mV, from a holding potential of −70 mV. Figure 8B shows the current–voltage (IV) relationship in INS-1 cells following overexpression of CSP1 (CSP1 and GFP, filled circles) or mock-transfection (GFP, open circles), using the lipofectamine transfection technique. No difference in Ca2+-channel activity between CSP1 and mock-transfected cells was obtained (Figure 8B). Example current traces from the descending phase of the IV-curve are shown in Figure 8B. Figure 8.(A) CSP1-overexpressing INS-1 cells identified by GFP fluorescence. Confocal image of a cell group reveals a single GFP-expressing cell. Phase-contrast of the cell group having undergone the transfection procedure. Overlay of the confocal and phase-contrast images shows distribution of GFP within the cell cluster. Bar = 10 μm. (B) Effects of CSP1 overexpression on voltage-dependent Ca2+-channel activity in INS-1 cells. Integrated Ca2+-currents were recorded during voltage steps (100 ms) to membrane potentials between −60 and +50 mV from a holding potential of −70 mV in INS-1 cells overexpressing CSP1 (open circles; n = 32) and mock-transfected INS-1 cells (filled circles; n = 29). Currents are corrected for cell size by dividing the results in each recording by cell capacitance. No difference in cell capacitance between mock (4.18 ± 0.27 pF; n = 18) and CSP1-transfected INS-1 cells (4.18 ± 0.25 pF; n = 34) could be seen. Data are expressed as mean ± SEM. Examples of whole-cell Ca2+-current traces obtained during 100 ms depolarizations from a holding potential of −70 to +10 mV (10 mV steps) in INS-1 cells overexpressing CSP1 (left) and in mock-transfected (right) INS-1 cells. (C) Identification of cells that accumulate Cy3-conjugated secondary antibodies. Cy3-conjugated secondary antibodies (red fluorescence) were perfused into individual mouse β-cells via the patch pipette using the whole-cell configuration and visualized via confocal microscopy. A group of mouse β-cells is seen in transmission microscopy. Overlay of the confocal and transmission images shows a single Cy3-fluorescent (red) cell. Bar = 10 μm. (D) Effects of CSP-antibodies on integrated Ca2+-currents in mouse β-cells. Membrane currents were recorded using the whole-cell configuration of the patch–clamp technique. Repetitive depolarizing voltage steps (100 ms) were applied to 0 mV from a holding potential of −70 mV. Test pulses were given every 20 s. The whole-cell configuration was established ∼1 min before starting the pulse protocol. Examples of current traces under control conditions and when CSP antibodies (dilution 1:100) were included in the pipette solution. The two top traces are from the same control cell (Ctr) and show the resulting current traces at 1 and 15 min after starting the pulse protocol. Below are corresponding traces in the presence of CSP antibodies (CSPab). This experimental procedure was repeated four times with identical results. Download figure Download PowerPoint In a second approach, we studied the effects of a CSP antiserum on voltage-dependent Ca2+-channel currents. Cy3-conjugated secondary antibodies were allowed to perfuse into individual cells via the patch pipette in order to verify accumulation of antibody in the cells (Figure 8C). Mouse β-cells were perfused with CSP antibodies (diluted 1:100) via the recording patch pipette. The cells were then depolarized for 100 ms, from −70 to 0 mV every 20 s. No effect of the CSP antibodies (Figure 8D, bottom traces) on depolarization-induced Ca2+-channel currents was obtained during a time period of 15 min, as compared with control cells perfused with mouse IgG (Figure 8D, top traces). Effect of CSP overexpression on single-cell insulin secretion To investigate the effects of CSP on β-cell secretory capacity, we applied an amperometric technique to study release of serotonin from INS-1 cells preloaded with 5-hydroxy-DL-tryptophan 5-HTP. It is well-established that 5-HTP is converted to serotonin in the β-cell and that serotonin is loaded into the secretory vesicles and co-secreted with insulin by exocytosis (Smith et al., 1995; Aspinwall et al., 1997). Again, CSP1-overexpressing cells were identified by co-expression of GFP. The secretory response to K+-stimulation was significantly decreased in cells overexpressing CSP1 (Figure 9A) as compared with mock-transfected cells (Figure 9B). The number of spikes decreased from 24.8 ± 3.6 (n = 6) in mock-transfected cells to 8.0 ± 1.1 (n = 8) in cells overexpressing CSP1 (P <0.001; Figure 9C). The area of current spikes (measured in coulombs) can be used to quantitate the moles of detected hormone or transmitter released per vesicle using Faraday's law (Wightman et al., 1991). No difference in mean spike area between CSP1 and mock-transfected cells could be demonstrated (Figure 9D), indicating that the quality of released serotonin was unaffected by CSP1 overexpression. Figure 9.Amperometric recordings from single, CSP1-transfected or mock-transfected, INS-1 cells preloaded with 5-HTP. (A) and (B) show typical examples of recordings following stimulation of CSP1-transfected (A) and mock-transfected (B) INS-1 cells with 25 mM KCl. Current spikes were recorded by a carbon fiber near the cell and each spike represents a single quantum of released catecholamine. The cells were perfused with 3 mM glucose and the arrows above the recordings indicate the application of KCl. Compiled data of number of spikes (C) and mean spike area (D) during 30 s periods under each experimental condition. Note that there is a significant decrease in number of spikes after CSP transfection as compared with mock-transfection. Data are presented as mean ± SEM. In (C), n = 4–7; in (D), a total number of 64 spikes in eight different recordings were analyzed in CSP1-transfected cells and 149 spikes in six different recordings following mock-transfection. (E) Example of spikes in CSP1- (top) and mock- (bottom) transfected INS-1 cells. Ten representative spikes from each group were superimposed
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