Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M510528200 on November 21, 2005

J. Biol. Chem., Vol. 281, Issue 9, 5522-5531, March 3, 2006
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
281/9/5522    most recent
M510528200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lajus, S.
Right arrow Articles by Lang, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lajus, S.
Right arrow Articles by Lang, J.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

{alpha}-Latrotoxin Induces Exocytosis by Inhibition of Voltage-dependent K+ Channels and by Stimulation of L-type Ca2+ Channels via Latrophilin in beta-Cells*

Sophie Lajus{ddagger}, Pierre Vacher§, Denise Huber, Mathilde Dubois{ddagger}, Marie-Noëlle Benassy{ddagger}, Yuri Ushkaryov||, and Jochen Lang{ddagger}1

From the {ddagger}Institut Européen de Chimie et Biologie, JE 2390 and §INSERM E347, 2 rue Robert Escarpit, 33607 Pessac/Bordeaux, France, the Centre Médical Universitaire, Université de Genève, 1211 Genève, Switzerland, and the ||Department of Biological Sciences, Imperial College London, London SW7 2AY, United Kingdom

Received for publication, September 26, 2005 , and in revised form, November 15, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The spider venom {alpha}-latrotoxin ({alpha}-LTX) induces massive exocytosis after binding to surface receptors, and its mechanism is not fully understood. We have investigated its action using toxin-sensitive MIN6 beta-cells, which express endogenously the {alpha}-LTX receptor latrophilin (LPH), and toxin-insensitive HIT-T15 beta-cells, which lack endogenous LPH. {alpha}-LTX evoked insulin exocytosis in HIT-T15 cells only upon expression of full-length LPH but not of LPH truncated after the first transmembrane domain (LPH-TD1). In HIT-T15 cells expressing full-length LPH and in native MIN6 cells, {alpha}-LTX first induced membrane depolarization by inhibition of repolarizing K+ channels followed by the appearance of Ca2+ transients. In a second phase, the toxin induced a large inward current and a prominent increase in intracellular calcium ([Ca2+]i) reflecting pore formation. Upon expression of LPH-TD1 in HIT-T15 cells just this second phase was observed. Moreover, the mutated toxin LTXN4C, which is devoid of pore formation, only evoked oscillations of membrane potential by reversible inhibition of iberiotoxin-sensitive K+ channels via phospholipase C, activated L-type Ca2+ channels independently from its effect on membrane potential, and induced an inositol 1,4,5-trisphosphate receptor-dependent release of intracellular calcium in MIN6 cells. The combined effects evoked transient increases in [Ca2+]i in these cells, which were sensitive to inhibitors of phospholipase C, protein kinase C, or L-type Ca2+ channels. The latter agents also reduced toxin-induced insulin exocytosis. In conclusion, {alpha}-LTX induces signaling distinct from pore formation via full-length LPH and phospholipase C to regulate physiologically important K+ and Ca2+ channels as novel targets of its secretory activity.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The black widow spider venom {alpha}-latrotoxin ({alpha}-LTX)2 induces massive exocytosis of synaptic vesicles and of large dense core vesicles. This property has been extensively exploited to investigate the molecular mechanisms underlying exocytosis (1, 2). Toxin action requires first the binding to a surface receptor, and three distinct receptors for {alpha}-LTX have been identified: the latrophilins (LPHs), which contain a large extracellular adhesion molecule domain and a C-terminal portion bearing the signature of G-protein-coupled receptors (3, 4), neurexin Ia and beta (2), and the receptor-like protein-tyrosine phosphatase {sigma} (5). It is generally accepted that the toxin inserts subsequently as a tetramer into membranes to form a stable, cation-permeable pore (6), and the ensuing Ca2+ influx plays a major role in the activation of exocytosis. Indeed, expression of a C-terminally truncated form of LPH lacking all except the first transmembrane domain is sufficient to establish toxin-induced pores leading to calcium influx in epithelial HEK293 cells and sensitization of exocytosis in chromaffin cells (7–9).

Although these findings indicate that receptor-mediated signal transduction is not required for the action of {alpha}-LTX, other observations suggest that pore-mediated Ca2+ influx is not sufficient to explain the action of the toxin. {alpha}-LTX sensitizes exocytosis to Ca2+ in chromaffin cells and in synaptosomes (10, 11). Moreover, a point mutated toxin increases exocytosis in the absence of ion fluxes through a toxin-induced pore (12). The intracellular actions provoked by {alpha}-LTX to induce exocytosis are not fully resolved, apart from pore-mediated Ca2+ influx. Depending on the system, they may implicate phospholipase C with subsequent activation of protein kinase C and of release of Ca2+ from the intracellular stores (10, 11, 13–15).

We have previously demonstrated that {alpha}-LTX receptors are also expressed on primary beta-cells and the toxin induces exocytosis of the peptide hormone insulin (16). Clonal beta-cells cell lines provide a useful model for toxin-induced exocytosis of large dense core vesicles, because they differentially express LPH: whereas high levels of LPH are found in the toxin-sensitive MIN6 cells, HIT-T15 cells express only very low amounts and are toxin-insensitive (16). This situation is clearly distinct from PC12, chromaffin, or HEK293 cells, which are toxin-sensitive (7–9). beta-Cells therefore provide a very suitable model and, moreover, ion channels, signal transduction events and the molecular mechanism underlying insulin exocytosis are relatively well characterized (17–19).

Using this model in combination with truncated or full-length receptors, we addressed the issue of receptor-mediated signaling and pore formation-dependent events in the action of latrotoxin on intracellular calcium, membrane conductances, and exocytosis. To obtain a better understanding of the underlying events, we also compared the effect of native {alpha}-LTX to the action of recombinant mutated latrotoxin, LTXN4C, which is devoid of pore formation (12). Our data demonstrate for the first time a regulation of K+ channels and of L-type Ca2+ channels by latrotoxin that is mediated by its receptor latrophilin via phospholipase C and contributes to the secretory effect.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Monoclonal anti-Myc and anti-HA antibodies were purified from culture medium of myeloma cells generously provided by Dr. K. Matter (Université de Genève), polyclonal anti-Myc antibodies were obtained from Research Diagnostics (Flanders, NJ). Anti-Cav1.2 and anti-Cav1.3 antibodies were obtained from Alomone (Jerusalem, Israel). {alpha}-LTX was prepared and iodinated in Dr. Ushkaryov's laboratory (12). Commercial preparations used (Calbiochem) gave qualitatively similar results. Recombinant LTXN4C was produced and purified as described previously (12). Protein kinase and phospholipase C inhibitors, A23187 [GenBank] , apamin, and iberiotoxin were from Calbiochem, calciseptine from Latoxan (Apt, France). The m9 subclone of MIN6 cells (20) was kindly provided by Dr. S. Seino (Chiba, Japan) and was used throughout this study. PC12 cells were generously provided by Dr. B. Rudkin (Ecole Nationale Supérieure, Lyon, France) and used between passages 2 and 9.

Molecular Cloning—The N-terminal HA-affinity epitope was obtained by inserting a sequence coding for HRQDLPGNDNSTAGNS between amino acids 24 and 25. To this end the adjacent sequences were amplified from full-length LPH using the primers pairs N1 (5'-GCGGTACCTGGATAGCGGTTTGACTC-3'; 5-GCGGTACCTGGATAGCGGTTTGACTC-3') and N2 (5'-CGGGATCCAGGGCATAACGTAGATACGG-3'; 5'-GGAATTCCCTGAGCCGGGCTGGAC-3'). The amplicons were subcloned into pKS+ at the restriction sites ClaI/KpnI and BamHI/EcoRI, respectively. Two synthetic oligonucleotides (5'-AATTACCTGCTGTACTGTTATCATTTCCCGGGAGATCTTGT-3'; 5'-CGACAAGATCTCCCGGGAAATGATAACAGTACAGCAGGT-3') were annealed and inserted into the EcoRI/ClaI sites. The construct was subsequently excised by restriction with HindIII and BamHI and subcloned into the HindIII/BamHI sites of LPH constructs. LPH-TD1 Myc (LPH1–890) was generated by PCR from LPH-HA using the primer 5'-GTGGTATATGATGGTGCC-3' and 5'-ATCGAATTCGTGGATGGTGTTGCGGTCGG-3'. The amplicon was digested with XhoI/EcoRI and subcloned into the corresponding sites of pcDNA3+ encoding an in-frame 3'-Myc tag and complemented by insertion of the 5'-portion of LPH into the HindIII/XhoI sites. To introduce a C-terminal Myc tag, a C-terminal fragment of LPH was amplified to replace the stop codon by an EcoRI site using the primers 5'-CGAATCCGGAGGATGTGG-3' and 5'-ATCGAATTCGAGACTAGTGACCAACTGC-3'. The amplicon was digested with KpnI/EcoRI, subcloned into the KpnI/EcoRI sites of pcDNA3+ encoding an in-frame 3'-Myc tag and complemented by insertion of the 5'-portion of LPH into the HindIII/KpnI sites. All sequences were verified by sequencing of both strands. The generation of LPH-TD1–7 and of LPH-TD1–5 has been described before (21). The fusion protein syt2-C2AB-eGFP was constructed by PCR amplification of syt2-C2AB (amino acids 101–422) and insertion in-frame into peGFP.

Cell Culture, Transient Transfection, Secretion, and Immunoblotting—Cell culture, transient transfection, and secretion assays for endogenous insulin or human C-peptide were performed as described previously (22, 23) using enzyme-linked immunosorbent assay. Human growth hormone was used as reporter gene in the case of MIN6m9 cells. To this end the open reading frame of human growth hormone was excised by EcoRI from the plasmid pMMTV-GH (Nichols Institute, San Clemente, CA) and inserted into the corresponding sites of pcDNA3.1+. In control transfections plasmids encoding for LPH or its truncated forms were replaced by peGFP. For immunoblotting, cells were detached (23), resuspended in cold phosphate-buffered saline with 1% Triton X-100, incubated for 30 min on ice, and disrupted by brief sonication. Proteins were solubilized in sample buffer for 30 min at 37 °C and separated on 8% SDS-PAGE. Protein blotting, antibody incubation, and detection were performed as described previously (23) except that liquid transfer was used (8 h, 150 mA).

Immunohistochemistry and Imaging—To observe surface labeling, cells were incubated with primary antibody at 4 °C on ice, washed and fixed for 5 min with 5% paraformaldehyde on ice before adding the second antibody. To observe intracellular labeling under the same conditions, primary antibodies were added after fixation. All procedures were carried out on ice. Imaging was performed using an inverted microscope (Nikon TD300 equipped with a Z-drive) coupled to a monochromator (Till Photonics) and appropriate emission filters. Images were recorded by a charge-coupled device camera (Micromax 1300Y HS, Roperts Scientific) using Metamorph software (Universal Imaging) and deblurred by deconvolution (Autodeblur, Universal Imaging). The same set-up was used for imaging of living cells. In this case cells on coverslips were kept in 1 ml Krebs-Ringer buffer (KRB) (23) supplemented with 0.05% BSA (KRB-BSA) at 37 °C on a heated stage during acquisition. Toxin in KRB-BSA was pressure ejected (5 p.s.i., 10 s) from a micropipette held at ~20 mm from the cell.

Carbon Fiber Amperometry—Pheochromocytoma PC12 cells (50,000 cells per 35-mm dish) were transfected for 8 h using Lipofectamine and assayed 48 h later. Prior to recording, cells were loaded for 1 h (1 mM dopamine, 1 mM ascorbic acid, pH 7.4, in culture medium) and washed twice. During amperometry cells were kept at ambient temperature (23 °C) in modified Ringer buffer (145 mM NaCl, 5 mM KCl, 1.3 mM MgCl2, 3 mM CaCl2, 20 mM Hepes, pH 7.4, 10 mM glucose) on the stage of the inverted microscope described above and cotransfected cells identified by fluorescence of eGFP. Cells were stimulated by a 20-s ejection at 5 p.s.i. from a micropipette mounted on an Eppendorf micro-injector (Femtojet) held at ~2 cell diameters (40 µm) from the cell to be recorded. Only single, round cells were used, and carbon fibers were positioned using a piezoelectric driver (PCS-1000, Burleigh Instruments). The micropipette and the carbon fiber (ProCFE, 5 µm, Axon Instruments) held at 700 mV were kept at approximately the same distance throughout experiments. Fibers were used only if the root mean square was <0.5 nA at 700 mV. Currents were recorded using an EPC9 at 2.5 kHz, low pass filtered at 1 kHz, and spikes were analyzed with a program generously provided by Dr. F. Borges (Universidad de La Laguna, Tenerife, Spain) (24).

Measurements of [Ca2+]i—HIT-T15 cells grown on coverslips were cotransfected with LPH/CIRL constructs and a plasmid expressing DsRed fluorescent protein to identify transfected cells. 72 h after transfection, cells were loaded with 13 mM of the fluorescent probe indo pentaacetoxymethyl ester (indo-1/AM, Sigma) in KRB (3 mM glucose) for 45 min at 37 °C, washed and maintained at room temperature in KRB (3 mM glucose and 0.05% BSA) before fluorescence measurements. MIN6 cells were handled identically except that transfection was omitted. [Ca2+]i measurements were carried out as already described (25). Test substances were applied in KRB (3 mM glucose and 0.05% BSA) through a "pouring" pipette with a tip opening of 10–20 mm and positioned at a distance of 50–100 mm from the cell. The absence of mechanical artifacts due to drug application was confirmed by applying external medium (KRB) to the cells.

Electrophysiological Recordings—The whole cell recording mode of the patch clamp technique was used as already described (25). Transiently transfected cells were identified as given above. KRB was used as standard extracellular solution contained, and the osmolality was adjusted to 300–310 mosM/kg with sucrose. The recording pipette was filled with an artificial intracellular saline containing (in mM): 150 potassium chloride, 2 MgCl2, 1.1 EGTA, and 5 HEPES (pH 7.3 ± 0.01 with KOH; osmolality: 290 mosM/kg). Drugs were applied as described for calcium measurements, and all experiments were performed at room temperature (20–22 °C). To isolate voltage-dependent Ca2+ currents, K+ currents were eliminated by replacing K+ gluconate used to formulate the intracellular electrolyte with isomolar N-methylglucamine gluconate. The solution was then buffered to pH 7.3 with HEPES-gluconate. Because Ca2+ currents were not always stable and often disappeared during whole cell recording, current-voltage (intensity-voltage) relations for calcium currents were constructed using cells that showed little or no rundown within 10–15 min after impalement. Results are expressed as mean ± S.E. Each experiment was repeated several times.

Toxin Binding—Cells were transiently transfected in 24 wells and detached 48 h later by the use of 10 mM EDTA in KRB at 37 °C. Cells were centrifuged (5 min, 2,000 x g, 4 °C) and resuspended in KRB containing 1.3 mM CaCl2 but no EGTA. Aliquots were kept for cell counting and protein determination. 200 ml of cell suspension was transferred to 1.5-ml Eppendorf tubes and binding initiated by addition of radioiodinated toxin (26) for 15 min at 37 °C. The binding was stopped by the addition of ice-cold buffer and immediate centrifugation at 10,000 x g for 5 min at 4 °C. The pellets were washed once with ice-cold KRB, centrifuged at 10,000 x g for 2 min at 4 °C, and counted for radioactivity in a {gamma}-counter. Nonspecific binding was determined in the presence of 50 nM native toxin.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Surface Expression of Full-length and C-terminally Truncated LPH Constructs—C-terminal truncations of LPH, which lack the domains homologous to G-protein-coupled receptors, have been reported to suffice for actions of {alpha}-LTX in several cell systems, although these constructs are not always efficiently expressed (7, 8, 21). In our attempt to test their function in toxin-mediated exocytosis of insulin-containing large dense core vesicles we transiently expressed full-length and truncated LPH in toxin-insensitive hamster HIT-T15, or in mouse MIN6 insulinoma cells, which express endogenous LPH and are toxin-sensitive (16). Three truncated constructs were made: LPH-TD1, terminated after the first transmembrane domain; LPH-TD1–5 (amino acids 1–1020), limited to five transmembrane domains; and LPH-TD1–7 (amino acids 1–1099), truncated after the last transmembrane domain.

We examined first their surface expression after transient transfection using constructs, which carry an HA tag at the extracellular N terminus and an Myc tag at their intracellular C termini (Fig. 1A). To identify surface expression, antibody binding to HA tags was conducted on ice prior to fixation. The results observed confirmed surface expression of LPH and LPH-TD1 in HIT-T15 cells as well as LPH-TD1 in MIN6 cells (Fig. 1B). Surface expression was also observed for LPH-TD1–7, whereas LPH-TD1–5 was visualized only in a small number of cells (data not shown). As shown in Fig. 1C, expression levels of total LPH and LPH-TD1 were comparable. The antibody used is directed against the N-terminal hemagglutinin epitope. Latrophilin is processed in the endoplasmic reticulum into two non-covalently bound subunits, N-terminal p120 and C-terminal p85 (25). Only the 120-kDa extracellular N-terminal form was detected on Western blots, which indicated that transiently expressed full-length LPH was completely cleaved in HIT-T15 cells.

To further quantify the expression, we analyzed binding of 125I-{alpha}-LTX to transiently expressed constructs in intact HIT-T15 cells (Fig. 1D). Similar affinities (Kd values of ~0.25 nM) were observed for LPH, LPH-TD1, LPH-TD1–7, and LPH-TD1–5, whereas only negligible binding was detected in control cells. Moreover, the amount of binding sites expressed was comparable for all constructs except for LPH-TD1–5. These observations indicated that LPH, LPH-TD1, and LPH-TD1–7 were efficiently expressed and bound the toxin with comparable characteristics in insulin-secreting cells. To assess whether these proteins are capable to mediate {alpha}-LTX-induced effects, we determined functional responses of transiently expressed LPH or its truncated forms first on exocytosis of large dense core vesicles.


Figure 1
View larger version (56K):
[in this window]
[in a new window]
  		FIGURE 1.
Full-length latrophilin and truncated latrophilin are expressed on the surface of HIT-T15 or MIN6 cells and bind {alpha}-LTX to comparable extent. A, scheme of constructs used (with N-terminal HA tags and C-terminal Myc tags). B, HIT-T15 or MIN6 cells were transiently transfected with plasmids encoding full-length LPH (LPH, LPH1–1468) or LPH-TD1 (LPH1–890). 48 h later, cells were immunostained at 4 °C with (PERM) or without permeabilization (NP) using anti-Myc (C terminus) or anti-HA (N terminus) antibodies. C, immunoblot of control (CON) or transiently transfected HIT-T15 cells (with indicated constructs) using an antibody directed against the N-terminal HA-epitope. Molecular masses are given in kDa, and p120 is indicated by an arrow. D, Scatchard analysis of binding of 125I-{alpha}-LTX to HIT-T15 cells transfected with plasmids encoding the following constructs: full-length LPH/CIRL ({blacksquare}), LPH lacking the intracellular C terminus (LPH-TD1–7; LPH1–1099, {triangleup}), LPH truncated after the fifth (LPH 1–5, LPH1–1020, {square}) or after the first transmembrane domain (LPH-TD1, {circ}). Mean values are given from three separate experiments. Inset: total binding (4 nM 125I-{alpha}-LTX, filled bars) and unspecific binding (in the presence of a 25-fold excess of unlabeled toxin, open bars).

 
Full-length LPH Mediates Exocytosis Induced by {alpha}-LTX or LTXN4C—Next we determined the effects of LPH and LPH-TD1 in {alpha}-LTX-induced exocytosis and transiently cotransfected these receptors in HIT-T15 cells with a plasmid coding for human prepro-insulin as reporter gene (Fig. 2A). Membrane depolarization by 48 mM KCl induced a 3- to 4-fold increase, which was not altered by expression of the different constructs. As expected, {alpha}-LTX up to 2 nM did not induce release of human C-peptide in control transfected HIT-T15 cells, whereas cells expressing full-length LPH increased human C-peptide release more potently than KCl to ~10-fold of basal secretion. The LPH construct LPH-TD1–7 behaved similarly though a slightly greater efficacy was constantly observed. Cells expressing LPH-TD1 demonstrated only a marginal increase in toxin-evoked hormone secretion (Fig. 2A). This observation stems from a large series of experiments done at different passage numbers. Similar results were obtained using LPH-TD1 devoid of HA or Myc tags (data not shown).


Figure 2
View larger version (33K):
[in this window]
[in a new window]
  		FIGURE 2.
Effect of native {alpha}-LTX or recombinant LTXN4C on secretion from HIT-T15 cells and MIN6 cells expressing LPH or its truncated forms. A, HIT-T15 cells were transiently cotransfected with indicated constructs (or control plasmid) and a plasmid encoding human preproinsulin. The release of human insulin C-peptide was measured in the absence or the presence of indicated concentrations of {alpha}-LTX (filled symbols) or in the presence of 48 mM KCl (open symbols). Values are expressed as percentage of release in the absence of toxin. n = 8–23 for each point. *, 2p < 0.05 as compared with control transfected cells. B, MIN6 cells were transiently cotransfected with indicated constructs and a plasmid encoding human growth hormone. The release of growth hormone was measured as in A. n = 3–8 for each point. *, 2p < 0.05 as compared with the absence of toxin;+, 2p < 0.05 as compared with control transfected cells. C, effect of recombinant LTXN4C on secretion from HIT-T15 cells transiently cotransfected with a plasmid encoding human prepro-insulin and with a control plasmid (open circles) or with a plasmid encoding LPH (closed circles). n = 3–6 for each point; *, as in B. D, effect of recombinant LTXN4C (closed circles) or KCl (48 mM, open circle) on secretion from native MIN6 cells. n = 3–6 for each point; *, as in B.

 
LPH-TD1 has been shown to suffice for {alpha}-LTX binding and induction of exocytosis in neuroendocrine PC12 cells (7, 8). One major difference between PC12 and HIT-T15 cells resides in the expression levels of endogenous LPH. For this reason we also tested the effect of LPH-TD1 expression in MIN6 cells, known to express endogenous LPH receptor (16) (Fig. 2B). In control cells, {alpha}-LTX increased the release of reporter gene product. Co-expression of full-length LPH sensitized secretion to {alpha}-LTX, and this effect was also apparent in the case of expression of LPH-TD1.

We also tested whether a mutant toxin, LTXN4C, can induce secretion, because this protein does not assemble in pore-forming tetramers (21). As shown in Fig. 2 (C and D), LTXN4C induced insulin secretion in the subnanomolar range. Again transient expression of LPH was required in HIT-T15 cells to observe an effect (Fig. 2C), whereas responses similar to those induced by 48 mM KCl were observed in native MIN6 cells (Fig. 2D).

Our LPH-TD1 construct differed by one amino acid from those truncated forms, which have been shown to enhance sensitivity to {alpha}-LTX in pheochromocytoma cell line PC12 cells (7, 8). To control further for expression and function of our construct we also measured exocytosis in PC12 cells. Exocytosis assessed by amperometry in non-transfected cells demonstrated a vigorous secretory response after application of 1 nM {alpha}-LTX (Fig. 3, A and C) but only a spurious response in the case of 0.1 nM toxin (concentrations in the pipette). We therefore used this concentration to test whether LPH or our LPH-TD1 construct may sensitize these cells to the effects of the toxin. Transient expression of either construct sensitized cells to the toxin to similar extent (Fig. 3, A and C). In addition, mutant toxin LTXN4C induced exocytosis albeit to a lesser extent than wild-type toxin (Fig. 3, B and C). This demonstrates that the construct used by us behaved similar to the ones previously reported in PC12 cells (7, 8) and that LPH-TD1 is indeed sufficient to sensitize exocytosis in cells expressing already endogenous LPH.


Figure 3
View larger version (30K):
[in this window]
[in a new window]
  		FIGURE 3.
Effect of {alpha}-LTX or recombinant LTXN4C on secretion from PC12 cells as determined by amperometry. A, representative traces of native cells stimulated with 1 or 0.1 nM {alpha}-LTX (concentration in pipette solution) and cells expressing either LPH or LPH-TD1 and exposed to 0.1 nM {alpha}-LTX. B, LTXN4C (1 nM) stimulates secretion in native cells. C, statistical evaluation of release parameters (15–721 spikes in 8–16 cells were analyzed for each condition; *, 2p < 0.05 as compared with control transfected cells).

 
The requirement of full-length LPH for {alpha}-LTX-induced secretion in HIT-T15 cells as well as the effects of LTXN4C in clonal beta-cells and PC12 cells clearly indicated the presence of signaling pathways distinct from pore-induced influx of calcium and other ions. We therefore investigated these putative pathways by examining first the effects of the toxins on [Ca2+]i.

Full-length LPH, but Not C-terminally Truncated LPH-TD1, Mediates {alpha}-LTX-induced Oscillations in [Ca2+]i—We first measured whether transient expression of the constructs may alter basal levels of [Ca2+]i. A number of HIT-T15 or MIN6 cells showed spontaneous fluctuations of varying amplitude and frequency in basal [Ca2+]i (active cells). These oscillations were completely blocked in Ca2+-free extracellular medium or in the presence of Ca2+ channel inhibitors. Others exhibited a stable [Ca2+]i (silent cells) over a prolonged time period. The values of intracellular calcium were comparable in MIN6 cells (119.5 ± 2.4 nM; n = 30), to native HIT-T15 cells (115.5 ± 2.4 nM, n = 14), or HIT-T15 cells expressing full-length LPH (115.3 ± 2.5 nM, n = 45) or LPH-TD1 (114.7 ± 2.4 nM, n = 21). Therefore expression of full-length or truncated LPH did not affect basal [Ca2+]i.

Application of 2 nM {alpha}-LTX to native HIT-T15 cells did not alter [Ca2+]i (n = 14, data not shown). HIT-T15 cells expressing full-length LPH exhibited an increase in [Ca2+]i upon exposure to 2 nM {alpha}-LTX (Fig. 4A), and two different patterns were observed. First, a transient effect, termed phase I, appeared (Fig. 4A). This phase is characterized by either the appearance of calcium spikes or an increase in calcium spikes amplitude and frequency in already active cells. It lasted from 40 to 550 s with a mean of 211 ± 162 s (n = 26). Second, a long-lasting increase was observed with a clear plateau-phase, termed phase II (Fig. 4A). This response was still present at the end of the recordings and lasted always more than 550 s (n = 17).


Figure 4
View larger version (22K):
[in this window]
[in a new window]
  		FIGURE 4.
Transiently expressed full-length LPH, but not C-terminally truncated LPH-TD1, mediates oscillatory and constant increase in [Ca2+]i upon exposure to {alpha}-LTX. Determination of cytosolic free calcium in individual HIT-T15 or MIN6 cells loaded with indo-1AM. {alpha}-LTX was used at 2 nM. Selected traces represent typical cases observed for each cell model: phase I (oscillatory pattern, Ca2+ spikes), phase II (monophasic pattern, long-lasting plateau), and phase I+II (phase I followed by phase II). A, HIT-T15 cells expressing full-length LPH. B, HIT-T15 cells expressing LPH-TD1. C, native MIN6 cells. D, effect of Ni2+ on {alpha}-LTX effects in MIN6 cells. E, effect of KCl (48 mM) on MIN6 cell and on HIT-T15 cells expressing LPH.

 
In cells responding by both phases (I+II) the extent of the [Ca2+]i rise above baseline (370 ± 12 nM, n = 13) was comparable to that induced by KCl-evoked depolarization (381.5 ± 26.5 nM, n = 7). In cells responding by type I or type II alone the amplitude of the response was lower (phase I alone: 130 ± 11.5 nM, n = 13; type II alone: 85 ± 8.5 nM, n = 4). 30 out of 43 HIT-T15 cells expressing LPH responded, and the absence of response may in part be due to the method employed to detect cotransfected cells (see "Experimental Procedures"). 13 cells showed only phase I, 4 only phase II, and 13 phase I and II.

In the case of transient expression of the LPH-TD1, phase I responses were never observed and 4 of 11 cells responded with phase II (Fig. 4B). The amplitude of the responses was comparable to that observed in HIT-T15 cells expressing LPH (phase II alone): 97.5 ± 8.5 nM (n = 4). Exposure to {alpha}-LTX increased [Ca2+]i also in MIN6 cells, which express endogenous LPH (Fig. 4C). All cells tested (18) responded to the toxin, and a large majority (12) demonstrated only the transient phase I response, whereas concomitant phase I and II were present in 6 cells (phase I: 191.5 ± 10 nM, n = 12; phase II: 336.5 ± 24 nM, n = 6).

In MIN6 cells nickel (Ni2+, 0.5 mM), a calcium channel inhibitor, completely abrogated the phase I response and inhibited partially phase II (Fig. 4D). Ni2+ also reduced both KCl- and {alpha}-LTX-evoked insulin release by 97.1 ± 8.3 and 92.3 ± 6.1%, respectively (n = 6). As a comparison, the effects of KCl (48 mM) are also given for HIT-T15 and MIN6 cells (Fig. 4E).

LTXN4C Increases [Ca2+]i in MIN6 Cells by Calcium Mobilization and by Influx Through L-type Voltage-dependent Ca2+ Channels—Our preceding observations suggested that the effect of {alpha}-LTX includes two distinct phases and that phase I differs from pore formation. We therefore examined the effects of LTXN4C in MIN6 cells, because this recombinant toxin should not induce pore formation. As shown in Fig. 5A, the mutated toxin provoked a sharp, transient rise in [Ca2+]i in all cells tested (332 ± 8 nM above baseline; n = 46). In stark contrast to the native toxin, LTXN4C never induced a plateau phase (compare Figs. 5A and 4C). We sometimes observed a slowly decreasing phase as shown in Fig. 5A (trace a; 14 out of 46 cells). In the absence of extracellular calcium [Ca2+]i still increased by 91 ± 13 nM in 15 out of 28 cells (see traces a and b). This suggests that LTXN4C is capable of inducing the mobilization of Ca2+ from intracellular stores.

To determine the implication of Ca2+ influx and mobilization we employed the L-type channel blocker calciseptine (27) and the inositol 1,4,5-trisphosphate-receptor antagonist xestospongin C (28). Both inhibited largely the rise in [Ca2+]i (Fig. 5A; calciseptine 28 ± 7 nM, n = 7; xestospongin C 88 ± 8 nM, n = 8). Changes in [Ca2+]i induced by {alpha}-LTX have been linked previously to the activation of phospholipase C (13). Phospholipase C can be inhibited in insulinoma cells by the compound U73122 [GenBank] (29, 30). Indeed, U73122 [GenBank] , but not its inactive stereoisomer U73343 [GenBank] abolished the increase in [Ca2+]i evoked by LTXN4C (32 ± 9 nM, n = 11 and 288 ± 10 nM, n = 6, respectively). We subsequently tested whether protein kinase C (PKC) may be implicated as a downstream target. Two reagents that inhibit PKC, BIS I and Gö 6983 (31, 32), considerably reduced the effect of LTXN4C on [Ca2+]i (56 ± 9 nM, n = 9 and 149 ± 40 nM, n = 14). Note that in the case of Gö 6983 we observed mainly shortening of the increase in [Ca2+]i in several cells (trace b, 4 out of 14 cells).

A rise in [Ca2+]i induces exocytosis, and binding of C2-domain-containing proteins, such as the cytosolic domain of synaptotagmin, to the plasma membrane is thought to be important for this process (33, 34). We employed this mechanism to compare further the effect of {alpha}-LTX and LTXN4C in living MIN6 cells. To this end MIN6 cells were transiently transfected with a plasmid expressing the two cytosolic C2-domains of synaptotagmin 2 linked to eGFP (syt2-C2AB-eGFP). As shown in Fig. 5B (upper panel), exposure of these cells to 1 nM {alpha}-LTX resulted in complete translocation to the plasma membrane, whereas in the case of LTXN4C the extent of translocation was less prominent (n = 4 for each condition). Analysis of the time course revealed that {alpha}-LTX provoked first a partial translocation followed by complete translocation of syt2-C2AB-eGFP. In contrast, LTXN4C induced only partial translocation with clear oscillatory patterns (Fig. 5B, lower panel).

Because inhibitors of L-type calcium channels and PKC abolished the effect of LTXN4C on [Ca2+]i, we asked whether these agents might altered toxin-induced insulin secretion. As expected, the Ca2+ channel blockers nifedipine and calciseptine reduced secretion subsequent to membrane depolarization by KCl (Fig. 5C, upper panel). The blockers also reduced secretion evoked by LTXN4C indicating the role of L-type channels in its action. Nifedipine did not alter hormone release induced by the calcium-ionophore A23187 [GenBank] underscoring the specificity of the inhibitors at the concentrations used. Similarly the PKC inhibitors Gö 6983, BIS I, and staurosporine reduced LTXN4C-evoked insulin secretion, although relatively high concentrations had to be used (Fig. 5C, lower panel). It has to be noted that these reagents increased KCl-evoked release in MIN6 cells already at concentrations of 1–5 mM (data not shown).


Figure 5
View larger version (36K):
[in this window]
[in a new window]
  		FIGURE 5.
LTXN4C evokes transient Ca2+-influx, Ca2+-release from intracellular stores, translocation of synaptotagmin C2AB-domains, and secretion in MIN6 cells via phospholipase C and PKC. A, determination of cytosolic free calcium (in nanomolar) in individual MIN6 cells loaded with indo-1. Native MIN6 cell were exposed to LTXN4C alone (0.2 nM; as indicated by horizontal bar) or in the presence of indicated agents (calciseptine, 1 µM; xestospongin C, 3.3 µM; U73122, 1 µM; U733343, 1 µM; Gö 6983, 5 µM; BIS I, 1 µM). In certain conditions two types of responses were observed as indicated by a and b. B, MIN6 cells were transiently transfected with the calcium-sensing C2AB domain of synaptotagmin 2 (linked to eGFP) and stimulated by 1 nM {alpha}-LTX or LTXN4C. Images were recorded before and during stimulation (upper panel) and translocation measured (lower panel). In C: upper panel, insulin secretion from MIN6 cells was stimulated by either KCl (35 mM), LTXN4C (0.3 nM), or the calcium ionophore A23187 (calcimycin, 10 µM) in the absence or presence of nifedipine or calciseptine. Lower panel, MIN6 cells were kept in KRB or stimulated with LTXN4C (0.3 nM) in the absence or presence of Gö 6983 (10 µM), BIS I (10 µM), or staurosporine (15 µM). n = 4–6 for each point. *, 2p < 0.05 as compared with the absence of channel blockers or PKC inhibitors.

 
{alpha}-LTX Induced Membrane Depolarization Distinct from Pore Formation and Inhibited K+ Currents in HIT-T15 Cells Expressing LPH—Because the toxin provoked oscillatory increases in [Ca2+]i and translocation of syt2-C2AB-eGFP, we investigated more closely its effects on membrane potential and the induction of ionic currents by patch-clamp recordings (Fig. 6). Immediately after establishment of whole cell recording, the mean-resting membrane potential of HIT/full-length LPH, HIT/LPH TD1, and MIN6 cells was –52 ± 2.8 mV (n = 11), –54 ± 1.9 mV (n = 11), and –56 ± 1.5 mV (n = 12), respectively.

As could be expected for excitable cells, HIT-T15 and MIN6 cells exhibit spontaneous action potentials of various amplitude and kinetics at rest (see Figs. 6A and 7A). Application of 48 mM K+ solution elicited a rapid and reversible membrane depolarization in HIT-T15 cells (Fig. 6A). In native HIT-T15, application of 2 nM {alpha}-LTX did not elicit any appreciable response (Fig. 6A). In contrast, in HIT-T15 cells expressing LPH, application of 2 nM {alpha}-LTX induced a slower but stronger membrane depolarization (45–60 mV) than KCl. Toxin effects on membrane potential established more slowly than the observed effects on [Ca2+]i, probably due to differences in recording conditions imposed here by the patch-clamp approach, including dilution of cytosolic components in the patch pipette. However, similar to responses in [Ca2+]i, two phases were clearly present (as indicated by vertical dashed lines). Action potentials were transiently elicited during the onset of the depolarization indicated as phase I in Fig. 6A. In 3 out of 4 cells, this membrane depolarization was followed by a transient membrane hyperpolarization of various amplitudes (from –5 to –30 mV). Then, membrane potential became depolarized and remained so for the rest of the recordings (phase II). In HIT-T15 cells expressing LPH-TD1, {alpha}-LTX (2 nM) elicited membrane depolarization, although of lower amplitude (10–20 mV), and membrane potential slowly recovered. In contrast, action potentials indicative of phase I were never recorded (Fig. 6A). Toxin-induced membrane depolarization was insensitive to diazoxide, excluding an involvement of the KATP channel (data not shown).

We investigated subsequently the time course in more depth in HIT-T15 cells expressing full-length LPH (Fig. 6B). To monitor membrane conductance and K+ current, respectively, hyperpolarizing and depolarizing voltage steps of constant amplitude were repeatedly applied (every 10 s, Fig. 6B, trace a). During an initial period {alpha}-LTX did not induce any significant change on steady-state current (Fig. 6B, trace a, phase I) but slowly reduced the K+ current evoked by electrical depolarization from –40 to +40 mV (Fig. 6B, trace b). The maximal effect was obtained 80 s after the beginning of toxin ejection. During this time no significant effect on basal membrane conductance could be detected (Fig. 6B, trace c). Second, 60–120 s after application of the toxin a huge inward current appeared (from 600 pA to >1 nA, Fig. 6B, trace a; phase II as indicated by vertical dashed line) and was associated with an increase in membrane conductance (from 1.1 ± 0.1 nS to 5.35 ± 0.2 nS, n = 7; Fig. 6B, trace c). This current slowly developed in stages and was not reversible within 15 min. It could be responsible for the phase II of the calcium response described above. Furthermore, the driving force (Vh – Erev) underlying the long-lasting inward current (I) response to the toxin was estimated to obtain an indication on the nature of the ions implicated in this phase II event. Using Ohm's law as applied to membranes (I = G(Vh – Erev), where Erev is reversal potential), the conductance (G) can be derived by measuring throughout the time course changes in current responses to brief hyperpolarizing commands (20 mV from a holding potential, Vh, of –40 mV). This in turn permitted repeated estimates of Erev. For example, the 24 estimates of the driving force calculated for the cell illustrated in Fig. 6B (trace d) yielded an average reverse potential Erev of +35 ± 2 mV. This value is consistent with the contribution of Ca2+ ions to this current.

Because K+ channels participate in the control of the membrane potential (31), we investigated the effect of {alpha}-LTX on these channels more closely (Fig. 6C). Cells were voltage-clamped at –40 mV, close to the mean resting membrane potential. Contamination of K+ current recordings with Na+ was avoided by the use of the Na+ channel blocker tetrodotoxin (2 mM) in the external solution. Under these conditions, voltage steps of increasing intensity elicited an outward current of increasing amplitude. This outward current was greatly reduced by external application of 48 mM KCl (data not shown) confirming that it represents a pathway for K+ efflux. In HIT-T15 cells expressing LPH, a prominent inhibition of outward currents was observed (Fig. 6C, upper panels) in response to {alpha}-LTX (~70%), which was observed over the whole voltage range (–40 to +60 mV). In contrast, in cells expressing LPH-TD1 (Fig. 6C, lower panels), {alpha}-LTX was ineffective on K+ currents elicited by depolarization from –40 to –10 mV, and it only slightly reduced K+ current (~30%) for higher potentials. These data are in agreement with the current-clamp and [Ca2+]i experiments showing that {alpha}-LTX produces phase I events only in HIT-T15 cells expressing full-length LPH.


Figure 6
View larger version (32K):
[in this window]
[in a new window]
  		FIGURE 6.
{alpha}-LTX-induced membrane depolarization correlates with inhibition of voltage-dependent K+ currents in HIT-T15 cells expressing LPH. A, effects of KCl (48 mM) or {alpha}-LTX (2 nM) on the membrane potential of native HIT-15 cells (HIT) or HIT-T15 cells expressing LPH or LPH-TD1. Recordings were obtained under zero current conditions (current clamp). The two different phases of{alpha}-LTX effects are indicated (I and II). B, time course of{alpha}-LTX effects on ion currents. A HIT-T15 cell expressing LPH was held at –40 mV and repetitive depolarizing steps (to +20 mV) and hyperpolarizing steps (to –60 mV) were applied to monitor voltage-dependent K+ current and membrane conductance, respectively. Separation between phase I and II is indicated by a vertical dotted line in each panel (set at the beginning of changes in steady-state current and conductance). Panel a, steady-state current trace. Baseline (horizontal dotted line) and voltage steps (vertical lines) are given. Panel b, plot of K+ current amplitude (I, pA) versus time. Panel c, plot of conductance (G, nS) versus time. Panel d, plot of reversal potential (Erev, mV) versus time. Calculated Erev (see text) is +35 mV. Recordings were obtained under zero current conditions (current clamp). In C: left panels, a delayed outward current was elicited during a sustained depolarizing voltage step from –40 mV to various test potentials (10-mV increment). Patch clamp recordings were carried out under control condition (CON) or during application of toxin ({alpha}-LTX, 2 nM). Right panels, amplitudes of the current responses at the end of voltage steps before (open squares, CON) and after the application of the toxin (filled squares, {alpha}-LTX) were plotted as a function of the step potential (I-V curves).

 
LTXN4C Induced Oscillation in Membrane Potential and Inhibited BK-type K+ Channels in MIN6 Cells via Phospholipase C—We next examined the behavior {alpha}-LTX and of LTXN4C in MIN6 cells. The K+ channel blocker TEA (5 mM) is capable to elicit oscillations of the membrane potential as shown in Fig. 7A. Application of wild-type {alpha}-LTX induced initially similar oscillations, and only later constant membrane depolarization was observed. The two events were clearly distinct and therefore again indicative for the presence of two phases. In stark contrast, the mutated toxin LTXN4C provoked only oscillations of the membrane potential of considerable amplitude (Fig. 7A).

Using the same protocol as described for Fig. 6B, we investigated the time course and compared it to other inhibitors of K+ channels. A run-down of the K+ outward current was evident under our experimental conditions when comparing the earliest and latest time points in Fig. 7B (trace a). LTXN4C produces a pronounced and transient inhibition of the K+ outward current from 455 ± 24 to 98 ± 8 pA(n = 11). This was similar to the first phase observed in LPH-expressing HIT-15 cells upon exposure to {alpha}-LTX (see Fig. 6B). However, LTXN4C did not produce a second phase as demonstrated by the return to the base line (Fig. 7B, trace a). A similar effect was produced by iberiotoxin, a peptide inhibitor of BK-type K+ channels, whereas apamin, an inhibitor of SK-type K+ channels, was ineffective in MIN6 cells (Fig. 7B, trace b). Indeed, iberiotoxin reduced outward currents to 94 ± 10 pA (n = 10). Moreover, inhibition of BK-type K+ channels by iberiotoxin completely abolished the effect of LTXN4C (Fig. 7Bc; n = 5) indicating that the effects of LTXN4C and of iberiotoxin are mediated by the same type of channels. We next examined whether phospholipase C is implicated in the action of LTXN4C on K+ channels (Fig. 7B, trace c). Indeed, U73122 [GenBank] blocked the effect of the recombinant toxin on K+ currents, whereas the stereoisomer U73343 [GenBank] was ineffective. Therefore, regulation of K+ currents and of [Ca2+]i (see above) implied phospholipase C.

The inhibition of the voltage-dependent K+ outward current was further characterized in MIN6 cells (Fig. 7D) as before in HIT-T15 cells (see above). LTXN4C provoked a considerable inhibition of the currents and a right-shift of the I-V curve. We next asked whether inhibition of the K+ outward currents are per se sufficient to induce insulin secretion in MIN6 cells and may contribute to the secretory activity of the toxin. Indeed, TEA and iberiotoxin alone were capable to induce a significant increase in insulin secretion (Fig. 7E). TEA and iberiotoxin also augmented insulin secretion evoked by 35 mM KCl by 70.3 ± 8.8 and 95 ± 36%, respectively (n = 4).

LTXN4C Transiently Increases L-type Voltage-dependent Ca2+ Currents Independently from Its Action on Membrane Potential—The LTX-induced events on membrane potential are seemingly sufficient to induce opening of voltage-dependent Ca2+ channels and to provoke influx of the cation. To our surprise, LTXN4C had still an effect on calcium currents when MIN6 cells were clamped to 0 mV thus obliterating any contribution from LTX-induced changes in membrane potential (Fig. 8A). LTXN4C enhanced inward currents both at the beginning and throughout the applied pulse. This led to a downward shift of the I-V curve indicating that the effect was not voltage-dependent (Fig. 8B). Note that this increase is considerable and amounts to some 50%. Time-course experiments using voltage jumps, shown in Fig. 8C, demonstrate the transient nature of the effect induced by LTXN4C as its application is accompanied by a short increase in Ca2+ currents. Insulin-secreting cells express a variety of voltage-dependent Ca2+ channels. In addition to the well established role of L-type channels, R-type channels may also control insulin exocytosis to a certain extent (35). To further determine the nature of the Ca2+-current involved, we employed the L-type-specific blocker calciseptine, which abolished oscillations in Ca2+ and in insulin secretion evoked by LTXN4C (Fig. 5, A and C). Calciseptine completely blocked the voltage-evoked inward Ca2+ current and abrogated the effect of LTXN4C (Fig. 8D). This blockade of calcium currents and LTXN4C effects was observed throughout the time course (Fig. 8E).


Figure 7
View larger version (61K):
[in this window]
[in a new window]
  		FIGURE 7.
LTXN4C induces oscillations in membrane potential and inhibits iberiotoxin-sensitive K+ currents in MIN6 cells via phospholipase C. A, effects of TEA (5 mM), {alpha}-LTX (2 nM), or LTXN4C (0.2 nM) on the membrane potential of native MIN6 cells. Applications of toxins or drugs are indicated by horizontal bars. Recordings were obtained under zero current conditions (current clamp), baselines (dotted lines) and phases (I and II) are indicated. B, time course of toxin effects on K+ currents. The cells were held at –40 mV, and depolarizing commands (450 ms/100 mV) were repeatedly applied to evoke outward K+ currents. Panel a, LTXN4C (0.1 nM) reduced K+ current amplitude (arrow) from 455 ± 24 to 98 ± 8 pA (n = 11). The decline in the baseline is indicated by a dotted line. Panel b, iberiotoxin (arrows, 50 nM), an inhibitor of BK K+ channels, reduced K+ current amplitude to 94 ± 10 pA (n = 10), whereas apamin (100 nM), an inhibitor of SK K+ channels, was ineffective. Panel c, in the presence of iberiotoxin (50 nM), LTXN4C (0.1 nM) did not induce any further inhibition (n = 5). Panel d, the phospholipase inhibitor U73122 but not the inactive stereoisomer U73343 (both at 1 mM) inhibited LTXN4C effects on K+ current amplitude (U73122: 379 ± 34 pA, n = 9; U73343: 122 ± 15 pA, n = 8). In C: upper panels, a delayed outward current was elicited during a sustained depolarizing voltage step from –40 mV to various test potentials (10-mV increments). Patch clamp recordings were carried out under control condition (CON) and during application of toxin (LTXN4C, 0.1 nM). Lower panel, the amplitudes of the current responses at the end of voltage steps before (open squares) and after application of LTXN4C (filled squares) were plotted as a function of the step potential (I-V curves). D, insulin secretion from MIN6 cells was increased by KCl (35 mM), LTXN4C (0.3 nM), iberiotoxin (0.2 nM), or TEA (5 mM). n = 6 for each point; *, 2p < 0.05 as compared with KRB.

 
The molecular identity of L-type Ca2+ channels involved in insulin secretion is a matter of debate and may be formed by Cav1.2 ({alpha}1C) or Cav1.3 ({alpha}1D) (36). As the expression pattern has not been established in the MIN6 subclone used here (m9), we have performed immunoblots using isoform-specific antibodies. As shown in Fig. 8F, both proteins are expressed and may be regulated by LTXN4C.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Pancreatic beta-cells secrete the peptide hormone insulin by exocytosis and require a machinery similar to that of neuroendocrine cells (17). Our analysis of {alpha}-LTX action in these cells has now revealed previously unidentified and physiologically important targets, i.e. K+ channels and L-type Ca2+ channels. Their regulation required the G-protein-coupling moiety of latrophilin and stimulates exocytosis.


Figure 8
View larger version (32K):
[in this window]
[in a new window]
  		FIGURE 8.
LTXN4C transiently increases L-type voltage-dependent Ca2+ currents in MIN6 cells independently from toxin-induced membrane depolarization. A, a cell was held at –40 mV and depolarizing commands (450 ms/40 mV) were applied to evoke inward Ca2+ currents. LTXN4C (0.2 nM; filled symbols) enhances the inward current at the beginning (Ipeak, circles) and at the end (Isteady state, squares) of the pulse. B, current-voltage (I-V) relationships for the cell shown in A of steady-state Ca2+ currents under control conditions (CON, open squares) and in the presence of 0.2 nM LTXN4C (filled squares). C, in another cell, we have repeatedly applied the same voltage jump (from –40 to 0 mV, every 20 s) to determine the time course of the LTXN4C (0.2 nM) effects. Note the transient increase of the inward current (arrow). D, as previously, a cell was held at –40 mV and depolarizing commands (450 ms/40 mV) were applied to elicit inward Ca2+ currents. Calciseptine (1 mM), an inhibitor of the L-type Ca2+ channel, completely and irreversibly blocked the inward current, and 2 nM LTXN4C was no longer capable of affecting this current. E, time course (as described in B) of the responses to calciseptine (1 mM) and LTXN4C (0.2 nM) of the cell shown in D. F, expression of Cav1.2 and Cav1.3. Post-nuclear supernatants from rat brain (RB, 20 mg/lane), MIN6 (60 mg/lane), or HIT-T15 cells (60 mg/lane) were separated by SDS-PAGE, blotted, and incubated with corresponding antibodies. Molecular masses are given by bars (200 and 150 kDa). Preincubation of antibodies with the antigenic peptides completely abolished the immunoreactivity (data not shown).

 
Various ionic events have been observed previously in native endocrine and neuroendocrine cells after application of {alpha}-LTX, including "spikes" and "plateaus" in primary chromaffin and beta-cells, but they were mainly interpreted in terms of pore formation (9, 37, 38). We have now been able to clearly define two distinct phases by the use of different cell lines, receptor constructs, and wild-type or mutated toxin. In phase I oscillatory membrane depolarizations appear as well as calcium spikes. In contrast, during phase II a large and mixed transmembrane conductance appeared representing known pore formation (39) and provoked a sustained increase in [Ca2+]i as had been reported for secretory and non-secretory cells (7, 9, 40). Several observations demonstrated that phase I was independent from pore formation. First, this phase required the full-length receptor and was linked to the inhibition of voltage-dependent K+ fluxes. The inhibition of these K+ fluxes cannot be explained by pore formation, because it occurred prior to them (see Fig. 6B). Moreover, toxin-induced pores are known to be poorly selective and should induce increases but not reduction in ion fluxes (41). Most notably, phase I did not exhibit any significant effect on steady-state current or basal membrane conductance, which would have been indicative of pore formation. Finally, LTXN4C only induced phase I effects; they were of transient nature and sensitive to an inhibitor of phospholipase C.

Further support for a two-step event is given by observations on {alpha}-LTX-induced exocytosis of glutamate from synaptosomes that can also be divided into two phases (11). An initial, receptor- and phosphorylation-dependent secretion of neurotransmitters is followed by release depending on pore formation. Therefore, not only mutant, but also wild-type toxin activates specific signaling pathways distinct from pore formation in neuronal and endocrine cells.

The specific phase I events required full-length latrophilin, including its G-protein coupling domains in HIT-T15 cells that are naturally toxin-insensitive. LPH-TD1 did not suffice to support efficiently toxin-induced stimulation of insulin exocytosis despite increases in [Ca2+]i. We do not think that reduced increases in [Ca2+]i in LPH-TD1-expressing cells (as compared with LPH-expressing ones, see Fig. 4) explain the absence of measurable exocytosis. Although changes in [Ca2+]i were observed only in one-third of the cells expressing LPH-TD1, their amplitude corresponded approximately to those induced by KCl over the stimulation period. Ca2+ channels and exocytotic sites are coupled in beta-cells (42), and it is feasible that pores induced during phase II in HIT-T15 cells expressing LPH-TD1 may not be coordinated with release sites.

Previous reports suggested that expression of the extracellular domain of LPH (LPH-TD1), if anchored to the plasma membrane, is sufficient for toxin-induced increases in [Ca2+]i or enhancement of exocytosis (4, 7, 8, 43). In contrast to HIT-T15 cells, the cellular systems used in these reports (PC12, HEK293, or chromaffin cells) express endogenous LPH and are sensitive to the toxin (7, 10, 44). Similar to those observations we have observed enhancement of {alpha}-LTX effects upon overexpression of our LPH-TD1 construct in cells that harbor endogenous LPH, such as PC12 and MIN6 cells. However, this was clearly not the case in HIT-T15 cells that do not contain functional endogenous toxin receptors. This suggests complementation between endogenous full-length and transiently expressed truncated receptor as the base for LPH-TD1 effects. Indeed, G-protein-coupled receptors form oligomers, and truncated receptors can be salvaged by interaction with full-length receptors (45). Cooperativity is also indicated by co-immunoprecipitation of different toxin receptors provided that {alpha}-LTX is present (5). Moreover, toxin-induced direct interactions have been reported between the extracellular N-terminal portion of LPH, as generated here by LPH-TD1, and its C-terminal membrane-spanning portions (15). Our data also suggest that neurexins may not compensate for truncations in LPH as HIT-T15 cells, but not MIN6 cells, express considerable levels of endogenous neurexin I{alpha} and Ibeta (16).

Our data demonstrate for the first time that two types of ion channels are regulated as downstream effectors by binding of toxin to latrophilin: inward-rectifying K+ channels and voltage-dependant Ca2+ channels (VDCCs). As inhibition of phospholipase C abolished membrane depolarization and increases in [Ca2+]i, the following sequence of events can be established. Activation of phospholipase C leads to closure of inward-rectifying K+ channels, and the ensuing membrane depolarization will induce opening of VDCCs followed by Ca2+ influx. In addition, toxin binding to LPH further stimulated VDCCs independently from its action on membrane depolarization. Clearly, combining both effects will considerable enhance the efficiency of the toxin in terms of exocytosis.

Pharmacological approaches using specific agents identify the VDCC as L-type, which constitutes the major but not only VDCC in beta-cells in terms of exocytosis (35, 36). It is currently still a matter of debate whether Cav1.2 or Cav1.3 constitutes the major molecular form implicated in insulin secretion (46). Similar to primary cells, HIT-T15 and MIN6 cells express Cav1.2 and Cav1.3 channels (see Ref. 36 and our study). Thus, both may be targets of LPH-mediated signaling, although Cav1.3 seems to be expressed in these cells to a greater extent.

The observed increase in [Ca2+]i required the activation of protein kinase C according to pharmacological criteria. L-type VDDCs are subject to phosphorylation by PKC subsequent to the activation of receptors coupled to the G-protein Gq, and the functional outcome depends on the splice variant present (47, 48). Such a mechanism could eventually underlie the stimulation of L-type VDDCs by LTXN4C observed here, although other pathways may apply (36). In beta-cells an intriguing parallel to LPH-mediated signaling is given by the recent description on fatty-acid-induced insulin secretion via the G-protein-coupled receptor GPR40 (29). Similar to LPH, GPR40 regulates phospholipase C and L-type VDCCs.

Different K+ currents with specific characteristics and roles at distinct stages modulate secretion in beta-cells, mainly the ATP-sensitive KATP, the voltage-dependent KV and the calcium- and voltage sensitive KCa (49). Glucose metabolism alters the ATP/ADP ratio, which leads to the closure of KATP channels, membrane depolarization, Ca2+ influx via VDCCs, and insulin exocytosis (18). Repolarization of beta-cells implies mainly voltage-dependent (Kv) and perhaps Ca2+-activated KCa channels (50, 51). Our observations of voltage-dependent effects and their resistance to diazoxide exclude the KATP channel as target. Genetic or pharmacological inhibition of Kv leads to increased stimulated insulin secretion in primary, HIT-T15, and MIN6 cells (52, 53). In addition, HIT-T15 and primary cells express functional voltage-dependent Ca2+-activated KCa channels (54). As demonstrated here for MIN6 cells by the use of iberiotoxin and apamin, these cells express the BK-, but not the SK-type of these KCa channels. The observed inhibition of latrotoxin effects on K+ currents by iberiotoxin suggests that latrotoxin specifically inhibits BK-type channels. Moreover, inhibition of repolarization by BK channels using iberiotoxin was sufficient to induce insulin exocytosis. Regulation of BK channels by {alpha}-LTX therefore contributes to the stimulation of insulin exocytosis and may well explain the enhancement of depolarization-induced secretion by the native toxin in chromaffin and primary beta-cells (37, 38, 55).

Using pharmacological approaches and taking advantage of the mutant toxin LTXN4C we have in part delineated the downstream signaling of latrophilin in clonal beta-cells. The increase in [Ca2+]i depends on activation of phospholipase C, and similar pathways have been described previously in synaptosomes, in neurons, in neuroblastoma cells transiently expressing latrophilin, as well as in the case of the recently identified latrophilin-like orthologue in Caenorhabditis elegans (12, 13, 15, 56). In concordance with recordings from pyramidal neurons, the observed release from intracellular stores in MIN6 cells implied the inositol 1,4,5-trisphosphate receptor (13). Latrotoxin-induced inhibition of K+ channels or Ca2+ influx through VDCCs has not been reported in these neuronal preparations. L-type VDDCs are of physiological importance in neurons, however, they are not directly linked to neuroexocytosis in contrast to exocytosis in endocrine cells (57). As far as BK channels are concerned, their potential role in neuroexocytosis is not fully established (58, 59).

Inhibition of calcium influx only partially inhibited toxin-induced exocytosis of insulin. This suggests that LPH-mediated stimulation implies downstream targets in addition to ion channels. Indeed, subnanomolar concentrations of {alpha}-LTX stimulate exocytosis even in the absence of changes in [Ca2+]i (60) and on top of Ca2+ in permeabilized cells (10). Protein kinases A and C are known to augment exocytosis by increasing the size of the releasable pool of vesicles in insulin-secreting cells (61). A most recent detailed analysis demonstrated that both {alpha}-LTX and LTXN4C increase exocytosis in beta-cells by sensitizing the release process to calcium via PKC (62).

Collectively, our data support the view that {alpha}-LTX activates exocytosis via latrophilin employing specific signaling mechanisms in addition to formation of a cation-permeable pore. Most importantly, the receptor-mediated mechanisms are mediated by phospholipase C and impinge on targets relevant for the physiological regulation of insulin exocytosis, namely repolarizing K+ currents and L-type calcium channels.


   FOOTNOTES
 
* This work was supported by the Region of Aquitaine and the University of Bordeaux I (Grants BQR2003 and 2004). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Tel.: 33-540-00-3349; Fax: 33-540-00-3348; E-mail: j.lang{at}iecb.u-bordeaux.fr.

2 The abbreviations used are: {alpha}-LTX, {alpha}-latrotoxin; LTXN4C, recombinant mutated {alpha}-LTX; BSA, bovine serum albumin; COS, African green monkey kidney cells; DMEM, Dulbecco's modified Eagle's medium; HEK293, human embryonic kidney 293 cells; HIT-T15, hamster insulinoma cells; KATP, ATP-dependent K+ currents; Kv, voltage-dependent K+ currents; KCa, calcium- and voltage-dependent K+ currents, KRB, Krebs-Ringer buffer; LPH, latrophilin; MIN6, mouse insulinoma cells; PC12, rat pheochromocytoma cells; PKC, protein kinase C; TEA, tetraethylammonium; VDCC, voltage-dependent Ca2+ channel; GFP, green fluorescent protein; eGFP, enhanced GFP; BK, large conductance Ca2+- and voltage-activated K+ channel; SK, small conductance Ca2+- and voltage-activated K+ channel. Back


   ACKNOWLEDGMENTS
 
We are grateful to Dr. R. D. Burgoyne (University of Liverpool), Dr. A. Kuhn, and Dr. N. Sojic (Ecole Nationale Supérieure de Chimie et Physique de Bordeaux, Bordeaux) for their help in installing the amperometry and to Drs. O. Nuesse and N. Moustaid-Moussa for helpful comments on the manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Ushkaryov, Y. A., Volynski, K. E., and Ashton, A. C. (2004) Toxicon 43, 527–542[Medline] [Order article via Infotrieve]
  2. Südhof, T. C. (2001) Annu. Rev. Neurosci. 24, 933–962[CrossRef][Medline] [Order article via Infotrieve]
  3. Lelianova, V. G., Davletov, B. A., Sterling, A., Rahman, M. A., Grishin, E. V., Totty, N. F., and Ushkaryov, Y. A. (1997) J. Biol. Chem. 272, 21504–21508[Abstract/Free Full Text]
  4. Krasnoperov, V. G., Bittner, M. A., Beavis, R., Kuang, Y., Salnikow, K. V., Chepurny, O. G., Little, A. R., Plotnikov, A. N., Wu, D., Holz, R. W., and Petrenko, A. G. (1997) Neuron 18, 925–937[CrossRef][Medline] [Order article via Infotrieve]
  5. Krasnoperov, V., Bittner, M. A., Mo, W., Buryanovsky, L., Neubert, T. A., Holz, R. W., Ichtchenko, K., and Petrenko, A. G. (2002) J. Biol. Chem. 277, 35887–35895[Abstract/Free Full Text]
  6. Orlova, E. V., Rahman, M. A., Gowen, B., Volynski, K. E., Ashton, A. C., Manser, C., van Heel, M., and Ushkaryov, Y. A. (2000) Nat. Struct. Biol. 7, 48–53[CrossRef][Medline] [Order article via Infotrieve]
  7. Krasnoperov, V., Bittner, M. A., Holz, R. W., Chepurny, O., and Petrenko, A. G. (1999) J. Biol. Chem. 274, 3590–3596[Abstract/Free Full Text]
  8. Sugita, S., Ichtchenko, K., Khvotchev, M., and Südhof, T. C. (1998) J. Biol. Chem. 273, 32715–32724[Abstract/Free Full Text]
  9. Hlubek, M. D., Stuenkel, E. L., Krasnoperov, V. G., Petrenko, A. G., and Holz, R. W. (2000) Mol. Pharmacol. 57, 519–528[Abstract/Free Full Text]
  10. Bittner, M. A., and Holz, R. W. (2000) J. Biol. Chem. 275, 25351–25357[Abstract/Free Full Text]
  11. Ashton, A. C., Volynski, K. E., Lelianova, V. G., Orlova, E. V., Van Renterghem, C., Canepari, M., Seagar, M., and Ushkaryov, Y. A. (2001) J. Biol. Chem. 276, 44695–44703[Abstract/Free Full Text]
  12. Volynski, K. E., Capogna, M., Ashton, A. C., Thomson, D., Orlova, E. V., Manser, C. F., Ribchester, R. R., and Ushkaryov, Y. A. (2003) J. Biol. Chem. 278, 31058–31066[Abstract/Free Full Text]
  13. Capogna, M., Volynski, K. E., Emptage, N. J., and Ushkaryov, Y. A. (2003) J. Neurosci. 23, 4044–4053[Abstract/Free Full Text]
  14. Tsang, C. W., Elrick, D. B., and Charlton, M. P. (2000) J. Neurosci. 20, 8685–8692[Abstract/Free Full Text]
  15. Volynski, K. E., Silva, J. P., Lelianova, V. G., Atiqur Rahman, M., Hopkins, C., and Ushkaryov, Y. A. (2004) EMBO J. 23, 4423–4433[CrossRef][Medline] [Order article via Infotrieve]
  16. Lang, J., Ushkaryov, Y., Grasso, A., and Wollheim, C. B. (1998) EMBO J. 17, 648–657[CrossRef][Medline] [Order article via Infotrieve]
  17. Lang, J. (1999) Eur. J. Biochem. 259, 3–17[Medline] [Order article via Infotrieve]
  18. Rorsman, P. (1997) Diabetologia 40, 487–495[CrossRef][Medline] [Order article via Infotrieve]
  19. Henquin, J. (2000) Diabetes 49, 1751–1760[Abstract]
  20. Minami, K., Yano, H., Miki, T., Nagashima, K., Wang, C. Z., Tanaka, H., Miyazaki, J. I., and Seino, S. (2000) Am. J. Physiol. 279, E773–E781
  21. Volynski, K. E., Meunier, F. A., Lelianova, V. G., Dudina, E. E., Volkova, T. M., Rahman, M. A., Manser, C., Grishin, E. V., Dolly, J. O., Ashley, R. H., and Ushkaryov, Y. A. (2000) J. Biol. Chem. 275, 41175–41183[Abstract/Free Full Text]
  22. Zhang, H., Kelley, W. L., Chamberlain, L. H., Burgoyne, R. D., and Lang, J. (1999) J. Cell Sci. 112, 1345–1351[Abstract]
  23. Boal, F., Zhang, H., Tessier, C., Scotti, P. A., and Lang, J. (2004) Biochemistry 43, 16212–16223[CrossRef][Medline] [Order article via Infotrieve]
  24. Machado, J. D., Morales, A., Gomez, J. F., and Borges, R. (2001) Mol. Pharmacol. 60, 514–520[Abstract/Free Full Text]
  25. Sorin, B., Goupille, O., Vacher, A. M., Paly, J., Djiane, J., and Vacher, P. (1998) J. Biol. Chem. 273, 28461–28469[Abstract/Free Full Text]
  26. Davletov, B. A., Shamotienko, O. G., Lelianova, V. G., Grishin, E. V., and Ushkaryov, Y. A. (1996) J. Biol. Chem. 271, 23239–23245[Abstract/Free Full Text]
  27. Sekine, N., Ullrich, S., Regazzi, R., Pralong, W. F., and Wollheim, C. B. (1996) Endocrinology 137, 1841–1850[Abstract]
  28. Tsuboi, T., da Silva Xavier, G., Holz, G. G., Jouaville, L. S., Thomas, A. P., and Rutter, G. A. (2003) Biochem. J. 369, 287–299[CrossRef][Medline] [Order article via Infotrieve]
  29. Fujiwara, K., Maekawa, F., and Yada, T. (2005) Am. J. Physiol. 289, E670–E677
  30. Sheu, L., Pasyk, E. A., Ji, J., Huang, X., Gao, X., Varoqueaux, F., Brose, N., and Gaisano, H. Y. (2003) J. Biol. Chem. 278, 27556–27563[Abstract/Free Full Text]
  31. Gschwendt, M., Dieterich, S., Rennecke, J., Kittstein, W., Mueller, H. J., and Johannes, F. J. (1996) FEBS Lett. 392, 77–80[CrossRef][Medline] [Order article via Infotrieve]
  32. Davies, S. P., Reddy, H., Caivano, M., and Cohen, P. (2000) Biochem. J. 351, 95–105[CrossRef][Medline] [Order article via Infotrieve]
  33. Lang, J., Fukuda, M., Zhang, H., Mikoshiba, K., and Wollheim, C. B. (1997) EMBO J. 16, 5837–5846[CrossRef][Medline] [Order article via Infotrieve]
  34. Gerber, S. H., and Sudhof, T. C. (2002) Diabetes 51, S3–11[Abstract/Free Full Text]
  35. Jing, X., Li, D. Q., Olofsson, C. S., Salehi, A., Surve, V. V., Caballero, J., Ivarsson, R., Lundquist, I., Pereverzev, A., Schneider, T., Rorsman, P., and Renstrom, E. (2005) J. Clin. Invest. 115, 146–154[CrossRef][Medline] [Order article via Infotrieve]
  36. Yang, S. N., and Berggren, P. O. (2005) Am. J. Physiol. 288, E16–E28
  37. Liu, J., and Misler, S. (1998) J. Neurosci. 18, 6113–6125[Abstract/Free Full Text]
  38. Silva, A. M., Liu-Gentry, J., Dickey, A. S., Barnett, D. W., and Misler, S. (2005) J. Physiol. (Lond.) 565, 783–799[Abstract/Free Full Text]
  39. Filippov, A. K., Tertishnikova, S. M., Alekseev, A. E., Tsurupa, G. P., Pashkov, V. N., and Grishin, E. V. (1994) Neuroscience 61, 179–189[CrossRef][Medline] [Order article via Infotrieve]
  40. Van Renterghem, C., Iborra, C., Martin-Moutot, N., Lelianova, V., Ushkaryov, Y., and Seagar, M. (2000) Eur. J. Neurosci. 12, 3953–3962[CrossRef][Medline] [Order article via Infotrieve]
  41. Hurlbut, W. P., Chieregatti, E., Valtorta, F., and Haimann, C. (1994) J. Membr. Biol. 138, 91–102[Medline] [Order article via Infotrieve]
  42. Wiser, O., Trus, M., Hernandez, A., Renstrom, E., Barg, S., Rorsman, P., and Atlas, D. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 248–253[Abstract/Free Full Text]
  43. Ichtchenko, K., Bittner, M. A., Krasnoperov, V., Little, A. R., Chepurny, O., Holz, R. W., and Petrenko, A. G. (1999) J. Biol. Chem. 274, 5491–5498[Abstract/Free Full Text]
  44. Grasso, A., Pelliccia, M., and Alema, S. (1982) Toxicon 20, 149–156[Medline] [Order article via Infotrieve]
  45. Terrillon, S., and Bouvier, M. (2004) EMBO Rep. 5, 30–34[CrossRef][Medline] [Order article via Infotrieve]
  46. Bourinet, E., Mangoni, M. E., and Nargeot, J. (2004) J. Clin. Invest. 113, 1382–1384[CrossRef][Medline] [Order article via Infotrieve]
  47. Weiss, S., Doan, T., Bernstein, K. E., and Dascal, N. (2004) J. Biol. Chem. 279, 12503–12510[Abstract/Free Full Text]
  48. Yang, L., Liu, G., Zakharov, S. I., Morrow, J. P., Rybin, V. O., Steinberg, S. F., and Marx, S. O. (2005) J. Biol. Chem. 280, 207–214[Abstract/Free Full Text]
  49. Dukes, I. D., and Philipson, L. H. (1996) Diabetes 45, 845–853[Abstract]
  50. Henquin, J. C. (1990) Pflugers Arch. 416, 568–572[CrossRef][Medline] [Order article via Infotrieve]
  51. MacDonald, P. E., Wang, X., Xia, F., El-kholy, W., Targonsky, E. D., Tsushima, R. G., and Wheeler, M. B. (2003) J. Biol. Chem. 278, 52446–52453[Abstract/Free Full Text]
  52. MacDonald, P. E., Sewing, S., Wang, J., Joseph, J. W., Smukler, S. R., Sakellaropoulos, G., Saleh, M. C., Chan, C. B., Tsushima, R. G., Salapatek, A. M., and Wheeler, M. B. (2002) J. Biol. Chem. 277, 44938–44945[Abstract/Free Full Text]
  53. MacDonald, P. E., Ha, X. F., Wang, J., Smukler, S. R., Sun, A. M., Gaisano, H. Y., Salapatek, A. M., Backx, P. H., and Wheeler, M. B. (2001) Mol. Endocrinol. 15, 1423–1435[Abstract/Free Full Text]
  54. Eddlestone, G. T., Ribalet, B., and Ciani, S. (1989) J. Membr. Biol. 109, 123–134[CrossRef][Medline] [Order article via Infotrieve]
  55. Michelena, P., de la Fuente, M. T., Vega, T., Lara, B., Lopez, M. G., Gandia, L., and Garcia, A. G. (1997) J. Physiol. (Lond.) 502, 481–496[Abstract/Free Full Text]
  56. Willson, J., Amliwala, K., Davis, A., Cook, A., Cuttle, M. F., Kriek, N., Hopper, N. A., O'Connor, V., Harder, A., Walker, R. J., and Holden-Dye, L. (2004) Curr. Biol. 14, 1374–1379[CrossRef][Medline] [Order article via Infotrieve]
  57. Trimmer, J. S., and Rhodes, K. J. (2004) Annu. Rev. Physiol. 66, 477–519[CrossRef][Medline] [Order article via Infotrieve]
  58. Wang, Z. W., Saifee, O., Nonet, M. L., and Salkoff, L. (2001) Neuron 32, 867–881[CrossRef][Medline] [Order article via Infotrieve]
  59. Hu, H., Shao, L.-R., Chavoshy, S., Gu, N., Trieb, M., Behrens, R., Laake, P., Pongs, O., Knaus, H. G., Ottersen, O. P., and Storm, J. F. (2001) J. Neurosci. 21, 9585–9597[Abstract/Free Full Text]
  60. Tse, F. W., and Tse, A. (1999) Endocrinology 140, 3025–3033[Abstract/Free Full Text]
  61. Yang, Y., and Gillis, K. D. (2004) J. Gen. Physiol. 124, 641–651[Abstract/Free Full Text]
  62. Liu, J., Wan, Q., Lin, X., Zhu, H., Volynski, K., Ushkaryov, Y., and Xu, T. (2005) Traffic 6, 756–765[CrossRef][Medline] [Order article via Infotrieve]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 EndocrinologyHome page
M. Dubois, P. Vacher, B. Roger, D. Huyghe, B. Vandewalle, J. Kerr-Conte, F. Pattou, N. Moustaid-Moussa, and J. Lang
Glucotoxicity Inhibits Late Steps of Insulin Exocytosis
Endocrinology, April 1, 2007; 148(4): 1605 - 1614.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
281/9/5522    most recent
M510528200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lajus, S.
Right arrow Articles by Lang, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lajus, S.
Right arrow Articles by Lang, J.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2006 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement