α-Latrotoxin Induces Exocytosis by Inhibition of Voltage-dependent K+ Channels and by Stimulation of L-type Ca2+ Channels via Latrophilin in β-Cells
2005; Elsevier BV; Volume: 281; Issue: 9 Linguagem: Inglês
10.1074/jbc.m510528200
ISSN1083-351X
AutoresSophie Lajus, Pierre Vacher, Denise Huber, Mathilde Dubois, Marie‐Noëlle Benassy, Yuri A. Ushkaryov, Jochen Lang,
Tópico(s)Erythrocyte Function and Pathophysiology
ResumoThe spider venom α-latrotoxin (α-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 β-cells, which express endogenously the α-LTX receptor latrophilin (LPH), and toxin-insensitive HIT-T15 β-cells, which lack endogenous LPH. α-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, α-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, α-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. The spider venom α-latrotoxin (α-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 β-cells, which express endogenously the α-LTX receptor latrophilin (LPH), and toxin-insensitive HIT-T15 β-cells, which lack endogenous LPH. α-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, α-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, α-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. The black widow spider venom α-latrotoxin (α-LTX) 2The abbreviations used are: α-LTX, α-latrotoxin; LTXN4C, recombinant mutated α-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. 2The abbreviations used are: α-LTX, α-latrotoxin; LTXN4C, recombinant mutated α-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. 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 (1Ushkaryov Y.A. Volynski K.E. Ashton A.C. Toxicon. 2004; 43: 527-542Crossref PubMed Scopus (108) Google Scholar, 2Südhof T.C. Annu. Rev. Neurosci. 2001; 24: 933-962Crossref PubMed Scopus (169) Google Scholar). Toxin action requires first the binding to a surface receptor, and three distinct receptors for α-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 (3Lelianova V.G. Davletov B.A. Sterling A. Rahman M.A. Grishin E.V. Totty N.F. Ushkaryov Y.A. J. Biol. Chem. 1997; 272: 21504-21508Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 4Krasnoperov 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. Petrenko A.G. Neuron. 1997; 18: 925-937Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar), neurexin Ia and β (2Südhof T.C. Annu. Rev. Neurosci. 2001; 24: 933-962Crossref PubMed Scopus (169) Google Scholar), and the receptor-like protein-tyrosine phosphatase σ (5Krasnoperov V. Bittner M.A. Mo W. Buryanovsky L. Neubert T.A. Holz R.W. Ichtchenko K. Petrenko A.G. J. Biol. Chem. 2002; 277: 35887-35895Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). It is generally accepted that the toxin inserts subsequently as a tetramer into membranes to form a stable, cation-permeable pore (6Orlova E.V. Rahman M.A. Gowen B. Volynski K.E. Ashton A.C. Manser C. van Heel M. Ushkaryov Y.A. Nat. Struct. Biol. 2000; 7: 48-53Crossref PubMed Scopus (121) Google Scholar), 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 (7Krasnoperov V. Bittner M.A. Holz R.W. Chepurny O. Petrenko A.G. J. Biol. Chem. 1999; 274: 3590-3596Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 8Sugita S. Ichtchenko K. Khvotchev M. Südhof T.C. J. Biol. Chem. 1998; 273: 32715-32724Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 9Hlubek M.D. Stuenkel E.L. Krasnoperov V.G. Petrenko A.G. Holz R.W. Mol. Pharmacol. 2000; 57: 519-528Crossref PubMed Scopus (36) Google Scholar). Although these findings indicate that receptor-mediated signal transduction is not required for the action of α-LTX, other observations suggest that pore-mediated Ca2+ influx is not sufficient to explain the action of the toxin. α-LTX sensitizes exocytosis to Ca2+ in chromaffin cells and in synaptosomes (10Bittner M.A. Holz R.W. J. Biol. Chem. 2000; 275: 25351-25357Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar, 11Ashton A.C. Volynski K.E. Lelianova V.G. Orlova E.V. Van Renterghem C. Canepari M. Seagar M. Ushkaryov Y.A. J. Biol. Chem. 2001; 276: 44695-44703Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Moreover, a point mutated toxin increases exocytosis in the absence of ion fluxes through a toxin-induced pore (12Volynski K.E. Capogna M. Ashton A.C. Thomson D. Orlova E.V. Manser C.F. Ribchester R.R. Ushkaryov Y.A. J. Biol. Chem. 2003; 278: 31058-31066Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). The intracellular actions provoked by α-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 (10Bittner M.A. Holz R.W. J. Biol. Chem. 2000; 275: 25351-25357Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar, 11Ashton A.C. Volynski K.E. Lelianova V.G. Orlova E.V. Van Renterghem C. Canepari M. Seagar M. Ushkaryov Y.A. J. Biol. Chem. 2001; 276: 44695-44703Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 13Capogna M. Volynski K.E. Emptage N.J. Ushkaryov Y.A. J. Neurosci. 2003; 23: 4044-4053Crossref PubMed Google Scholar, 14Tsang C.W. Elrick D.B. Charlton M.P. J. Neurosci. 2000; 20: 8685-8692Crossref PubMed Google Scholar, 15Volynski K.E. Silva J.P. Lelianova V.G. Atiqur Rahman M. Hopkins C. Ushkaryov Y.A. EMBO J. 2004; 23: 4423-4433Crossref PubMed Scopus (78) Google Scholar). We have previously demonstrated that α-LTX receptors are also expressed on primary β-cells and the toxin induces exocytosis of the peptide hormone insulin (16Lang J. Ushkaryov Y. Grasso A. Wollheim C.B. EMBO J. 1998; 17: 648-657Crossref PubMed Scopus (73) Google Scholar). Clonal β-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 (16Lang J. Ushkaryov Y. Grasso A. Wollheim C.B. EMBO J. 1998; 17: 648-657Crossref PubMed Scopus (73) Google Scholar). This situation is clearly distinct from PC12, chromaffin, or HEK293 cells, which are toxin-sensitive (7Krasnoperov V. Bittner M.A. Holz R.W. Chepurny O. Petrenko A.G. J. Biol. Chem. 1999; 274: 3590-3596Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 8Sugita S. Ichtchenko K. Khvotchev M. Südhof T.C. J. Biol. Chem. 1998; 273: 32715-32724Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 9Hlubek M.D. Stuenkel E.L. Krasnoperov V.G. Petrenko A.G. Holz R.W. Mol. Pharmacol. 2000; 57: 519-528Crossref PubMed Scopus (36) Google Scholar). β-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 (17Lang J. Eur. J. Biochem. 1999; 259: 3-17Crossref PubMed Scopus (280) Google Scholar, 18Rorsman P. Diabetologia. 1997; 40: 487-495Crossref PubMed Scopus (249) Google Scholar, 19Henquin J. Diabetes. 2000; 49: 1751-1760Crossref PubMed Scopus (939) Google Scholar). 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 α-LTX to the action of recombinant mutated latrotoxin, LTXN4C, which is devoid of pore formation (12Volynski K.E. Capogna M. Ashton A.C. Thomson D. Orlova E.V. Manser C.F. Ribchester R.R. Ushkaryov Y.A. J. Biol. Chem. 2003; 278: 31058-31066Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). 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. 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). α-LTX was prepared and iodinated in Dr. Ushkaryov’s laboratory (12Volynski K.E. Capogna M. Ashton A.C. Thomson D. Orlova E.V. Manser C.F. Ribchester R.R. Ushkaryov Y.A. J. Biol. Chem. 2003; 278: 31058-31066Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Commercial preparations used (Calbiochem) gave qualitatively similar results. Recombinant LTXN4C was produced and purified as described previously (12Volynski K.E. Capogna M. Ashton A.C. Thomson D. Orlova E.V. Manser C.F. Ribchester R.R. Ushkaryov Y.A. J. Biol. Chem. 2003; 278: 31058-31066Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Protein kinase and phospholipase C inhibitors, A23187, apamin, and iberiotoxin were from Calbiochem, calciseptine from Latoxan (Apt, France). The m9 subclone of MIN6 cells (20Minami K. Yano H. Miki T. Nagashima K. Wang C.Z. Tanaka H. Miyazaki J.I. Seino S. Am. J. Physiol. 2000; 279: E773-E781Crossref PubMed Google Scholar) 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 (21Volynski 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. Ushkaryov Y.A. J. Biol. Chem. 2000; 275: 41175-41183Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). 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 (22Zhang H. Kelley W.L. Chamberlain L.H. Burgoyne R.D. Lang J. J. Cell Sci. 1999; 112: 1345-1351Crossref PubMed Google Scholar, 23Boal F. Zhang H. Tessier C. Scotti P.A. Lang J. Biochemistry. 2004; 43: 16212-16223Crossref PubMed Scopus (30) Google Scholar) 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 (23Boal F. Zhang H. Tessier C. Scotti P.A. Lang J. Biochemistry. 2004; 43: 16212-16223Crossref PubMed Scopus (30) Google Scholar), 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 (23Boal F. Zhang H. Tessier C. Scotti P.A. Lang J. Biochemistry. 2004; 43: 16212-16223Crossref PubMed Scopus (30) Google Scholar) 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) (23Boal F. Zhang H. Tessier C. Scotti P.A. Lang J. Biochemistry. 2004; 43: 16212-16223Crossref PubMed Scopus (30) Google Scholar) 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) (24Machado J.D. Morales A. Gomez J.F. Borges R. Mol. Pharmacol. 2001; 60: 514-520PubMed Google Scholar). 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 (25Sorin B. Goupille O. Vacher A.M. Paly J. Djiane J. Vacher P. J. Biol. Chem. 1998; 273: 28461-28469Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). 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 (25Sorin B. Goupille O. Vacher A.M. Paly J. Djiane J. Vacher P. J. Biol. Chem. 1998; 273: 28461-28469Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). 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 × 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 (26Davletov B.A. Shamotienko O.G. Lelianova V.G. Grishin E.V. Ushkaryov Y.A. J. Biol. Chem. 1996; 271: 23239-23245Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar) for 15 min at 37 °C. The binding was stopped by the addition of ice-cold buffer and immediate centrifugation at 10,000 × g for 5 min at 4 °C. The pellets were washed once with ice-cold KRB, centrifuged at 10,000 × g for 2 min at 4 °C, and counted for radioactivity in a γ-counter. Nonspecific binding was determined in the presence of 50 nm native toxin. 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 α-LTX in several cell systems, although these constructs are not always efficiently expressed (7Krasnoperov V. Bittner M.A. Holz R.W. Chepurny O. Petrenko A.G. J. Biol. Chem. 1999; 274: 3590-3596Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 8Sugita S. Ichtchenko K. Khvotchev M. Südhof T.C. J. Biol. Chem. 1998; 273: 32715-32724Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 21Volynski 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. Ushkaryov Y.A. J. Biol. Chem. 2000; 275: 41175-41183Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). 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 (16Lang J. Ushkaryov Y. Grasso A. Wollheim C.B. EMBO J. 1998; 17: 648-657Crossref PubMed Scopus (73) Google Scholar). 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 (25Sorin B. Goupille O. Vacher A.M. Paly J. Djiane J. Vacher P. J. Biol. Chem. 1998; 273: 28461-28469Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). 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-α-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 α-LTX-induced effects, we determined functional responses of transiently expressed LPH or its truncated forms first on exocytosis of large dense core vesicles. Full-length LPH Mediates Exocytosis Induced by α-LTX or LTXN4C—Next we determined the effects of LPH and LPH-TD1 in α-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, α-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). LPH-TD1 has been shown to suffice for α-LTX binding and induction of exocytosis in neuroendocrine PC12 cells (7Krasnoperov V. Bittner M.A. Holz R.W. Chepurny O. Petrenko A.G. J. Biol. Chem. 1999; 274: 3590-3596Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 8Sugita S. Ichtchenko K. Khvotchev M. Südhof T.C. J. Biol. Chem. 1998; 273: 32715-32724Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). 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 (16Lang J. Ushkaryov Y. Grasso A. Wollheim C.B. EMBO J. 1998; 17: 648-657Crossref PubMed Scopus (73) Google Scholar) (Fig. 2B). In control cells, α-LTX increased the release of reporter gene product. Co-expression of full-length LPH sensitized secretion to α-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 (21Volynski 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. Ushkaryov Y.A. J. Biol. Chem. 2000; 275: 41175-41183Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). 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 α-LTX in pheochromocytoma cell line PC12 cells (7Krasnoperov V. Bittner M.A. Holz R.W. Chepurny
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