Introduction

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Since the demonstration that black widow spider venom and its active component -latrotoxin (Ltx) produce massive exocytosis at the frog neuromuscular junction (Longenecker et al., 1970; Frontali et al., 1976; Pumplin and Reese, 1977; Fesce et al., 1986), the toxin has been the focus of intense investigation. Ltx also causes release from a variety of other neurons and cells [for reviews see Meldolesi et al. (1986) and Surkova (1994)]. An early hypothesis for the action of Ltx was that the toxin itself produces divalent ion-permeable channels in the plasma membrane. This was based on the observation that Ltx inserts into artificial bilayers to form high conductance, divalent ion-permeable channels (Finkelstein et al., 1976). The flux of Ca²⁺ through such channels could stimulate exocytosis and thus contribute to the actions of Ltx. [At the neuromuscular junction, Mg²⁺ can substitute for Ca²⁺ to support Ltx-induced secretion (Misler and Hurlburt, 1979).] Indeed, Ltx produces channels in PC12 cells (Wanke et al., 1986), neuroblastoma cells (Hurlbut et al., 1994), and rat adrenal chromaffin cells (Barnett et al., 1996).

However, already in the early studies there was evidence that in biological membranes the toxin was not acting alone on the bilayer but interacting with specific receptors. Ltx was found to bind in a saturable manner with nanomolar affinity to synaptosomal membranes (Tzeng and Siekevitz, 1979; Rosenthal et al., 1990). The binding was both Ca²⁺-dependent and Ca²⁺-independent. These findings raised the possibility that receptors may contribute to the insertion or conductance properties of Ltx or by themselves form channels when the ligand bound. Recently, two families of high affinity Ltx receptors have been cloned. Calcium-independent receptor for -latrotoxin (CIRL), or latrophilin, binds Ltx in a Ca²⁺-independent manner (Krasnoperov et al., 1997; Lelianova et al., 1997), and neurexin 1 binds Ltx in a Ca²⁺-dependent manner (Petrenko et al., 1990). Both are able to support Ltx-induced, Ca²⁺-dependent secretion from chromaffin or PC12 cells (Krasnoperov et al., 1997; Bittner et al., 1998; Sugita et al., 1999).

We have focused on the function of CIRL in Ltx-induced secretion. The primary sequence of CIRL predicts a G-protein-coupled receptor with significant homology to members of the secretin receptor family. To date, three members of the CIRL family of receptors have been identified (Sugita et al., 1998; Krasnoperov et al., 1999). CIRL-1 and CIRL-3 are expressed primarily in brain, whereas CIRL-2 is ubiquitously expressed. No endogenous ligand or G-protein-activated effector has yet been identified for any of the CIRL receptors. Immunoblotting indicates that bovine chromaffin cells express CIRL-1 or a closely related protein (M. A. Bittner and R.W.H., submitted). Binding of toxin to the endogenous receptor occurs in the absence of Ca²⁺, and subsequent addition of Ca²⁺-containing medium results in Ca²⁺ influx and secretion. Overexpression of CIRL-1 by transient transfection increased 10-fold the sensitivity of chromaffin cells to the effects of Ltx. These observations raise the possibility that the Ltx-induced channels previously observed in rat chromaffin cells (Barnett et al., 1996) directly result from the interaction of Ltx with CIRL-1 or a closely related receptor. Alternatively, a recent report has suggested a mechanism whereby the interaction of Ltx with CIRL (Latrophilin) activates endogenous neuronal channels to elicit secretion (Davletov et al., 1998).

There are several key findings in this study that provide insight into the mechanism by which Ltx affects biological membranes. Using patch clamp techniques and microspectrofluorimetry with bovine chromaffin cells and HEK293 cells, we demonstrate that the interaction of Ltx with transiently expressed CIRL-1 results in a high conductance channel that permits a rise in cytosolic Ca²⁺. Channel formation can occur when CIRL-1 or neurexin 1 is expressed in a nonexcitable cell. Importantly, experiments with CIRL-1 mutants and neurexin 1 indicate that the distinctive channels produced by Ltx interaction with receptor occur with different extracellular binding domains and do not require specific membrane anchoring domains on the receptor. The data suggest that Ltx receptors recruit and tether the toxin to the membrane to facilitate its ability to create channels. The experiments also identify distinct steps in the kinetics of channel formation.

	Materials and Methods

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Plasmids. The construction of the plasmids encoding CIRL-1 (pCDR7) and the CIRL-1 carboxyl-terminal deletion mutants (pCDR-7TMR and pCDR-1TMR) has been described previously (Krasnoperov et al., 1997; Ichtchenko et al., 1999). To construct the pCDR-p120/vesicular stomatitis virus glycoprotein (VSV-G) plasmid encoding the extracellular region of CIRL-1 (p120; residues 1 through 855) and a single transmembrane domain of VSV-G (Guan and Rose, 1984; Guan et al., 1985), the pCDR7 plasmid was partially digested with Bsplu11I and PmeI and the resulting linear fragment encoding p120 was purified. The VSV-G fragment was produced by PCR amplification of the plasmid encoding VSV-G (pSVGL) using a Bsplu11I-tagged forward primer and a PmeI-tagged reverse primer. The PCR product was then cut by Bsplu11I and PmeI, and the purified fragment was ligated to the p120-encoding fragment described above. A synthetic linker corresponding to amino acids 833 through 855 of CIRL-1 was then inserted into the Bsplu11I site between the p120 and VSV-G inserts to complete the pCDR-p120/VSV-G plasmid. The structures of CIRL-1 and the CIRL-1 mutant receptors are shown schematically in Fig. 1. The plasmid encoding neurexin 1 (pCMVbN1-1) was a gift from Thomas C. Sudhof (The University of Texas Southwestern Medical Center) and has been described previously (Sugita et al., 1999). The plasmid encoding VSV-G (pSVGL) was a gift from Dr. John K. Rose (The Salk Institute) and has been described previously (Guan and Rose, 1984). The plasmid encoding a mutant green fluorescent protein (GFP; S65T) was a gift from Dr. Ian Macara (University of Virginia) and has been described previously (Helm et al., 1995). The plasmid encoding muscarinic M₃ receptor (pCMVM3) was a gift from Dr. Stephen K. Fisher (University of Michigan).

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Structure of CIRL-1 and CIRL-1 mutant receptors. CIRL-1, wild-type receptor; 7TMR, CIRL-1 without its carboxyl-terminal tail; 1TMR, extracellular domain of CIRL-1 (p120) anchored by only one CIRL-1 transmembrane domain; p120/VSV-G, extracellular domain of CIRL (p120) anchored to the membrane by the transmembrane domain of VSV-G. [Modified from Krasnoperov et al. (1999).] Note: the discontinuity between the extracellular domains and the transmembrane domains indicates that each receptor consists of two heterologous subunits resulting from endogenous proteolytic processing of the precursor proteins. Although the p120/VSV-G construct shows similar discontinuity, cleavage of this receptor is unknown.

Cell Culture and Transfection. Bovine adrenal chromaffin cell cultures were prepared and maintained using methods identical with those described in previous studies (Bittner and Holz, 1992). Cells were cultured as monolayers on collagen-coated glass coverslips, which formed the bottoms of 35-mm culture dishes at a density of 600,000 cells per dish and were transfected by calcium phosphate precipitate (Wilson et al., 1996) 14 to 18 h after plating. The cells were cotransfected with precipitates containing equal mass amounts of experimental (pCDR7) or control plasmid (pCMVneo), along with soluble GFP-encoding plasmid (p7sGFP), which served as a marker for transfection.

Immunocytochemistry. Three consecutive sequences encoding the Influenza hemagglutinin (HA) epitope tag, YPYDVPDYA, were introduced using a PCR-based technique into the CIRL-1 coding sequence at the amino terminus. HEK293 cells were plated on poly(L-lysine)-coated glass coverslips that formed the well bottoms of 12-well culture dishes. Cells were maintained and cotransfected with plasmids encoding epitope-tagged CIRL-1 and GFP as described above. Immunocytochemistry was performed 36 to 48 h after transfection. For surface staining, transfected cells were rinsed twice with Dulbecco's phosphate-buffered saline (DPBS) and fixed with 4% (w/v) paraformaldehyde in 0.1 M cacodylic acid (pH 7.0) for 30 min at room temperature. The cells were then rinsed once with DPBS, quenched with 50 mM NH₄Cl in DPBS for 30 min, and rinsed twice with DPBS at room temperature. Nonspecific binding was blocked with 0.1% (w/v) gelatin in DPBS for 20 min, followed by 4% donkey serum in DPBS for 20 min at room temperature. After a single rinse with DPBS at room temperature, the cells were incubated with mouse anti-HA (1:500; 12CA5, Berkeley Antibody Co., Berkeley, CA) in DPBS for 2 h at 4°C. The cells were then rinsed five times with DPBS and incubated with rhodamine-conjugated donkey anti-mouse antibody (1:100) and 5 mg/ml BSA in DPBS for 70 min at 4°C. After this final incubation, the cells were rinsed five times with DPBS at 4°C and mounted in 0.1% paraphenyldiamine, 90% glycerol, and 0.1 M PBS (pH 9.0). Cell surface fluorescence was examined by collecting images with a confocal imaging system (Bio-Rad Laboratories, Hercules, CA) with a 100× objective.

Electrophysiology. Patch electrodes were pulled from 1.5-mm A × 1.12-mm i.d. borosilicate glass (Corning 7740) capillaries (A-M Systems, Carlsborg, WA), coated with elastomer (Sylgard; Dow Corning), and fire-polished to 3 to 8 m. The standard extracellular solution was a physiological salt solution (PSS) consisting of (in millimolar): NaCl, 145; KCl, 5; CaCl₂, 2; MgCl₂, 1; glucose, 10; and HEPES, 10 (pH 7.3 at room temperature). Ca²⁺-free PSS and divalent ion-free PSS were prepared by excluding the appropriate divalent ion-containing salts and including 1 mM EGTA. Electrodes were filled with a solution consisting of (in millimolar): CsOH, 120; CH₄O₃S, 120; CsCl, 20; MgCl₂, 1; Mg-ATP, 2; Li-GTP, 0.5; EGTA, 0.25; and HEPES, 20 (pH 7.3). Whole cell or outside-out patch clamp recordings of ionic current were made using an amplifier (Axopatch 200A; Axon Instruments, Burlingame, CA) with a computer interface (ITC-16; Instrutech Corp., Great Neck, NY). Membrane conductance and series resistance were compensated electronically to 75%. Current signals were filtered at 5 kHz (8-pole Bessel) and stored directly onto the computer hard disk for later analysis. Voltage protocols, data acquisition, and analyses were performed using software (Pulse Control; Richard Bookman, University of Miami) developed as an extension of the numerical/graphics program Igor (WaveMetrics, Lake Oswego, OR). Unless otherwise indicated, recordings were made at room temperature from cells or membrane patches voltage clamped at a holding potential of 70 mV.

Calcium Measurements. Measurements of intracellular free Ca²⁺ concentration ([Ca²⁺]_i) were made using Fura-2 and dual-wavelength microspectrofluorimetry (Stuenkel, 1994). Loading of cells with Fura-2 was accomplished by incubation at 37°C for 30 min in PSS containing 1 µM acetoxymethyl ester of Fura-2 (Fura-2 AM; Molecular Probes, Eugene, OR) in dimethyl sulfoxide carrier (0.1% final concentration). Emission signals of Fura-2 at alternating excitation wavelengths of 340 and 380 nm were monitored at 500 nm using a photomultiplier-based AR-CM system (SPEX Industries, Edison, NJ). The ratio of emitted light (340/380 excitation) was used as a readout for changes in [Ca²⁺]_i as described previously (Fajtova et al., 1991). Contamination of the Fura-2 signal by GFP was insignificant.

Reagents and Cell Perfusion. Ltx was purified as previously described (Petrenko et al., 1990). Carbachol (CCh) was purchased from Sigma Chemical Co. (St. Louis, MO). Drugs were dissolved in PSS, Ca²⁺-free PSS, or divalent ion-free PSS and applied by pressure ejection from fused-silica tubing (300-µm i.d.; Poly Micro Technologies, Phoenix, AZ) positioned immediately adjacent to the cell from which membrane current or Fura-2 fluorescence was being recorded.

	Results

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Transfection with a Plasmid Encoding CIRL-1 Renders Chromaffin Cells Supersensitive to Channel Formation by Ltx. Bovine adrenal chromaffin cells were transiently cotransfected with plasmids encoding GFP (p7sGFP) and either CIRL-1 (pCDR7) or control plasmid (pCMVneo). Cells in the whole-cell patch clamp configuration were continuously voltage clamped at a holding potential (V_h) of 70 mV and Ltx was applied by local perfusion. In the initial experiments, Ltx was applied under Ca²⁺-free conditions to ensure that only those receptors that bind Ltx in the absence of Ca²⁺ were being studied. Cells overexpressing CIRL-1 exhibited large changes in membrane current when challenged with 5 pM Ltx (Fig. 2A; n = 4). In contrast, control cells without overexpressed CIRL-1 showed no change in membrane current when challenged with the same concentration of toxin (Fig. 2B; n = 6). Higher concentrations of Ltx (50 or 100 pM) produced channel formation in control cells due to interaction with endogenous Ltx receptors (see below).

View larger version (12K): [in this window] [in a new window]   Fig. 2.   Overexpression of CIRL-1 in bovine chromaffin cells facilitates Ltx-induced channel formation. CIRL-1- (A) and control-transfected (B) chromaffin cells were voltage clamped at a holding potential of 70 mV and whole-cell membrane current (Im) was monitored before and during continuous application of 5 pM Ltx under Ca2+-free conditions (indicated by open bars). A large inward current developed in CIRL-1-transfected cells (n = 4) but not in control-transfected cells (n = 6).

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Channels resulting from interaction of Ltx with transfected CIRL-1 and with the endogenous Ltx receptor in chromaffin cells exhibit similar characteristics. CIRL-1- (A, B) and control-transfected (C, D) chromaffin cells were voltage clamped at a holding potential of 70 mV and whole-cell membrane current (Im) was monitored before and during continuous application of 5 pM Ltx (CIRL-1-transfected cells) or 50 pM Ltx (control cells) in the absence (left) or presence (right) of extracellular Ca2+. Current records represent expanded time segments surrounding the initial current responses and do not indicate the onset of each toxin application. In Ca2+-free solution, large (20 to 40 pA) unitary current steps were seen in both CIRL-1- (n = 4) and control-transfected (n = 6) cells. Increased current fluctuation was associated with the current steps when Ca2+ was present (n = 6 and n = 4, respectively).

Low Concentrations of Ltx Induce Channels in HEK293 Cells Transiently Transfected with Plasmid Encoding CIRL-1. HEK293 cells were transiently transfected with a plasmid encoding HA-tagged CIRL-1. Immunocytochemical staining with HA antibody revealed plasma membrane expression of HA-tagged CIRL-1 in transfected cells (Fig. 4). Little or no protein was detected in intracellular compartments. Nontransfected cells or cells transfected with pCMVneo were unstained (data not shown).

View larger version (73K): [in this window] [in a new window]   Fig. 4.   CIRL-1 is expressed as a membrane protein in HEK293 cells. Confocal fluorescence image of HA epitope-tagged CIRL-1 in transfected HEK293 cells. Note the increased fluorescence intensity along the cell surface. Bar, 7 µm.

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Expression of CIRL-1 in HEK293 cells facilitates Ltx-induced channel formation. CIRL-1- (A) and control-transfected (B) HEK293 cells were voltage clamped at a holding potential of 70 mV and whole-cell membrane current (Im) was monitored before and during continuous application of 5, 50, or 500 pM Ltx in the absence of extracellular Ca2+ (open bars indicate periods of specified treatments under Ca2+-free conditions). The current record in A, right, represents an expanded time segment surrounding the initial current response depicted in A, left. As in chromaffin cells, low concentrations (5 pM) of Ltx produced a large inward current in CIRL-1-transfected HEK293 cells (n = 12), whereas in control cells (n = 6), a higher concentration (500 pM) of Ltx was required to elicit a current response.

View larger version (13K): [in this window] [in a new window]   Fig. 6.   Extracellular calcium changes the conductance effects of Ltx in CIRL-1-transfected HEK293 cells. A, Ltx (50 pM) was applied 10 s before the beginning of the record in the presence of 2 mM Ca2+. B, Ltx (5 pM) was first applied in the absence of Ca2+ (open and solid bars indicate periods of specified treatments in the absence and presence of extracellular Ca2+, respectively). Discrete current increases were observed (difficult to see with the expanded Im scale). Where indicated, the perfusion solution was changed to 2 mM Ca2+ (without Ltx) and back to 0 Ca2+ (without Ltx). Note the difference in the time scales.

Increases in Cytosolic Calcium Induced by Ltx Interaction with CIRL-1 Require Extracellular Calcium. It has been demonstrated in chromaffin cells that Ltx interaction with CIRL-1 or with endogenous receptors causes an increase in cytosolic Ca²⁺ ([Ca²⁺]_i) (Bittner et al., 1998). Because CIRL-1 has seven transmembrane domains and is thought to be a G-protein-coupled receptor, increases in [Ca²⁺]_i could result from Ca²⁺ influx through the channel and/or from a phospholipase C-mediated increase in IP₃ and release of Ca²⁺ from intracellular stores. Measurement of Fura-2 fluorescence was used to detect Ltx-induced changes of [Ca²⁺]_i in HEK293 cells transiently transfected with CIRL-1-encoding plasmid (pCDR7). When the extracellular solution contained 2 mM Ca²⁺, local application of 50 pM Ltx to CIRL-1-transfected cells elicited robust increases in [Ca²⁺]_i (Fig. 7A; n = 12) that were characterized by a rapid rise followed by a sustained plateau. Under Ca²⁺-free conditions, local application of 50 pM Ltx to CIRL-1-transfected cells did not elicit changes in [Ca²⁺]_i in any of the 14 cells investigated (Fig. 7B). Addition of Ca²⁺ to the extracellular solution produced immediate increases in [Ca²⁺]_i (Fig. 7B, n = 3) despite extended wash periods (up to 100 s) during which the Ltx stimulus was absent. The latter response was likely due to Ca²⁺ influx through channels previously formed by the Ltx-CIRL-1 interaction. The lack of a rise in [Ca²⁺]_i in the absence of extracellular Ca²⁺ was not due to the inability of the cells to respond to phospholipase C activation. Application of the muscarinic agonist carbachol in the absence of extracellular Ca²⁺ increased [Ca²⁺]_i in HEK293 cells transiently transfected with a muscarinic receptor (M₃ receptor, Fig. 7C). Intracellular Ca²⁺ rose rapidly and then smoothly declined toward resting level in 7 of 10 transfected cells. Thus, although HEK293 cells permit functional coupling between transiently transfected G-protein-coupled receptors and phospholipase C, Ltx acting through CIRL-1 does not increase [Ca²⁺]_i by such a mechanism. More likely the increase in [Ca²⁺]_i occurs via influx through the channel.

View larger version (20K): [in this window] [in a new window]   Fig. 7.   Ltx causes increases in cytosolic Ca2+only in the presence of extracellular calcium in CIRL-1-transfected HEK293 cells. Intracellular Ca2+ was monitored by microspectrofluorimetry of Fura-2. A, Ltx (50 pM) was applied to a CIRL-1-transfected cell in the presence of 2 mM Ca2+. Similar responses were obtained in 12 cells. B, Ltx (50 pM) was applied to a CIRL-1-transfected cell in the absence of Ca2+. There was no change in the Fura-2 fluorescence until solution containing 2 mM Ca2+ was perfused. Similar results were obtained in 14 cells. C, An HEK293 cell transfected with a plasmid encoding a muscarinic receptor (M3) was perfused with a Ca2+-free solution. At the indicated time, CCh (100 µM) was perfused in the continuing absence of Ca2+. Application of CCh produced elevations in [Ca2+]i in 7 of 10 cells. Open and solid bars indicate periods of specified treatments in the absence and presence of extracellular Ca2+, respectively.

Effects on Conductance of Varying Ltx Concentration Applied to CIRL-1-Transfected HEK293 Cells. Following Ltx application in the absence of Ca²⁺, there was a latency before the onset of current response that varied inversely with the Ltx concentration (Fig. 8A). Ltx-induced current was detected almost immediately (by 3.4 ± 1.0 s; n = 5) following the application of 500 pM Ltx. In contrast, latencies of 16.4 ± 3.4 s (n = 8) and 37.4 ± 4.2 s (n = 9), respectively, were observed before channel formation following the application of 50 and 5 pM Ltx. The latencies were highly reproducible with an approximately linear relationship between the reciprocal of the lag time and toxin concentration (Fig. 8B). Despite the effect of toxin concentration on the latency, the average sizes of single channel current steps were virtually independent of toxin concentration (Fig. 8, A and C).

View larger version (19K): [in this window] [in a new window]   Fig. 8.   The effects of various concentrations of Ltx on the time course of appearance and the size of the current steps in the absence of Ca2+ in HEK293 cells. A, CIRL-1-transfected HEK293 cells were voltage clamped at 70 mV and whole-cell current (Im) responses were monitored during continuous perfusion (starting at 0 s) of 5 (n = 8), 50 (n = 7), or 500 pM (n = 5) Ltx in the absence of Ca2+. The means ± S.E. of the time of occurrence and the cumulative size of the first four to five discrete current jumps are plotted. B, the reciprocal of the latency of the first current step from A is plotted versus Ltx concentration. C, the average current steps at the different Ltx concentrations are compared. D, the effects of application of a 5-s pulse of Ltx (50 pM) in the absence of extracellular Ca2+ (indicated by the open bar) to a CIRL-1-transfected cell. Note that, after a delay, channel formation occurs after removal of the Ltx. This was observed in three of three cells.

Ltx Causes Channel Formation in Outside-Out Patches from CIRL-1-Transfected HEK293 Cells. Because the primary sequence of CIRL-1 predicts that it is a G-protein-linked receptor, Ltx binding to the extracellular domain may activate an intracellular signal transduction pathway that regulates channel formation. To test this possibility, the effects of Ltx on outside-out membrane patches from CIRL-1-transfected HEK293 cells were investigated. In five of eight patches, local perfusion of Ca²⁺-free, Ltx (50 pM)-containing solution resulted in the appearance of distinct, unitary current steps (Fig. 9A). Patches that did not respond to 50 pM Ltx were also unresponsive to subsequent application of 500 pM Ltx, suggesting that the patches lacked CIRL-1. The average amplitude (27.8 ± 1.3 pA; n = 20) of the Ltx-induced current steps recorded from patches was nearly identical with the average amplitude of steps recorded in the whole-cell configuration. These results suggest that formation of the conductance pathway depends on Ltx-CIRL-1 interaction but not on a consequent activation of a cytosolic signal transduction cascade. In each patch exhibiting Ltx-induced channel activity, a cascade of many current steps as observed in whole-cell recordings always followed the first current step. A plot of the time course of this phenomenon is shown in Fig. 9B.

View larger version (11K): [in this window] [in a new window]   Fig. 9.   Ltx-induced channel formation occurs in outside-out patches from CIRL-1-transfected HEK293 cells. Outside-out membrane patches excised from CIRL-1-transfected HEK293 cells were voltage clamped at 70 mV and current (Im) was monitored before and during continuous application of 50 pM Ltx in the absence of extracellular Ca2+. A, treatment with Ltx produced a large inward current in five of eight membrane patches (the current record shown represents an expanded time segment surrounding an initial Im response and does not indicate the onset of toxin application). Note the appearance of large unitary current steps, which were similar to those seen in whole-cell recordings. B, plot of the average time course of Ltx-induced current responses of outside-out membrane patches (n = 5).

Mutants of CIRL-1 with Carboxyl-Terminal Deletions Support Ltx-Dependent Conductance Increases. The roles of the long cytosolic tail and the transmembrane domains of CIRL-1 in mediating Ltx-induced channel formation were investigated with two carboxyl-terminal CIRL-1 deletion mutants (see Fig. 1). One mutant, 7TMR, consisted of the extracellular domain (p120) along with the seven transmembrane domains and connecting loops but without the long cytosolic, carboxyl tail. The other mutant, 1TMR, consisted of the extracellular domain (p120) and only the first transmembrane domain and the first intracellular segment. Large increases in membrane conductance were observed following application of 50 pM Ltx in cells expressing either mutant (Fig. 10). However, there were differences in the resulting conductances. In HEK293 cells expressing the CIRL-1 deletion mutant without the carboxyl-terminal tail (7TMR), Ltx produced stepwise increases in current when extracellular Ca²⁺ was absent (Fig. 10A; n = 4) and responses characterized by rapid current fluctuations when extracellular Ca²⁺ was present (Fig. 10B; n = 3). The average amplitude of the individual steps was 29.2 ± 0.5 pA (n = 14). These results were similar to those obtained following Ltx interaction with wild-type CIRL-1. In contrast, the currents induced by Ltx in cells expressing the deletion mutant with only the single transmembrane domain (1TMR) exhibited rapid current fluctuations even when extracellular Ca²⁺ was absent (Fig. 10C; n = 7). Sustained individual current steps were not observed. Nevertheless, the current responses were reversibly suppressed by extracellular calcium (Fig. 10D; n = 3), a result similar with that produced following Ltx interaction with wild-type CIRL-1.

View larger version (35K): [in this window] [in a new window]   Fig. 10.   Effects of mutations in the transmembrane domain and cytoplasmic tail of CIRL-1 on Ltx-induced channels in HEK293 cells. HEK293 cells were transiently transfected with plasmids encoding the extracellular domain (p120) of CIRL-1 and various mutations in the transmembrane or cytosolic domains. Transfected cells were then voltage clamped at a holding potential of 70 mV and whole-cell membrane current (Im) was monitored. In all panels except D, the current records shown represent expanded time segments surrounding initial Im responses and do not indicate the onset of each toxin application. A and B, 7TMR, the extracellular and transmembrane domains without the long cytoplasmic tail. (A) Ltx (50 pM) in the absence of extracellular Ca2+ produced discrete stepwise increases in current in a HEK293. B, Ltx (50 pM) application in the presence of extracellular Ca2+ produced rapidly fluctuating current increases. C and D, 1TMR, the extracellular domain with only the first transmembrane and cytosolic domains. C, Ltx (50 pM) application in the absence of extracellular Ca2+ caused rapidly fluctuating current increases in a HEK293 cell. D, the same cell as in C. Open and solid bars indicate periods of specified treatments in the absence and presence of extracellular Ca2+, respectively. Ltx was first perfused in the absence of Ca2+. Where indicated, solution containing 0 Ltx and either 2 mM Ca2+or 0 Ca2+was subsequently perfused. E and F, p120/VSV-G, the extracellular domain of CIRL-1 with all the transmembrane domains and the cytoplasmic tail replaced with the single transmembrane domain and cytosolic segment of VSV-G. E, Ltx (50 pM) application in the absence of extracellular Ca2+ produced discrete stepwise increases in current. F, Ltx (50 pM) application in the presence of extracellular Ca2+ produced rapidly fluctuating current increases.

Extracellular Domain of CIRL-1 Fused to Transmembrane Domain of Vesicular Stomatitus Virus Glycoprotein Supports Ltx-Induced Conductance Increases. To determine whether channels resulting from the interaction of Ltx with CIRL-1 require a specific transmembrane domain, a chimeric protein was constructed consisting of the extracellular domain of CIRL-1, p120, fused to the carboxyl-terminal 49 amino acids of VSV-G. This segment of VSV-G consists of a 20-amino acid membrane-spanning domain and a 29-amino acid cytoplasmic domain (Guan and Rose, 1984; Guan et al., 1985). In the absence of extracellular Ca²⁺, application of 50 pM Ltx to HEK293 cells transfected with p120/VSV-G resulted in stepwise increases in inward current (Fig. 10E, n = 5). The average amplitude of the current steps was 29.3 ± 0.9 pA (n = 11). In the presence of extracellular Ca²⁺, currents fluctuated rapidly and individual channels were difficult to resolve (Fig. 10F, n = 3). These Ltx-induced conductance effects in the absence and presence of extracellular Ca²⁺ were nearly identical with those produced by Ltx in HEK293 cells transiently expressing wild-type CIRL-1 or 7TMR.

Neurexin 1 Interaction with Ltx Facilitates Channel Formation. Neurexin 1 is a neuronal Ltx receptor that binds the toxin in a Ca²⁺-dependent manner (Petrenko et al., 1990; Ushkaryov et al., 1992). It is an integral membrane glycoprotein that is structurally unrelated to CIRL-1. The electrophysiological effects of the interaction of Ltx with neurexin 1 have not been described. For the sake of comparison with CIRL-1, the effects on conductance of the interaction of Ltx with neurexin 1 were investigated. In the presence of extracellular Ca²⁺, application of 50 pM Ltx to HEK293 cells transfected with a plasmid encoding neurexin 1 produced a large inward current characterized by rapid fluctuations (Fig. 11A, n = 4). The same concentration of Ltx produced no change in membrane current when extracellular Ca²⁺ was absent (Fig. 11B, upper trace, n = 5). To observe single channel current steps, local perfusion of HEK293 cells (n = 5) for 5 s with 50 pM Ltx in the presence of extracellular Ca²⁺ (to permit toxin binding) was followed immediately by perfusion with a solution that lacked both Ltx and Ca²⁺ (Fig. 11B, lower trace). Channel formation occurred several seconds after removal of Ltx and Ca²⁺, and the current responses were characterized by sustained individual steps that had an average amplitude of 28.8 ± 0.7 pA (n = 14). These data suggest that toxin binding to neurexin 1, but not channel formation, was dependent on extracellular Ca²⁺. Once formed, the channel produced from Ltx interaction with neurexin 1 exhibited characteristics nearly identical with that of the channel produced by Ltx interaction with CIRL-1.

View larger version (17K): [in this window] [in a new window]   Fig. 11.   Expression of neurexin 1 in HEK293 cells facilitates Ltx-induced channel formation. HEK293 cells were transiently transfected with plasmid encoding neurexin 1. Cells were then voltage clamped at a holding potential of 70 mV and whole-cell membrane current (Im) was monitored. A, continuous Ltx (50 pM) application in the presence of extracellular Ca2+ produced an inward current characterized by rapid fluctuations. B (upper trace), Ltx (50 pM) in the absence of extracellular Ca2+ was applied continuously. No current response was observed after 60 s of Ltx application. B (lower trace), the same cell as in B, upper trace. After a period of Ltx (50 pM) application in the absence of extracellular Ca2+, a 5 sec pulse of Ltx (50 pM) was applied to the cell in the presence of extracellular Ca2+. Immediately following this pulse, the cell was perfused with a solution that lacked both Ltx and Ca2+. Discrete inward current steps were observed. At the indicated time, the cell was perfused with a solution containing 2 mM Ca2+. Extracellular Ca2+ reduced the amplitude of the current response. Open and solid bars indicate periods of specified treatments in the absence and presence of extracellular Ca2+, respectively.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Ltx Produces High Conductance Channels on Interaction with CIRL-1 or Neurexin 1. This study demonstrates that the interaction of Ltx with transiently expressed CIRL-1 produced 400-pS channels in chromaffin and HEK293 cells. An analysis of CIRL-1 mutant receptors indicated that the transmembrane structure of CIRL-1 is not critical for channel formation. Ltx interaction with neurexin 1 transiently expressed in HEK293 cells produced channels that were nearly identical with those produced by Ltx interaction with CIRL-1, demonstrating that channel formation does not require a specific extracellular binding domain of the Ltx receptor. As discussed below, our findings suggest that the Ltx receptors CIRL-1 and neurexin 1 act to recruit and tether Ltx to the membrane to facilitate its insertion into the membrane and formation of high conductance channels and that the properties of the conductance pathway itself are determined primarily by Ltx.

Receptor Tethers Ltx to the Membrane to Facilitate Channel Formation. The structural basis by which CIRL-1 facilitates Ltx-induced channel formation and regulates channel behavior was investigated by determining the effects on Ltx-induced conductances of mutations in the membrane domain and cytosolic tail of CIRL-1. One of these mutants, 7TMR, consisting of the extracellular and the entire transmembrane domains but lacking the long cytoplasmic tail, produced current responses identical with those produced by Ltx interaction with wild-type CIRL-1. The results suggest that the cytoplasmic tail does not contribute to channel behavior.

The Ltx-Induced Channel and the Rise in Cytosolic Ca²⁺ Do Not Require G-Protein Activation or Cytosolic Components. The prediction that CIRL-1 is a G-protein-coupled receptor raised the possibility that the channel produced by Ltx interaction with CIRL-1 results from a G-protein-requiring transduction cascade. As indicated above, such a mechanism in HEK293 cells is unlikely because of the similarity of the channels resulting from the interaction of Ltx with CIRL-1 and p120/VSV-G. Although the conductance effects are not caused by activation of a G-protein-linked pathway, the interaction of Ltx with CIRL-1 could, nevertheless, activate a G-protein-linked pathway that would be responsible for other cellular effects. It has also been suggested that Ltx interaction with CIRL-1 activates phospholipase C by a G-protein-linked mechanism, causing Ca²⁺ release from IP₃-sensitive Ca²⁺ stores (Davletov et al., 1998). However, we found that Ltx interaction with CIRL-1 in HEK293 cells in Ca²⁺-free medium did not alter [Ca²⁺]_i. This result was not simply due to an inability of our cells to couple transiently transfected G-protein-coupled receptors and phospholipase C, because application of carbachol under Ca²⁺-free conditions to cells transfected with muscarinic M₃ receptor produced a rise in [Ca²⁺]_i. It is likely that the Ltx-induced rise in [Ca²⁺]_i in Ca²⁺-containing medium is a direct effect of Ca²⁺ influx through the channel with a secondary effect of Ca²⁺-activated phospholipase C activity and IP₃ production. This conclusion is consistent with investigations in COS7 cells (Krasnoperov et al., 1997) and chromaffin cells that failed to demonstrate an increase in IP₃ production in response to Ltx in Ca²⁺-free medium (M. A. Bittner and R. W. Holz, submitted).

Evidence for Multiple Steps between Ltx Binding and Channel Formation. The kinetics of Ltx-CIRL-1-dependent channel formation revealed distinct steps in channel formation. Following onset of perfusion of 5 or 50 pM Ltx to CIRL-transfected HEK293 cells, the onset of channel formation had latencies of 37 or 16 s, respectively. Importantly, following brief application of Ltx, channels first appeared many seconds after extracellular Ltx had been washed away. Thus, following binding of toxin to CIRL-1, events that can require many seconds lead to channel formation. Indeed, there is evidence that suggests that there are two states of binding of Ltx to receptor. The dissociation of prebound Ltx from synaptosomes (Tzeng and Siekevitz, 1979) or from PC 12 cells (Rosenthal et al., 1990) is biphasic with half-times of dissociation of the order of 5 to 10 min (Tzeng and Siekevitz, 1979) and several hours. It has been suggested that the binding data reflect a sequence of steps in which Ltx binds with relatively low affinity followed by a higher affinity association (Tzeng and Siekevitz, 1979). This change in the state of binding of Ltx to its receptor could correspond to channel formation.