Calcium-Independent Receptor for a-Latrotoxin and Neurexin 1a Facilitate Toxin-Induced Channel Formation: Evidence That Channel Formation Results from Tethering of Toxin to Membrane

a-Latrotoxin binding to the calcium-independent receptor for a-latrotoxin (CIRL-1), a putative G-protein-coupled receptor, stimulates secretion from chromaffin and PC12 cells. Using patch clamp techniques and microspectrofluorimetry, we demonstrate that the interaction of a-latrotoxin with CIRL-1 produces a high conductance channel that permits increases in cytosolic Ca. a-Latrotoxin interaction with CIRL-1 transiently expressed in bovine chromaffin cells produced a 400-pS channel, which rarely closed under Ca-free conditions. The major effect of overexpressing CIRL-1 was to greatly increase the sensitivity of chromaffin cells to channel formation by a-latrotoxin. a-Latrotoxin interaction with CIRL-1 transiently overexpressed in non-neuronal human embryonic kidney 293 (HEK293) cells produced channels that were nearly identical with those observed in chromaffin cells. Channel currents were reduced by millimolar Ca. At a-latrotoxin concentrations below 500 pM, channel formation occurred many seconds after binding of toxin to CIRL-1 indicating distinct steps in channel formation. In all cases there was a rapid, sequential addition of channels once the first channel appeared. An analysis of CIRL-1 mutants indicated that channel formation in HEK293 cells is unlikely to be transduced by a G-protein-dependent mechanism. a-Latrotoxin interaction with a fusion construct composed of the extracellular domain of CIRL-1 anchored to the membrane by the transmembrane domain of vesicular stomatitis virus glycoprotein, and with neurexin 1a, an a-latrotoxin receptor structurally unrelated to CIRL-1, produced channels virtually identical with those observed with wild-type CIRL-1. We propose that a-latrotoxin receptors recruit toxin to facilitate its insertion across the membrane and that a-latrotoxin itself controls the conductance properties of the channels it produces. Since the demonstration that black widow spider venom and its active component a-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 Ca through such channels could stimulate exocytosis and thus contribute to the actions of Ltx. [At the neuromuscular junction, Mg can substitute for Ca 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; This work was supported by Grants to R.W.H. (RO1DK27959), E.L.S. (NS36227), A.G.P. (NS35098, NS34937), and M.D.H. (American Heart Association of Michigan Postdoctoral Fellowship). ABBREVIATIONS: Ltx, a-latrotoxin; CIRL, calcium-independent receptor for a-latrotoxin; HEK293, human embryonic kidney 293; TMR, transmembrane region; p120, CIRL-1 extracellular domain; VSV-G, vesicular stomatitis virus glycoprotein; GFP, green fluorescent protein; HA, hemagglutinin; PCR, polymerase chain reaction; DPBS, Dulbecco’s phosphate-buffered saline; PSS, physiological salt solution; CCh, carbachol; IP3, inositol trisphosphate. 0026-895X/00/010519-10$3.00/0 Copyright © The American Society for Pharmacology and Experimental Therapeutics All rights of reproduction in any form reserved. MOLECULAR PHARMACOLOGY, 57:519–528 (2000). 519 at A PE T Jornals on M ay 0, 2017 m oharm .aspeurnals.org D ow nladed from Rosenthal et al., 1990). The binding was both Ca-dependent and Ca-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 a-latrotoxin (CIRL), or latrophilin, binds Ltx in a Ca-independent manner (Krasnoperov et al., 1997; Lelianova et al., 1997), and neurexin 1a binds Ltx in a Cadependent manner (Petrenko et al., 1990). Both are able to support Ltx-induced, Ca-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 Ca, and subsequent addition of Ca-containing medium results in Ca 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 Ca. Channel formation can occur when CIRL-1 or neurexin 1a is expressed in a nonexcitable cell. Importantly, experiments with CIRL-1 mutants and neurexin 1a 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 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 1a (pCMVbN1a-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). 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