Saxitoxin and ,U-Conotoxins (Brain/Electric Organ/Heart/Tetrodotoxin) EDWARD MOCZYDLOWSKI*, BALDOMERO M
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Proc. Nati. Acad. Sci. USA Vol. 83, pp. 5321-5325, July 1986 Neurobiology Discrimination of muscle and neuronal Na-channel subtypes by binding competition between [3H]saxitoxin and ,u-conotoxins (brain/electric organ/heart/tetrodotoxin) EDWARD MOCZYDLOWSKI*, BALDOMERO M. OLIVERAt, WILLIAM R. GRAYt, AND GARY R. STRICHARTZt *Department of Physiology and Biophysics, University of Cincinnati College of Medicine, 231 Bethesda Avenue, Cincinnati, OH 45267-0576; tDepartment of Biology, University of Utah, Salt Lake City, UT 84112; and tAnesthesia Research Laboratories and the Department of Pharmacology, Harvard Medical School, Boston, MA 02115 Communicated by Norman Davidson, March 17, 1986 ABSTRACT The effect oftwo pL-conotoxin peptides on the 22 amino acids with amidated carboxyl termini (18). One of specific binding of [3H]saxitoxin was examined in isolated these toxins, GIIIA, has recently been shown to block muscle plasma membranes of various excitable tissues. pt-Conotoxins action potentials (18) and macroscopic Na current in a GITIA and GIHIB inhibit [3H]saxitoxin binding inlEkctrophorus voltage-clamped frog muscle fiber (19). At the single channel electric organ membranes with similar Kds of %50 x 10-9 M level, the kinetics of GIIIA block have been shown to in a manner consistent with direct competition for a common conform to a single-site binding model (Kd, 110 x 10-9 M at binding site. GITIA and GIIIB similarly compete with the 0 mV), from analysis of the statistics of discrete blocking majority (80-95%) of [3Hlsaxitoxin binding sites in rat skeletal events induced in batrachotoxin-activated Na channels from muscle with Kds of -25 and "140 x 10-9 M, respectively. rat skeletal muscle (18). These studies revealed close simi- However, the high-affinity saxitoxin sites in lobster axons, rat larities between the blocking action ofGIIIA and the classical brain, and rat heart are virtually insensitive to GIHA concen- guanidinium toxins TTX and STX, including similar voltage- trations up to 10 p&M. These results and previously published dependent binding to batrachotoxin-activated Na channels. data suggest that three Na-channel subtypes can be distin- These results suggested that ,u-conotoxin peptides might guished on the basis of toxin pharmacology: Na channels of share the same receptor site for TTX and STX. skeletal muscle andElectrophorus electroplax have high affinity Although Na-channel block by GIIIA can be readily for iz-conotoxins and tetrodotoxin, neuronal Na channels have demonstrated in frog and rat skeletal muscle, significant low affinity for p-conotoxins and high affinity for tetrodotoxin, Na-channel blocking effects with this toxin have not been while heart Na channels and a similar subtype also found in demonstrated in nerve or brain Na channels from these same denervated muscle have low affinity for both it-conotoxin and species (18). This discrimination between neuronal and tetrodotoxin. muscle Na channels suggested that Na channels in these two tissues might represent different channel subtypes. These Excitable tissues such as nerve, skeletal muscle, and heart implications are pursued in this paper by examining the effect contain voltage-dependent Na channels that mediate the of two purified pu-conotoxins on the specific binding of rapidly activating and inactivating inward Na' current of [3HISTX to Na channels in various tissues. The ,u-conotoxins action potentials in these cells. Purified preparations of Na studied are GIIIA and GIIIB, which differ by only 2 amino channels from electric organ, muscle, brain, and heart con- acids out of 22. GIIIB has substitutions of arginine for tain a similar large glycoprotein, while muscle and brain glutamine at residue 14 and methionine for glutamine at preparations also contain smaller peptides (1-4). Since the residue 18 of GIIIA (18). We find that GIIIA and GIJIB large glycoprotein from Electrophorus electric organ was first competitively inhibit the binding of [3H]STX to sites in rat purified as a toxin receptor and has now been shown to muscle and Electrophorus electric organ membranes but not produce functional Na channels (5), it appears that this in rat brain, rat heart, or lobster axon membranes. Some of protein contains both the channel and the external receptor these results have been presented in preliminary form (19, site for heterocyclic guanidinium toxins such as tetrodotoxin 20). (TTX) and saxitoxin (STX). In recent years, much effort has been devoted to the identification of Na-channel subtypes MATERIALS AND METHODS based on the binding affinity of TTX and STX (6-15). These studies have identified a subtype with low affinity for Native membranes from various sources were prepared at TTX/STX that is present in cardiac muscle and noninner- 0C-40C as follows. The final fraction of each preparation vated skeletal muscle cells of mammals. However, previous was resuspended at -10 mg ofprotein per ml in 0.3 M sucrose comparison studies of Na channels in normal adult skeletal buffer (10 mM Mops-NaOH, pH 7.4/0.2 mM EDTA, 3 mM muscle and nerve concluded that Na channels in these two NaN3) and stored frozen at -80'C. tissue types may be the same molecular species because of Crude microsomal fractions from rat skeletal muscle and their very similar high affinity for TTX/STX and similar Electrophorus electricus electric organ (World Wide Scien- electrophysiological properties (9, 16). tific Animals, Apoka, FL) were prepared according to a Recently, a new class of peptide toxins with blocking published method (21) except that Electrophorus membranes activity against Na channels has been identified in the venom were not extracted with KC1. Five milliliters of crude of Conus geographus, an Indo-Pacific mollusc capable of microsomes (10-20 mg of protein per ml in 0.3 M sucrose paralyzing fish by injecting them with small venomous barbs buffer) was layered over 30 ml of 0.95 M sucrose buffer and (17, 18). Seven homologs ofthe ,u-conotoxin class have been centrifuged at 85,000 x g for 15 hr. The resulting low-density purified, sequenced, and shown to consist ofa single chain of band was diluted in buffer, pelleted at 100,000 x g, and used in [3H]STX binding studies. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: STX, saxitoxin; NEO, neosaxitoxin; TTX, tetro- in accordance with 18 U.S.C. §1734 solely to indicate this fact. dotoxin. 5321 5322 Neurobiology: Moczydlowski et al. Proc. Natl. Acad. Sci. USA 83 (1986) Whole rat brains (20 g) plus 0.3 M sucrose buffer (150 ml) A were homogenized in a Teflon/glass homogenizer and cen- rat muscle trifuged at 2000 x g for 15 min. This supernatant was saved and the pellets were rehomogenized in 150 ml of 0.3 M 0 0.8 sucrose buffer and centrifuged as described above. The combined supernatant was pelleted at 100,000 x g for 40 min. V_ 0.2 The resulting pellets were rehomogenized in 150 ml of buffer without sucrose and centrifuged at 8000 x g for 10 min. The 0.4 b supernatant was pelleted at 100,000 x g and used in [3H]STX binding sites. ;0.20 NEO X TTX A membrane preparation from rat heart ventricular muscle was prepared according to procedure II of Jones et al. (22). 0.1 1 10 100 The Ca2' loading step was not used and a fraction sediment- ing at the interface of a 0.25 M/0.6 M sucrose gradient was saved at the final step. ~~~~~~~~~B F1. D A plasma membrane fraction from nerve axons of lobster 0 muscleo(A) and (B) ratmembranesbyNEOSTXandtTTX.brain walking legs was prepared by the method of Balerna et al. (23). 0~0.8A The standard assay for [3H]STX binding contained 0.2 M mM mg 0.6- choline Cl, 10 Mops-NaOH (pH 7.4), 0.5 of bovine [toxin] (nM) serum albumin per ml, 4.5 nM [3H]STX, and various mem- NEO STX TTX brane preparations at 0.3-2 mg ofprotein per ml in a final vol b 0.4 t of 125 or 250 ,Al. [3H]STX binding was allowed to equilibrate for 30-60 min at 0°C before separating the bound ligand at 4°C I- on 1-ml columns of Dowex 50X-200 (Tris+ form) cation 0 exchange resin in Pasteur pipettes. The assay was performed by rapidly layering 50 or 100 ,ul of the assay mixture on the 0.1 1 10 100 column, briefly injecting the sample into the bed with a [toxin] (nM) 0.5 ml of 20 mM HCl tight-fitting syringe, adding Tris (pH FIG. 1. Displacement of specific [3H]STX binding to rat skeletal 7.2), and rapidly forcing this eluant into a scintillation vial for muscle (A) and rat brain (B) membranesTTTX.by NEO, STX, and counting. The processing time ofeach sample was 10-15 sec. The ordinate is defined as the fraction of specifically bound [3H]STX Specific [3H]STX binding was determined from the differ- that remains in the presence of increasing concentrations of free ence between a control for nonspecific binding that included toxins, plotted along the abscissa. The initial specific binding of 20 ,uM TTX (Calbiochem). Data points are the means of [3H]STX in the absence of other toxins is definedasmfc= 1, with> = duplicate measurements. [3H]STX used in these experiments 0 equivalent to the level of nonspecific binding measured in the of 20 TTX. The initial levels of was prepared according to Ritchie et al. (24). ,u-Conotoxins presence aaM specific [3H]STX binding in the absence of other toxins were 0.9-1.2 pmiol/mg in GIIIA and GIIIB were purified from crude venom of C.