Proc. Natl. Acad. Sci. USA Vol. 96, pp. 754–759, January 1999 Neurobiology Pseudechetoxin: A peptide blocker of cyclic nucleotide-gated ion channels (Pseudechis australis, Australian king brown snakeypeptide toxinsysnake venomypatch-clamp techniqueypeptide sequencing) R. LANE BROWN*†,TAMMIE L. HALEY*, KAREN A. WEST‡, AND JOHN W. CRABB‡ *Neurological Sciences Institute, Oregon Health Sciences University, 1120 NW 20th Avenue, Portland, OR 97209; and ‡Eye Institute, Department of Ophthalmic Research, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195 Edited by Denis Baylor, Stanford University School of Medicine, Stanford, CA, and approved November 20, 1998 (received for review October 21, 1998) ABSTRACT Ion channels activated by the binding of CNG channel proteins are tetramers containing a- and cyclic nucleotides first were discovered in retinal rods where b-subunits (9–15). Each subunit contains an amino-terminal they generate the cell’s response to light. In other systems, transmembrane domain and pore region (16) followed by a however, it has been difficult to unambiguously determine single cyclic nucleotide-binding domain near the carboxyl whether cyclic nucleotide-dependent processes are mediated terminus (17). The cooperative binding of four cyclic nucleo- by protein kinases, their classical effector enzymes, or cyclic tide molecules activates a nonspecific cation conductance (18). Although expression of the a-subunits alone generates cyclic nucleotide-gated (CNG) ion channels. Part of this difficulty b has been caused by the lack of specific pharmacological tools. nucleotide-dependent currents, coexpression of the -subunits Here we report the purification from the venom of the creates channels that more faithfully reproduce the properties Australian King Brown snake of a peptide toxin that inhibits of the native channel (13, 14). current through CNG channels. This toxin, which we have The lack of specific pharmacological tools has made it difficult to unambiguously determine whether cyclic nucleoti- named Pseudechetoxin (PsTx), was purified by cation ex- de-dependent processes are mediated by PKs, their classical change and RP-HPLC and has a molecular mass of about 24 effector enzymes, or CNG channels. Although there is a kDa. When applied to the extracellular face of membrane a formidable array of specific blockers for sodium, calcium, and patches containing the -subunit of the rat olfactory CNG potassium channels, specific blockers for CNG channels are channel, PsTx blocked the cGMP-dependent current with a Ki scarce. A number of pharmaceuticals, including L-cis-diltiazem of 5 nM. Block was independent of voltage and required only (19), tetracaine (20), pimozide (21), and LY-58358 (22), have a single molecule of toxin. PsTx also blocked CNG channels been reported to block current through CNG channels, but containing the bovine rod a-subunit with high affinity (100 these agents have significant drawbacks. First, they exert their nM), but it was less effective on the heteromeric version of the effects from the cytoplasmic face of the channel, making their ' m rod channel (Ki 3 M). We have obtained N-terminal and utility questionable in more intact preparations. Second, all of partial internal sequence data and the amino acid composi- these blockers have multiple targets, making results somewhat tion of PsTx. These data indicate that PsTx is a basic protein difficult to interpret. An alternative route to determine the that exhibits some homology with helothermine, a toxin identity of the effector enzyme is through the development of isolated from the venom of the Mexican beaded lizard. PsTx cyclic nucleotide analogs with differential effects on CNG promises to be a valuable pharmacological tool for studies on channels and PKG. Some progress has been made in this the structure and physiology of CNG channels. direction in the past few years. Wei and colleagues (23) have produced a series of cGMP derivatives that combine phospho- rothioate modification of the cyclic phosphate with phenyle- For many years cyclic nucleotides were presumed to exert their theno-(PET) derivatization of the 2-amino group. Specifically, numerous cellular effects primarily through the activation of they have shown that the SP-8-Br-PET-cGMPS isomer is a the cyclic nucleotide-dependent protein kinases, PKA and potent activator of PKG, but a competitive inhibitor of the rod PKG (1, 2). Recently, however, this assumption has been CNG channel (23). Conversely, the RP-8-Br-cGMPS activates challenged by the advent of ion channels directly activated by the CNG channel, but antagonizes activation of PKG (24). the binding of cyclic nucleotides. These channels are emerging Although these derivatives show some promise as selective as important components of signaling systems throughout the pharmacological agents, they bind to the channel with rather body (3). Cyclic nucleotide-gated (CNG) channels first were low affinity and their ability to permeate the cell membrane is identified in the sensory epithelium of the visual and olfactory unclear. systems where they generate the receptor’s electrical signal in In this manuscript, we report the purification of a peptide response to light and odorants (for a review see ref. 4). In the toxin that blocks current through CNG channels when applied past few years, CNG channels have been discovered in other to the extracellular face of excised outside-out membrane excitable tissues such as the brain, heart, and skeletal muscle, patches. This toxin was purified from the venom of Pseudechis as well as nonexcitable tissues such as the liver, kidneys, and australis, the Australian king brown snake, and promises to be testes. Their roles in nonsensory cells, however, remain ob- a useful pharmacological tool to study the structure and scure. In the retina, CNG channels have been shown to function of CNG channels. stimulate synaptic transmission in response to nitric oxide cues (5, 6), and recent results suggest that they may play a role in MATERIALS AND METHODS long-term potentiation (7). In nonexcitable tissues they may provide a regulated pathway for the entry of extracellular Snake venoms were obtained from Venom Supplies (Tanunda, calcium (8). South Australia). Spider venoms were from Spider Pharm The publication costs of this article were defrayed in part by page charge This paper was submitted directly (Track II) to the Proceedings office. Abbreviations: CNG, cyclic nucleotide-gated; PsTx, Pseudechetoxin; payment. This article must therefore be hereby marked ‘‘advertisement’’ in PK, protein kinase; TFA, trifluoroacetic acid. accordance with 18 U.S.C. §1734 solely to indicate this fact. †To whom reprint requests should be addressed. e-mail: brownrl@ PNAS is available online at www.pnas.org. otolith.lhs.org. 754 Downloaded by guest on September 29, 2021 Neurobiology: Brown et al. Proc. Natl. Acad. Sci. USA 96 (1999) 755 (Feasterville, PA). Other venoms were from Sigma. Calcium 4.6 mm) C4 Jupiter column (Phenomenex) by using 40% channel blockers were from Alamone Labs (Jerusalem, Isra- acetonitrileywater (containing 0.1% TFA). el). Cyclic nucleotides and tosylamidophenylethyl chlorom- A second purification method used an initial step of cation- ethyl ketone-treated trypsin were from Sigma. cDNAs encod- exchange chromatography. Fifty milligrams of dried P. aust- ing the a-subunits of the olfactory and retinal CNG channels ralis venom was dissolved in 50 mM Hepes, pH 7.5, and applied were the kind gifts of R. R. Reed (Johns Hopkins University), to a Bioscale S-5 column (Bio-Rad). The venom components Emily Liman (Harvard University), and William Zagotta were eluted from this strong cation-exchange matrix by using (University of Washington). The cDNA encoding the rod a 60-min linear gradient from 0–2 M KCl with 50 mM Hepes, b-subunit was the gift of Robert Molday (University of British pH 7.5 throughout. Each fraction was screened for the pres- Columbia). L-cis-Diltiazem was the gift of E. Boehme at ence of PsTx by analytical RP-HPLC (as described below) and Hoechst-Marion-Roussel (Cincinnati, OH). All other reagents SDSyPAGE. PsTx subsequently was purified to apparent were of the highest purity available. homogeneity by RP-HPLC on the analytical Jupiter C4 HPLC Purification of Pseudechetoxin (PsTx). We have purified column. Proteins were eluted by usinga1mlymin linear PsTx by using two methods. In the first method, 100 mg of gradient (2% per min) from 0.1% TFA to acetonitrile (con- dried venom from Pseudechis australis was dissolved in 1 ml of taining 0.08% TFA). PsTx eluted from this chromatography HPLC starting buffer (water containing 0.1% trifluoroacetic system at approximately 32.5 min, well resolved from all other acid, TFA); insoluble material was removed by a 5-min cen- proteins. trifugation at maximum speed in an Eppendorf microcentri- Expression of CNG Channels. cRNAs encoding the olfac- fuge. The clarified venom was loaded onto a Jupiter C5 column tory and rod CNG channels were transcribed by using the (250 3 21.2 mm; Phenomenex, Torrance, CA) by using a mMessage mMachine kit (Ambion, Austin, TX) as described Hewlett-Packard 1100 HPLC system. PsTx was eluted with a (25). Xenopus oocytes were injected with '25 ng of cRNA. For 120-min gradient from 0.1% TFA to 80% acetonitrile (con- experiments using both the a- and b-subunits of the rod taining 0.08% TFA) witha5mlymin flow rate. Fractions channel, the respective cRNAs were injected in a 1:2 molar containing PsTx inhibitory activity were pooled, and PsTx was ratio. Patch-clamp experiments were performed 3–5 days after further purified by isocratic elution from an analytical (250 3 injection. FIG.1. (A) RP-HPLC separation of PsTx from P. australis venom. The components in 100 mg of venom were separated on a C5 column by using a wateryacetonitrile gradient as described. (Upper) The column profile at 280 nm. Ten-milliliter fractions were dried and redissolved in 1 ml of water. After a 1:30 dilution into control buffer, the fractions were applied to excised outside-out patches expressing the olfactory CNG channel a-subunit.