Pseudechetoxin: a peptide blocker of cyclic nucleotide-gated ion channels

Abstract

Ion channels activated by the binding of cyclic nucleotides first were discovered in retinal rods where they generate the cell's response to light. In other systems, however, it has been difficult to unambiguously determine whether cyclic nucleotide-dependent processes are mediated by protein kinases, their classical effector enzymes, or cyclic nucleotide-gated (CNG) ion channels. Part of this difficulty has been caused by the lack of specific pharmacological tools. Here we report the purification from the venom of the Australian King Brown snake of a peptide toxin that inhibits current through CNG channels. This toxin, which we have named Pseudechetoxin (PsTx), was purified by cation exchange and RP-HPLC and has a molecular mass of about 24 kDa. When applied to the extracellular face of membrane patches containing the alpha-subunit of the rat olfactory CNG channel, PsTx blocked the cGMP-dependent current with a Ki of 5 nM. Block was independent of voltage and required only a single molecule of toxin. PsTx also blocked CNG channels containing the bovine rod alpha-subunit with high affinity (100 nM), but it was less effective on the heteromeric version of the rod channel (Ki approximately 3 microM). We have obtained N-terminal and partial internal sequence data and the amino acid composition of PsTx. These data indicate that PsTx is a basic protein that exhibits some homology with helothermine, a toxin isolated from the venom of the Mexican beaded lizard. PsTx promises to be a valuable pharmacological tool for studies on the structure and physiology of CNG channels.

Figure 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 water/acetonitrile 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 α-subunit. (Lower) Histogram depicts the fractional inhibition of current at +80 mV (upward, black fill) and at −80 mV (downward, gray fill). The PsTx eluted at 118 min. (indicated by an arrow, Upper); the corresponding inhibitory activity was found in fractions 59 and 60. These data suggest that PsTx is a minor component of P. australis venom, perhaps <0.1%. (B) Block of CNG channel currents by 10 mM Mg²⁺ and crude PsTx (reversed-phase fraction 59). CNG channel currents in excised outside-out patches were evoked by 20-ms steps from a holding potential of mV to a voltage ranging from −100 to +100 mV in 20-mV steps. Shown are current families in control solution and in the presence of 10 mM MgCl₂ and PsTx applied to the extracellular face of the membrane. (C) SDS/PAGE analysis of reversed-phase fractions 56–64. A 10-μl sample of each reconstituted fraction was applied to a SDS-15% polyacrylamide gel. After separation, the gel was stained with Coomassie brilliant blue R250. Inhibitory activity was strongly correlated with the presence of a 24-kDa protein in fractions 59 and 60.

Figure 2

(A) Cation exchange chromatography of crude P. australis venom. Fifty milligrams of venom was applied to a Bio-Rad S-50 cation exchange matrix and eluted with a KCl gradient. Shown is the column profile at 280 nm. Inhibitory activity eluted near the end of the gradient. The indicated fractions were pooled for RP-HPLC. (B) Reversed-phase chromatography of pooled cation exchange fractions. Cation exchange fractions were pooled from 100 mg of raw venom and applied to a C4 reversed-phase column. Components were differen tially eluted with a water/acetonitrile gradient (0.1% TFA throughout). (Upper) The column profile at 280 nm. The 24-kDa component, PsTx, eluted at 32–34 min as a broad peak as indicated by the arrow. One-minute fractions were collected and dried. After being redissolved in 1 ml of water, a portion of each fraction was diluted 1:30 into control buffer containing BSA and applied to the extracellular face of a membrane patch expressing the rat olfactory channel. (Lower) the fraction current inhibition at +80 mV (upward, black) and at −80 mV (downward, gray). (C) SDS/PAGE analysis of a final RP-HPLC purified PsTx preparation. Shown is a SDS–15% polyacrylamide gel containing 1 μg of PsTx after staining with Coomassie brilliant blue.

Figure 3

Inhibitory activity of PsTx is sensitive to trypsin proteolysis. Shown are currents recorded from outside-out patches containing the α-subunit of the rat olfactory CNG channel. Pipette contains 2 mM cGMP; the extracellular face of the patch was exposed to control solution containing the substances indicated. Voltage pulses were given as described in Fig. 1B. For the panel labeled “Trypsin,” 20 nM PsTx was incubated in control solution containing 100 μg/ml of trypsin for 36 hr. at 37°C. At that time, a 2-fold molar excess of soybean trypsin inhibitor (STI) was added to halt digestion. In the panel labeled “Trypsin + STI,” trypsin and STI were premixed before incubation.

Figure 4

(A) PsTx blocks CNG channels with nM affinities. Outside-out patches containing the α-subunit of the rat olfactory CNG channel (rOLF; •), the α-subunit of the bovine rod CNG channel (bRET; ○), or both the α- and β-subunits of the bovine rod CNG channel (bRETαβ; ⧫) were tested for their sensitivity to block by PsTx at −60 mV. Dose–response relations were fit by the Hill equation: I_+PsTx/I_−PsTx = K_iⁿ/([PsTx]ⁿ + K_iⁿ) to determine apparent affinities. The fit parameters for the curves shown were: rOLF −K_i = 5 nM, n = 1.2; bRET − K_i = 100 nM; n = 1.2; bRETαβ − K_i = 3.5 μM; n = 1.2. (B) Block by PsTx was slow to develop and reverse. Voltage pulses to +50 mV were given every 2.5 or 5 sec. to outside-out patches expressing the rat olfactory CNG channel α-subunit. Shown is the fractional current as a function of time after the application and removal of 125 nM PsTx (arrows). Thin lines show single exponential fits with rate constants k_on = 4 × 10⁵ M⁻¹ sec⁻¹ and k_off = 2.1 × 10⁻³ sec⁻¹.