Inhibition of CFTR channels by a peptide toxin of scorpion venom

Matthew D. Fuller,1,2 Zhi-Ren Zhang,2 Guiying Cui,2 Julia Kubanek,2 and Nael A. McCarty2

1Program in Molecular and Systems Pharmacology, Emory University, Atlanta 30322-3090; and 2School of Biology, Georgia Institute of Technology, Atlanta, Georgia 30332-0230

Submitted 26 March 2004 ; accepted in final form 1 July 2004


    ABSTRACT
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 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
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Peptide toxins have been valuable probes in efforts to identify amino acid residues that line the permeation pathway of cation-selective channels. However, no peptide toxins have been identified that interact with known anion-selective channels such as the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR channels are expressed in epithelial cells and are associated with several genetic disorders, including cystic fibrosis and polycystic kidney disease. Several organic inhibitors have been used to investigate the structure of the Cl permeation pathway in CFTR. However, investigations of the wider cytoplasmic vestibule have been hindered by the lack of a high-affinity blocker that interacts with residues in this area. In this study we show that venom of the scorpion Leiurus quinquestriatus hebraeus reversibly inhibits CFTR, in a voltage-independent manner, by decreasing single-channel mean burst duration and open probability only when applied to the cytoplasmic surface of phosphorylated channels. Venom was able to decrease burst duration and open probability even when CFTR channels were locked open by treatment with either vanadate or adenosine 5'-({beta},{gamma}-imido)triphosphate, and block was strengthened on reduction of extracellular Cl concentration, suggesting inhibition by a pore-block mechanism. Venom had no effect on ATP-dependent macroscopic opening rate in channels studied by inside-out macropatches. Interestingly, the inhibitory activity was abolished by proteinase treatment. We conclude that a peptide toxin contained in the scorpion venom inhibits CFTR channels by a pore-block mechanism; these experiments provide the first step toward isolation of the active component, which would be highly valuable as a probe for CFTR structure and function.

cystic fibrosis; anion channel; cystic fibrosis transmembrane conductance regulator; ion channel block


THE CYSTIC FIBROSIS (CF) transmembrane conductance regulator (CFTR) is a member of the ATP-binding cassette (ABC) transporter superfamily (44), which forms small-conductance Cl channels in apical membranes of epithelial cells. Loss of function mutations in the CFTR gene lead to CF (11, 13). However, diseases such as secretory diarrhea and polycystic kidney disease are thought to be caused by unwarranted CFTR Cl channel activity (17). The CFTR protein is composed of two predicted motifs, each containing a membrane-spanning domain as well as a nucleotide-binding domain (NBD) that contains sequences that interact with ATP. A regulatory domain containing multiple sites that are phosphorylated by protein kinases A (PKA) and C (PKC) (49) links the two motifs. Although much is known about the domain architecture of CFTR, the structure of the channel pore is not well defined.

Various experimental approaches have been used to identify residues in CFTR that line the Cl permeation pathway (27, 32, 35). One approach relies on small, organic pore-blocking drugs used as molecular probes (34, 4648, 56, 6163). Point mutations that changed sensitivity to the blocking agents provided information about the location of residues in the pore (22, 36). The current model of the CFTR channel pore suggests that four transmembrane {alpha}-helices (TMs 5, 6, 11, and 12) line the Cl permeation pathway (12, 33, 50). However, many regions of the pore remain uninvestigated, particularly the cytoplasmic vestibule, because of the lack of probes that specifically interact with residues in these areas. The cytoplasmic vestibule is predicted to be much larger than the extracellular vestibule, which suggests that the CFTR pore has an orientation within the plasma membrane opposite to that of the pore of ligand-gated anion channels (33) and Ca2+-activated Cl [Cl(Ca)] channels (42). This prediction is consistent with studies of selectivity and open channel block (35, 61) as well as the recent structural determination of the Escherichia coli MsbA transporter, a homolog of the human multidrug resistance transporter and a fellow member of the ABC transporter superfamily (10).

Peptide toxins such as charybdotoxin (ChTx), agitoxin 2 (AgTx), and µ-conotoxin have been shown to be very potent inhibitors of cation-selective ion channels (19). The use of peptide inhibitors has provided substantial information about the structure of cation channels, including the localization of the K+ channel selectivity filter (1, 9, 21, 43). Peptide toxins that are specific for anion-selective channels have been much more difficult to identify. Chlorotoxin (ClTx), a 36-amino acid peptide toxin from the scorpion Leiurus quinquestriatus quinquestriatus (Lqq), has been shown to inhibit endogenously expressed Cl channels in rat colonic epithelial cells and human glial cells (15, 54, 55), although the molecular identities of those channels are not known. However, further studies have shown that ClTx does not inhibit CFTR or Ca2+-activated or volume-regulated Cl channels when applied to the extracellular surface of these channels in Xenopus oocytes (31). Therefore, peptide toxins that are specific for Cl channels of known molecular identity are yet to be identified.

Discovery of a peptide toxin inhibitor of CFTR could aid in the identification of residues that contribute to the inner vestibule of the channel pore. This toxin may also be useful for identifying residues that comprise the cytoplasmic loops that connect the transmembrane helices used to form the pore, provided that the loops form a "pore turret" similar to that found in K+ channels (18). To search for a novel peptide inhibitor, we studied CFTR Cl channels in excised inside-out membrane patches from Xenopus oocytes expressing wild-type (wt) human CFTR. Here we show that the venom of the scorpion Leiurus quinquestriatus hebraeus (Lqh) reversibly inhibits CFTR channels. The results show that the venom contains a peptide toxin that inhibits CFTR, in a voltage-independent manner, when applied to the channel's cytoplasmic surface. However, neither native nor synthetic ClTx had any effect, suggesting that the component of venom active at CFTR channels is not ClTx. Single-channel studies show that the peptide toxin blocks CFTR channel conductance in an all-or-none manner with mean block times on the order of hundreds of milliseconds, suggestive of a high-affinity interaction. These studies describe a bioassay, and provide initial mechanistic information, for the isolation of the active component from scorpion venom.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Reagents and chemicals. Unless otherwise noted, all reagents were obtained from Sigma (St. Louis, MO). L-15 medium was purchased from GIBCO BRL (Gaithersburg, MD). Scorpion venom was purchased from Latoxan (Valence, France). PKA was from Promega (Madison, WI). ClTx was purchased from Sigma, Latoxan, Alexis Biochemical (San Diego, CA), and Calbiochem (San Diego, CA). HPLC-grade acetonitrile was purchased from Fisher (Fair Lawn, NJ).

Preparation of oocytes and cRNA injections. Animal procedures were performed in accordance with the "Guiding Principles for Research Involving Animals and Human Beings" of the American Physiological Society. The methods used were similar to those described previously (36, 61). Briefly, stage V–VI oocytes were isolated from Xenopus and incubated at 18°C in a modified Liebovitz's L-15 medium with the addition of HEPES (pH 7.5), gentamicin, and penicillin-streptomycin. For single-channel recordings and two-electrode voltage-clamp (TEVC) recordings, cRNA was prepared from a construct carrying the full coding region of CFTR in the pAlter vector (Promega). For macropatch recordings, cRNA was prepared from a high-expression construct, generously provided by D. Gadsby (Rockefeller University, New York, NY). For CFTR expression, oocytes were injected with 5–20 ng of wt-CFTR cRNA along with 0.4 ng of cRNA for the {beta}2-adrenergic receptor ({beta}2-AR) for TEVC and single-channel experiments or 50–100 ng of CFTR cRNA for macropatch experiments. Recordings were made at room temperature 2–5 days after injection.

Electrophysiology. The electrophysiological techniques used were similar to those described previously (61). TEVC electrodes were pulled in four stages from borosilicate glass (Sutter Instruments, Novato, CA) and filled with 3 M KCl. Pipette resistances measured 0.5–0.9 M{Omega} in bath solution. Bath solutions (ND96) for whole cell TEVC experiments contained (in mM) 96 NaCl, 2 KCl, 1 MgCl2, and 5 HEPES (pH 7.5; adjusted with NaOH). CFTR channels were activated by perfusion of ND96 containing isoproterenol at a final concentration of 0.5–5 µM. Noninjected oocytes were used for study of endogenously expressed Cl(Ca) channels. TEVC data were acquired with a GeneClamp 500B amplifier and pCLAMP software (Axon Instruments, Union City, CA); currents were filtered at 500 Hz. All CFTR data were background subtracted by using as background the currents measured in the absence of isoproterenol in the same oocyte.

Single-channel and macropatch recordings were obtained using excised inside-out patches. Oocytes were prepared for study by manually removing the vitelline membrane after shrinking in hypertonic solution (34). Pipettes were pulled in four stages from borosilicate glass (Sutter) and fire polished. Pipette solution contained (in mM) 150 N-methyl-D-glucamine (NMDG)-Cl, 5 MgCl2, and 10 TES (pH 7.4; adjusted with Tris). Intracellular bath solution for excised patches contained (in mM) 150 NMDG-Cl, 1.1 MgCl2, 2–10 Tris-EGTA, 0.2–2 MgATP, and 10 TES (pH 7.4; adjusted with Tris). In some experiments, 0.1–0.2 mg/ml venom, 5 mM VO4, and/or 2.75 mM adenosine 5'-({beta},{gamma}-imido)triphosphate (AMP-PNP) was added to the intracellular solution. CFTR channels were activated by the catalytic subunit of PKA (50 U/ml) after excision. Patch pipette resistances were from 8 to 14 M{Omega} for single-channel recordings and from 1 to 4 M{Omega} for macropatch experiments. Typical seal resistances ranged from 100 to >300 G{Omega}. Single-channel experiments were performed with an Axopatch 200B amplifier (Axon Instruments) and recorded at 10 kHz to DAT tape (model DTC-EZ700; Sony). The membrane potential (Vm) was held at either –80 or –100 mV. Data were subsequently played back and filtered with a four-pole Bessel filter (Warner Instruments, Hamden, CT) at a corner frequency of 100 Hz and acquired with a Digidata 1322A interface (Axon Instruments) and computer at 500 Hz with pCLAMP. Digitized Clampex records were analyzed with both Clampfit 9.0 and Igor Pro 4.02 (WaveMetrics, Lake Oswego, OR). In single-channel experiments, recordings of channel activity with venom present began <30 s after treatment, at which time block had developed fully.

Macropatch recordings for determination of the voltage dependence of inhibition used a holding potential of 0 mV. The Vm was then stepped to –100 mV for 50 ms before ramping to +100 mV over the course of 135 ms. Voltage ramps were run in triplicate and averaged. For recordings examining the macroscopic opening rate on rapid introduction of ATP, Vm was stepped from 0 to –100 mV and held throughout the entire course of fast solution exchange. Macropatch recordings were also performed with an Axopatch 200B amplifier operated with pCLAMP software, filtered at 100 Hz, acquired at 1 kHz with pCLAMP, and analyzed with Clampfit 9.0.

Vanadate stock solutions were prepared by adjusting a 100 mM Na3VO4 solution to pH 10 with NaOH followed by storage at 4°C. Aliquots of stock solution were boiled for 15 min before dilution into buffered bath solution immediately before use. In macropatch experiments, bath solutions with 0.2–2 mM MgATP, no MgATP, 0.1 mg/ml venom, or 0.2–2 mM MgATP plus 0.1 mg/ml venom were applied by a fast perfusion system (model SF-77B; Warner Instruments) to phosphorylated CFTR channels. Solution exchange was complete within ~25 ms based on the activation of endogenous Cl(Ca) channels in oocytes by exposure to bath solution containing 10 mM Ca2+ (Refs. 14, 20; see Fig. 2C).



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Fig. 2. Effects of Lqh venom on CFTR and Ca2+-activated Cl [Cl(Ca)] current. A: whole cell current response to voltage steps in an oocyte expressing wt-CFTR 10 min after start of experiment (basal), background-subtracted currents after plateau activation after addition of 5 µM isoproterenol (activated), and background-subtracted currents after 5-min application of 5 µM isoproterenol with 0.1 mg/ml partially fractionated Lqh venom (activated + Lqh venom). Membrane potential was stepped from a holding potential of –30 mV to potentials ranging from –140 to +80 mV, with steps of 75-ms duration. B: 2-electrode voltage clamp currents for Cl(Ca) and CFTR, in representative experiments. Cl(Ca) I-V relationship for 1 uninjected oocyte in the absence ({bullet}) and presence ({lozenge}) of 0.1 mg/ml partially fractionated Lqh venom (traces overlie each other) is shown. Cl(Ca) currents are non-background-subtracted basal leak currents from endogenously expressed Cl(Ca) channels. CFTR channel I-V relationship for 1 cRNA-injected oocyte in the absence ({blacksquare}) and presence ({triangledown}) of 0.1 mg/ml partially fractionated Lqh venom (traces overlie each other) is also shown. C: experimental protocol used to assess potential inhibition of Cl(Ca) channels by venom applied to the cytoplasmic surface; representative traces from 2 excised inside-out macropatches are shown. Each patch was exposed to solution containing 10 mM Ca2+ 3 times, with a fast perfusion apparatus, with a time constant of ~25 ms. The resulting Cl(Ca) current is shown without (top) and with (bottom) application of 0.1 mg/ml partially fractionated Lqh venom. Vm = –50 mV. D: with each subsequent exposure to Ca2+, the peak current was reduced by ~20%. Open bars compare the %desensitization of peak current in the 2nd exposure compared with the 1st, or the 3rd exposure compared with the 2nd, for experiments without venom. Filled bars show similar data for experiments in which the patch was exposed to venom during the time indicated. Data shown are means ± SE for n = 3 observations for each condition.

 
Analysis of single-channel and macropatch experiments. Each patch served as its own control before venom or toxin exposure in all experiments. Transition analysis for single-channel experiments used a 50% cutoff between the open and closed current levels. Burst duration analysis was completed on records from patches containing one to three active CFTR channels; we used a minimum interburst duration cutoff of 100 ms to discriminate between interburst gating and fast intraburst closings (61). The mean burst duration was estimated as previously described (57, 61) with the following formula:

(1)
where tj is the time that j channels are simultaneously open and n is the total number of transitions from an open burst to an interburst closed state. Thus a multichannel open burst event is transformed to n single-channel open events with burst duration t for each event. The number of active channels in a given patch was determined as the maximum number of channels simultaneously open at any point during the recording.

All-points amplitude histograms had bin widths of 0.01 pA and were fit with Gaussian distributions with Clampfit 9.0. Open- and closed-time histograms had bin widths of 50 ms and were constructed from recordings that were a minimum of 360 s in length and contained only single CFTR channels; most records used for dwell time analysis were ~30 min in duration. Because venom-induced blockade introduced a new population of closed dwell times not represented in control recordings, we were able to estimate the apparent rate constant for venom dissociation (koff) from the mean blocked intervals ({tau}c) in a given channel record:

(2)
Macroscopic opening rate was calculated as the inverse of the time constant resulting from a single-exponential fit of the time course of development of current on rapid application of ATP or ATP plus venom to patches containing previously phosphorylated channels.

Venom processing and HPLC fractionation. Leiurus quinquestriatus hebraeus (Lqh) venom was dispersed in intracellular bath solution or ND96, at 2.5 mg dry venom/ml, by extensive vortexing followed by brief homogenization with a Potter-Elvenhjem tissue grinder. The mucous component was pelleted by centrifugation at 6,000 g for 30 min at 22°C. Assuming that the active component of venom would likely be of low molecular weight, as is the case for most peptide toxins of ion channels, we recovered the upper, relatively mucus-free, solution and then filtered with Biomax-10 Micropartition System filters (Millipore, Bedford, MA) with a 10-kDa cutoff and centrifuged at 2,000 g at 22°C in a fixed-angle rotor to remove the higher-molecular-weight components of venom. The demucused, filtered venom was stored at –80°C and diluted to given concentrations, based on equivalent dry venom weight, immediately before use. Hereafter, this component of venom is referred to as "partially fractionated venom"; this material would be expected to contain a number of low-molecular-weight peptides.

Reversed-phase HPLC separation was performed with a Waters 1525 binary HPLC coupled to a Waters 2487 dual-wavelength absorbance detector, utilizing a Zorbax 300SB-C3 analytic column (4.6 mm x 250 mm). Partially fractionated Lqh venom was prepared in intracellular solution at 5 mg/ml and applied to the column in 400-µl aliquots. Peptide components of the venom were eluted with a gradient of solution A (100% H2O) to solution B (100% acetonitrile) run up to 95% solution B in 50 min at a flow rate of 1 ml/min.

Statistics. Results are expressed as means ± SE for n observations. Values represent mean burst duration or channel open probability (Po) recorded during venom application normalized to that measured under control conditions in the same experiment. Recordings were compared using paired Student's t-test. Differences were considered statistically significant when P < 0.05. All tests were performed with SigmaStat 2.03 (Jandel Scientific, San Rafael, CA).


    RESULTS
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 EXPERIMENTAL PROCEDURES
 RESULTS
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Fractionated Lqh venom inhibits wt-CFTR channels from cytoplasmic side only. DeBin and Strichartz (16) have shown that crude venom from the related scorpion Lqq inhibits the endogenous colonic epithelial Cl channels (molecular identity unknown), but only when applied to the cytoplasmic surface of these channels. Hence, we asked whether the partially fractionated Lqh venom might affect CFTR activity when applied to its cytoplasmic surface. In excised inside-out macropatches from Xenopus oocytes expressing wt-CFTR (see EXPERIMENTAL PROCEDURES), no currents were observed during voltage ramps performed after macropatch excision into bath solution lacking ATP [symmetric Cl: 150 mM extracellular Cl concentration ([Cl]o)/150 mM intracellular Cl concentration ([Cl]i)]. CFTR channels were activated by adding 50 U/ml PKA and 1 mM MgATP to the bath, which induced the appearance of a large current that reached steady state in 4–5 min. Removal of PKA and ATP from the bath caused a depletion of the CFTR current (Fig. 1B, control), which was partially reversed on reexposure to MgATP (Fig. 1B, activated). Subsequent application of 1 mM MgATP solution containing 0.1 mg/ml equivalent of partially fractionated Lqh venom (based on the dry weight of the crude, unfractionated venom) resulted in a decrease in steady-state CFTR current. Because the partially fractionated Lqh venom was applied to the patch rapidly, it is unlikely that the observed reduction in current represents further channel dephosphorylation in the absence of PKA. Current-voltage (I-V) relationships for a representative patch in the presence and absence of venom are shown in Fig. 1C. Interestingly, the relationship between voltage and the mean fraction of control current remaining after addition of partially fractionated Lqh venom (I/Io, Fig. 1D) suggests that inhibition of CFTR by venom has no voltage dependence (n = 4). However, decreasing the [Cl] on the extracellular side of the patch (30 mM [Cl]o/300 mM [Cl]i) resulted in an increase in block by 0.1 mg/ml venom from 25.2 ± 2.3% with symmetric [Cl] to 58.1 ± 9.8% with asymmetric [Cl] (n = 3).



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Fig. 1. Leiurus quinquestriatus hebraeus (Lqh) venom inhibited cystic fibrosis transmembrane conductance regulator (CFTR) channels from the cytoplasmic side. A: voltage (V) ramp protocol used to determine CFTR current-voltage (I-V) relationships. B: representative currents from voltage ramps in an inside-out macropatch excised from an oocyte expressing wild-type (wt)-CFTR after phosphorylation by protein kinase A (PKA) and ATP. Bath solution with no MgATP (control), 1 mM MgATP (activated), or 1 mM MgATP + 0.1 mg/ml partially fractionated Lqh venom (activated + Lqh venom) was applied to CFTR channels that were previously phosphorylated, with a fast perfusion system. C: macroscopic I-V relationship for the same patch as in B, before (solid line) and during (dashed line) addition of 0.1 mg/ml partially fractionated Lqh venom. Vm, membrane potential. D: mean fraction of control macroscopic current remaining (I/Io) after addition of Lqh venom. Symbols and error bars indicate means ± SE; n = 4.

 
Previous studies showed that the purified form of the known Cl channel peptide toxin inhibitor ClTx does not inhibit CFTR when applied to the extracellular surface of the channels (31). However, these negative results could reflect improper folding or oxidation status of the purified toxin. Therefore, we asked whether Lqh venom had any effect on CFTR when applied to the extracellular side of the channel. To determine how partially fractionated Lqh venom affects CFTR activity when applied to the extracellular surface of the channels, studies were performed using TEVC recordings in Xenopus oocytes expressing wt-CFTR and the {beta}2-AR. Figure 2A shows background currents, background-subtracted currents after activation of CFTR, and CFTR currents in the presence of 0.1 mg/ml Lqh venom. The steady-state I-V relationships in the presence or absence of partially fractionated Lqh venom overlie each other and are shown in Fig. 2B for a representative oocyte. These results show that the active component of Lqh venom is ineffective when applied to the extracellular face of CFTR channels in the whole cell configuration.

Lqh venom does not indiscriminately inhibit every type of Cl channel. To determine whether inhibition of CFTR from the cytoplasmic side is due to a nonspecific effect of venom, we asked whether venom had any effect on the endogenous Cl(Ca) channels of oocytes (29). The partially fractionated Lqh venom had no effect on spontaneously active endogenous channels when applied to the extracellular solution in TEVC experiments (Fig. 2B). To investigate block of endogenous channels from the cytoplasmic surface of the membrane, we used excised inside-out macropatches from uninjected oocytes and repeatedly exposed the patch surface to solution containing 10 mM Ca2+ with or without venom (Fig. 2C). Although the magnitude of the Cl(Ca) current in control patches decreased during the experiment because of channel desensitization, no added inhibition was observed in the presence of venom (Fig. 2C). Cl(Ca) current had an average decay, on the second Ca2+ treatment, of 22.4 ± 2.8% under control conditions and 26.8 ± 4.1% with venom present (P = 0.491, n = 3). A similar average decay was seen on the third Ca2+ treatment (23.8 ± 9.0% in control vs. 28.2 ± 7.0% with venom washout; P = 0.722, n = 3; Fig. 2D). Hence, partially fractionated Lqh venom does not appear to inhibit the Xenopus Cl(Ca) channel from either the cytoplasmic or the extracellular side. In other studies, we have also found (52) that the partially fractionated venom selectively inhibits ClC-2 voltage-gated Cl channels but does not inhibit ClC-0 or ClC-1. However, these ClC channels are not expressed endogenously in oocytes. Hence, the reduction in Cl current in oocytes expressing CFTR on exposure to venom reflects selective inhibition of CFTR activity. Together, these findings suggest that the component of the Lqh venom that is active at CFTR does not indiscriminately inhibit every type of Cl channel and that the active component cannot reach its binding site from the extracellular end of the CFTR pore.

Lqh venom reversibly inhibits CFTR by decreasing channel burst duration and Po. To determine the mechanism of inhibition, excised inside-out patch recordings of phosphorylated CFTR channels were studied in the absence and presence of venom; Fig. 3 shows a representative experiment. The control record shows current through wt-CFTR channels in a patch held at a Vm of –80 mV. Before application of partially fractionated Lqh venom channel Po was high, with up to five CFTR channels open concurrently. Addition of partially fractionated Lqh venom (0.1 mg/ml equivalent) resulted in a decrease in Po, which was reversed after removal of venom by flushing the chamber with solution containing 1 mM MgATP and PKA only. Recordings of patches with only single channels suggested that the reduction in Po is caused by decreased open burst durations, apparently due to the introduction of discrete blocking events during application of venom (Figs. 4 and 5). There was no change in single-channel current amplitude (i) on exposure to venom (control –0.58 ± 0.02 pA vs. venom –0.58 ± 0.02 pA; Vm = –80 mV; n = 3; P = 0.580), suggesting that the single-channel conductance (g) was not altered (Fig. 4). The blocked states exhibited in records with partially fractionated Lqh venom were many times longer in duration than blocked states caused by the organic pore blockers currently used in CFTR research. The blocked states were similar in duration to those induced by peptide toxin pore block of voltage- and ligand-gated cation channels (3, 19, 38, 40), as well as long closings induced by the recently identified CFTR gating modifier CFTRinh-172 (28, 51). The increase in blocked events indicated in the all-points histogram (Fig. 4) is also consistent with a venom-induced decrease in Po.



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Fig. 3. Fractionated Lqh venom reversibly inhibited wt-CFTR. Representative single-channel recording from an excised inside-out patch is shown. Control conditions before application of partially fractionated Lqh venom (A), immediately after addition of 0.1 mg/ml partially fractionated Lqh venom to the intracellular bath (5 ml total; B), and immediately after perfusion of the bath with 10 ml of fresh buffered solution (C) are shown. The gaps in the record are ~1 min each. All measurements were made in the presence of PKA (50 U/ml) and 1 mM MgATP, with Vm clamped at –80 mV and symmetric Cl [150 mM extracellular Cl concentration ([Cl]o)-150 mM intracellular Cl concentration ([Cl]i)]. Dashed lines represent the closed current level, and downward deflections represent channel openings.

 


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Fig. 4. Lqh venom introduced blocked events into the single-channel record. Left: representative single-channel recordings from excised inside-out patches before (A) and immediately (<30 s) after (B) treatment with 0.1 mg/ml partially fractionated Lqh venom. All measurements were made in the presence of PKA (50 U/ml) and 1 mM MgATP with Vm clamped at –80 mV and symmetric Cl (150 mM [Cl]o-150 mM [Cl]i). Dashed lines represent the closed current level, and downward deflections represent channel openings. Expanded segments of the trace in each condition are shown at bottom. Right: representative all-points amplitude histograms in the presence or absence of partially fractionated Lqh venom. Smooth lines represent fits to Gaussian function.

 


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Fig. 5. Lqh venom reduces wt-CFTR mean burst duration (MBD) and channel open probability (NPo). A: apparent MBD with and without partially fractionated Lqh venom. B: apparent NPo with and without partially fractionated Lqh venom. In A and B, each symbol represents a separate experiment. In B, recovery is shown on washout of venom by solution that does not contain PKA. C and D: effect of 0.1 mg/ml partially fractionated Lqh venom on wt-CFTR MBD (C) and NPo (D). Columns and error bars indicate means ± SE of n = 6 observations at each condition. Values represent MBD and NPo recorded during venom application and those measured in the same patch under control conditions at the beginning of each experiment. Significant difference from control value: *P < 0.003, **P < 0.008.

 
To better quantify the effects of partially fractionated Lqh venom, we measured CFTR channel apparent mean burst duration and NPo [N (number of channels in patch) x Po] in patches with a small number of active channels. Both the apparent mean burst duration and Po were reduced on exposure to 0.1 mg/ml of partially fractionated Lqh venom (Fig. 5, A and B). The inhibitory effects of the fractionated Lqh venom were reversible on washout. Figure 5C shows that the venom caused a 67 ± 8% decrease in mean burst duration (calculated with Eq. 1) from 4,593 ± 572 ms under control conditions to 1,299 ± 180 ms when the partially fractionated Lqh venom was applied (n = 6; P = 0.003). The reduction in burst duration also resulted in a 32 ± 9% decrease in NPo (Fig. 5D) from 0.79 ± 0.12 under control conditions to 0.49 ± 0.09 when the partially fractionated Lqh venom was applied (n = 6; P = 0.008).

Analysis of the blocking events could produce an estimate of the dissociation rate of the active component from the CFTR channel. We used kinetic analysis of CFTR channels in the presence and absence of venom, assuming a closed-open-blocked scheme, but excluded from the analysis the brief closures <10 ms in duration. According to this model we expect the open-time and closed-time distributions to follow a single-exponential function for recordings in the absence of venom, representing gating-induced interburst closed states; a single closed duration was expected in control records because filtering the data at 100 Hz eliminates the flickery intraburst closures (62). We constructed dwell time histograms from the single-channel recordings in the absence and presence of 0.1 mg/ml partially fractionated Lqh venom (data for a representative experiment are shown in Fig. 6). The mean open time decreased when partially fractionated Lqh venom was applied to the channel, consistent with the reduction in apparent mean burst duration, due to introduction of long blocked states (Fig. 5). Open-time distributions before and after fractionated Lqh venom treatment were fit with single-exponential functions giving mean open duration ({tau}o) = 3,302 ± 682 ms for control and {tau}o = 788 ± 86 ms in presence of venom (n = 5), respectively (Fig. 6A).



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Fig. 6. Dwell time analysis of CFTR channel activity from the patch shown in Fig. 4. A: Open-time histograms of a single CFTR channel with 1 mM MgATP and 50 U/ml PKA continuously present before (black lines) and during (gray lines) treatment with 0.1 mg/ml partially fractionated Lqh venom; Vm = –80 mV. Data were fit to a single-exponential function to determine time constants ({tau}o = mean open duration). Note that the mean open time decreased with application of venom. B: closed-time histograms of a single CFTR channel with 1 mM MgATP and 50 U/ml PKA continuously present before (black lines) and during (gray lines) treatment with partially fractionated Lqh venom; Vm = –80 mV. Data were fit to a single-exponential function ({tau}c = mean closed duration). Note the greater number of blocked events with 0.1 mg/ml partially fractionated venom present compared with control. Dwell time histograms shown were constructed from 20–25 min of current recordings in each condition.

 
Closed-time histograms of single CFTR channels before and during treatment with 0.1 mg/ml partially fractionated Lqh venom were also analyzed to determine the mean blocked time (Fig. 6B). Under control conditions the closed-time distributions were also fit by a single-exponential function with {tau}c = 1,909 ± 1,106 ms (n = 5), which represents interburst closures. Channel gating for wt-CFTR under control conditions is distinguished by bursts of activity separated by long closures between bursts (59). Therefore, even recordings of long duration resulted in a low number of closed events that were used for the construction of the dwell time histograms and consequently resulted in a calculated mean with large variance. Venom treatment resulted in the introduction of a new population of blocked events in addition to the long closings seen in control conditions (Fig. 6B). According to our model we would expect the closed-time distribution in the presence of venom to follow a double-exponential function with one component representing the venom-induced blocked events and the other representing the closed events resulting from normal channel gating. However, in the presence of partially fractionated venom, the closed-time distributions were still fit best with a single exponential, giving {tau}c = 674 ± 253 ms (n = 5). The closed-time histograms in the presence of venom were fit by a single-exponential function simply because the large number of venom-induced blocked events greatly overwhelmed the limited number of open-closed gating transitions seen during the recording. Therefore, the estimated Lqh venom mean blocked time is {tau}c = 674 ms, giving an estimated koff of ~1.48 s–1 using Eq. 2. This value is similar to dissociation rates calculated for peptide toxin block of other ion channels (3, 15, 19, 40) and is around tenfold lower than dissociation rates of known CFTR pore blockers (61, 63).

If the active component of venom blocked channels via the same mechanism as the open-channel blockers diphenylamine-2-carboxylate (DPC) and 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), the closed-open-blocked scheme predicts that channel burst durations would be lengthened after treatment with venom because of the added time the channels spend in the blocked state; however, the integrated mean open time per burst in the conducting state is unaltered (39). This is predicted to occur only when the blocking molecule interacts in the pore in such a way that residence at its binding site precludes channel closure (33). With fast-acting CFTR pore blockers like DPC and NPPB, the drug-induced blocked states can be distinguished easily from the closings induced by CFTR gating so that determination of when a burst starts and stops is possible, allowing for precise measurements of burst duration. Venom-induced block could also lead to lengthened burst durations; however, venom-induced closings are similar in length to those seen with normal channel gating, making calculations of the effect of venom on channel burst durations impossible. Therefore, in our analysis, because the venom completely blocks Cl conductance when bound, burst durations are defined as the length of time that a channel is in a Cl-conducting state before shifting to a nonconducting state, either by gating-induced closings or by venom-induced block. It is important to note that the closed-open-blocked model assumes that the active component can reach its binding site only when the channels are open, although such state dependence of inhibition has not yet been determined. Hence, the current scheme that we are using for kinetic analysis may not be an exact representation of the kinetic scheme.

Inhibitory activity of Lqh venom is abolished by trypsinization. Proteolytic treatment has been shown to abolish the activity of peptide inhibitors (60). The partially fractionated Lqh venom was subjected to a 5-h treatment with 0.0028% trypsin and then boiled for 30 min to inactivate the protease. Application of 0.1 mg/ml trypsinized venom to single-channel patches had no apparent effect on CFTR activity (Fig. 7, A and B). As shown in Fig. 7C, we compared NPo in the absence (1.18 ± 0.26) and presence (1.23 ± 0.16) of trypsinized Lqh venom and found no significant effect (n = 3; P = 0.871). Similar results were obtained when we determined the mean burst duration in the absence and presence of trypsinized Lqh venom (control 4,641 ± 1,236 ms vs. venom 3,596 ± 1,069 ms; n = 3; P = 0.576). Trypsinized venom treatment also did not cause a change in single-channel current amplitude (control –0.68 ± 0.01 pA vs. venom –0.70 ± 0.03 pA; Vm = –100 mV; n = 3; P = 0.483). As an experimental control, boiling the partially fractionated Lqh venom solution without trypsin treatment did not alter the inhibitory property, suggesting that the active component is heat stable (not shown). These results suggest that the active component of venom that inhibits CFTR is a peptide.



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Fig. 7. Venom activity is abolished by trypsinization. A and B: representative continuous traces from excised, inside-out patches before (A) and immediately (<30 s) after (B) treatment with 0.1 mg/ml trypsinized venom; Vm = –100 mV. Solutions contained 1 mM MgATP and 50 U/ml PKA at all times. Boiling the solution for 30 min before conducting experiments inactivated the trypsin. C and D: effect of trypsinized venom (0.1 mg/ml) on wt-CFTR channel NPo (C) and MBD (D). Columns and error bars indicate means ± SE of n = 4 observations at each condition. Values represent NPo and MBD recorded before and during trypsinized venom application in the same patch.

 
ClTx is not the active component in partially fractionated Lqh venom. DeBin et al. (15) showed that ClTx purified from whole Lqq venom blocks endogenous colonic Cl channels with a Kd of ~600 nM. We applied purified ClTx to the cytoplasmic surface of CFTR channels in an attempt to determine whether ClTx is the peptide toxin inhibitor of CFTR contained in the partially fractionated Lqh venom. Recordings before and during ClTx application were performed after channel phosphorylation by PKA. Application of 600 nM ClTx to the cytoplasmic surface of the channels did not cause a decrease in CFTR activity (Fig. 8). The mean burst duration (Fig. 8B) in the presence of ClTx was 100 ± 7% of the control value (n = 4; P = 0.998), whereas NPo (Fig. 8C) in the presence of ClTx was 102 ± 12% of the control value (n = 4; P = 0.902). Also, ClTx treatment did not alter single-channel current amplitude (control –0.72 ± 0.02 pA vs. venom –0.73 ± 0.03 pA; Vm = –100 mV; n = 3, P = 0.718; data not shown). Experiments were performed with synthetic ClTx (n = 5; Sigma, Calbiochem) as well as natural ClTx purified from crude scorpion venom (n = 6; Latoxan, Alexis Biochemical) at concentrations as high as 1.2 µM. The natural form of the toxin was used to ensure that ClTx was in its proper conformation; this form of ClTx was shown previously to block Cl channels expressed in rat glioma cells (45). Injection of ClTx into whole oocytes did not inhibit CFTR currents when tested by TEVC (data not shown). These results suggest that ClTx is not the active peptide contained in venom that inhibits CFTR.



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Fig. 8. Chlorotoxin (ClTx) does not inhibit CFTR. A: representative single-channel recordings from excised inside-out patches in the presence of 1 mM MgATP and 50 U/ml PKA before and immediately (<30 s) after application of 600 nM chlorotoxin, with Vm = –100 mV; expanded segments are shown at bottom. B and C: summaries of the effect of chlorotoxin on wt-CFTR channel MBD (B) and NPo (C). Columns and error bars indicate means ± SE of n = 4 observations at each condition. Values represent NPo and MBD recorded during venom application normalized to those measured under control conditions at the beginning of the experiments.

 
Fractionated scorpion venom blocks the channel permeation pathway. Ion channel inhibition can be caused by altering the gating process or by blocking the channel pore. To determine whether toxin-induced inhibition of CFTR represents pore block, we used two experimental agents, VO4 and AMP-PNP, which have been shown to increase CFTR channel mean open times by severalfold. VO4 is a transition-state analog of inorganic phosphate that interrupts the ATP hydrolysis cycle by binding tightly to CFTR channels in place of the released hydrolysis product (6). Alternatively, AMP-PNP is a hydrolysis-resistant ATP analog that can cause channels opened by ATP to remain open for extended durations (24). Studies were performed with excised inside-out patches of wt-CFTR with 1 mM MgATP, PKA (50 U/ml), and either 5 mM VO4 or 2.75 mM AMP-PNP continuously present before and during venom treatment. In the absence of venom, channel activity was increased by VO4 (Fig. 9A) and AMP-PNP (Fig. 9B) (compare to Fig. 4A). Application of 0.2 mg/ml partially fractionated Lqh venom to the cytoplasmic surface of the patches in each condition resulted in a decrease in CFTR activity by introducing additional blocked states. The partially fractionated Lqh venom (0.2 mg/ml) significantly decreased CFTR NPo to 68 ± 6% of the VO4 control value (n = 3; P = 0.036) and decreased NPo to 70 ± 5% of the AMP-PNP control value (n = 3; P = 0.031). In addition, dwell time histograms from single-channel patch recordings in the presence of VO4 or AMP-PNP before and during treatment with partially fractionated Lqh venom gave similar mean dwell time estimates for channel block by venom ({tau}c = 453 ± 302 ms; n = 2; not shown). There was no change in i on exposure to venom when channels were locked open either with VO4 (control –0.67 ± 0.01 pA vs. venom –0.68 ± 0.05 pA; Vm = –100 mV; n = 2, P = 0.866; Fig. 9C) or with AMP-PNP (control –0.57 ± 0.04 pA vs. venom –0.59 ± 0.03 pA; Vm = –80 mV; n = 2, P = 0.559; Fig. 9D). Channels in these experiments were locked in the open conformation by VO4 or AMP-PNP, suggesting that the continued ability of Lqh venom to inhibit CFTR is due to pore block.



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Fig. 9. Lqh venom inhibits wt-CFTR in the presence of either VO4 or adenosine 5'-({beta},{gamma}-imido)triphosphate (AMP-PNP). A: representative traces from excised inside-out patches with 1 mM MgATP, 50 U/ml PKA, and 5 mM VO4 continuously present before and immediately (<30 s) after application of 0.2 mg/ml partially fractionated venom; Vm = –100 mV. B: representative traces from excised inside-out patches with 1 mM MgATP, 50 U/ml PKA, and 2.75 mM AMP-PNP continuously present before and immediately (<30 s) after application of 0.2 mg/ml partially fractionated venom; Vm = –80 mV. Expanded segments for each condition are shown. C and D: representative all-points amplitude histograms of channels locked open with VO4 (C) or AMP-PNP (D) in the presence or absence of partially fractionated Lqh venom. Smooth lines represent fits to Gaussian function.

 
Fractionated scorpion venom does not alter channel gating. Peptide toxins, such as the scorpion {alpha}- and {beta}-toxins and the spider {omega}-grammotoxin-SIA, have been shown to alter gating of voltage-activated cation channels (23, 37). The toxin binds to and stabilizes the closed conformation of the channel that 1) requires stronger depolarizations for activation, 2) causes a decrease in opening rate, and/or 3) stabilizes inactivation. Populations of phosphorylated CFTR channels in oocyte macropatches open to a steady state of activity at a constant rate when 1 mM MgATP is applied (58). To determine whether venom-induced inhibition of CFTR is due to inhibition of gating, we asked whether partially fractionated Lqh venom alters the macroscopic opening rate. If the peptide toxin component of venom preferentially binds to the closed state of the channel and inhibits channel opening, we would expect to see a decrease in macroscopic opening rate. When 0.1 mg/ml partially fractionated Lqh venom was applied to the macropatch, the ATP-dependent opening rate was not reduced whereas the steady-state current was decreased as expected (Fig. 10A). If ATP-dependent gating were slowed by venom, we would expect to see an increase in the time constant for the fit of the relaxation. On the other hand, if inhibition by venom caused introduction of a new kinetic rate representing transitions from the open state to the blocked state, the time constant should be reduced (23), although this would only be discernible if the two rates were quite dissimilar in magnitude. The data in Fig. 10 show an insignificant trend toward this effect.



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Fig. 10. Lqh venom does not modify CFTR channel gating. A: representative trace from 1 excised inside-out macropatch experiment is shown before and during application of 0.1 mg/ml partially fractionated Lqh venom; Vm = –100 mV. Bath solution contained 1 mM MgATP, no MgATP, 0.1 mg/ml partially fractionated venom, or 1 mM MgATP + 0.1 mg/ml partially fractionated Lqh venom and was changed in ~25 ms by a fast perfusion system. CFTR channels were phosphorylated before the experiment began. At positions indicated by the lowercase letters, the ATP-induced current rise is shown at a higher time resolution in insets. The red dotted lines represent single-exponential fits of the traces shown in the insets. B: summary of the effects of partially fractionated Lqh venom (0.1 mg/ml equivalent) on wt-CFTR apparent macroscopic opening rate on rapid application of 0.2, 1, or 2 mM MgATP. Columns and error bars indicate means ± SE of n = 3–6 observations for each condition. Open bars are apparent macroscopic opening rates in the presence of MgATP only, and filled bars are apparent macroscopic opening rates in the presence of MgATP + 0.1 mg/ml partially fractionated Lqh venom.

 
Figure 10B shows that the apparent macroscopic opening rates of CFTR on exposure to 1 mM MgATP in the absence and presence of 0.1 mg/ml partially fractionated Lqh venom were 0.836 ± 0.20 and 0.920 ± 0.25 s–1, respectively (n = 4, P = 0.237). Similar results were seen at both higher and lower MgATP concentrations (Fig. 10B). Exposure to 0.2 mM MgATP activated CFTR current with an apparent opening rate of 0.417 ± 0.19 s–1 before and 0.639 ± 0.26 s–1 during application of partially fractionated Lqh venom (n = 3, P = 0.099), whereas wash on of 2 mM MgATP resulted in opening rates of 0.992 ± 0.24 and 1.02 ± 0.48 s–1, respectively (n = 6, P = 0.648), suggesting that the peptide toxin component contained in the venom does not interfere with ATP binding or hydrolysis. Therefore, Lqh venom-induced inhibition of CFTR apparently is not due to altered gating. These results also show that the active component of venom reaches its binding site within the <30-s exposure before the second treatment with MgATP.

Scorpion venom separation by reversed-phase HPLC. To show that the inhibitory activity of the venom was due to a toxin that can potentially be purified, the partially fractionated scorpion venom was further fractionated by means of reversed-phase HPLC using a C3 column (Fig. 11A). The active toxin was eluted with a gradient of acetonitrile in water. Fractions were collected at 10-min intervals and examined for activity on wt-CFTR in excised inside-out macropatches. A summary of the inhibitory activity of each of the fractions is shown in Fig. 11B. The fraction collected from 21 to 30 min, corresponding to an acetonitrile concentration between 25% and 50%, resulted in 23.7 ± 3.4% (n = 3) inhibition of macroscopic current when diluted to 0.1 mg/ml (equivalent to dry venom). However, fractions collected at other times during the HPLC had less significant effects: 0–10% acetonitrile caused a 6.4 ± 6.4% decrease (n = 2), 10–25% acetonitrile caused a 8.7 ± 2.6% decrease (n = 2), and 50–75% acetonitrile caused a 13.2 ± 6.6% decrease (n = 3) in CFTR current. These results suggest that the active toxin contained in the venom elutes from the C3 column in >25% acetonitrile and retains its activity.



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Fig. 11. Fractionation of Lqh venom by reversed-phase HPLC. A: 2 mg of partially fractionated venom was applied to a reversed-phase C3 analytic column in a Waters HPLC system and separated by using a gradient of acetonitrile as described in EXPERIMENTAL PROCEDURES. Fractions were collected every 10 min from the beginning. Dashed line indicates the acetonitrile elution gradient. B: summary of the inhibitory effects of each of the HPLC fractions. Fractions were diluted to the equivalent of 0.1 mg/ml dry venom and applied to the cytoplasmic surface of wt-CFTR in macropatches. Columns and error bars indicate means ± SE of n = 2–3 observations at each condition.

 

    DISCUSSION
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 ABSTRACT
 EXPERIMENTAL PROCEDURES
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The goal of this study was to complete the foundational steps for identifying and characterizing the activity of a novel peptide inhibitor of CFTR that potentially can be used as a molecular probe of the channel pore. A number of organic agents have been shown to inhibit CFTR channels. Several small organic drugs inhibit CFTR by blocking the channel pore (36, 4648, 56, 6163). These drugs appear to bind to the channels in a nonspecific, low-affinity manner, with typical Kd values between 50 and >100 µM at –100 mV. Our data suggest that the venom of the scorpion L. quinquestriatus hebraeus contains a low-molecular-weight peptide toxin that inhibits wt-CFTR channels exclusively from the cytoplasmic side. Venom application altered CFTR activity by decreasing channel Po and burst duration by introducing blocked states hundreds to thousands of milliseconds in duration. Lowering [Cl]o resulted in increased venom blocking activity, which strongly suggests that the venom acts by blocking the channel pore. There was no effect on CFTR activity when the venom was applied to the channel's extracellular surface. Protease treatment eliminated the venom's activity, suggesting that the active component is a peptide. Boiling did not affect the activity of the venom, suggesting that the toxin is heat stable (5, 41). The toxin continued to inhibit the CFTR channels when they were locked in the open conformation, which further suggests that the toxin works through a pore block mechanism. Additionally, this active component of scorpion venom appears to have no effect on the ATP-dependent channel gating mechanism of CFTR.

The known Cl channel inhibitor ClTx had no effect on CFTR activity when applied to either side of the channel. Recent studies have identified many low-molecular-mass peptide toxins from Lqh and other scorpion venoms. Sequence comparisons have determined that Lqh-8/6 from L. quinquestriatus hebraeus (2), Bs8 and Bs14 from Buthus sindicus (4), and PBITx1 from Parabuthus schlechteri (53) are similar to ClTx from Lqq venom. Therefore, there is a possibility that this Lqh toxin will be one previously identified; alternatively, this study will result in the identification of a novel peptide toxin that selectively inhibits CFTR.

Generally, small organic pore blockers of CFTR exhibit stronger voltage dependence of blockade than larger blockers, suggesting that the small blockers are able to reach further across the electric field and permeation pathway into the narrower regions of the pore. However, all of these compounds appear to reach their binding sites from the cytoplasmic side of the channel (33, 48). These findings suggest that the cytoplasmic vestibule is larger than the extracellular vestibule (63); however, at this time there are no identified pore blocking agents that bind specifically to the wide cytoplasmic vestibule of the CFTR channel. If we presume that the active toxin in Lqh venom that inhibits CFTR is similar to other known pore-blocking peptide toxins, such as apamin (25), we would not anticipate that this toxin would reach its cytoplasmic binding site from the extracellular end of the channel, because it would have to traverse the Cl permeation pathway through the narrow region from Ser341 to Phe337, which has been described as a selectivity filter for CFTR (26, 35), before reaching the wide cytoplasmic vestibule.

Although both types of pore blockers, organic and peptide toxin, are active from the same side of the pore, their kinetics of block are quite different. The smaller organic blockers bind to the channel rapidly; however, they bind with low affinity, so their interactions with the channel are usually brief. Normal blocked dwell times range from tenths of a millisecond for DPC to tens of milliseconds for glibenclamide (46, 6163). In contrast, the calculated dwell time for our toxin is in the range of hundreds of milliseconds. These longer blocked times could be reflective of high-affinity interactions between the toxin and potentially multiple amino acids in the channel (30). However, the on rate of toxin block is not known, because we do not know the molar contribution this specific toxin makes to the full venom. Therefore, the affinity of the toxin for CFTR cannot be calculated accurately at this time.

We found that the venom inhibits CFTR channels with little or no voltage dependence. One possible explanation for this result is that the toxin's binding site is outside the electric field, where it is feasible that the voltage change across the pore would not be sensed. Pseudechetoxin (PsTx) has been shown to bind to residues in the pore turret that forms the wide outer vestibule of cyclic nucleotide-gated ion channels and resulted in block that was voltage independent (7, 8). Because so little is known about the structure of the CFTR cytoplasmic vestibule, it is possible that the {alpha}-helices that line the pore or the loops that connect them extend cytoplasmically beyond the electric field. If the toxin interacts with these portions of the pore, little voltage dependence of block would be expected. A second possible explanation is that the peptide toxin as a whole has a neutral net charge. Known peptide toxins have groups of positively or negatively charged amino acids located on their surfaces. It is these charged residues that lead to electrostatic interactions between the toxin and the channel when the toxin binds within the pore, resulting in a voltage dependence of toxin-induced inhibition. If the new toxin that inhibits CFTR does not have a net charge, then a voltage change across the membrane would not significantly alter the blocking activity.

An additional possibility is toxin-channel interactions that lead to allosteric inhibition rather than pore block. It is possible that the peptide inhibitor binds to the channel in an area away from the pore that in some way inhibits the conformational changes necessary for channel opening. Because the venom is only active from the cytoplasmic side of the channel, it is conceivable that either of the NBDs or the R domain could be the toxin's binding site. However, the results shown in Figs. 9 and 10 argue against this possibility, as does the increase in efficacy under conditions of reduced [Cl]o. Finally, the venom could contain a compound that activates the phosphatases that are endogenously expressed. If this were the case, one could argue that the decrease in CFTR activity is due to channel dephosphorylation. In this scenario we would not expect to see a recovery of CFTR current on venom washout in the macropatch experiments where PKA is removed from the bath before recordings begin. However, we have seen complete or near-complete recovery of current during venom washout in multiple patches (see, e.g., Fig. 5B). Therefore, we conclude that the peptide component blocks the CFTR channel pore directly.

Scorpion peptide toxins have been shown to inhibit many types of cation channels. However, no peptide toxins have been shown to inhibit Cl channels of known molecular identity. In this report we show that there is a peptide toxin in scorpion venom that inhibits CFTR Cl channels. However, this toxin works from the cytoplasmic side of the channel. It is highly unlikely that the scorpion developed this toxin to intentionally inhibit CFTR channels in this way. It is possible that another channel or transporter is the toxin's natural target. At this point we know that this toxin is a peptide that inhibits CFTR channels. More importantly, the toxin has the potential to be used as a new probe for studies of CFTR channel structure. Understanding the pore structure of the CFTR Cl channel will likely provide important information for the rational design of CFTR-activating (for CF) or -inhibiting (for secretory diarrhea and polycystic kidney disease) agents. This is also the first report describing peptide toxin inhibition of an ABC transporter. Therefore, this report represents a novel direction for CFTR, Cl channel, and ABC transporter research in general.


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This work was supported by grants from the National Science Foundation (MCB-0077575) and the National Institute of Diabetes and Digestive and Kidney Diseases (DK-56481 and DK-066409). N. A. McCarty is an Established Investigator of the American Heart Association.


    ACKNOWLEDGMENTS
 
We thank H. Lester and H. C. Hartzell for reviewing an early version of the manuscript. We also thank B. Song for assistance with the preparation of the constructs used in this study.


    FOOTNOTES
 

Address for reprint requests and other correspondence: N. A. McCarty, School of Biology, Georgia Institute of Technology, 310 Ferst Dr., Atlanta, GA 30332-0230 (E-mail: nael.mccarty{at}biology.gatech.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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