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
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ABSTRACT |
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cystic fibrosis; anion channel; cystic fibrosis transmembrane conductance regulator; ion channel block
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 -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.
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EXPERIMENTAL PROCEDURES |
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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 VVI 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 520 ng of wt-CFTR cRNA along with 0.4 ng of cRNA for the 2-adrenergic receptor (
2-AR) for TEVC and single-channel experiments or 50100 ng of CFTR cRNA for macropatch experiments. Recordings were made at room temperature 25 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 0.50.9 M 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 0.55 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, 210 Tris-EGTA, 0.22 MgATP, and 10 TES (pH 7.4; adjusted with Tris). In some experiments, 0.10.2 mg/ml venom, 5 mM VO4, and/or 2.75 mM adenosine 5'-(,
-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
for single-channel recordings and from 1 to 4 M
for macropatch experiments. Typical seal resistances ranged from 100 to >300 G
. 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 0.22 mM MgATP, no MgATP, 0.1 mg/ml venom, or 0.22 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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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 (
c) in a given channel record:
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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).
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RESULTS |
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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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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 (o) = 3,302 ± 682 ms for control and
o = 788 ± 86 ms in presence of venom (n = 5), respectively (Fig. 6A).
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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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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: 010% acetonitrile caused a 6.4 ± 6.4% decrease (n = 2), 1025% acetonitrile caused a 8.7 ± 2.6% decrease (n = 2), and 5075% 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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DISCUSSION |
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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 -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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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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