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* Program in Molecular and Systems Pharmacology, Emory University, Atlanta, Georgia; and School of Biology, Georgia Institute of Technology, Atlanta, Georgia
Correspondence: Address correspondence to Nael A. McCarty, Georgia Institute of Technology, School of Biology, 310 Ferst Dr., Atlanta, GA 30332-0230. Tel.: 404-385-2955; Fax: 404-894-0519; E-mail: nael.mccarty{at}biology.gatech.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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). Loss of function mutations in the gene encoding CFTR cause cystic fibrosis, the most common lethal, autosomal recessive genetic disease in Caucasians, which can result in severe lung disease, pancreatic insufficiency, and infertility (2). However, inappropriate CFTR channel activity is associated with diseases such as secretory diarrhea and polycystic kidney disease (3). Therefore, CFTR is an important medicinal target for therapeutics aimed at correcting channel activity.
CFTR is a member of the adenosine triphosphate (ATP)-binding cassette (ABC) transporter superfamily (4). The channel is a functional monomer with a single polypeptide required to form a single-channel pore (5,6). The polypeptide is composed of two halves, each containing a transmembrane domain and a cytosolic nucleotide binding domain (NBD). The two homologous halves are linked by a regulatory (R) domain. CFTR channel activity requires the presence of hydrolysable nucleoside triphosphates at the NBDs, and the R-domain must be phosphorylated by protein kinase A (PKA) and/or protein kinase C (7,8). Dimerization of NBD1 and NBD2 is an important step during CFTR gating (9). The NBD1/NBD2 dimer configuration promotes the formation of two ATP binding pockets NBD-A and NBD-B, identified according to which portion of the primary sequence contributes the catalytic lysine (9–12); hence, NBD-B includes K1250.
The use of reagents such as 3-isobutyl-1-methylxanthine resulted in a better understanding of the cAMP-dependent regulatory mechanism for CFTR modulation (13,14). These compounds alter channel activity indirectly by inhibiting phosphodiesterase activity causing a decrease in the rate of cAMP degradation, thereby resulting in the potentiation of cAMP-dependent CFTR activity; 3-isobutyl-1-methylxanthine may also alter ATP-dependent gating directly (13,15). Alternatively, non- or poorly-hydrolysable ATP analogs such as adenylyl-imidodiphosphate (AMP-PNP) and adenosine 5'-O-(3-thio)triphosphate (ATP
S), or analogs of ATP hydrolysis products such as vanadate (VO4) or pyrophosphate, have been used to study ATP-dependent gating of CFTR (11,16–18). These compounds directly interact with the channel in the active sites formed by NBD dimerization and alter channel gating. However, since these compounds work by mimicking the activity of ATP or its products, they have not been as useful in answering the following question: Which regions of the CFTR protein change conformation during the gating process subsequent to binding and/or hydrolysis of ATP? The ideal probe for answering this question would be one that interacts with the NBDs during the gating cycle, but can also sense conformational changes that occur outside of the NBDs, which may lead to opening of the channel pore.
We recently reported that a peptide toxin or toxins contained in the venom of the scorpion Leirus quinquestriatus hebraeus (Lqh) reversibly inhibits WT-CFTR only when applied to the cytoplasmic surface of the channels (19). Here we report the complete characterization of the Lqh venom inhibitory activity. In macropatch configuration, the degree of inhibition showed strong dependence upon experimental protocol, particularly with regard to whether the venom and ATP were applied separately or concurrently. In addition, either increasing or decreasing the MgATP concentration used to activate the channels altered the level of inhibition seen with a single concentration of venom. We also found that the potency of venom for intraburst inhibition was reduced in single-channel recordings of WT-CFTR channels with very high open probability or when the venom was applied to K1250A-CFTR channels. In combination with the observation that single channels locked open by treatment with either VO4 or AMP-PNP are less sensitive to inhibition by venom (19), these findings suggest that the inhibition of CFTR is due to interaction of venom with the NBDs in a state-dependent manner. We conclude that the venom alters channel activity by binding during interburst or intraburst closed states, and could be useful for reporting the structural changes that occur subsequent to or during binding and hydrolysis of ATP.
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MATERIALS AND METHODS |
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,20). Oocytes were harvested from mature Xenopus laevis (Xenopus 1, Ann Arbor, MI). The animals were anesthetized by immersion in tricaine (1.5 mg/mL) and several ovarian lobes were surgically removed under sterile conditions. The follicle cell layer was removed by incubating oocytes for 2 h with 1 mg/mL collagenase in calcium-free OR2 solution: 82.5 mM NaCl, 2.5 mM KCl, 1 mM MgCl2, and 5 mM HEPES (pH 7.4). The oocytes were washed with calcium-free OR2 and transferred to a modified Liebovitz's L-15 medium with the addition of HEPES (pH 7.5), gentamicin, and penicillin-streptomycin, and incubated at 18°C. For single-channel recordings, cRNA was prepared from a construct (20) carrying the full coding region of CFTR in the pAlter vector (Promega, Madison, WI). The mutant K1250A-CFTR construct was prepared with the QuikChange protocol (Stratagene, La Jolla, CA) using oligonucleotide-mediated mutagenesis. The construct was verified by sequencing across the entire open reading frame before use. For macropatch recordings, cRNAs were prepared from high expression constructs encoding WT-CFTR (pGEMHE-WT) and Flag-cut-
R-CFTR (pGEMHE-Flag3-633 and pGEMHE-837-1480), generously provided by Dr. David Gadsby (The Rockefeller University, NY). Oocytes were injected with 5–20 ng of CFTR cRNA along with 0.4 ng of cRNA for the ß2-adrenergic receptor for single-channel experiments, allowing activation of CFTR currents by exposure to isoproterenol, or 25–100 ng CFTR cRNA alone for macropatch experiments. Recordings were made 2–5 days after injection.
Electrophysiology
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 (21). Pipettes were pulled from borosilicate glass (Sutter Instruments, Novato, CA). Pipette solution contained 150 mM NMDG-Cl, 5 mM MgCl2, and 10 mM TES (pH 7.4; adjusted with Tris). Intracellular bath solution for excised patches contained 150 mM NMDG-Cl, 1.1 mM MgCl2, 2–10 mM Tris-EGTA, 0.2–5 mM MgATP, and 10 mM TES (pH 7.4; adjusted with Tris). In some experiments 0.1–0.2 mg/mL Lqh venom, 200 µM DPC, 5 mM VO4, and/or 2.75 mM AMP-PNP was added to the intracellular solution. CFTR channels were activated by the catalytic subunit of PKA (50 U/mL) after patch excision into solution containing 1 mM MgATP. 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. All recordings were performed at room temperature (22–25°C).
Single-channel experiments were performed using an Axopatch 200B amplifier (Axon Instruments, Union City, CA) and recorded at 10 kHz to DAT tape (# DTC-EZ700, Sony, San Diego, CA). The membrane potential was held at either –80 mV or –100 mV. In some cases, 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 using a Digidata 1322A interface (Axon Instruments) and computer at 400 Hz using the Clampex program of pClamp (Axon Instruments). For intraburst closed-time analysis, experiments were played back and filtered at a corner frequency of 500 Hz and digitized at 2 kHz. Digitized Clampex records were analyzed using Clampfit 9.0 (Axon Instruments) and Igor Pro 4.02 (WaveMetrics, Lake Oswego, OR). In single-channel experiments, recordings of channel activity with venom present began 
30 s after solution change (19).
Macropatch recordings were also performed with an Axopatch 200B amplifier operated by pClamp software, filtered at 100 Hz, and acquired at 1 kHz with Clampex followed by analysis using Clampfit 9.0. Macropatch currents were obtained by applying one of four voltage protocols. In experiments to measure the on-rate of CFTR inhibition by either DPC or partially fractionated Lqh venom (Lqh-pf venom), the membrane potential was set to 0 mV and then stepped to –80 mV for 150 ms and repeated once per second for 30 s. To determine the maximal inhibitory effect of DPC or venom, the membrane potential was set to 0 mV and then either stepped to various membrane voltages from –100 mV to +100 mV for 150 ms in 20-mV increments, or stepped to –100 mV and held for 50 ms, then ramped to +100 mV over 150 ms. All voltage ramps were run in triplicate and averaged. In the fourth voltage protocol, the membrane potential was stepped from 0 mV to either –80 mV or –100 mV and held throughout the entire course of the experiment. Bath solutions with MgATP, no MgATP, venom, DPC, or MgATP plus venom or DPC were applied to the patches by a fast perfusion system (Warner Instruments, model SF-77B). Solution exchange was complete within <25 ms as judged by the activation of endogenous Cl(Ca) channels in oocytes by exposure to bath solution containing 10 mM Ca2+ (19). WT-CFTR channels were phosphorylated by PKA (50 U/mL) and 1 mM MgATP until the macroscopic current had reached the maximum level for a given patch. After apparent complete CFTR activation, but before employing the fast solution exchange system, the PKA and ATP were flushed from the bath using the ATP-free bath solution until the ATP-dependent current had completely subsided.
Analysis of single-channel and macropatch recordings
Each patch served as its own control before exposure to venom or DPC in all experiments. Transition analysis for single-channel experiments used a 50% cutoff between the open and closed current levels. Open duration analysis was performed on records from patches containing 1–3 active CFTR channels. For initial open duration analysis, records were filtered at 100 Hz and a 100 ms minimum interburst duration cutoff was used to discriminate between interburst gating and brief intraburst closings (22). Subsequent records were filtered at 500 Hz where a 1-s minimum interburst duration cutoff was applied, and a 5-ms minimum intraburst duration cutoff was used to discriminate between brief intraburst closings and transitions to the closed current level due to block by the buffer TES (23). The mean open duration in multichannel patches was determined as previously described (22,24) with the formula
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j is the apparent number of active channels, tj is the time that at least j channels are simultaneously open and n is the total number of transitions from an open state to a closed state during the multichannel open event. Thus, a multichannel open burst event is transformed to n single-channel open events with duration t. For the determination of channel open probability (Po), only those patches containing apparently five or fewer open channels were included. Po was calculated as NPo/N, where N is the apparent number of active channels in the patch, determined as the maximum number of channels that were simultaneously open at any point during the control recording of at least 3-min duration; channel number was assumed to be the same upon subsequent exposure to venom. Similar techniques have been used previously to determine CFTR channel number in a given patch (25). Open- and closed-time histograms had bin widths of 2 ms and were constructed from recordings that were a minimum of 360 s in length. Most records used for Po or dwell-time analysis were 30 min in duration. Only apparent intraburst closings (or blocked events, in the presence of venom) were included in construction of closed duration histograms; hence, no closings >1000 ms were used in that analysis, because closings of this length were assumed to be due to true channel closure. Since venom-induced inhibition introduced a new population of intraburst closed dwell times not present in control recordings, we were able to estimate the apparent rate constant of venom dissociation (koff) from the mean intraburst blocked intervals (
c) in a given channel record as
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Venom processing
Lqh venom was dispersed into intracellular bath solution at 2.5 mg dry venom/mL by extensive vortexing followed by brief homogenization using a Potter-Elvenhjem tissue grinder (19). The mucous component was pelleted by centrifugation at 6000 x 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, the upper, relatively mucous free solution was recovered and then filtered using a Biomax-10 Micropartition System filter (Millipore, Bedford, MA) with a 10-kDa cutoff, and centrifuged at 2000 x g at 22°C in a fixed angle rotor, to remove the higher molecular-weight components of venom. The resulting mucous-free, filtered venom was stored at –80°C and diluted to given concentrations, based on equivalent dry venom weight, immediately before use. This fraction of venom, which contains several peptide components (19), is referred to as Lqh-pf venom.
Statistics
Results are expressed as mean ± SE for n observations. Comparisons are by paired and unpaired Student's t-test. Differences were considered statistically significant when p < 0.05. Linear regression analysis was performed using SigmaPlot (Jandel Scientific, San Rafael, CA). All statistical tests were performed using SigmaStat 2.03 (Jandel Scientific).
Reagents
Unless otherwise noted, all reagents were obtained from Sigma Chemical (St. Louis, MO). L-15 media was purchased from Gibco/BRL (Gaithersburg, MD). Scorpion venom was purchased from Latoxan (Valence, France). PKA was from Promega. 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 (17). DPC was from Aldrich Chemical (Milwaukee, WI), and was dissolved in DMSO at stock concentrations of 0.1–0.5 M and diluted to a 200 µM final concentration immediately before use. The final concentration of DMSO varied up to a maximum of 0.1%, which had no effect on CFTR currents.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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). In the experiment shown in Fig. 1 A (middle), Lqh-pf venom was applied to phosphorylated CFTR channels that had been allowed to close upon removal of cytosolic ATP. When ATP was returned to the bath, the resulting current density was reduced compared to currents in the absence of venom; application of 0.1 mg/mL Lqh-pf venom to closed CFTR channels in this way led to inhibition of macroscopic current by 24.5 ± 2.5% (n = 9, p 
0.001) at Vm = –80 mV. This result could arise from interaction of the toxin with closed channels before reintroduction of ATP, thus inhibiting their opening, and/or from inhibition of open channels in the presence of ATP. As a control, we examined the effects of DPC when applied to the closed channels in the same way (Fig. 1 A, bottom). DPC is a known CFTR channel pore blocker (20–22), which has not been shown to have any state-dependence of action. Application of 200 µM DPC to closed CFTR channels led to 26.1 ± 2.6% block (n = 5, p 0.001) upon subsequent activation with ATP, suggesting that, like Lqh-pf venom, DPC can effectively bind to the closed channels. However, since the on-rate of DPC is very high (21), this experimental protocol does not preclude binding of DPC to open channels, as well.
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R-CFTR channels lacking the regulatory R-domain (Fig. 1 B), which does not require PKA-mediated phosphorylation (26). The use of this CFTR variant therefore effectively eliminates the potential for dephosphorylation during the recording, suggesting that the decrease in macroscopic current observed in WT-CFTR is due to venom-induced inhibition or DPC-induced block, respectively. Flag-cut-R-CFTR was inhibited by venom to approximately the same degree as WT-CFTR.
To determine whether venom can inhibit open channels, experiments were performed in which Lqh-pf venom was rapidly applied to CFTR channels that were already fully activated. ATP was rapidly applied to patches containing previously phosphorylated channels for 30 s before addition of venom or DPC (Fig. 1 C). Exposure to 200 µM DPC in the continuing presence of 1 mM MgATP led to 31.9 ± 3.1% block of macroscopic current (n = 5, p 0.001), suggesting that DPC can block CFTR channels in the open state equally well as closed channels (Fig. 1 D). However, application of 0.1 mg/mL Lqh-pf venom onto activated CFTR channels led to only 5.13 ± 1.3% decrease in macroscopic current (n = 14, p = 0.002), indicating that venom is less effective at inhibiting CFTR channels in the open state compared to in the closed state (p 0.001, Fig. 1 D). These results suggest that Lqh-pf venom inhibits CFTR by preferentially interacting with channels in the closed state. Because the Po of individual CFTR channels in the presence of 1 mM ATP is <0.5 (19,23), venom may interact with open channels that eventually close even in the continued presence of ATP, leading to a slow increase in fractional inhibition through time, as observed (Fig. 1 E). However, the majority of DPC block occurs very rapidly.
Effectiveness of macroscopic inhibition depends on the level of CFTR channel activity
The data presented thus far suggest that Lqh-pf venom may preferentially bind to the closed CFTR channels and then inhibit development of Cl– conductance upon channel activation by ATP. The ATP-dependent gating cycle of WT-CFTR includes several closed states that could be the target of Lqh-pf venom binding (11,18) (Fig. 2 (thick arrow pathway)). Phosphorylated channels reside in the resting (C1) closed state before initial interactions with ATP. Upon binding ATP at NBD-A, channels reside in state C2, which represents the closed state between open bursts (the interburst closed state). The rate of ATP release from NBD-A has been shown to be extremely low (10). Hence, CFTR gating under physiological conditions typically involves transitions from the C2 state through the open states and back to C2, by binding and hydrolysis of ATP at NBD-B, along the path described by the solid rectangle in Fig. 2. In addition, there are two potentially short-lived closed states that occur during the ATP gating cycle. The C3 and C4 closed states occur either immediately before NBD dimerization after ATP binding at NBD-B (C3) or after NBD de-dimerization (C4) as a prerequisite for release of ADP from NBD-B. Three open states are shown, which differ according to what is bound at NBD-B. CFTR activity can be defined by the fraction of time the channel spends in each of these states.
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,18,27). We measured the effect of Lqh-pf venom on phosphorylated WT-CFTR channels in macropatches activated by four concentrations of MgATP ranging from 0.2 mM to 5 mM (Fig. 3 A). In the presence of 1 mM MgATP, treatment with 0.1 mg/mL Lqh-pf venom produced 24.5 ± 2.5% inhibition of macroscopic currents (n = 9). The same concentration of venom had a reduced effect when the channels were activated with 2 mM MgATP (18.4 ± 2.5% inhibition, n = 7). At high [MgATP] (5 mM), 0.1 mg/mL Lqh-pf venom produced only very modest inhibition (14.4 ± 0.6%, n = 3). However, with 0.2 mM MgATP, inhibition of macroscopic currents by Lqh-pf venom was enhanced to 30.8 ± 2.1% (n = 4, p 0.001 compared to inhibition with 5 mM ATP present). These results suggest that the ability of Lqh-pf venom to inhibit CFTR is dependent on CFTR channel activity, where conditions that promote high channel activity diminish the efficacy of inhibition.
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The data in Fig. 3, A and B, suggest that ATP may compete with venom at the NBDs, because venom is ineffective at inhibiting macroscopic current in the presence of high [ATP]. However, because CFTR channel activity is also increased as a saturating function of [ATP], the results may also indicate that efficacy of inhibition by venom is dependent upon channel activity more directly, without invoking competition at the ATP binding site. To test this idea, we used the macropatch data to compare fractional inhibition as a function of estimated Po (Fig. 3 C). Because Po in the absence of venom is known for single-channel experiments but not for macropatch experiments, we transformed the macropatch data using true values for Po measured from single channels under defined conditions, making use of the relationship between the Po of phosphorylated CFTR channels and [MgATP] (18). To perform this transformation, we first measured macropatch current as a function of [MgATP]. Studies have shown that 5 mM MgATP can fully activate WT-CFTR macroscopic current (18); thus the relative current at MgATP concentrations <5 mM was determined by normalizing the macropatch current at those concentrations to the current in the presence of 5 mM MgATP (Fig. 3 C, top). Because the mean Po of WT-CFTR with 1 mM MgATP in single-channel patches is known, we then normalized macroscopic currents to those measured with 1 mM MgATP (Fig. 3 C, middle). This transformation allowed us to assign estimated open probabilities to each of the macropatch recordings at various MgATP concentrations (Fig. 3 C, bottom), based on the measured Po of WT-CFTR single channels with 1 mM MgATP (Po = 0.339 ± 0.029, n = 51). A three-parameter Hill equation was used to fit the data, giving p 0.001, R2 = 0.99. From these calculations we find that CFTR channels in macropatches activated by 0.2 mM MgATP had an estimated Po of 0.170, whereas those recorded with 5 mM MgATP in the bath had an estimated Po of 0.525.
A plot of inhibition of WT-CFTR macroscopic currents by 0.1 mg/mL Lqh-pf venom as a function of estimated Po is shown in Fig. 3 D, where each point represents an individual macropatch experiment at 0.2, 1, 2, or 5 mM MgATP (n = 23). Linear regression analysis suggested that there is a significant relationship between the magnitude of inhibition by 0.1 mg/mL Lqh-pf venom and CFTR estimated channel Po (p 0.001, R2 = 0.45). It is clear that channels with high activity before application of venom were inhibited poorly by venom, while inhibition of channels with low activity was significantly stronger. This result suggests that the level of channel activity is the determining factor in the ability of the Lqh-pf venom to inhibit, rather than competition at the ATP binding site.
If venom competitively inhibits ATP binding at NBD-B, we also would expect there to be a lengthening of the C2 interburst closings during channel gating. One manifestation of the effect would be a reduction in the macroscopic opening rate of channels in the presence of ATP. However, in our previous study we found no significant change in ATP-induced apparent macroscopic CFTR opening rate upon rapid introduction of ATP (19). A possible explanation for the lack of an effect on apparent macroscopic opening rate is that the Lqh-pf venom remains bound to the channels in the C2 state for very long periods resulting in an extremely low off-rate, so low that the active toxin does not dissociate from the channel within the 30-s wash-on of ATP in the presence of venom during the macropatch protocol. In other words, competitive inhibition at the ATP binding site should only be evident as a change in macroscopic opening rate if the intrinsic rates of binding and unbinding are similar for ATP and the active toxin.
To determine if Lqh-pf venom treatment locked channels into the interburst closed state, we used excised, inside-out multichannel patches. Fig. 4 A shows an example of currents from WT-CFTR channels activated with 50 U/mL PKA and 1 mM MgATP before (left) and during treatment with 0.1 mg/mL Lqh-pf venom (right). Lqh-pf venom treatment resulted in apparent complete knockout of many of the channels that were active in the control recordings; this reduced activity was maintained over several minutes of recording, suggesting that the apparent rate of venom dissociation is very low. Our previous results showed that this effect is reversible (19). Consistent with the lock of channels into the C2 interburst closed state, we often found in patches containing a single WT-CFTR channel at low to moderate Po that exposure to venom rapidly led to disappearance of that channel, despite the continued presence of ATP and PKA (Fig. 4 B). We reasoned that the venom-induced lengthening of the time that the channel spent in the C2 closed state would be more evident if an extremely active variant of CFTR were used in this type of experiment. Removal of Walker A lysines that are important for ATP hydrolysis can greatly increase Po (16). K1250A-CFTR channels exhibit a greatly reduced rate of ATP hydrolysis, resulting in channels that are open for tens of seconds; however, K1250A-CFTR channels also exhibit a reduced opening rate such that they remain in the C2 closed state longer than WT-CFTR channels (18). Studies were performed with K1250A-CFTR in multichannel patches with 50 U/mL PKA and 1 mM MgATP continuously present. Upon treatment with 0.1 mg/mL Lqh-pf venom, similar results were observed in that many of the active channels disappeared from the patch within a few minutes (Fig. 4 C). The very large increase in interburst closed duration as a result of venom binding can be clearly seen since there are no multichannel open events at any point during the recording with venom (2 min). These results, along with those from the macropatch recordings, suggest that the venom interacts with CFTR when the channels are in the C2 closed state, shifting them to the C2B state (Fig. 2). Also, the extremely low rate of dissociation of venom from the channel after binding during C2 closings could explain why there is no change in the apparent ATP-dependent macroscopic opening rate in macropatch recordings (19).
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). Analysis of those recordings showed that 0.1 mg/mL venom caused a 67% decrease in mean burst duration, resulting in a 32% decrease in apparent Po of those channels that were still visible in the patch, in records filtered at 100 Hz. Therefore, a second effect of venom treatment was that the mean duration of CFTR open bursts was apparently shortened because sojourns at the closed current level, representing new blocked states, were introduced into the record. Fig. 5 shows an example of currents from WT-CFTR single channels activated with 50 U/mL PKA and 1 mM MgATP before (top) and during treatment with 0.1 mg/mL Lqh-pf venom (bottom). Upon venom treatment, open bursts appeared to be shortened by the introduction of new closures; the venom-induced closures seemed to be the result of open channel block. However, results from studies with WT-CFTR in macropatch recordings (Fig. 1 C), as well as K1250A-CFTR in recordings of only a few channels (Fig. 4 B), suggested that the venom does not interact with the open state. To clarify this discrepancy, we studied the intraburst behavior of CFTR channels in the presence and absence of venom, in recordings with limited filtering (500 Hz).
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,28); such closings are filtered out of most CFTR channel records published. It is the brief FC intraburst closures that give WT-CFTR gating its flickery character. We reasoned that the FC intraburst closed state may represent a target for venom binding. To determine if the Lqh-pf venom does interact with CFTR during the intraburst closings we recorded (at 500 Hz) WT-CFTR in the absence and presence of 0.1 mg/mL Lqh-pf venom (Fig. 5). The expanded records clearly indicate that there are numerous intraburst closings during open bursts of WT-CFTR in control conditions and during venom treatment. For analysis of the CFTR activity under these conditions we used a 5-ms minimum duration cutoff for intraburst closings and a minimum 1-s cutoff for interburst closings; these criteria should help to distinguish FC intraburst closings from TES-induced pore block (closed state transitions <5 ms in duration) and gating-induced channel closures to the C2 state (closed state transitions >1000 ms in duration). If the Lqh-pf venom interacts with the FC intraburst closings under these conditions we would anticipate the following results: 1), there would be no change in the mean open burst duration; and 2), there would be an increase in the mean intraburst closed duration, because the brief intraburst (FC) closed state would be transformed into a longer (i.e., FCB; see Fig. 2) inhibited state. Hence, venom binding during these brief closings would appear to have the effect of prolonging the duration of the intraburst closed state until the toxin dissociates from the channel, which would be evident as an increase in the occurrence of closures with durations of >100 ms and a decrease in the number of brief closings.
We analyzed channel open and closed events in records of WT-CFTR in the absence and presence of 0.1 mg/mL Lqh-pf venom, using records filtered at 500 Hz. Only intraburst closings were used in this analysis; therefore, no closings of >1000 ms duration were included. Treatment of WT-CFTR with 0.1 mg/mL Lqh-pf venom did not result in a significant change in channel open time (overall mean open duration = 670.3 ± 64.7 ms in control versus 582.1 ± 44.6 ms with Lqh-pf venom, p = 0.328, n = 6) (Fig. 6 A). These results suggest that venom does not interact with the open CFTR channel. However, Lqh-pf venom treatment did result in a significant 424% increase in overall mean intraburst closed duration from 36.4 ± 9.4 ms in control conditions to 154.5 ± 28.9 ms during exposure to Lqh-pf venom (p < 0.01, n = 6) (Fig. 6 B). Dwell-time histograms were then constructed from these records (Fig. 6 C). In the absence of venom, the closed-time distribution was fit best by a single-exponential function giving mean c = 5.2 ms. However, addition of Lqh-pf venom resulted in a closed-time distribution that was best fit by the sum of two exponentials giving mean c1 = 12.7 ms and mean c2 = 729.9 ms. This analysis provides an estimated Lqh-pf venom mean block time of 730 ms, giving an estimated koff of 1.37 s–1 using Eq. 2. Importantly, the number of brief closed states (5–30 ms in duration) was reduced in the presence of venom. These results suggest that Lqh-pf venom also binds to active CFTR channels while in the FC intraburst closed state, shifting channels to the longer FCB blocked state.
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,29). Under standard conditions (50 U/mL PKA and 1 mM MgATP continuously present), Po in our records varied from patch to patch usually in a range between 0.15 and 0.45; the mean Po was 0.339 ± 0.029 (n = 51). The amount of venom-induced inhibition also appeared to vary depending on the level of CFTR channel activity in a given patch before addition of venom. Therefore, to quantify this phenomenon we separated the analysis of venom-induced inhibition in experiments with high activity (Po > 0.35) from that in experiments with low activity (Po < 0.35). In the trace shown in Fig. 5, representative of a low activity experiment, treatment with Lqh-pf venom resulted in 25.5% inhibition (apparent Po changed from 0.231 to 0.172). In a series of experiments of this sort, treatment with 0.1 mg/mL Lqh-pf venom resulted in a 31.4 ± 6.96% decrease in mean apparent Po of WT-CFTR channels with low activity before venom (Po = 0.181 ± 0.031 vs. 0.118 ± 0.018, n = 6, p = 0.029). Channels that displayed elevated activity under control conditions were not as extensively inhibited by the same concentration of venom (Po = 0.584 ± 0.091 vs. 0.554 ± 0.087, n = 2). Visual examination of recordings of highly active WT-CFTR channels indicated that the elevated Po was a result of extremely long open bursts (data not shown). However, inhibition of these highly active channels was improved by exposure to an increased concentration of Lqh-pf venom (0.2 mg/mL). As summarized in Table 1, the amount of inhibition by 0.1 mg/mL venom was significantly different, depending on the level of channel activity under standard conditions with PKA and ATP continuously present. These results suggest that the venom-mediated inhibition of CFTR is dependent on the channel open probability.
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) (Fig. 2, upper dashed pathway). A second treatment that can be used to lock CFTR open is application of vanadate, a transition-state analog of the ATP hydrolysis product inorganic phosphate, which binds tightly in place of Pi and interrupts the ATP hydrolysis cycle and channel gating (17) (Fig. 2, lower dashed pathway). In addition, we also employed the Walker A mutant K1250A-CFTR, which gates open much like WT-CFTR, although the rate of ATP binding to NBD-B is somewhat reduced, whereas hydrolysis of ATP is greatly reduced, resulting in open bursts that are many tens of seconds in duration (Fig. 2, boxes).
Single WT-CFTR channels were studied with 50 U/mL PKA, 1 mM MgATP, and either 5 mM vanadate or 2.75 mM AMP-PNP continuously present in the bath solution. All recordings were filtered at 500 Hz to enable examination of the short intraburst FC closings. Fig. 7 A shows a representative trace from one recording where channel activity was stimulated by AMP-PNP. Under these conditions, CFTR Po was elevated as compared to that of channels which are allowed to proceed through the normal gating cycle, as indicated by the long open burst duration (compare Fig. 5, top). In the presence of AMP-PNP (or vanadate, Fig. 7 B), periods of normal channel gating can be seen before channels became locked in the open state. After entry into the locked-open state, there are occasional brief intraburst closures that have durations of 5–100 ms. Hence, even in the presence of vanadate or AMP-PNP, CFTR Po remains <1.0. Application of 0.2 mg/mL Lqh-pf venom to the patch in the presence of ATP + AMP-PNP (Fig. 7 A, bottom) or ATP + vanadate (Fig. 7 B, bottom) resulted in the appearance of additional closed states. In control conditions, K1250A-CFTR channels remained almost entirely in the open state (Fig. 7 C, top), with few brief closures. After treatment with Lqh-pf venom, a few intraburst closed/blocked events were identified, although their frequency was low (Fig. 7 C, bottom). Treatment of single K1250A-CFTR channels with 0.1 mg/mL Lqh-pf venom resulted in only 2.66 ± 0.59% inhibition of channel activity (Po = 0.772 ± 0.079 vs. 0.751 ± 0.074, n = 3). Notably, the durations of the venom-induced intraburst blocked states in K1250A-CFTR and WT-CFTR with ATP + AMP-PNP, or WT-CFTR with ATP + vanadate, were similar to those observed in WT-CFTR with ATP alone (400–700 ms). However, the frequency that the blocked events occurred and the total amount of inhibition of Po were decreased, even though the venom concentration was doubled compared to that shown in Fig. 5 (see Table 1).
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5 s in duration were used in our analysis of experiments with those reagents. All K1250A-CFTR openings were used. Fig. 8 D shows the observed mean open time between FC intraburst closings, in the absence of venom, which clearly varied depending on experimental conditions. WT-CFTR channels transitioned to FC intraburst closures once every 627.6 ± 25.7 ms under standard control conditions (n = 3 recordings, n = 47 bursts, n = 433 open duration events). It is important to note that, in each of these recordings, the control channel Po was between 0.174 and 0.281, indicating that these channels would have been greatly inhibited by 0.1 mg/mL Lqh-pf venom (Table 1). Channels locked open by AMP-PNP (mean open time between FC closings 1485.2 ± 98.2 ms; n = 2 recordings, n = 27 bursts, n = 176 open duration events) or vanadate (1010.3 ± 58.9 ms; n = 2 recordings, n = 29 bursts, n = 234 open duration events) transitioned to intraburst closures significantly less frequently (p 0.001 each, compared to WT-CFTR). The largest increase in mean open time between intraburst closings was seen in K1250A-CFTR where the channels remained open for an average of 1580.9 ± 106.8 ms (n = 3 recordings, n = 3 bursts, n = 177 open duration events; p 0.001 compared to WT-CFTR) between intraburst closings. These results suggest that the frequency at which CFTR moves to the FC state is dependent upon the status of binding and hydrolysis events at NBD-B. Comparing the data in Fig. 8 D with that in Table 1 shows that as these manipulations increased Po and decreased the frequency of transitions to the FC state, venom-mediated inhibition was reduced. Hence, intraburst inhibition of open channels by venom appears to occur upon binding of the active toxin at the FC state. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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). WT-CFTR channels that exhibited high activity under control conditions were very poorly inhibited by venom. Additionally, WT-CFTR channels that were locked in the open conformation by vanadate or AMP-PNP, and CFTR channels bearing the K1250A mutation, were inhibited by venom only at higher concentrations. These observations suggested that the level of CFTR activity could affect the efficacy of channel inhibition by venom. Data in the present study indicate that the toxin or toxins contained in Lqh venom preferentially bind to closed CFTR channels, suggesting that venom-induced inhibition is state-dependent. Single-channel studies, where CFTR activity was altered by either changing the cytoplasmic [MgATP], treating the channels with vanadate or AMP-PNP, or by eliminating ATP hydrolysis by mutation and thereby slowing channel gating, suggest that the amount of inhibition by Lqh-pf venom is dependent on the channel's Po. Lastly, the data suggest that the toxin or toxins can effectively inhibit CFTR channels only if allowed to interact in either of two closed states.
State-dependent inhibition of cation channels by either a peptide toxin or a small organic compound has been described on several occasions (31–33). N-type Ca2+ channel block by 
-conotoxin GVIA, Shaker K+ channel block by 
-conotoxin PVIIA, and HERG K+ channel block by ergtoxin, are examples of ion channel block by peptide toxins that show state-dependence (34–36). However, few examples of state-dependent inhibition of anion channels have been documented. Accardi and Pusch (37) have shown that p-chlorophenoxy-acetic acid has higher affinity for the closed state of ClC-0 channels than for the open state. Similarly, the buffer 3-(N-morpholino) propanesulfonic acid was shown to block the major subconductance state more readily than the full conductance state of CFTR when applied to the cytoplasmic surface of the channel (28). However, at this time there are no CFTR gating modifier reagents that have been shown to bind in a state-dependent manner.
Allosteric CFTR gating modifiers such as CFTRinh-172 and genistein are believed to bind to the NBDs. However, their usefulness as probes to study CFTR conformational changes during gating is limited due to their complex modes of action. Genistein is a CFTR activator at low µM concentrations, but at high µM concentrations, genistein decreases channel Po due to a prolongation of the closed time (14,24); at high concentrations, genistein also blocks the channel pore (38). CFTRinh-172 is thought to work as a gating modifier by interacting with NBD-1; however, this mechanism has not been rigorously tested (39).
Data presented here show that venom inhibits CFTR channel activity in a closed-state dependent manner by binding to a site that prohibits channel opening from the C2 state and that also inhibits channel reopening from the FC state; the former effect leads to a lengthening of the interburst duration, whereas the latter effect introduces new intraburst closed states. A conservative view would consider that these two behaviors represent binding to the same site, while the kinetics of interaction with that site depend upon the status of ATP binding and hydrolysis at NBD-B. However, as these experiments were performed with only partially fractionated venom we must consider that these two mechanisms may represent the activities of two different toxins, which may bind to two different sites. Individual toxins isolated from venom will be required to address this possibility.
For the following reasons, we propose that the active component or components of Lqh-pf venom interact(s) with CFTR at the NBDs adjacent to but not within the active site of NBD-B:
R-CFTR channels (Fig. 1) suggest that the venom does not inhibit CFTR by interacting with the R-domain. ). This mode of interaction explains the dependence upon [ATP]. Channels that bind toxin in the C2 state are conformationally excluded from opening after binding ATP at NBD-B, and channels that have bound this second ATP and entered into an open burst cannot bind the toxin until they close to the C2 state. ), and because binding at the C3 state would be enhanced at high [MgATP], which is in contrast to our findings. Hence, our data are most consistent with the notion that interaction of venom with channels in the C2 closed state underlies the lengthening of interburst closed durations in an ATP concentration-dependent manner.
The apparent dependence of the efficacy of venom-mediated inhibition in the C2 state upon the cytosolic ATP concentration (Fig. 3 A) is suggestive of simple competitive inhibition of ATP binding at NBD-B. However, we favor instead a relationship between efficacy of inhibition and channel activity and therefore the availability of the C2 state, which is partly controlled by ATP binding at NBD-B, for the following reasons:
). A combination of mutational analysis and binding studies using labeled ATP will be helpful for clarifying these possible mechanisms.
Venom-mediated inhibition of CFTR channels appears to occur via two mechanisms: one that lengthens interburst durations, by locking channels into the C2 closed state as discussed above, and another that affects intraburst kinetics. The effect on interburst kinetics cannot fully explain the reduced efficacy of inhibition of single K1250A-CFTR channels or single WT-CFTR channels in the presence of AMP-PNP or vanadate. To incorporate those findings, we must include another state amenable to interaction with the toxin. It is well known that brief, flickery closures, with durations between 5 and 100 ms, are seen in WT-CFTR under standard conditions and even in channels that have been locked into an openable state by events in the NBDs such as binding nonhydrolyzable nucleotides. Hence, these closed events likely involve changes in conformation of the pore domain, or in the structures that connect the pore domain and the NBDs, and do not reflect binding and hydrolysis events in the NBDs. To distinguish these closures from those that occur during ATP-dependent gating, we label them FC for flickery closings (Fig. 2). Our previous experiments, analyzing records that were heavily filtered (100 Hz), showed that exposure of WT-CFTR channels to Lqh-pf venom led to a decrease in mean closed durations (c) as well as a decrease in mean open burst durations (o) (19). The decrease in c arose because the population of venom-induced closed states, with durations 400–700 ms, greatly outnumbered the population of long, interburst closed states, with typical durations > 1 sec. These data suggested that the toxin bound to the open state, because the new population of venom-induced closed states appeared to arise from the open current level. This was most apparent in experiments with channels locked open by vanadate, AMP-PNP, or mutation K1250A. However, binding of toxin to the open state is not consistent with the lack of venom-mediated inhibition of current when venom was applied to open channels in macropatch experiments such as that shown in Fig. 1 C. Furthermore, filtering the data at 100 Hz precluded analysis of the brief intraburst closures.
To determine whether venom interacts with CFTR during an intraburst closure, we recorded channel activity with reduced filtering of the data (500 Hz). These records (Figs. 7 and 8) indicate numerous FC closings during open bursts of WT-CFTR under standard conditions and, to a lesser extent, in bursts from WT-CFTR locked open by AMP-PNP or vanadate. Careful examination of the closed time histograms compiled from records with limited filtering indicated that exposure to venom resulted in a decrease in the number of intraburst FC events with durations <20 ms and a concomitant increase in the number of intraburst closed states with duration in the hundreds of milliseconds, which represent the venom-induced intraburst closed states. Hence, the brief FC states appeared to be converted into the much longer FCB states upon binding the toxin (Fig. 2).
As described in Results, the expected Po of individual CFTR channels in the presence of 1 mM ATP is <0.5. One would expect that the Lqh-pf venom would have ample opportunity to achieve significant inhibition of macroscopic currents with this level of channel activity by binding in the C2 state. However, as shown in Fig. 1 C, a 30-s application of Lqh-pf venom onto fully activated CFTR resulted in only minimal channel inhibition. If the C2 interburst and FC intraburst closings are, in effect, the targets for the Lqh-pf venom, then how can we explain these results? We suggest that the level of Lqh-pf venom-mediated inhibition is dependent on the amount of time that CFTR is in the proper conformation during venom application; this is the definition of state-dependence. Multichannel records suggest that the dissociation rate of the Lqh-pf venom from the C2 closed state is extremely slow; however, we have no accurate measure of the venom on-rate to the C2 interburst closed state. It is plausible that the venom on-rate is also relatively slow and that C2 closings in WT-CFTR channels undergoing normal gating are not of sufficient length to warrant venom binding when the channels are fully activated by high concentrations of MgATP; this is likely the case since we continued to see single CFTR channel openings for tens of minutes while Lqh-pf venom was continuously present (for example, see Fig. 5). One would expect that at some point Lqh-pf venom would bind to the channel when it is transiently in the C2 closed state resulting in channel knockout unless the C2 closings are not of sufficient duration when ATP is present in the bath solution.
Which mechanism, interburst inhibition in the C2 state or intraburst inhibition in the FC state, is more important for the overall inhibition of CFTR channel current? The data shown in Fig. 1 suggest that the former accounts for the majority of the inhibitory efficacy of venom at CFTR. The 25% inhibition of macroscopic current at 0.1 mg/mL Lqh-pf venom should represent the result of both mechanisms of action. When venom is allowed to interact with channels in the absence of ATP, some fraction of channels bind the active toxin and become locked in the C2 state such that they cannot open upon reintroduction of ATP, but the extent of inhibition by this mechanism cannot be determined without exposure to ATP. However, application of venom to channels that have not bound toxin in the C2 state, and are undergoing normal gating, leads to, at most, 5% inhibition (see Fig. 1, C–E). Hence, the remainder, i.e., 20% inhibition, must arise from the block of channels in the interburst C2 closed state.
What determines the development of intraburst inhibition? Analysis of single-channel records that were from 200 to 1300 s in duration indicates that WT-CFTR channels reside in a conformation reflecting intraburst closings of 5–20 ms (the FC state) only 0.39 ± 0.09% of the time (n = 7; data not shown). From these results, we can calculate that WT-CFTR channels will reside in the FC state for a total of only 117 ms during the 30-s applications of 1 mM MgATP in the macropatch configuration. These results suggest that a 30-s application of 0.1 mg/mL Lqh-pf venom to open CFTR channels will not likely result in maximum inhibition of channel activity by either mechanism, although if the venom were applied to the activated channels for an extended period then a greater level of inhibition would develop through time; the data in Fig. 1 E are consistent with this.
The previous results could also explain why Lqh-pf venom is significantly less effective when applied to K1250A-CFTR, since the frequency of short intraburst closings in this mutant is much less than that seen with WT-CFTR (Fig. 8 D). Similar results are also seen in recordings of WT-CFTR that were locked open with either vanadate or AMP-PNP, which would explain why an increased concentration of venom was needed to achieve levels of inhibition similar to that seen during venom treatment of WT-CFTR under standard conditions (Table 1).
What determines the frequency of the FC intraburst closings? We hypothesize that the frequency of these events depends on the association between the CFTR NBD dimer and the intracellular loops that connect the transmembrane helices on the cytoplasmic side of the protein. Several studies suggest that the conformation of the CFTR pore changes between the open and closed states, including evidence that changes in anion selectivity and susceptibility to blockade are linked to the ATP hydrolysis cycle (40,41). Where this conformational change takes place is unknown since the regions within the CFTR polypeptide that link binding and hydrolysis of ATP at the NBDs to control of the pore conformation have not yet been identified. However, it seems safe to speculate that the intracellular loops identified in the structures of several ABC transporter proteins, including CFTR, may serve this role (42,43
