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Department of Pharmacology, University of Washington, Seattle, Washington (S.S., T.S., W.A.C.) and Merck Research Laboratories, West Point, Pennsylvania (R.L.K.)
Received August 17, 2007; accepted December 21, 2007
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Abstract |
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; Cestèle and Catterall, 2000). Sodium channels are composed of one pore-forming 
subunit of approximately 2000 amino acid residues associated with one or two smaller auxiliary subunits, β1to β4 (Catterall, 2000). The subunit consists of four homologous domains (I-IV), each containing six transmembrane segments (S1-S6), and a reentrant pore loop (P) between S5 and S6 (Catterall, 2000). The S4 transmembrane segments are positively charged and serve as voltage sensors to initiate channel activation (Armstrong, 1981; Catterall, 1986; Stühmer et al., 1989; Yang and Horn, 1995; Chanda and Bezanilla, 2002). The "sliding helix" (Catterall, 1986) or "helical screw" (Guy and Seetharamulu, 1986) models for voltage sensing propose that the S4 segments, which have positively charged amino acids at intervals of three residues, transport gating charge outward to activate sodium channels in response to depolarization by moving along a spiral pathway through the protein structure (Yarov-Yarovoy et al., 2006). This transmembrane movement of the S4 gating segments is a molecular target for neurotoxin action via voltage-sensor trapping (Rogers et al., 1996; Cestèle et al., 1998, 2001, 2006).
Scorpion venoms contain two groups of polypeptide toxins that alter sodium channel gating. The -scorpion toxins, as well as sea anemone toxins and some spider toxins, bind to neurotoxin receptor site 3 and slow or block inactivation (Catterall, 1977, 1979; Catterall and Beress, 1978; Nicholson et al., 1994). Amino acid residues that contribute to neurotoxin receptor site 3 are localized in the S3-S4 linker in domain IV (Rogers et al., 1996; Benzinger et al., 1998) and in the large extracellular loops in domains I and IV (Tejedor and Catterall, 1988; Thomsen and Catterall, 1989). Binding of toxins to IVS3-S4 is thought to slow inactivation by preventing the normal outward movement of the IVS4 transmembrane segment during channel gating (Rogers et al., 1996; Sheets et al., 1999). In contrast to these toxins that inhibit inactivation gating, β-scorpion toxins bind to neurotoxin receptor site 4 on sodium channels and enhance activation by shifting its voltage dependence to more negative potentials (Cahalan, 1975; Jover et al., 1980; Jaimovich et al., 1982). Our previous results implicate the extracellular loops S1-S2 and S3-S4 in domain II in formation of neurotoxin receptor site 4 (Cestèle et al., 1998). Moreover, a voltage sensor-trapping mechanism, in which the bound β-scorpion toxin holds the IIS4 segment in its outward, activated position was proposed to account for enhancement of activation (Cestèle et al., 1998, 2001, 2006). Voltage-sensor trapping by both - and β-scorpion toxins inhibits gating currents generated by the transmembrane movement of the S4 segments, providing a mechanistic signature for voltage-sensor trapping (Nonner, 1979; Meves et al., 1982).
The polypeptide toxins from the tarantula Thrixopelma pruriens (Protoxins) are members of the inhibitory cysteine-knot family of protein toxins, consisting of 30 to 35 amino acid residues with three disulfide bridges (Norton and Pallaghy, 1998; Middleton et al., 2002; Priest et al., 2007). This family includes toxins that inhibit activation of sodium channels, such as ProTx-II, and potassium channels, such as hanatoxin, by interfering with the normal function of the voltage sensors (Swartz and MacKinnon, 1997; Middleton et al., 2002). In this study, we have probed the mechanism of ProTx-II action with combined measurements of its effects on sodium currents and gating currents conducted by NaV1.2 and NaV1.5 channels. Our results show that ProTx-II inhibits gating currents conducted during voltage-dependent activation of sodium channels. The inhibitory effects of the toxin can be reversed by strong, long-lasting positive voltage pulses, which drive the voltage sensor into its activated conformation. Voltage-dependent reversal of ProTx-II effects was more rapid for NaV1.5 channels, the primary sodium channel in the heart. Mutations in the S3-S4 linker in domain II reduce toxin affinity, and mutations in the IIS4 voltage sensor prevent voltage-dependent reversal of toxin action. Our results indicate that ProTx-II impedes the normal gating function of the IIS4 voltage sensor by a voltage-sensor trapping mechanism and can be knocked off its receptor site on the extracellular side of the voltage sensor by voltage-driven movement of the IIS4 voltage sensor into its activated conformation.
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Materials and Methods |
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). Restriction endonucleases and other molecular biology reagents were purchased from New England Biolabs (Ipswich, MA) and Roche Applied Science (Indianapolis, IN). Bovine serum albumin was from Sigma (St. Louis, MO). pCDM8 vector and the MC1061 Escherichia coli bacterial strain were from Invitrogen (Carlsbad, CA). cDNAs encoding rat Nav1.2a -subunit (Auld et al., 1990) and rat Nav1.5 -subunit subcloned into pCDM8 vector (Rogers et al., 1996) were used for expression in Xenopus laevis oocytes. Point mutations L833C, L833N, E837Q, L838C, L840C, N842A, and G845N in the DIIS3-S4 linker as well as R850Q, R853Q and a double mutation RR850,853QQ in DIIS4 of Nav1.2a channel were produced in our lab previously (Cestèle et al., 1998, 2001; Sokolov et al., 2005). X. laevis frogs were purchased from Nasco (Fort Atkinson, WI).
Expression in X. laevis Oocytes. pCDM8 plasmids encoding rat Nav1.5, Nav1.2a, and mutant Nav1.2a sodium channel subunits were linearized with XbaI or ClaI, respectively, and plasmids encoding β1 subunits were linearized with HindIII. Transcription was performed with T7 RNA polymerase (Ambion Inc., Austin, TX). Isolation, preparation, and maintenance of X. laevis oocytes were carried out as described previously (McPhee et al., 1995). Healthy stage V-VI oocytes selected manually were pressure-injected with 50 nl of a solution containing a 1:1 molar ratio of /β1 subunit RNA. Electrophysiological recordings were carried out 4 to 7 days after injection.
Cut-Open Oocyte Voltage Clamp. Cut-open oocyte voltage-clamp experiments were performed as described by Stefani and Bezanilla (1998), except that access to the cytoplasm was obtained by rupturing the vegetal pole membrane of the oocyte. The oocyte capacitative transients were partially compensated with the voltage clamp amplifier (CA-1; Dagan Corporation). Online P/-4 leak subtraction was used. Microelectrodes were pulled from borosilicate glass capillary tubes 1.5 mm OD (A-M Systems, Carlsborg, WA) and had resistances of 250 to 350 k
when filled with 3 M KCl. Extracellular solution contained 120 mM sodium methanesulfonate (MeSO3), 10 mM HEPES, 1.8 mM Ca-MeSO3, and 1% bovine serum albumin, pH 7.4. Internal solution consisted of 110 mM K-MeSO3, 10 mM Na-MeSO3, 10 mM EGTA, and 10 mM HEPES, pH 7.4. ProTx-II was prepared as a 100 µM stock in 120 mM Na-MeSO3, 10mM HEPES, and 0.2 mg/ml bovine serum albumin, aliquoted at 5 µM, and stored at -20°C. Aliquots containing toxin were thawed immediately before experiments and diluted in extracellular solution.
The starting solution volume in the upper boat of the recording chamber (Dagan Corporation, Minneapolis, MN) was typically 150 µl. Volumes of 37.5 or 50 µl of solution containing 5 µM ProTx-II were typically added to the recording chamber, resulting in final concentrations of 1 to 1.25 µM, except where other concentrations are noted in the figure legends, and the cells were allowed to equilibrate for 3 to 5 min before recording. After each experiment, the recording and guard chambers were rigorously washed to remove all traces of ProTx-II in a multistep washing procedure using 75% EtOH and 95% MeOH.
All experiments were performed at room temperature. Currents were filtered at 5 kHz with a low-pass Bessel filter, and then digitized at 20 kHz. Voltage commands were generated using Pulse 8.5 software (HEKA, Lambrecht/Pfalz, Germany) and ITC18 analog-to-digital interface (Instrutech, Port Washington, NY).
Data Analysis. Data were analyzed with Igor Pro 4.0 WaveMetrics, (Lake Oswego, OR). Voltage-clamp protocols are described in the figure legends. Voltage dependences of activation and inactivation were fit by Boltzmann functions of the form Gmax/{1 + exp[(V - Va)/k]}, where Gmax is the maximum conductance, Va is the half-activation or inactivation potential, and k is a slope factor. Rates of inactivation in Fig. 1C were determined by single exponential fitting of current traces. Pooled data are reported as means ± S.E. Statistical comparisons were performed using Student's t test, with p < 0.05 as the criterion for significance.
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Results |
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). Ionic currents were recorded in 120 mM Na+ control solution before and 5 min after adding 1 µM ProTx-II to the recording chamber (Fig. 1). ProTx-II (1 µM) inhibited 65.3 ± 4.1% of ionic current in Nav1.2 channels at a test pulse potential of -10 mV (Fig. 1A). Previous results (Middleton et al., 2002) showed that the effect of ProTx-II as a function of concentration can be fit by a single binding site model. Plotting the effect of ProTx-II on sodium current versus concentration yielded an inhibition curve that was fit by a single binding site model with an EC50 of 540 nM and inhibition of essentially 100% of the sodium current at saturation (Fig. 1B). The EC50 of ProTx-II for sodium channels expressed in X. laevis oocytes is significantly higher than previously observed in mammalian cells (Middleton et al., 2002).
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Effects of ProTx-II on Gating Currents. Gating currents of Nav1.2a channels were recorded in the presence of 1 µM tetrodotoxin to block ionic conductance through the central pore of the channel. Families of Qon and Qoff gating currents were recorded in control solution and after 1 µM ProTx-II was applied (Fig. 2). ProTx-II substantially reduced the amplitude of gating currents (Fig. 2A). Integration of Qon currents revealed the voltage dependence of gating charge movement across the membrane during sodium channel activation (Fig. 2B). ProTx-II inhibited the gating charge movement across a wide range of membrane potentials. Inhibition of gating charge movement was 24.6 ± 6.4% at +100 mV, the most positive membrane potential studied. On average, 65% reduction of sodium current is caused by 24.6% reduction in gating current (Fig. 2C). By extrapolation, assuming the same concentration-response relationship for gating and ionic current (Fig. 1B), a concentration of ProTx-II that inhibits 100% of sodium channels would block 38% of gating charge movement. Evidently, ProTx-II blocks an essential component of gating charge movement, which greatly reduces activation of NaV1.2 channels.
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Voltage-Dependent Reversal of ProTx-II Inhibition. Binding of -scorpion toxins is reversed by strong depolarization, indicating that voltage sensor activation can reverse toxin binding (Catterall, 1977; Rogers et al., 1996). We investigated whether the effects of ProTx-II on ionic currents and gating currents are also reversed by strong depolarization. In the first type of experiment, a strong conditioning depolarization (+100 mV) of increasing duration was followed by 20 ms at the holding potential (-100 mV) to allow recovery from fast inactivation and then by a test pulse to either -10 mV to measure ionic currents or +50 mV to measure gating currents (Fig. 4). The conditioning pulses reversed ProTx-II inhibition of both ionic and gating currents (Fig. 4B). The amplitudes of both ionic currents and gating currents increased because of the depolarizing pulses, and reversal of ProTx-II inhibition followed an exponential time course with similar kinetics for both ionic and gating currents (Fig. 4C). However, even the strongest conditioning depolarizations gave incomplete relief of inhibition of ionic current, reaching 60 to 65% of control at +100 mV and 630 ms.
Applying conditioning pulses of different magnitude ranging from +40 to +100 mV allowed us to measure the kinetics of reversal of protoxin inhibition at these voltages (Fig. 5). Reversal of ProTx-II inhibition was fastest and most prominent at +100 mV (
+100mV = 260 ms, 130% maximum increase in ionic current amplitude) and was very slow at +40 mV (+40mV = 1.5 s, 25% maximum increase in amplitude). These results indicate that strong depolarizations are required to overcome the energy of ProTx-II interaction with the resting state of the voltage sensor, drive it into the activated conformation, and cause dissociation of ProTx-II.
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The energy required to cause dissociation of the toxin at positive potentials, and thereby permit activation of the channel, shifts the effective activation curve toward more positive potentials for toxin-bound channels, as observed in the voltage dependence of recovery of the sodium current during long depolarizations in the presence of toxin. Plotting the normalized percentage increase in test pulse current after 630-ms conditioning depolarizations from the experiments of Fig. 5B versus the conditioning pulse potential results in an isochronal activation curve for toxin-bound channels, which is strongly shifted toward more positive potentials relative to activation of toxin-free channels (Fig. 5C). After a 630-ms conditioning depolarization to +20 mV, there is no relief of toxin block, suggesting that toxin-bound channels cannot activate at this potential. Conversely, after 630 ms at +100 mV, the current increases 2.33-fold. Further relief is not achieved with increased depolarization. Thus, toxin-bound channels can undergo voltage-dependent activation, but that activation is greatly slowed and positively shifted because of the energy required to dissociate the toxin from the channel before it can activate.
Protoxin Action on Cardiac NaV1.5 Channels. ProTx-II inhibits sodium currents conducted by NaV1.5 channels to a similar extent as NaV1.2 channels (Middleton et al., 2002; Fig. 6A). However, the reversal of inhibition during strong depolarizations is faster than for NaV1.2 channels (Fig. 6B). This difference in the rate of reversal of ProTx-II inhibition is observed across a wide range of prepulse potentials (Fig. 6C). These results indicate that the dissociation rate for ProTx-II from the activated state of NaV1.5 channels is approximately 2.5-fold faster than for NaV1.2 channels, suggesting subtype-specific effects of ProTx-II on sodium channel gating.
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). A rapidly moving component of gating current is selectively inhibited (Fig. 3), suggesting that a kinetically distinct voltage sensor movement is blocked. As the voltage sensor in domain IV moves slowly (Chanda and Bezanilla, 2002), primarily the voltage sensors in domains I, II, and III contribute to the measured gating charge movement. Only 38% of gating charge movement is blocked when channel activation is completely inhibited, consistent with complete inhibition of movement of one of the voltage sensors in domains I, II, or III by ProTx-II. Gating modifier toxins bind to the S3-S4 linkers of sodium channels (Rogers et al., 1996). The β-scorpion toxin Css-IV binds to IIS3-S4 (Cestèle et al., 1998), whereas the -scorpion toxin LqTx binds to IVS3-S4 (Rogers et al., 1996). Even though ProTx-II stabilizes sodium channels in their closed state and β-scorpion toxins stabilize sodium channels in their open state, they both affect channel activation via a voltage-sensor trapping mechanism. Because β-scorpion toxins bind to IIS3-S4, we investigated this region as a potential receptor site for ProTx-II.
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= -26.7 mV;L833C, V = -23.8, n = 12). The L833C mutation was surprisingly specific, because mutating the same amino acid to Asn in L833N had no effect on ProTx-II action. In contrast, mutations of the other amino acid residues in the IIS3-S4 loop had lesser or no effect.
If ProTx-II modifies voltage-dependent conformational changes by binding to the outer end of the IIS4 voltage sensor, neutralizing the gating charges in the outer end of the IIS4 segment may affect the toxin-channel interaction considerably. Therefore, we tested whether the double mutant RR850,853QQ, in which the two outer gating charges in IIS4 are neutralized, interacts differently with ProTx-II. Previous studies have shown that these mutations do not substantially alter activation and inactivation of sodium channels (Cestèle et al., 2001; Sokolov et al., 2005). Sodium currents conducted by the double mutant RR850,853QQ were almost as sensitive to 1.25 µM ProTx-II as wild-type Nav1.2 channels (Fig. 8A; mean values, 44 ± 5.7%, n = 8 for RR850,853QQ versus 63 ± 5.7%, n = 7 for WT). These results indicate that there was no marked reduction in the affinity of the toxin for the resting channel and that the toxin was still able to trap the mutant IIS4 voltage sensor in its resting state.
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In contrast to the lack of major effects of these mutations on the function of NaV1.2 channels and the inhibition of ionic current by ProTx-II, the block of gating current by ProTx-II was nearly completely lost in the RR850,853QQ mutant (Fig. 8B). Moreover, voltage-dependent reversal of inhibition during conditioning depolarizations was almost completely abolished in the double mutant RR850,853QQ channel (Fig. 8, C and D). No significant voltage-dependent reversal of inhibition was detected for RR850,853QQ with conditioning depolarizations as strong as +150 mV (data not shown). These results indicate that the gating movement of Arg850 and Arg853 at the outer end of the IIS4 segment is responsible for voltage-dependent reversal of protoxin inhibition and support the conclusion that ProTx-II interacts specifically with the IIS4 voltage sensor to inhibit gating current.
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Discussion |
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; Meves et al., 1982). This effect probably reflects binding to a specific conformation of the voltage sensor and stabilizing that conformation according to a voltage-sensor trapping mechanism (Rogers et al., 1996; Cestèle et al., 1998, 2001, 2006). Our results show that ProTx-II shares this signature effect with other gating modifier toxins. By binding to a single site, ProTx-II reduces ON gating charge movement by 24.6 ± 6.4% at 1 µM and by approximately 38% when extrapolated to saturation. The toxin specifically reduces a rapidly moving component of gating charge, suggesting that it selectively impairs the movement of one or more voltage sensors that contribute to the rapid component of gating charge movement.
Voltage-Dependent Reversal of ProTx-II Action. A second hallmark of gating modifier toxins is voltage-dependent enhancement or reversal of toxin action, first demonstrated for reversal of the binding and action of -scorpion toxins (Catterall, 1977; Rogers et al., 1996). This effect is thought to represent the voltage-driven outward movement of the IVS4 voltage sensor pushing the bound toxin off its binding site. As for -scorpion toxins, our results show that reversal of ProTx-II action is dramatically accelerated by strong positive prepulses. These results are most consistent with a voltage-sensor trapping model in which ProTx-II binds to one of the four S4 voltage sensors in its resting conformation and holds it in this inward position. Strong positive prepulses provide sufficient electrical energy to force outward movement of the voltage sensor into its activated conformation and are able to overcome the chemical energy of ProTx-II binding and push it off its receptor site. We observe this effect as depolarization-dependent reversal of toxin action.
Protoxin Acts on the IIS4 Voltage Sensor. ProTx-II inhibits sodium channels with a concentration dependence that is fit by a single binding site model (Middleton et al., 2002; Fig. 1B). Because the voltage sensor in domain IV moves slowly (Chanda and Bezanilla, 2002), the voltage sensors in domains I, II, and III are probably primarily responsible for the gating charge movement measured in our experiments. Our findings suggest that approximately 38% of gating current would be blocked by ProTx-II at toxin concentrations at which all sodium channels are inhibited. They also show that a rapidly activating component of gating current is preferentially blocked. These results are consistent with the hypothesis that binding of a single ProTx-II molecule inhibits the function of a single voltage sensor.
Three independent lines of investigation implicate the IIS4 voltage sensor in ProTx-II action. First, we found that the mutation L833C in the IIS3-S4 linker reduces the affinity for ProTx-II action, consistent with involvement of this amino acid residue in toxin binding. Second, we found that neutralization of the first two gating-charge-carrying arginine residues in IIS4, Arg850 and Arg853, completely prevents the effects of ProTx-II on gating current, indicating that these residues are required for the charge movement blocked by ProTx-II. Neutralization of these gating charges may inhibit movement of the entire IIS4 segment, including the potential gating charges carried by Arg856 and Lys859, and it may further impede the movement of gating charges in other S4 segments through the allosteric interactions among voltage sensors described previously (Chanda et al., 2004). Third, the loss of depolarization-dependent reversal of block by ProTx-II also requires Arg850 and Arg853 at the outer end of segment IIS4. These results suggest that Arg850 and Arg853 interact either sterically or electrostatically with ProTx-II as they move outward during activation and thereby push the toxin off its receptor site. All together, these three lines of evidence provide strong support for the conclusion that ProTx-II inhibits sodium channels by inhibiting the normal activation of the voltage sensor in domain II.
ProTx-II and β-scorpion toxins both affect sodium channel activation but have opposing effects on that process. According to our hypothesis, ProTx-II impedes gating charge movement and channel activation by trapping the IIS4 voltage sensor in its resting conformation. β-Scorpion toxins facilitate channel activation by trapping the IIS4 voltage sensor in its activated conformation. The specific effects of both β-scorpion toxin and ProTx-II on the IIS4 voltage sensor suggest that this voltage sensor may have a privileged role in the actions of gating modifier toxins that affect sodium channel activation, whether they enhance or inhibit it. Further studies are required to completely map the amino acid residues that form the receptor site for ProTx-II on NaV1.2 channels.
In a recent study of site-directed mutants of NaV1.5 channels, Smith et al. (2007) analyzed a large number of amino acid residues for involvement in the action of ProTx-II and found no major effects. They did not analyze the equivalent of mutant L833C, but they found that different mutations of residues in the IIS3-S4 loop had no effect on ProTx-II action, in agreement with our work. Based on this extensive analysis, it seems that ProTx-II has a unique site of action compared with other gating modifier toxins. It will be of great interest to define its receptor site and determine how it impedes movement of the IIS4 voltage sensor.
Subtype-Specific Reversal of ProTx-II Action. Brain and cardiac Na+ channels display similar sensitivity for inhibition of channel activation by ProTx-II at hyperpolarized potentials where the channels are in their resting conformations (Fig. 1) (see also Middleton et al., 2002). However, the rates for voltage-dependent reversal of protoxin-II inhibition differ substantially between Nav1.2 and Nav1.5. The cardiac Nav1.5 isoform has faster and more prominent relief of inhibition at conditioning potentials ranging from +40 mV to +100 mV. These results indicate that at least part of the ProTx-II receptor site differs between brain and cardiac sodium channels and that the release of ProTx-II after voltage-dependent activation is more rapid for the Nav1.5 channel. This suggests that ProTx-II has a lower affinity for the activated state of the Nav1.5 channel than for that of Nav1.2. Determination of the amino acid residues responsible for the difference in ProTx-II action between NaV1.2 and NaV1.5 channels may give insight into the molecular basis for the substantially different voltage dependence of activation of these channel subtypes.
Comparison of Voltage-Sensor Trapping by ProTx-II and Hanatoxin. Hanatoxin is another gating modifier toxin that inhibits KV2.1 channels (Swartz and MacKinnon, 1995). This channel is composed of four independent, identical subunits that form a noncovalently associated tetramer, in contrast to sodium channels whose four nonidentical but homologous domains are covalently linked in a single polypeptide chain. Hanatoxin blocks gating charge movement more completely than ProTx-II (Lee et al., 2003), as expected because hanatoxin binds to all four subunits of KV2.1 channels and prevents gating movement of all four S4 segments. Like Protoxin-II, hanatoxin binding to only one of the four homologous subunits/domains is sufficient to impair channel opening (Swartz and MacKinnon, 1997; Lee et al., 2003), and hanatoxin affinity for KV2.1 is reduced after prolonged depolarization (Phillips et al., 2005). Hanatoxin-blocked channels can open during long depolarizing pulses with toxin bound, resulting in accelerated deactivation and in strongly shifted voltage dependence of activation (Swartz and MacKinnon, 1997; Phillips et al., 2005) that resembles the strong positive shift of the voltage dependence of activation observed for ProTx-II-blocked sodium channels (Fig. 5). Thus, hanatoxin and ProTx-II have similar mechanisms of action that depend on trapping a voltage sensor in its resting conformation. Differences in the details of their mechanisms of action probably reflect the ability of KV2.1 channels to bind four hanatoxin molecules to their four voltage sensor domains.
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Footnotes |
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ABBREVIATIONS: ProTx-II, protoxin II; MeSO3, methanesulfonate.
Address correspondence to: William Catterall, University of Washington School of Medicine Department of Pharmacology, Box 357280, Seattle, WA 98195-7280. E-mail: wcatt{at}u.washington.edu
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