Address correspondence to Heinrich Terlau, Max-Planck-Institute for Experimental Medicine, Molecular and Cellular Neuropharmacology Group, Hermann-Rein-Str.3, D-37075 Göttingen, Germany. Fax: 49-551-3899-475. email: hterlau{at}gwdg.de
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ABSTRACT |
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Key Words: voltage-gated potassium channels permeation conotoxins pore block Xenopus oocytes
Abbreviations used in this paper: CTX, charybdotoxin;
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INTRODUCTION |
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The K+ binding sites cannot be occupied simultaneously, and at high concentrations of K+, there appear to be two isoenergetic configurations of maximal occupancy: the K+ ions either occupy the first, third, and fifth (cavity) sites, or the second and fourth sites and the extracellular site. During permeation, the ions in the pore shift their positions, and with the entrance of a "new" ion either from the extracellular or intracellular side, the last ion at the opposite end of the pore exits (Morais-Cabral et al., 2001; Zhou et al., 2001
). Although the KcsA channel contains only two transmembrane segments and is found in a prokaryotic cell, it has been adopted as a model system for the general mechanism by which ion channels achieve rapid permeation accompanied by strong ion selectivity. Recent data on the structure of KvAT that has six transmembrane helices (as do the majority of voltage-dependent K+ channels) indicated that the selectivity filter is essentially identical to that of KcsA (Jiang et al., 2003
).
Diverse ion species have been used to study gating and permeation mechanisms; it has been shown that for K+ channels, both K+ and Rb+ are both highly permeable. The presence of permeant cations in the external solution slow the closing of K+ channels, while activation kinetics are relatively unaffected (Swenson and Armstrong, 1981; Matteson and Swenson, 1986
). The experiments with different ion species suggest that gating and permeation of K+ channels are not totally uncoupled, and that the cations in the pore affect channel gating, particularly deactivation and inactivation rates. It has been hypothesized that Rb+, which enters the channel as well as K+, binds more tightly and therefore has a larger effect on the closing rate than the more loosely bound K+. In addition, the rate of C-type inactivation is affected by occupancy at a binding site near the external mouth of the pore (López-Barneo et al., 1993
).
Surprisingly, the distribution of Rb+ and K+ in the selectivity filter differs even though both ions are highly permeable (Morais-Cabral et al., 2001). The structures of MacKinnon and coworkers reveal that Rb+ strongly prefers sites 1 and 3 (starting from the outer side of the selectivity filter) (Morais-Cabral et al., 2001
) over a site 2 and 4 occupancy configuration. In this study, we used a specific toxin to investigate K+ channels; as the close interaction of toxins with channel proteins can be sensitive to ion occupancy and, in principle, provide information about the dynamic activity of the channel during gating.
-Conotoxin PVIIA (
-PVIIA), isolated from the fish-hunting Conus species C. purpurascens, selectively blocks the voltage-gated Shaker K+ channel (Terlau et al., 1996
) and binds in a bimolecular fashion to the extracellular mouth of the pore (Shon et al., 1998
). The peptide interacts strongly with amino acid residues known to be at the outer pore vestibule (Terlau et al., 1996
; Shon et al., 1998
; Jacobsen et al., 2000
).
-PVIIA shares common features with the scorpion toxin charybdotoxin (CTX) in its binding to K+ channels, such as dependence of block on the ionic composition of the medium (for CTX, Anderson et al., 1988
; MacKinnon and Miller, 1988
; Goldstein et al., 1994
; for
-PVIIA, Scanlon et al., 1997
; Garcia et al., 1999
; Terlau et al., 1999
). The previous analysis of
-PVIIA block of the Shaker channel demonstrated a state dependence: the potency of toxin block in the open conformation decreased with increasing potential; the off-rate was strongly voltage dependent while the on-rate was voltage insensitive. This voltage dependence was explained by interaction with K+ in the pore, first proposed for CTX block of large-conductance Ca2+-activated K+ channels (BK) and Shaker channels (MacKinnon and Miller, 1988
; Goldstein and Miller, 1993
). When K+ is the only permeant ion present, open channel block is not affected by the extracellular K+ concentration, [K+]o, and complete removal of intracellular K+ is necessary to relieve the voltage dependence of the block (Garcia et al. 1999
). In contrast, block of the closed state by the toxin is not voltage dependent and decreases upon increasing [K+]o, a result consistent with competition between
-PVIIA and K+ for a binding site at the outer end of the pore (Terlau et al. 1999
).
Thus, when toxin binds, it has a strong interaction with ions in the pore. In this study, we show that -PVIIA binding to Shaker channels in the presence of K+ and Rb+ exhibits properties that can be rationalized by differences in the selectivity filter occupancy of these two ions, validating the structural data also in 6TM potassium channels. It is demonstrated that the interaction of
-PVIIA with the Shaker channel in the closed and open conformation is different in the presence of two permeant cations, K+ and Rb+. When K+ is partially or completely replaced by Rb+, the kinetics and voltage dependence of open-channel block depend on both intracellular and extracellular ionic composition. Thus, the results reveal that
-PVIIA is sensitive to both differences in the occupancy of the channel pore as well as which specific ion is present in the pore. These data indicate that the ion(s) in the pore functionally affects the receptor site for the toxin.
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MATERIALS AND METHODS |
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Oocyte Expression System
Oocytes from Xenopus laevis were prepared as described previously (Stühmer, 1992). RNA was injected into stage VVI oocytes, and currents were recorded 17 d after injection after mechanical removal of the vitelline membrane. For outside-out patch-clamp recordings (Hamill et al., 1981
), the aluminum silicate glass pipettes had resistances between 0.8 and 1.2 M
. The pipette solution was (in mM) 115 XCl, 1.8 EGTA, 10 HEPES (X indicates either K or Rb). The bath solution contained (in mM) 115 XCl, 1.8 CaCl2, 10 HEPES. The pH was adjusted to 7.2 with TrisBase.
Currents were measured with an EPC-9 patch clamp amplifier driven by the Pulse+PulseFit software package (HEKA Elektronik). Current records were low-pass filtered at 5 kHz and sampled at a rate of 20 kHz.
Leak and capacitive currents were corrected on-line by using a P/n method. Toxin solution was added to the bath chamber by using a Gilson tip pipette. The indicated toxin concentrations correspond to the final concentration in the bath chamber.
Off-line analysis was performed with a Macintosh G3 microcomputer (Apple Computer, Inc.) with self-written programs in the Igor (Wavemetrics) environment.
Kinetic Analysis
To interpret the experimental data, we used a simple bimolecular reaction scheme to describe the interaction between the toxin and the channel, represented by
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where {U} represents the toxin-free channels and {B} the channels bound to a toxin molecule; kon is the second order association rate constant, [T] is the toxin concentration, and koff is the first order dissociation rate constant (Terlau et al.,1999). Following a step depolarization that opens the channels, U(t) is expected to follow a single-exponential relaxation with time constant
and asymptotic value U, whose values are related to the open-channel kinetic parameters KO, koffO, and konO.
From the experimental parameters and U, it is possible to evaluate KO, koffO, and konO according to the inverse relationships:
![]() | (1) |
![]() | (2) |
![]() | (3) |
For the open channels, the unblock probability U(t) is calculated at any potential step from the ratio between the current in the presence of toxin and under control conditions (Figs. 1 and 2)
. The kinetics of -PVIIA binding to the closed channels was evaluated with a double pulse protocol, where each stimulus consisted of two identical pulses (at 20 or 30 mV for symmetric K+, at 30 or +40 mV for symmetric Rb+) separated by a time interval, Ti, at the holding potential (between 80 and 100 mV), increasing from 5 ms to 5 s. With such a protocol, one measures the time course of the reequilibration of the binding to closed channels after the perturbance produced by the first pulse (Terlau et al., 1999
).
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RESULTS |
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When similar experiments are performed in the presence of symmetrical 115 mM RbCl (Fig. 1, bottom), the most obvious difference is that the -PVIIA block of open channels is much weaker and much less voltage dependent, although the initial values of toxin to control currents (Fig. 1, bottom right) indicate a toxin binding affinity to closed channels similar to that observed in symmetric K+ conditions (KC
270 nM). As a consequence, channel opening entrains
-PVIIA unbinding at all activating voltages, and in particular the inward current at 20 mV lacks the hump that in symmetrical K+ reveals an increase in toxin affinity (compare the scaled traces in the top and bottom middle panels of Fig. 1). In the experiment of Fig. 1, 500 nM
-PVIIA blocked only
38% of the steady-state inward current at 20 mV and
17% of the outward current at +60 mV, with apparent KO values of 820 nM at 20 mV and three times larger (
2.4 µM) at +60 mV.
We also notice that in symmetrical Rb+, toxin binding relaxations, as revealed by the ratios of toxin to control currents, have faster kinetics. From the data of Fig. 1 (bottom right), the estimated time constants are 5.65 ms at 20 mV and 4.6 ms at +60 mV; together with the KO values reported above, these time constants yield koffO estimates of 109 s1 at 20 mV and 180 s1 at +60 mV, and konO estimates of 133 s1µM1 at 20 mV and 74 s1µM1 at +60 mV. Thus, the main effect of replacing K+ for Rb+ appears to be an increase of koffO that makes toxin binding weaker and with faster kinetics; in addition, koffO is much less voltage dependent and a significant decrease of konO appears to contribute to the increase of KO with voltage.
The differences observed for -PVIIA binding to open and closed channels in the presence of these two cations were unexpected, because Rb+ is almost as permeable as K+ and readily enters the pore.
Comparison with -PVIIA Block Under Bi-ionic Conditions
To further investigate the very different interaction of -PVIIA with the Shaker channel depending on the type of ion that occupies the pore, similar experiments were also performed under asymmetric bi-ionic conditions. Fig. 2 shows a comparison of the current recordings obtained from outside-out patches under symmetrical K+ and Rb+ conditions with the currents from similar experiments with Kext/Rbint or Rbext/Kint (IV protocols: VH = 100 mV, VP from 50 mV to +70 mV, every 20 mV). As can be seen directly, the inward currents are blocked by
-PVIIA more than the outward currents measured at high depolarizations under all four conditions. Nevertheless, the hump observed for the inward currents in the presence of toxin for symmetrical K+ is only present also for Kext/Rbint but not for the other two conditions. To further compare these data, the unblock probability for the current records from all four conditions were calculated and are shown in Fig. 3
.
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The top left panel of Fig. 3 shows data for symmetrical K+ and 200 nM -PVIIA. It is seen that for depolarizations below +30 mV, U decreases during the pulse, while above this potential, an unblock is observed. At about +30 mV, the closed channel block appears to be equal to the open channel block, so that the ratio U(t) = ITx/Ict remains constant during the pulse. The horizontal dotted line shows the estimated unblock probability of the early currents, which is independent of the pulse potential and corresponds to a dissociation constant for
-PVIIA binding to closed channels, KC, of
270 nM (Terlau et al., 1999
). Fitting U(t) records with single exponentials (continuous lines) yields time constants that decrease monotonically with voltage from 24 ms at 30 mV to 8 ms at +70 mV, and estimates of the asymptotic toxin-free probabilities that increase with voltage from 0.26 at 30 mV to 0.85 at +70 mV. Using Eqs. 13, we estimate that between 30 and +70 mV, KO increases
16-fold, from 69 nM to 1.13 µM; konO decreases by
60%, from 155 to 94 s1µM1; and koffO increases
10-fold, from 11 to 106 s1, and is the main determinant of the destabilization of toxin binding at increasing depolarizations. At variance with kinetic parameters, steady-state toxin-free probabilities can also be obtained from tail currents, even for voltages near the reversal potential (unpublished data). Thus, KO estimates are in general more accurate and detailed in the whole voltage range of 50 to +80 mV. In the Kext/Kint experiment of Fig. 3 A, the voltage dependence of KO was fitted by a simple exponential, KO(V) = KO(0)*exp(V/vs), with KO(0) = 159 nM and vs = 37 mV. A semilogarithmic plot of mean KO(V) estimates (±SD) is given in Fig. 3 B as open squares. The straight line fitting the mean data corresponds to KO(0) = 148 nM and vs = 41 mV (Table I).
Analogous data for symmetrical Rb+ and 500 nM -PVIIA are shown in the top right panel of Fig. 3 A and as open squares in Fig. 3 B. As described in reference to Fig. 1 and 2, we notice a much faster and more extensive unblock at all potentials with a much weaker voltage dependence. When converted into toxin binding parameters through Eqs. 13, the time constants and the steady state of the unblock indicate that the replacement of K+ with Rb+ has little effect on the konO rate, which is weakly decreasing with voltage and has a mean value close to that of symmetrical K+ conditions (Table I). The replacement appears to affect predominantly the koffO rate, which is increased about 10-fold at low depolarizations. In addition, toxin binding to a Rb+-filled channel is not so much further destabilized at larger voltages as when K+ is the charge carrier (Fig. 3 B, filled squares; Table I).
U(t) records for Kext/Rbint conditions in the presence of 300 nM -PVIIA are shown in the bottom left panel of Fig. 3 A. It is seen that these data are quite similar to those obtained in symmetrical K+: the inward currents are blocked much more than the outward currents, and toxin block increases for V < +20 mV, whereas an unblock occurs at higher potentials. At +20 mV, the open channel block is about the same as the closed channel block and no unblock nor reblock is observed. The most significant difference with the case of symmetrical K+ is that toxin block gets increasingly weaker at positive voltages so that the apparent KO approaches the larger values observed in symmetrical Rb+ (Fig. 3 C, open circles).
The bottom right panel of Fig. 3 A shows U(t) records for Rbext/Kint conditions in the presence of 400 nM -PVIIA. It is seen that, similarly to the case of symmetrical Rb+, channel opening entrains toxin unblock at all voltages. However, the unblock does not increase with voltage as strongly as in Rbext/Rbint conditions. Indeed, comparing the respective U(t) records shows that
-PVIIA blocks the outward currents at +30 mV more than the inward currents at 30 mV. This behavior results in a clearly nonmonotonic voltage dependence of KO (Fig. 3 C, filled circles), which suggests a rather sharp transition of toxin binding, from a relatively low Rb+-like to a much higher K+-like affinity, near the current reversal potential (5 mV in this experiment).
Kinetics of -PVIIA Binding to Closed Channels
The kinetics of -PVIIA binding to closed channels can be measured with a double pulse protocol, in which a conditioning pulse to VP is followed by an identical pulse, separated by an interpulse interval at holding potential of variable duration Ti. The resting period between successive stimulations was at least 5 s in order to restore "tonic" conditions. With such a protocol we tested the reequilibration of the binding to closed channels at different times after the first pulse. As shown by Terlau et al. (1999)
, the kinetics of the reequilibration of the binding to closed channels is slowed down by increasing the extracellular K+ concentration (and decreasing Na+), due to a reduction of the association rate.
Fig. 4
A shows the current responses to such a protocol in symmetrical 115 mM KCl (VP = 20 mV, Vh = 100 mV, Ti as indicated in figure). We observe that the conditioning pulse induces an increase of toxin block that appears as a reproducible hump in the first response of each successive double stimulation (Terlau et al., 1999). However, for small Ti values, the second response has a shape similar to control conditions in that no peak is observed (in figure Ti = 80 ms). This indicates that the fraction of blocked channels is near the equilibrium for open-channel block at 20 mV that was achieved by the end of the conditioning pulse and corresponded to UO = 0.23. A quantitative analysis of the relaxation process is shown in the inset of Fig. 4 A, by plotting, as a function of Ti, early amplitudes of conditioning responses in toxin normalized to the control value. These data are well fitted by a single exponential increase with Ti with a time constant of 0.72 s. Assuming this value as an estimate of
C and using the value of UC = 0.5 measured from the early fraction of unblocked currents (unpublished data), we estimate in this experiment KC = 200 nM, koffC = 0.69 s1, and konC = 3.5 µM1s1. The estimates given in Table I for konC and koffC are obtained considering the linear dependence of
1 on the toxin concentration, through the relationship
1 = konC * [T]+ koffC (Terlau et al. 1999
). For the symmetrical 115 mM KCl case, they agree with the values obtained by Terlau et al. (1999)
.
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These observations demonstrate that the occupancy of the pore by Rb+ appears to also hasten the kinetics of -PVIIA binding to closed channels. In 115 mM external Rb+, the rebinding of
-PVIIA to closed channels after an unblocking depolarization appears to be about as fast as channel closing, which in high [Rb+]o has time constants in the order of 10 ms.
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DISCUSSION |
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It has been proposed that the interaction with K+ ions in the permeation pathway is the major determinant of the properties of -PVIIA binding (Garcia et al., 1999
; Terlau et al., 1999
). Permeant ions penetrating the ion pore from the intracellular side destabilize an extracellularly bound toxin with a probability that increases with voltage. If the channel pore affords a single-file multiple ion occupancy, the destabilizing configuration likely results from the concerted movement of two or three ions, each traversing only a fraction of the electrical distance across the pore. The above mechanism has been supported by recent structural work demonstrating that K+ channels are indeed multi-ion pores (Doyle et al., 1998
; Morais-Cabral et al., 2001
; Zhou et al., 2001
). With K+ as the only permeant ion present, the "on" binding of
-PVIIA to the extracellular vestibule of an open channel may be little sensitive to [K]o and to voltage because any hindering ion at the outer pore entrance could easily be pushed inwardly by the incoming toxin, whereas the occupancy configuration of the ion sites within the pore is expected to have a strong effect on the toxin "off" rate and to be strongly voltage dependent (Terlau et al., 1999
). When Rb+ solutions are used on both sides of the membrane, the control current does not differ from traces recorded in symmetrical K+, indicating normal permeation for Rb+ through the Shaker channel. Under these conditions, the pore is loaded exclusively with Rb+, and when the toxin is applied, block potency is strongly reduced and the voltage dependence is much less steep (Figs. 13). Rb+ and K+ have similar radii and both readily enter the channel, as shown also by the fact that in bi-ionic conditions the current reversal potential is close to zero.
In the cartoons of Fig. 5
, the numbers 14 indicate the four sites that a permeant ion encounters within the selectivity filter while moving inward through the narrow region of the pore, and S0 indicates the external ion-binding site. For simplicity, only the most likely populated ion occupancy configurations revealed by the structural study of Morais-Cabral et al. (2001) are taken into account. At high concentrations of permeant ions, as occurring in the experimental conditions explored in this work, these configurations involve the occupancy of the inner pore sites by two ions. When the channel is closed, the occupancy of S0 plays a major role for toxin binding and is solely determined by the species and concentration of permeant ions in the extracellular solution. At high [K]o, the probability of S0 occupancy is very high, also because it is compatible with two more ions occupying positions 2 and 4, a configuration that is equally favored by potassium ions as the 1,3 alternative. The mutant cycle analysis data on the binding of
-PVIIA with Shaker (Jacobsen et al., 2000
) suggests that the toxin interacts intimately with the pore and an interaction of the toxin with an external ion (one of the external binding sites in Bernèche and Roux, 2001
, and Zhou et al., 2001
; here called S0) is very likely (see also Moran, 2001
). Therefore,
-PVIIA will compete with K+ for this site, and toxin binding to the channel will occur only for the configurations in which S0 is not occupied and the inner ions are in the 1,3 position, so that the "on" binding rate is strongly reduced at high [K]o. However, after
-PVIIA binding, the inner ions can easily shift to the 2,4 configuration, consolidating the binding.
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Quite different considerations apply for the rate of toxin dissociation, which depends on the interaction of the bound toxin with the ions inside the pore and is expected to be quite different for different site-occupancy configurations. For potentials that drive inward currents, the relatively high affinity of -PVIIA when K+ is the charge carrier can be explained by assuming
-PVIIA binding can push K+ into the 2/4 configuration in the open state, which leads to the observed increase in affinity measured as a hump in the currents. For outward currents,
-PVIIA most likely is pushed away by the permeant K+ (Fig. 5).
For Rb+ as charge carrier, unblock after opening of the channel was always observed (Fig. 5). The 1/3 preference of Rb+ for the closed state results in a low occupancy of S0; activation would lead to an increase in occupancy of this site, resulting in the observed unblock at all voltages. Interestingly, with Rb+ as a charge carrier, the block of -PVIIA is much less voltage dependent, and fast binding/unbinding kinetics are observed with both inward and outward currents. These data indicate that the 1/3 preference of Rb+ leads to destabilization of toxin binding to the open state, and that in the Rb+-occupied channel pore, the binding site for
-PVIIA has a different character (either due to slight conformational changes or because the ensemble of accessible states has a different distribution of charge) than for K+, resulting in a different interaction with the toxin. Thus,
-PVIIA binding to Shaker channels is sensitive not only to differences in ion occupancy within the pore but also to subtle differences in the channel-permeant ion complex.
Interestingly, the recent results of Thompson and Begenisish (2003) suggest that the effects of extracellular tetraethylammonium (TEA) on K+ channels are also affected differentially by K+ and Rb+, although the observed effects are somewhat smaller in magnitude than those documented above. The ion replacement studies showed that the identity of the permeant ion in the internal solution determined the slight voltage dependence of extracellular TEA block, believed to be due to the fact that the TEA binding site is within the membrane electric field. Voltage dependence of TEA block in K+ solutions arises through the movement of K+ ions through some part of the membrane electric field; this voltage dependence was lost with Rb+ as the permeant ion.
Previously, the same authors reported evidence for different patterns of occupancy of specific sites in the pore by permeant ions; K+ and Rb+ interfered differently with the blocking ability of intracellular and extracellularly applied TEA. In Shaker channels, external TEA antagonizes the effectiveness of internal TEA only when K+ ions are in the intracellular and extracellular solution (Thompson and Begenisish, 2000). On the contrary, when Rb+ is the permeant ion, external TEA is entirely ineffective in protecting the channels from block by internal TEA, even if external TEA binds slightly more efficiently in the presence of Rb+ in the extracellular solution than of K+. The ability of K+ and the inability of Rb+ to relay interaction between external and internal TEA seems fully consistent with the freedom of K+ ions, but not Rb+, to shift easily in either direction their position inside the narrow region of the pore.
Our data suggest also that having a bound toxin modifies channel gating. This conclusion must be drawn from the fact that closed or open channels have different toxin binding affinity at the equilibrium potential (0 mV and symmetric K+ or Rb+). The difference appears to be small in symmetrical K+ conditions, where closed and open channels have the same toxin affinity for potentials between 20 and 30 mV, but much larger and in the opposite direction in symmetrical Rb+. Due to obvious considerations of microscopic reversibility at equilibrium, these data imply that the binding of -PVIIA slightly favors channel opening if the pore is filled with K+, whereas it strongly opposes it if the pore is filled with Rb+. Along the view of interpreting toxin effects as a reflection of the toxin interaction with ions in the pore, this suggests in turn that the channel gating structures depend on ion occupancy. Recent structural data provided evidence that the pore is not rigid, but on the contrary, a dynamic structure (Perozo et al., 1999
; Liu et al., 2001
; Zhou et al., 2001
). Minor adjustments in the structure of the selectivity filter may occur in the different occupational states and with different ion species. By using electron paramagnetic resonance spectroscopy on the KcsA channel, Perozo et al. (1998)
(1999
; Liu et al. 2001
) showed that during gating, significant conformational changes take place near the intracellular end, producing a large increase in the diameter of the vestibule below the central water-filled cavity while the outer vestibule is not greatly affected by the transitions between open and closed state, (Perozo et al., 1999
; Liu et al., 2001
). However, a small increase in the diameter of the permeation pathway was observed (Liu et al., 2001
), supporting the possibility of a dynamic role of the selectivity filter in contributing to the gating mechanism. Structural data (Zhou et al., 2001
) showing that the selectivity filter has two significantly different structures, a nonconductive state in low [K+] and a conductive one in high [K+], has been obtained, consistent with some structure adjustments while the ions move along the selectivity filter (Zhou et al., 2001
). Our data demonstrate that for the binding of
-PVIIA to Shaker K+ channels, the toxin sees a different interaction partner depending on both the state of occupancy of the pore and the nature of the permeant ions, and the small movements or conformational changes due to ions in a pore can have significant pharmacological and functional impacts. These specific results with
-PVIIA and the Shaker channel may be more broadly relevant, and suggest that channel pores should not be conceptualized as rigid polypeptide cages, but as dynamic structural complexes that change dependent on the permeant ions present.
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ACKNOWLEDGMENTS |
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This work was supported by the Biofuture Prize from the German Ministry of Education and Research (Förderkennzeichen: 0311859) (to H. Terlau).
Olaf S. Andersen served as editor.
Submitted: 24 February 2004
Accepted: 1 June 2004
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REFERENCES |
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