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Evidence for a Trap Door Mechanism of Activation Gating
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ABSTRACT |
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 Top ABSTRACT introductionmaterials and methodsresultsdiscussionreferences |
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Key Words: potassium channels • tetraethylammonium compounds • ion channel gating • open channel blockade • use-dependent blockade
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introduction |
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TopABSTRACTintroductionmaterials and methodsresultsdiscussionreferences |
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, 1969, 1971) pioneered the use of QA compounds for the study of K+ channel permeation and gating. He discovered that tetraethylammonium (TEA) and its QA derivatives block the ionic current only after the channel has been open. Because dissociation of the blockers was made faster by increasing the external [K+], he proposed that QA compounds bind within the pore.
For the most part, QA compounds also prevent K+ channels from closing. Armstrong (1971) found that for squid axon K+ channels, the tail currents (indicative of K+ channel closing upon repolarization) are made slower by nonyltriethylammonium (C9) blockade, and they have a rising phase ("hook"). These kinetic changes are explained by assuming that most of the channels remain open until the blocker dissociates, and then they close to the original unblocked closed state. However, the blocker's ability to prevent closing is not absolute. Armstrong observed a slow component of recovery from blockade, that was more pronounced and slower at very negative voltages. Since very negative potentials are known to favor closing of the channel, he suggested that this slow recovery from blockade was due to some of the channels closing with a QA ion trapped inside the pore.
For cloned wild-type Shaker K+ channels, QA blockers behave much as they do in the squid axon channel, except that there is no indication of trapping. Decyltriethylammonium (C10) slows the closing of the channels upon repolarization (Choi et al., 1993). Moreover, at voltages where the channel open probability is significantly lower than one, TEA and C10 show a substantially reduced apparent affinity for the channels, consistent with a scheme In which these blockers bind only to the open state (Choi et al., 1993).
We have accidentally found a point mutation, I470C, that changes the Shaker K+ channel so that it can trap TEA and C10. This mutation is in the S6 membrane-spanning region of the channel, near the 469 site previously shown to influence the binding affinity of long-chain QAs (Choi et al., 1993). Because this mutant also does not C-type inactivate, we could easily measure the voltage dependence of gating of blocked channels. This voltage dependence was not very different from the gating in the absence of blocker, suggesting that the blockers bind to a site that does not itself change very much during gating, except that the site is covered by a trap door or hinged lid when the channel closes.
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materials and methods |
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TopABSTRACTintroductionmaterials and methodsresultsdiscussionreferences |
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) with the 
6-46 mutation that removes N-type inactivation (Hoshi et al., 1990). The channel cDNA was subcloned into the GW1-CMV expression vector (British Biotechnology, Oxford, UK; Choi et al., 1991). In addition, our "wild-type" channel had the following background mutations: C301S, C308S, and T449V (Holmgren et al., 1996); all references to wild-type data obtained in this paper refer to this channel. The T449V mutation reduces the amount of C-type inactivation (López-Barneo et al., 1993); with the additional I470C mutation, no inactivation at all was apparent. The mutation I470C was made by polymerase chain reaction (Ausubel et al., 1996) and was confirmed by sequencing the entire subcloned 230-bp insert between amino acids 435 and 509.
The expression system used was human embryonic 293 cell line (HEK 293; Amer. Type Culture Collection, Rockville, MD). The channel expression plasmid (20 µg) was cotransfected with the 
H3-CD8 plasmid (Seed and Aruffo, 1987) which expresses the 
subunit of the human CD8 lymphocyte antigen. Cells expressing the CD8 antigen were identified visually by decoration with antibody-coated beads (Jurman et al., 1994).
Solutions and Electrophysiological Recordings
The internal solution contained (mM): 160 KCl, 1 EGTA, and 10 HEPES (pH 7.4). We omitted Mg2+ from our internal solution because we found it to be a voltage-dependent open channel blocker. The external solution contained (mM): 150 NaCl, 10 KCl, 3 CaCl2, 1 MgCl2, 10 HEPES, pH 7.4.
The electrophysiological recordings were performed from inside-out excised patches (Hamill et al., 1981) using the same conditions described earlier (Holmgren et al., 1996).
Stabilization Energy Calculation
The stabilization energy associated with a blocker was calculated from the apparent equilibrium constant for gating of blocked channels, relative to the apparent equilibrium constant for gating of unblocked channels: Gstab = RT ln [Kg(B)/Kg]. This was calculated at the voltage for which the data for both curves were most reliable (i.e., closest to 0.5). This voltage was –50 mV for C10 and –40 mV for TEA.
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results |
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TopABSTRACTintroductionmaterials and methodsresultsdiscussionreferences |
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; Fig. 1, top row). The exponential decline in the current represents the association of the blocker with the channels, which begins only after the channels start to open (Armstrong, 1971; Choi et al., 1993). Although there was 78% blockade by the end of the 60-ms pulse, there was complete recovery in the 10-s interval between the first and second pulses in the presence of blocker, as seen from the identical appearance of the two pulses. This is consistent with the previous finding that Shaker channels close only after C10 dissociates from the channel, so that the closed channels are not blocked.
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Could this simply be due to very tight binding of C10 to the mutant? We measured the kinetics of C10 interaction with the open state by analyzing the rate and extent of the decay in the current at different concentrations of blocker (Murrell-Lagnado and Aldrich, 1993; Holmgren et al., 1996). As for the wild-type channels, C10 blockade of the mutant had bimolecular association kinetics: the on-rate was proportional to the concentration and the off-rate was independent of concentration (Fig. 2). At 0 mV, the association rate was 1.3 x 107 M–1 s–1 (compared to 3 x 107 M–1 s–1 for the wild type), and the dissociation rate was 6.9 s–1 (compared to 60 s–1 for the wild type). Clearly, these changes are not enough to account for the different behavior of the mutant. In particular, dissociation of the blocker between depolarizing pulses must be much slower than the 
7 s–1 dissociation from the open state.
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where C, O, OB, and CB represent different states of the channel: closed, open, open-blocked, and closed-blocked, respectively; Kg and Kg(B) are the effective equilibrium constants for gating in the absence or presence of blocker; B is the blocker (C10 in this case); and and β are the association and dissociation rate constants, respectively. We assume that the C O and the CB OB transitions occur much faster than the blocking and unblocking reactions. Therefore, when the membrane was repolarized to –90 mV in the presence of C10 (Fig. 3), all of the channels closed: the 75% in the OB state closed to the CB state, while the 25% remaining in the O state closed to the C state. After removing the blocker and reopening the channels, the fast component comes from channels that open rapidly from C to O, while the slow recovery corresponds to channels that move rapidly from CB to OB but then recover slowly to the O state. In support of the assumption that the CB to OB transition is fast compared with blocker dissociation (OB to O), we find that the time course of the slow recovery matches the open state dissociation rate measured in Fig. 2.
In this model, because the blocker can dissociate only when the gate is open (i.e., from the OB state), the rate of recovery from block (βapp) should be proportional to the open probability of the blocked channels:
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where P(open|blocked) is the conditional probability that a channel is open given that it is blocked (equal to POB/(POB + PCB)). This allows us to measure the voltage-dependent gating of blocked channels by measuring βapp at different voltages (Miller et al., 1987). We measured βapp in two ways, depending on the voltage. At more positive voltages where we could measure a significant K current, we monitored the dissociation continuously from the rise of the current after removing blocker, as in the third pulse of Fig 3. For more negative voltages, we first removed the blocker (maintaining the voltage at –90 mV to prevent escape) and then held at the test voltage and applied very brief pulses to +60 mV to monitor the time course of recovery from blockade (Fig. 4).
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10 mV more negative than the V1/2 of the C O transition. After Miller et al. (1987), we define a "stabilization energy" for the blocker effect on gating, equal to RT ln [Kg(B)/Kg]. For C10 we found that the blocker stabilized the open state by a rather small energy of 1.2 kcal/mol.
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20% into the membrane field from the intracellular side (data not shown; similar to Choi et al., 1993).2
Trapping and Untrapping of TEA
The characteristics of blockade by TEA were also altered in the mutant and showed evidence of trapping. TEA blockade of wild-type channels is fast enough to appear as an instantaneous reduction in current (Fig. 6 A, top), but in the mutant, it is slow enough to produce a relaxation after channel opening like that seen for C10 (Fig. 6 A, bottom). The kinetics of the relaxation were consistent with a bimolecular association reaction (Fig. 6 B).
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75% of the current appears with a slower time constant corresponding to blocker dissociation.
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12 mV compared with the voltage dependence of unblocked channel gating. This effect corresponds to a stabilization energy of 1.6 kcal/mol, with TEA favoring the closed state. No intrinsic voltage dependence of TEA dissociation was apparent in the voltage range explored.
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discussion |
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TopABSTRACTintroductionmaterials and methodsresultsdiscussionreferences |
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We found that TEA and C10 were trapped in the channels with little energetic perturbation of the C O transition. A molecule of C10 bound to the channel stabilized the open conformation by 1.2 kcal/mol, while a TEA molecule stabilized the closed conformation by 1.6 kcal/mol. These small and opposite effects of trapped organic blockers on the energetics of gating lead us to believe that gating mostly changes the accessibility of the blocker binding site, with very little change of the site itself. In other words, we suspect (as originally pictured by Armstrong in 1971) that the blockers bind in a relatively static intracellular vestibule that leads to the narrow ion-selective part of the pore, and that the activation gate acts like a trap door to prevent access between this vestibule and the intracellular solution (Fig. 9 A). Trapping can occur because behind the closed gate there is a cavity, which is probably filled with water in the normal closed state but can accommodate a blocker molecule. Structural studies of a mutagenized model protein (cytochrome c peroxidase) show an artificially enlarged, water-filled cavity at the active site (Fitzgerald et al., 1996). Access to this cavity is controlled by a hinged lid that can close and trap small ligands, although large ligands show a "foot in the door" effect and hold the lid open.
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; Grissmer and Cahalan, 1989), because Ba2+ has the approximate dimensions of K+. It seems less likely for the trapping of relatively large organic blockers like TEA and C10, particularly because we observe little or no energetic penalty for closing in the presence of a blocker. Nevertheless we cannot completely rule this model out. The net energetic effect of a bound blocker on channel gating is necessarily the sum of unfavorable and favorable effects on the conformational change. That is, a blocker might sterically hinder the movement of the protein to the normal closed position and force it to adopt an energetically less favorable conformation; on the other hand, that less favorable closed conformation might have a better interaction with the blocker than the open state, which would tend to stabilize the modified closed state. Therefore, even though the net effect of a blocker on closing is very small (on the order of a single hydrogen bond), we cannot be completely confident that there is no change in the binding site upon closing.
Comparison with Trapping in Other Channels
In the squid axon delayed rectifier K+ channel, Armstrong (1971) found that a long-chain QA compound, C9, could be trapped at very negative voltages. Even at these voltages trapping was very inefficient, suggesting that C9 made it much more difficult for the channel to close (unlike the case studied here). Smaller blockers may be trapped more efficiently in the squid channel: John Clay (1995) found that internally applied TEA did not change the time course of channel closing in squid axon K+ channels. This is consistent with the possibility that TEA is trapped by the channels, although there is no evidence for trapping because TEA kinetics are so fast.
Although trapping was discovered in K+ channels using quaternary ammonium derivatives, the process is not exclusive to QA compounds or to K+ channels. Trapping has been observed in Na+ channels for local anesthetics (Strichartz, 1973; Hille, 1977; Ragsdale et al., 1994; Qu et al., 1995) and for disopyramide analogues (in rabbit myocytes; Carmeliet, 1988). Nicotinic acetylcholine–activated channels at the neuromuscular junction can trap chlorisondamine (Neely and Lingle, 1986). In various voltage-activated K+ channels, trapping has been shown for antiarrhythmic drugs (in rabbit myocytes; Carmeliet, 1992, 1993) and for aminopyridine compounds (Wagoner and Oxford, 1990; Choquet and Korn, 1992; Kirsch and Drewe, 1993). Divalent ions are also known to be trapped inside K+ channels (Eaton and Brodwick, 1980; Armstrong et al., 1982; Miller, 1987; Miller et al., 1987; Grissmer and Cahalan, 1989).
Ba2+ ions can block squid axon delayed rectifier K+ channels from either the extracellular or the intracellular side (Eaton and Brodwick, 1980; Armstrong and Taylor, 1980; Armstrong et al., 1982), and Ba2+ seems to reach the same site regardless of which side it is applied to. Opening the activation gates apparently increases access of Ba2+ to the pore from the intracellular side, while extracellular Ba2+ can enter the channel even in the closed state (Armstrong et al., 1982). This result argues that the activation gate is only at the intracellular side (but compare Miller et al., 1987 and Grissmer and Cahalan, 1989, both of whom found that Ba2+ could not enter from the external side in the closed state and therefore concluded that the pore is gated at both ends).
The most detailed kinetic study of trapping is for Ba2+ ions in Ca2+-activated K+ channels (Miller, 1987; Miller et al., 1987; Neyton and Pelleschi, 1991). Miller and colleagues (1987) found that internally applied Ba2+ could be trapped by closing of the channels. They measured the probability of Ba2+ escape from a single channel as a function of the holding voltage, and thus inferred (as we have here) the open probability of a blocked channel. They found that a bound Ba2+ ion stabilized the open conformation by 1.5 kcal/mol. This effect of Ba2+ is highly dependent on the identity and concentration of the monovalent ion bathing the extracellular face of the channel (Neyton and Pelleschi, 1991). We have not extensively studied the effect of external monovalents on the TEA and C10 effects on the Shaker I470C mutant channels, but we did not observe a difference between 10 mM external K+ and zero external K+.
Other Forms of Use-dependent Blockade in Shaker K+ Channels
Baukrowitz and Yellen (1996) showed that C10 produces a use-dependent blockade in Shaker channels that was very dependent on external [K+]. In that case, use dependence does not occur through blocker trapping, but rather by a different mechanism in which the blocker promotes the intrinsic C-type inactivation gating process. Because C-type inactivation and recovery is slow compared with blockade, both the peak and steady-state current in each pulse get progressively smaller. For the channels in this study, C-type inactivation was reduced by the 449V mutation and further reduced by the I470C mutation, and it plays no part in the use dependence observed here, which arises from trapping.
Mechanism of the Mutation's Effect
We do not know why the I470C mutation permits the Shaker K+ channel to trap QA blockers. Perhaps the mutation of four amino acids in the pore (one per subunit) from isoleucine to cysteine, which has a smaller side chain, makes extra room that allows the blocker to be retained in the closed state without interfering with the closing of the door. We wondered whether this change might also allow the blockers to bind more deeply in the channel than they ordinarily do, but we found no evidence for a change in the voltage dependence of blocker binding (data not shown).
Pharmacological Implications
In any case, the fact that a point mutation can change this channel from one that does not trap blockers to one that traps them effortlessly is quite remarkable. It suggests that the difference between channels that do or do not trap blockers is not a major architectural difference, but instead it requires only minor remodeling. Furthermore, it suggests that there are likely to be substantial differences between channel subtypes in their ability to trap blockers; even within a channel subtype, there could be significant species differences. Because blocker trapping has important consequences for the use-dependent behavior of therapeutic channel blockers, it may be important to determine this property for the precise human molecular target of a drug, rather than using an animal homologue.
1 Abbreviations used in this paper: C9, nonyltriethylammonium; C10, decyltriethylammonium; QA, quaternary ammonium; TEA, tetraeth-ylammonium.
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ACKNOWLEDGMENTS |
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This work was supported by a Muscular Dystrophy Association postdoctoral fellowship (to M. Holmgren) and by National Institutes of Health grant NS29693 (to G. Yellen).
Submitted: 23 January 1997
Accepted: 25 February 1997
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J. Zheng, L. Vankataramanan, and F. J. Sigworth Hidden Markov Model Analysis of Intermediate Gating Steps Associated with the Pore Gate of Shaker Potassium Channels J. Gen. Physiol., November 1, 2001; 118(5): 547 - 564. [Abstract] [Full Text] [PDF]
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D. Guo and Z. Lu Kinetics of Inward-Rectifier K+ Channel Block by Quaternary Alkylammonium Ions: Dimension and Properties of the Inner Pore J. Gen. Physiol., May 1, 2001; 117(5): 395 - 406. [Abstract] [Full Text] [PDF]
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K. S. Shin, B. S. Rothberg, and G. Yellen Blocker State Dependence and Trapping in Hyperpolarization-Activated Cation Channels: Evidence for an Intracellular Activation Gate J. Gen. Physiol., February 1, 2001; 117(2): 91 - 102. [Abstract] [Full Text] [PDF]
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R. Olcese, D. Sigg, R. Latorre, F. Bezanilla, and E. Stefani A Conducting State with Properties of a Slow Inactivated State in a Shaker K+ Channel Mutant J. Gen. Physiol., February 1, 2001; 117(2): 149 - 164. [Abstract] [Full Text] [PDF]
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R. Horn, S. Ding, and H. J. Gruber Immobilizing the Moving Parts of Voltage-Gated Ion Channels J. Gen. Physiol., September 1, 2000; 116(3): 461 - 476. [Abstract] [Full Text] [PDF]
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N. Mitrovic, A. L. George Jr., and R. Horn Role of Domain 4 in Sodium Channel Slow Inactivation J. Gen. Physiol., June 1, 2000; 115(6): 707 - 718. [Abstract] [Full Text] [PDF]
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J. S. Mitcheson, J. Chen, and M. C. Sanguinetti Trapping of a Methanesulfonanilide by Closure of the Herg Potassium Channel Activation Gate J. Gen. Physiol., March 1, 2000; 115(3): 229 - 240. [Abstract] [Full Text] [PDF]
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E. Nasi and M. del Pilar Gomez Divalent Cation Interactions with Light-Dependent K Channels: Kinetics of Voltage-Dependent Block and Requirement for an Open Pore J. Gen. Physiol., November 1, 1999; 114(5): 653 - 672. [Abstract] [Full Text] [PDF]
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D. Immke, M. Wood, L. Kiss, and S. J. Korn Potassium-dependent Changes in the Conformation of the Kv2.1 Potassium Channel Pore J. Gen. Physiol., June 1, 1999; 113(6): 819 - 836. [Abstract] [Full Text] [PDF]
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 S. Zhang, Z. Zhou, Q. Gong, J. C. Makielski, and C. T. January Mechanism of Block and Identification of the Verapamil Binding Domain to HERG Potassium Channels Circ. Res., May 14, 1999; 84(9): 989 - 998. [Abstract] [Full Text] [PDF]
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 H. Samartzidou and A. H. Delcour Excretion of Endogenous Cadaverine Leads to a Decrease in Porin-Mediated Outer Membrane Permeability J. Bacteriol., February 1, 1999; 181(3): 791 - 798. [Abstract] [Full Text] [PDF]
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G. Eaholtz, A. Colvin, D. Leonard, C. Taylor, and W. A. Catterall Block of Brain Sodium Channels by Peptide Mimetics of the Isoleucine, Phenylalanine, and Methionine (IFM) Motif from the Inactivation Gate J. Gen. Physiol., February 1, 1999; 113(2): 279 - 294. [Abstract] [Full Text] [PDF]
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M C Sanguinetti and Q P Xu Mutations of the S4-S5 linker alter activation properties of HERG potassium channels expressed in Xenopus oocytes J. Physiol., February 1, 1999; 514(3): 667 - 675. [Abstract] [Full Text] [PDF]
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H. Zhang, B. Zhu, J.-A. Yao, and G.-N. Tseng Differential Effects of S6 Mutations on Binding of Quinidine and 4-Aminopyridine to Rat Isoform of Kv1.4: Common Site but Different Factors in Determining Blockers' Binding Affinity J. Pharmacol. Exp. Ther., October 1, 1998; 287(1): 332 - 343. [Abstract] [Full Text]
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S. Trapp, P. Proks, S. J. Tucker, and F. M. Ashcroft Molecular Analysis of ATP-sensitive K Channel Gating and Implications for Channel Inhibition by ATP J. Gen. Physiol., September 1, 1998; 112(3): 333 - 349. [Abstract] [Full Text] [PDF]
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M. Spassova and Z. Lu Coupled Ion Movement Underlies Rectification in an Inward-Rectifier K+ Channel J. Gen. Physiol., August 1, 1998; 112(2): 211 - 221. [Abstract] [Full Text] [PDF]
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E. Marban, T. Yamagishi, and G. F Tomaselli Structure and function of voltage-gated sodium channels J. Physiol., May 1, 1998; 508(3): 647 - 657. [Abstract] [Full Text] [PDF]
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F. S.P. Chen and D. Fedida On the Mechanism by which 4-Aminopyridine Occludes Quinidine Block of the Cardiac K+ Channel, hKv1.5 J. Gen. Physiol., April 1, 1998; 111(4): 539 - 554. [Abstract] [Full Text] [PDF]
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R. A. Bello and K. L. Magleby Time-irreversible Subconductance Gating Associated with Ba2+ Block of Large Conductance Ca2+-activated K+ Channels J. Gen. Physiol., February 1, 1998; 111(2): 343 - 362. [Abstract] [Full Text] [PDF]
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L. Kiss, D. Immke, J. LoTurco, and S. J. Korn The Interaction of Na+ and K+ in Voltage-gated Potassium Channels: Evidence for Cation Binding Sites of Different Affinity J. Gen. Physiol., February 1, 1998; 111(2): 195 - 206. [Abstract] [Full Text] [PDF]
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J. G. Starkus, L. Kuschel, M. D. Rayner, and S. H. Heinemann Ion Conduction through C-Type Inactivated Shaker Channels J. Gen. Physiol., November 1, 1997; 110(5): 539 - 550. [Abstract] [Full Text] [PDF]
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C. M. Armstrong A Closer Picture of the K Channel Gate from Ion Trapping Experiments J. Gen. Physiol., May 1, 1997; 109(5): 523 - 524. [Full Text] [PDF]
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C. Fahlke, R. R. Desai, N. Gillani, and A. L. George Jr. Residues Lining the Inner Pore Vestibule of Human Muscle Chloride Channels J. Biol. Chem., January 12, 2001; 276(3): 1759 - 1765. [Abstract] [Full Text] [PDF]
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) and extent (f = Isteady-state/Ictrl), measured in the first pulse following application of blocker. We computed kassoc as (1 – f)/
with values of β0 = 6.3 ± 0.1 s–1; z = 4.9 ± 0.5; 
= 0.15 ± 0.02; and Vmid = –58 ± 0.8 mV. The normalized g-V was fit by the function
with values of z = 5.1 ± 0.1 and Vmid = –48 ± 0.2 mV.
