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Sodium channel activators: Model of binding inside the pore and a possible mechanism of action
FEBS 29809
FEBS Letters 579 (2005) 4207–4212
Hypothesis
Sodium channel activators: Model of binding inside the pore and a possible mechanism of action Denis B. Tikhonov, Boris S. Zhorov* Department of Biochemistry and Biomedical Sciences, McMaster University, 1200 Main Street West, Hamilton, Ont., Canada L8N 3Z5 Received 30 May 2005; revised 3 July 2005; accepted 8 July 2005 Available online 26 July 2005 Edited by Maurice Montal
Abstract Sodium channel activators, batrachotoxin and veratridine, cause sodium channels to activate easier and stay open longer than normal channels. Traditionally, this was explained by an allosteric mechanism. However, increasing evidence suggests that activators can bind inside the pore. Here, we model the open sodium channel with activators and propose a novel mechanism of their action. The activator-bound channel retains a hydrophilic pathway for ions between the ligand and conserved asparagine in segment S6 of repeat II. One end of the activator approaches the selectivity filter, decreasing the channel conductance and selectivity. The opposite end reaches the gate stabilizing it in the open state. Ó 2005 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. Keywords: Batrachotoxin; Veratridine; Energy minimization; Monte Carlo minimization; Channel gating; Channel block; Ion permeation
1. Introduction Naturally occurring alkaloids such as batrachotoxin (BTX), veratridine (VTD), aconitine, grayanotoxin, and their synthetic derivatives cause Na+ channels to open more easily and stay open longer [1]. The action of these toxins has the following common features: (i) the voltage dependence of activation is shifted towards more negative potentials so that the channels can open at potentials at which normal channels remain in the resting state; (ii) the inactivation is reduced or even removed so that activated channels mediate sustained currents; (iii) the single-channel conductance is reduced; and (iv) the strong sodium selectivity is lost. An additional important feature of the activatorÕs action is the use-dependence: the intensive stimulation enhances binding. The unique and complex behavior of Na+ channel activators is traditionally explained by the allosteric actions of lipid-soluble toxins that are thought to bind at the lipid-exposed side of the channel protein or at the subunit interface. However, * Corresponding author. Fax: +1 905 522 9033. E-mail address: [email protected] (B.S. Zhorov).
Abbreviations: BTX, batrachotoxin; VTD, veratridine; MC, Monte Carlo
increasing evidence suggests the necessity to revise this theory. First, activator-sensing residues are found in the pore-lining inner helices of all four repeats suggesting that the ligands could bind inside the pore [2]. The binding site of activators overlaps significantly with the binding site for local anesthetics, which are known to block the Na+ channel pore in a voltageand use-dependent mechanism [3]. Second, the X-ray structures of the open bacterial K+ channels MthK [4] and KvAP [5], which are believed to share a common fold with the Na+ channel, have the inner pore wide enough to accommodate relatively large molecules, such as Na+ channel activators [6]. Third, channel gating occurs at the middle region of the inner helices, where according to mutational analysis the Na+ channel activators bind and where they could affect gating properties. Fourth, to affect channel conductance and selectivity, activators must influence the structure of the ion permeation pathways probably including the selectivity-filter region. To affect both gating and selectivity simultaneously, the activators should bind like a bridge between the gate and the selectivity filter. In view of structures of K+ channels, the only possibility to comply with all these requirements for the bulky and rigid ligands is to bind inside the pore. In the absence of the Na+ channel X-ray structure, the allosteric mechanism cannot be ruled out. However, the above arguments justify the timeliness of the following theoretical analysis of the possibility that Na+ channel activators could be directly involved in the ion permeation pathway.
2. Common binding region for activators and blockers Na+ channel activators and blockers have several common features. First, in some cases, their structures are not vastly different (Fig. 1). Thus, alkaloids VTD and aconitine are classical Na+ channel activators, while some of their derivatives block the Na+ channel [7–9]. Some Na+ channel blockers, e.g., strychnine and pancuronium resemble activators by size and general shape. Both activators and blockers may have charged groups as well as donors and acceptors of H-bonds. Second, both activators [10,11] and blockers [12,13] bind in a usedependent manner. Third, both activators [14] and blockers [15,16] can prevent channel closure and shift gating equilibrium of the channel. Action of activators and blockers on the channel conductance is obviously different, but the difference is quantitative
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Fig. 1. Chemical and 3D structures of activators and blockers of the sodium channel. Chemical structures do not allow discriminating activators from blockers. Both types of ligands have rigid polycyclic hydrophobic core decorated by polar and ionized/ionizable groups. 3D structures of the ligands are easily deducible for the semirigid molecules with known stereochemistry. Comparison of 3D structures of activators with the closeststructure blockers shows clear tendency. Both activators and blockers are similar in size and have elongated structures. In activators, polar groups form a string at one side of the molecule, while the opposite side is predominantly hydrophobic. In blockers, hydrophilic groups are sparse. Blocker lappaconitine seems to be an exclusion from this rule as it has too many oxygen atoms. However, these are located at the opposite sides of the molecule.
rather than qualitative. Indeed, the action of pure blockers that plug the pore is described in terms of affinity (the ability to bind), while action of activators is described in terms of affinity and efficacy (the ability to sustain the current through the channel). Practically all activators decrease the unitary current, having an efficacy <1. In the extreme, the activators having a zero efficacy are blockers. In other words, the binding of blockers completely abolishes channel conductance, whereas activators allow ions to flow, but at a reduced rate. The fact that activators and blockers share common structural features and differ in the efficacy suggests that both types of ligands can bind to a common channel region. Steroidal blockers like pancuronium are generally believed to bind inside the pore, while the binding site of activators remains uncertain. The competition between activators and blockers and the fact that certain mutations affect binding of both activators and
blockers support the idea that activators and blockers share a common binding region.
3. Models of Na+ channel with ligands To elaborate this idea, we created a model of the Nav1.4 channel in the open state and docked ligands in the pore to analyze their possible interactions with the channel. The X-ray structure of the open K+ channel MthK was used as a template. The sequences of MthK and Na+ channel were aligned as proposed earlier [6,17]. In this alignment, the ligand-sensing residues in the inner helices face the pore. The P-loop region was modeled as in [18]. The channel model was optimized by the protocol of Monte Carlo (MC) with energy minimization [19] using the AMBER force field [20] and implicit solvation [21].
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Fig. 2. Ligands in the Na+ channel. (A) VTD-channel interaction energy and its van der Waals, electrostatic, and solvation components plotted against the distance between the ligandÕs Csp3 carbon next to the ester group and the plane comprising alpha carbons of residues two positions upstream from the DEKA locus. The ligand was pulled from the cytoplasmic pore entrance towards the selectivity filter with the help of the constraint between the Csp3 carbon and a shifting plane, which is normal to the pore axis. The plane was translated normally to the pore axis with a ˚ , and at each position the energy of the ligand-channel complex was MC-minimized [25]. The complex with the minimal ligand-channel step of 1 A ˚ ) is shown at (B). (B–D) Side views of the Na+ channel with ligands. Repeat I is removed for clarity. The inner helices and pore energy (distance 4 A helices are shown as violet and green ribbons, respectively. The ligands are shown as thick sticks. Activators VTD (B) and BTX (C) approach the selectivity filter by the ester oxygen. The polar sides of the activators face the conserved Asn784, whereas opposite hydrophobic side binds to the conserved Phe1750 (space-filled). Phe1236 (magenta sticks) can form stacking contact with the aromatic ring of activators (B). Electrostatic attraction with the ester oxygen of activator can displace Lys (magenta sticks) of the DEKA locus (C). A quaternary ammonium group of the blocker pancuronium (D) is close to the focus of macrodipoles of the pore helices. The blocker lacks hydrophilic groups to form a hydrophilic pathway through the pore. (E) Cytoplasmic view at the VTD-bound channel. The space-filled inner and outer helices are blue and gray, respectively. Phe1750 is magenta and Asn784 is green. VTD easily fits the central pore, leaving some space for the permeation of Na+. (F) VTD in the closed KcsA-like conformation of the channel. Despite the weaker interaction energy, the activator could be trapped in the closed channel.
In the presence of activators, the Na+ channel permeates larger organic cations than the normal channel [22]. This suggests direct interaction between the activator and the channel selectivity filter. Elongated BTX and VTD have an aromatic ring
connected to the rigid polycyclic body via an ester group (Fig. 1). Since the aromatic ring is necessary for activity [23,24], we docked the activators with the initial orientation of the aromatic ring towards the selectivity filter.
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To find the energetically optimal binding mode of VTD, we first calculated the energy profile by translating the ligand along the pore and MC-minimizing energy at each step [25]. The energy profile (Fig. 2A) has a single minimum corresponding to VTD approaching the selectivity-filter region. In the next stage, the optimal orientation of VTD was found by rotating the ligand around its long axis and MC-minimizing energy at each step using the methodology described elsewhere [18] (data not shown). The inner pore of the Na+ channel is lined by predominantly hydrophobic residues. It also contains a ring formed by conserved hydrophilic residues Ser429, Asn784, and Ser1276 and a highly conserved Phe1750. The latter is a critical determinant of binding of activators and local anesthetics [26]. In the lowest-energy orientation, the hydrophobic surface of VTD binds to the hydrophobic side of the pore, which includes Phe1750 (Fig. 2B). The opposite nucleophilic side of VTD is oriented towards the conserved Asn784 in the inner helix of repeat II. The preferable binding mode of BTX is also stabilized by the hydrophobic interactions with Phe1750 (Fig. 2C). The low-energy complexes have several interesting features. In particular, activators approach the selectivity filter by their ester oxygens and aromatic rings (Fig. 2B and C), which are known as important determinants of the agonistÕs activity. In our models these moieties interact directly with the DEKA locus and residues in the preceding positions, especially with Phe1236 that forms a stacking motif with the aromatic rings of the activators. Most importantly, the open pore is not completely occluded by the bulky activators (Fig. 2E). The remaining lumen is lined on one side by the polar groups of the activators and on the other side it has the conserved Asn784. Such a pathway can be permeable for cations. The comparison of the 3D structures of other ligands shows that activators have multiple nucleophilic groups clustered at one face, whereas the other face is predominantly hydrophobic (Fig. 1). In contrast, blockers lack a continuous string of oxygen atoms. The importance of the nucleophilic groups and their clustering at one face of the molecule has been demonstrated in earlier structure– activity studies [27]. In particular, a model of the oxygen triad has been proposed to explain the action of activators [28]. In view of our hypothesis, the clustering of nucleophilic groups enables ions to permeate via the activator-bound channel. The blocker pancuronium also binds tightly to Phe1750 (Fig. 2E). The quaternary ammonium group of the blocker occurs at the focus of macrodipoles of the pore helices, the position where tetrabutylammonium binds in K+ channels [29]. Unlike activators, which expose their polar groups towards Asn784, the blocker exposes its hydrophobic groups towards this conserved residue. In the absence of the Na+ channel X-ray structure, the resolution of our model is obviously low. Details of the interactions that involve flexible residues of the channel and flexible fragments of the ligands cannot be predicted unambiguously. Therefore, subtype specificity of action of the ligands cannot be predicted now. However, the major results are determined by the well-resolved structure of the semirigid ligands and general architecture of the pore rather than by the atomic-level details. These results include: (i) possible binding of the activator inside the pore, (ii) its preferable orientation with hydrophilic groups exposed to the lumen, and (iii) existence of the hydrophilic pathway through the activator bound but not the blocker-bound channel.
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4. Ion permeation through ligand-bound channel The wide cytoplasmic vestibule of the open P-loop channel allows the free passage of hydrated ions, while the selectivity filter governs ion selectivity and permeability. In the agonistbound channel, the cytoplasmic vestibule is occluded by the ligand and the lumen becomes narrow. To reveal structural determinants of Na+ permeation through the narrow lumen of the ligand-bound channel, we calculated MC-minimized energy profiles for the ion pulled from the cytoplasm to the selectivity filter (Fig. 3). In these calculations, the ion was constrained to the plane normal to the pore axis, the plane was ˚ , and energy was MC-minimized translated with the step of 1 A at each step. Four parts of the ion trajectory in the VTDbound channel can be discriminated. A small energy barrier is seen at the initial part, where the ion enters the pore and loses its hydration shell. At the narrowest part of the lumen, the ion is stabilized by the attraction to the conserved Asn784. Closer to the P-loop, the ion occurs in the focus of macrodipoles of the pore helices. The deepest minimum is observed at the selectivity-filter region, where electrostatic interactions dominate. Noteworthy, VTD does not cause a significant energy barrier, since the unfavorable dehydration of the ion is compensated by the electrostatic attraction to the polar groups of the activator. The fact that VTD blocks rather than activates the Na+ channel mutant Asn784Cys [30] suggests the involvement of Asn784 in the ion permeation. In agreement with this experiment, the Na+ profile in the VTD-bound mutant has a higher energy barrier at the level of Cys784 (Fig. 3A). The Na+ profile through the pancuronium-bound channel (Fig. 3A) has a significant energy barrier caused by uncompensated dehydration of the ion and its repulsion from the blockerÕs ammonium group. Thus, our model shows a possibility of ion permeation via the VTD-bound Na+ channel. In contrast, binding of pancuronium blocks ion permeation. The ion permeation through the activator-bound channel is sensitive to the structural peculiarities of the hydrophilic pathway. This agrees with the data that mutations in the inner helix of repeat II affect the action of activators but do not affect the block by local anesthetics [2].
5. Selectivity of the activator-bound channel According to our hypothesis, activators bind inside the pore, at the turns of P-loops. Activators can directly interact with the selectivity filter and significantly affect spatial organization of the DEKA locus, the ring of highly conserved residues Asp, Glu, Lys, and Ala in repeats I–IV, respectively, which govern selective permeability of Na+ channels [31]. For example, carbonyl oxygen of BTX could interact with the amino group of Lys1237 (Fig. 2B) and repulse carboxylic groups of Asp and Glu, thus destabilizing the native structure of the DEKA locus. This would affect selectivity. Limited precision of the agonistbound models does not allow us to address this question systematically. Permeation of organic cations is also known to substantially depend on the selectivity-filter structure. However, the latter does not behave as a simple molecular sieve since, counterintuitively, the DEAA mutant of the DEKA locus permeates larger organic cations than the AAAA mutant [32]. Our recent model of the selectivity-filter region
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6. Gating of the activator-bound channel In our model, the activators extend from the selectivity filter to the intracellular parts of the inner helices. The latter region is significantly different between the open and closed K+ channels. Mutations in this region affect the Na+ channel gating [3]. To analyze the effect of VTD on Na+ channel gating, we simulated the closing of the VTD-bound channel using the approach described in [33]. Despite the presence of the bulky VTD, the Na+ channel was able to reach the closed KcsA-like conformation from the open state (Fig. 2F). However, the VTD-channel interaction energy increased from 28.0 kcal/mol in the open state to 16.8 kcal/mol in the closed state. The latter was destabilized by the repulsion between VTD and the gate region. Stabilization of the open Na+ channel by VTD explains why the activator-modified channels open more easily and stay open for a longer time than normal channels. This model is also consistent with the proposal that BTX reaches its receptor through the cytoplasmic end of the openchannel pore and can be trapped in the closed channel [34].
7. Conclusions
Fig. 3. Ion permeation via the ligand-bound Na+ channels. (A) Energy of Na+ in the ligand-bound channels. Each point corresponds to the MC-minimized structure calculated with Na+ constrained to a plane, which is normal to the pore axis. The abscissa shows distance of the Na+-constraining plane from the selectivity filter. The VTD-bound channel imposes only a small energy barrier for Na+ permeation, whereas a high barrier in the pancuronium-bound channel would obstruct the ion permeation. Mutation of Asn784 by Cys results in less favorable interactions of Na+ with the VTD-bound channel. (B and C) Side and cytoplasmic views of 25 superimposed MC-minimized complexes of Na+ with the VTD-bound Na+ channel. At different positions of Na+, the side chain of Phe1750, which strongly interacts with VTD, experiences only small fluctuations. In contrast, the side chain of Asn784 moves to preserve attractive contacts with Na+.
[18] has provided a structural explanation of this effect. Altered conformations of Lys1237 in the activator-bound channel (Fig. 2B) can cause permeation of larger ions, the effect similar to that observed in the DEAA mutant.
Our hypothesis provides the structural explanation for the key aspects of the action of Na+ channel activators. The activators fit in the open pore and interact with all four inner helices in agreement with mutational experiments. Ions can permeate through the activator-bound but not the blockerbound channel. The key determinant of this effect is the hydrophilic lining of the narrow pathway in the agonist-bound channel. Asn784 contributes importantly to this pathway. Obvious distinction of the pathways through the normal and activator-bound channels explains essentially different levels of conductance observed. The extreme case of the zero conductance corresponds to the classical block. Direct interaction of the activators with the selectivity filter could explain altered selectivity of the activator-modified channels. Binding of activators to the inner pore at the gate region stabilizes the open state of the channel. This results in the state-dependent action of the activators and causes easier and longer opening of the activator-modified channels. Predicted interactions between the activators and P-loops open a way for experimental testing of our hypothesis. Mutations of P-loop residues upstream from the DEKA locus should affect the action of activators. In particular, our models suggest that Phe1236 can significantly contribute to the binding of activators (Fig. 2B). Coordinates of the models are available upon request from BSZ. E-mail: [email protected]. Acknowledgments: We thank Boris Khodorov and Ging-Kuo Wang for helpful discussions and Iva Bruhova for reading the manuscript. This work was supported by the grant from the Canadian Institutes of Health Research (CIHR). B.S.Z. is a recipient of the CIHR Senior Scientist award. Computations were performed, in part, at the SHARCNET supercomputer center of McMaster University.
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4212 [2] Wang, S.Y., Barile, M. and Wang, G.K. (2001) Disparate role of Na+ channel D2-S6 residues in batrachotoxin and local anesthetic action. Mol. Pharmacol. 59, 1100–1107. [3] Yarov-Yarovoy, V., McPhee, J.C., Idsvoog, D., Pate, C., Scheuer, T. and Catterall, W.A. (2002) Role of amino acid residues in transmembrane segments IS6 and IIS6 of the Na+ channel alpha subunit in voltage-dependent gating and drug block. J. Biol. Chem. 277, 35393–35401. [4] Jiang, Y., Lee, A., Chen, J., Cadene, M., Chait, B.T. and MacKinnon, R. (2002) The open pore conformation of potassium channels. Nature 417, 523–526. [5] Jiang, Y., Lee, A., Chen, J., Ruta, V., Cadene, M., Chait, B.T. and MacKinnon, R. (2003) X-ray structure of a voltage-dependent K+ channel. Nature 423, 33–41. [6] Zhorov, B.S. and Tikhonov, D.B. (2004) Potassium, sodium, calcium and glutamate-gated channels: pore architecture and ligand action. J. Neurochem. 88, 782–799. [7] Ohta, M., Narahashi, T. and Keeler, R.F. (1973) Effects of veratrum alcaloids on membrane potential and conductance of squid and crayfish giant axons. J. Pharmacol. Exp. Ther. 184, 143–154. [8] Ameri, A. and Simmet, T. (1999) Interaction of the structurally related Aconitum alkaloids, aconitine and 6-benzyolheteratisine, in the rat hippocampus. Eur. J. Pharmacol. 386, 187–194. [9] Wright, S.N. (2001) Irreversible block of human heart (hH1) sodium channels by the plant alkaloid lappaconitine. Mol. Pharmacol. 59, 183–192. [10] Catterall, W.A. (1977) Activation of the action potential Na+ ionophore by neurotoxins. J. Biol. Chem. 252, 8669–8676. [11] Khodorov, B.I. and Revenko, S.V. (1979) Further analysis of the mechanisms of action of batrachotoxin on the membrane of myelinated nerve. Neuroscience 4, 1315–1330. [12] Strichartz, G.R. (1973) The inhibition of sodium currents in myelinated nerve by quaternary derivatives of lidocaine. J. Gen. Physiol. 62, 37–57. [13] Courtney, K.R. (1975) Mechanism of frequency-dependent inhibition of sodium currents in frog myelinated nerve by the lidocaine derivative GEA. J. Pharmacol. Exp. Ther. 195, 225–236. [14] Barnes, S. and Hille, B. (1988) Veratridine modifies open sodium channels. J. Gen. Physiol. 91, 421–443. [15] Yeh, J.Z. and Narahashi, T. (1977) Kinetic analysis of pancuronium interaction with sodium channels in squid axon membranes. J. Gen. Physiol. 69, 293–323. [16] Yamamoto, D. (1986) Dynamics of strychnine block of single sodium channels in bovine chromaffin cells. J. Physiol. 370, 395– 407. [17] Yamaguchi, S., Zhorov, B.S., Yoshioka, K., Nagao, T., Ichijo, H. and Adachi-Akahane, S. (2003) Key roles of Phe1112 and Ser1115 in the pore-forming IIIS5-S6 linker of L-type Ca2+ channel alpha1C subunit (CaV 1.2) in binding of dihydropyridines and action of Ca2+ channel agonists. Mol. Pharmacol. 64, 235–248. [18] Tikhonov, D.B. and Zhorov, B.S. (2005) P-loops domain of sodium channel: homology with potassium channels and interaction with ligands. Biophys. J. 88, 194–197.
D.B. Tikhonov, B.S. Zhorov / FEBS Letters 579 (2005) 4207–4212 [19] Li, Z. and Scheraga, H.A. (1987) Monte Carlo-minimization approach to the multiple-minima problem in protein folding. Proc. Natl. Acad. Sci. USA 84, 6611–6615. [20] Weiner, S.J., Kollman, P.A., Case, D.A., Singh, U.C., Chio, C., Alagona, G., Profeta, S. and Weiner, P.K. (1984) A new force field for molecular mechanical simulation of nucleic acids and proteins. J. Am. Chem. Soc. 106, 765–784. [21] Lazaridis, T. and Karplus, M. (1999) Effective energy function for proteins in solution. Proteins 35, 133–152. [22] Mozhayeva, G.N., Naumov, A.P., Negulyaev, Y.A. and Nosyreva, E.D. (1977) The permeability of aconitine-modified sodium channels to univalent cations in myelinated nerve. Biochim. Biophys. Acta 466, 461–473. [23] Warnick, J.E., Albuquerque, E.X., Onur, R., Jansson, S.E., Daly, J., Tokuyama, T. and Witkop, B. (1975) The pharmacology of batrachotoxin. VII. Structure–activity relationships and the effects of pH.J. Pharmacol. Exp. Ther. 193, 232–245. [24] Khodorov, B.I., Yelin, E.A., Zaborovskaya, L.D., Maksudov, M.Z., Tikhomirova, O.B. and Leonov, V.N. (1992) Comparative analysis of the effects of synthetic derivatives of batrachotoxin on sodium currents in frog node of Ranvier. Cell. Mol. Neurobiol. 12, 59–81. [25] Zhorov, B.S. and Bregestovski, P.D. (2000) Chloride channels of glycine and GABA receptors with blockers: Monte Carlo minimization and structure–activity relationships. Biophys. J. 78, 1786–1803. [26] Ragsdale, D.S., McPhee, J.C., Scheuer, T. and Catterall, W.A. (1994) Molecular determinants of state-dependent block of Na+ channels by local anesthetics. Science 265, 1724–1728. [27] Masutani, T., Seyama, I., Narahashi, T. and Iwasa, J. (1981) Structure–activity relationship for Grayanotoxin derivatives in frog skeletal muscle. J. Pharmacol. Exp. Ther. 217, 812–819. [28] Kosower, E.M. (1983) A hypothesis for the mechanism of sodium channel opening by batrachotoxin and related toxins. FEBS Lett. 163, 161–164. [29] Zhou, M., Morais-Cabral, J.H., Mann, S. and MacKinnon, R. (2001) Potassium channel receptor site for the inactivation gate and quaternary amine inhibitors. Nature 411, 657–661. [30] Wang, G.K. and Wang, S.Y. (2003) Veratridine block of rat skeletal muscle Nav1.4 sodium channels in the inner vestibule. J. Physiol. 548, 667–675. [31] Heinemann, S.H., Terlau, H., Stuhmer, W., Imoto, K. and Numa, S. (1992) Calcium channel characteristics conferred on the sodium channel by single mutations. Nature 356, 441–443. [32] Sun, Y.M., Favre, I., Schild, L. and Moczydlowski, E. (1997) On the structural basis for size-selective permeation of organic cations through the voltage-gated sodium channel. Effect of alanine mutations at the DEKA locus on selectivity, inhibition by Ca2+ and H+, and molecular sieving. J. Gen. Physiol. 110, 693–715. [33] Tikhonov, D.B. and Zhorov, B.S. (2004) In silico activation of KcsA K+ channel by lateral forces applied to the C-termini of inner helices. Biophys. J. 87, 1526–1536. [34] Li, H.L., Hadid, D. and Ragsdale, D.S. (2002) The batrachotoxin receptor on the voltage-gated sodium channel is guarded by the channel activation gate. Mol. Pharmacol. 61, 905–912.
FEBS Letters 579 (2005) 4207–4212
Hypothesis
Sodium channel activators: Model of binding inside the pore and a possible mechanism of action Denis B. Tikhonov, Boris S. Zhorov* Department of Biochemistry and Biomedical Sciences, McMaster University, 1200 Main Street West, Hamilton, Ont., Canada L8N 3Z5 Received 30 May 2005; revised 3 July 2005; accepted 8 July 2005 Available online 26 July 2005 Edited by Maurice Montal
Abstract Sodium channel activators, batrachotoxin and veratridine, cause sodium channels to activate easier and stay open longer than normal channels. Traditionally, this was explained by an allosteric mechanism. However, increasing evidence suggests that activators can bind inside the pore. Here, we model the open sodium channel with activators and propose a novel mechanism of their action. The activator-bound channel retains a hydrophilic pathway for ions between the ligand and conserved asparagine in segment S6 of repeat II. One end of the activator approaches the selectivity filter, decreasing the channel conductance and selectivity. The opposite end reaches the gate stabilizing it in the open state. Ó 2005 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. Keywords: Batrachotoxin; Veratridine; Energy minimization; Monte Carlo minimization; Channel gating; Channel block; Ion permeation
1. Introduction Naturally occurring alkaloids such as batrachotoxin (BTX), veratridine (VTD), aconitine, grayanotoxin, and their synthetic derivatives cause Na+ channels to open more easily and stay open longer [1]. The action of these toxins has the following common features: (i) the voltage dependence of activation is shifted towards more negative potentials so that the channels can open at potentials at which normal channels remain in the resting state; (ii) the inactivation is reduced or even removed so that activated channels mediate sustained currents; (iii) the single-channel conductance is reduced; and (iv) the strong sodium selectivity is lost. An additional important feature of the activatorÕs action is the use-dependence: the intensive stimulation enhances binding. The unique and complex behavior of Na+ channel activators is traditionally explained by the allosteric actions of lipid-soluble toxins that are thought to bind at the lipid-exposed side of the channel protein or at the subunit interface. However, * Corresponding author. Fax: +1 905 522 9033. E-mail address: [email protected] (B.S. Zhorov).
Abbreviations: BTX, batrachotoxin; VTD, veratridine; MC, Monte Carlo
increasing evidence suggests the necessity to revise this theory. First, activator-sensing residues are found in the pore-lining inner helices of all four repeats suggesting that the ligands could bind inside the pore [2]. The binding site of activators overlaps significantly with the binding site for local anesthetics, which are known to block the Na+ channel pore in a voltageand use-dependent mechanism [3]. Second, the X-ray structures of the open bacterial K+ channels MthK [4] and KvAP [5], which are believed to share a common fold with the Na+ channel, have the inner pore wide enough to accommodate relatively large molecules, such as Na+ channel activators [6]. Third, channel gating occurs at the middle region of the inner helices, where according to mutational analysis the Na+ channel activators bind and where they could affect gating properties. Fourth, to affect channel conductance and selectivity, activators must influence the structure of the ion permeation pathways probably including the selectivity-filter region. To affect both gating and selectivity simultaneously, the activators should bind like a bridge between the gate and the selectivity filter. In view of structures of K+ channels, the only possibility to comply with all these requirements for the bulky and rigid ligands is to bind inside the pore. In the absence of the Na+ channel X-ray structure, the allosteric mechanism cannot be ruled out. However, the above arguments justify the timeliness of the following theoretical analysis of the possibility that Na+ channel activators could be directly involved in the ion permeation pathway.
2. Common binding region for activators and blockers Na+ channel activators and blockers have several common features. First, in some cases, their structures are not vastly different (Fig. 1). Thus, alkaloids VTD and aconitine are classical Na+ channel activators, while some of their derivatives block the Na+ channel [7–9]. Some Na+ channel blockers, e.g., strychnine and pancuronium resemble activators by size and general shape. Both activators and blockers may have charged groups as well as donors and acceptors of H-bonds. Second, both activators [10,11] and blockers [12,13] bind in a usedependent manner. Third, both activators [14] and blockers [15,16] can prevent channel closure and shift gating equilibrium of the channel. Action of activators and blockers on the channel conductance is obviously different, but the difference is quantitative
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Fig. 1. Chemical and 3D structures of activators and blockers of the sodium channel. Chemical structures do not allow discriminating activators from blockers. Both types of ligands have rigid polycyclic hydrophobic core decorated by polar and ionized/ionizable groups. 3D structures of the ligands are easily deducible for the semirigid molecules with known stereochemistry. Comparison of 3D structures of activators with the closeststructure blockers shows clear tendency. Both activators and blockers are similar in size and have elongated structures. In activators, polar groups form a string at one side of the molecule, while the opposite side is predominantly hydrophobic. In blockers, hydrophilic groups are sparse. Blocker lappaconitine seems to be an exclusion from this rule as it has too many oxygen atoms. However, these are located at the opposite sides of the molecule.
rather than qualitative. Indeed, the action of pure blockers that plug the pore is described in terms of affinity (the ability to bind), while action of activators is described in terms of affinity and efficacy (the ability to sustain the current through the channel). Practically all activators decrease the unitary current, having an efficacy <1. In the extreme, the activators having a zero efficacy are blockers. In other words, the binding of blockers completely abolishes channel conductance, whereas activators allow ions to flow, but at a reduced rate. The fact that activators and blockers share common structural features and differ in the efficacy suggests that both types of ligands can bind to a common channel region. Steroidal blockers like pancuronium are generally believed to bind inside the pore, while the binding site of activators remains uncertain. The competition between activators and blockers and the fact that certain mutations affect binding of both activators and
blockers support the idea that activators and blockers share a common binding region.
3. Models of Na+ channel with ligands To elaborate this idea, we created a model of the Nav1.4 channel in the open state and docked ligands in the pore to analyze their possible interactions with the channel. The X-ray structure of the open K+ channel MthK was used as a template. The sequences of MthK and Na+ channel were aligned as proposed earlier [6,17]. In this alignment, the ligand-sensing residues in the inner helices face the pore. The P-loop region was modeled as in [18]. The channel model was optimized by the protocol of Monte Carlo (MC) with energy minimization [19] using the AMBER force field [20] and implicit solvation [21].
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Fig. 2. Ligands in the Na+ channel. (A) VTD-channel interaction energy and its van der Waals, electrostatic, and solvation components plotted against the distance between the ligandÕs Csp3 carbon next to the ester group and the plane comprising alpha carbons of residues two positions upstream from the DEKA locus. The ligand was pulled from the cytoplasmic pore entrance towards the selectivity filter with the help of the constraint between the Csp3 carbon and a shifting plane, which is normal to the pore axis. The plane was translated normally to the pore axis with a ˚ , and at each position the energy of the ligand-channel complex was MC-minimized [25]. The complex with the minimal ligand-channel step of 1 A ˚ ) is shown at (B). (B–D) Side views of the Na+ channel with ligands. Repeat I is removed for clarity. The inner helices and pore energy (distance 4 A helices are shown as violet and green ribbons, respectively. The ligands are shown as thick sticks. Activators VTD (B) and BTX (C) approach the selectivity filter by the ester oxygen. The polar sides of the activators face the conserved Asn784, whereas opposite hydrophobic side binds to the conserved Phe1750 (space-filled). Phe1236 (magenta sticks) can form stacking contact with the aromatic ring of activators (B). Electrostatic attraction with the ester oxygen of activator can displace Lys (magenta sticks) of the DEKA locus (C). A quaternary ammonium group of the blocker pancuronium (D) is close to the focus of macrodipoles of the pore helices. The blocker lacks hydrophilic groups to form a hydrophilic pathway through the pore. (E) Cytoplasmic view at the VTD-bound channel. The space-filled inner and outer helices are blue and gray, respectively. Phe1750 is magenta and Asn784 is green. VTD easily fits the central pore, leaving some space for the permeation of Na+. (F) VTD in the closed KcsA-like conformation of the channel. Despite the weaker interaction energy, the activator could be trapped in the closed channel.
In the presence of activators, the Na+ channel permeates larger organic cations than the normal channel [22]. This suggests direct interaction between the activator and the channel selectivity filter. Elongated BTX and VTD have an aromatic ring
connected to the rigid polycyclic body via an ester group (Fig. 1). Since the aromatic ring is necessary for activity [23,24], we docked the activators with the initial orientation of the aromatic ring towards the selectivity filter.
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To find the energetically optimal binding mode of VTD, we first calculated the energy profile by translating the ligand along the pore and MC-minimizing energy at each step [25]. The energy profile (Fig. 2A) has a single minimum corresponding to VTD approaching the selectivity-filter region. In the next stage, the optimal orientation of VTD was found by rotating the ligand around its long axis and MC-minimizing energy at each step using the methodology described elsewhere [18] (data not shown). The inner pore of the Na+ channel is lined by predominantly hydrophobic residues. It also contains a ring formed by conserved hydrophilic residues Ser429, Asn784, and Ser1276 and a highly conserved Phe1750. The latter is a critical determinant of binding of activators and local anesthetics [26]. In the lowest-energy orientation, the hydrophobic surface of VTD binds to the hydrophobic side of the pore, which includes Phe1750 (Fig. 2B). The opposite nucleophilic side of VTD is oriented towards the conserved Asn784 in the inner helix of repeat II. The preferable binding mode of BTX is also stabilized by the hydrophobic interactions with Phe1750 (Fig. 2C). The low-energy complexes have several interesting features. In particular, activators approach the selectivity filter by their ester oxygens and aromatic rings (Fig. 2B and C), which are known as important determinants of the agonistÕs activity. In our models these moieties interact directly with the DEKA locus and residues in the preceding positions, especially with Phe1236 that forms a stacking motif with the aromatic rings of the activators. Most importantly, the open pore is not completely occluded by the bulky activators (Fig. 2E). The remaining lumen is lined on one side by the polar groups of the activators and on the other side it has the conserved Asn784. Such a pathway can be permeable for cations. The comparison of the 3D structures of other ligands shows that activators have multiple nucleophilic groups clustered at one face, whereas the other face is predominantly hydrophobic (Fig. 1). In contrast, blockers lack a continuous string of oxygen atoms. The importance of the nucleophilic groups and their clustering at one face of the molecule has been demonstrated in earlier structure– activity studies [27]. In particular, a model of the oxygen triad has been proposed to explain the action of activators [28]. In view of our hypothesis, the clustering of nucleophilic groups enables ions to permeate via the activator-bound channel. The blocker pancuronium also binds tightly to Phe1750 (Fig. 2E). The quaternary ammonium group of the blocker occurs at the focus of macrodipoles of the pore helices, the position where tetrabutylammonium binds in K+ channels [29]. Unlike activators, which expose their polar groups towards Asn784, the blocker exposes its hydrophobic groups towards this conserved residue. In the absence of the Na+ channel X-ray structure, the resolution of our model is obviously low. Details of the interactions that involve flexible residues of the channel and flexible fragments of the ligands cannot be predicted unambiguously. Therefore, subtype specificity of action of the ligands cannot be predicted now. However, the major results are determined by the well-resolved structure of the semirigid ligands and general architecture of the pore rather than by the atomic-level details. These results include: (i) possible binding of the activator inside the pore, (ii) its preferable orientation with hydrophilic groups exposed to the lumen, and (iii) existence of the hydrophilic pathway through the activator bound but not the blocker-bound channel.
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4. Ion permeation through ligand-bound channel The wide cytoplasmic vestibule of the open P-loop channel allows the free passage of hydrated ions, while the selectivity filter governs ion selectivity and permeability. In the agonistbound channel, the cytoplasmic vestibule is occluded by the ligand and the lumen becomes narrow. To reveal structural determinants of Na+ permeation through the narrow lumen of the ligand-bound channel, we calculated MC-minimized energy profiles for the ion pulled from the cytoplasm to the selectivity filter (Fig. 3). In these calculations, the ion was constrained to the plane normal to the pore axis, the plane was ˚ , and energy was MC-minimized translated with the step of 1 A at each step. Four parts of the ion trajectory in the VTDbound channel can be discriminated. A small energy barrier is seen at the initial part, where the ion enters the pore and loses its hydration shell. At the narrowest part of the lumen, the ion is stabilized by the attraction to the conserved Asn784. Closer to the P-loop, the ion occurs in the focus of macrodipoles of the pore helices. The deepest minimum is observed at the selectivity-filter region, where electrostatic interactions dominate. Noteworthy, VTD does not cause a significant energy barrier, since the unfavorable dehydration of the ion is compensated by the electrostatic attraction to the polar groups of the activator. The fact that VTD blocks rather than activates the Na+ channel mutant Asn784Cys [30] suggests the involvement of Asn784 in the ion permeation. In agreement with this experiment, the Na+ profile in the VTD-bound mutant has a higher energy barrier at the level of Cys784 (Fig. 3A). The Na+ profile through the pancuronium-bound channel (Fig. 3A) has a significant energy barrier caused by uncompensated dehydration of the ion and its repulsion from the blockerÕs ammonium group. Thus, our model shows a possibility of ion permeation via the VTD-bound Na+ channel. In contrast, binding of pancuronium blocks ion permeation. The ion permeation through the activator-bound channel is sensitive to the structural peculiarities of the hydrophilic pathway. This agrees with the data that mutations in the inner helix of repeat II affect the action of activators but do not affect the block by local anesthetics [2].
5. Selectivity of the activator-bound channel According to our hypothesis, activators bind inside the pore, at the turns of P-loops. Activators can directly interact with the selectivity filter and significantly affect spatial organization of the DEKA locus, the ring of highly conserved residues Asp, Glu, Lys, and Ala in repeats I–IV, respectively, which govern selective permeability of Na+ channels [31]. For example, carbonyl oxygen of BTX could interact with the amino group of Lys1237 (Fig. 2B) and repulse carboxylic groups of Asp and Glu, thus destabilizing the native structure of the DEKA locus. This would affect selectivity. Limited precision of the agonistbound models does not allow us to address this question systematically. Permeation of organic cations is also known to substantially depend on the selectivity-filter structure. However, the latter does not behave as a simple molecular sieve since, counterintuitively, the DEAA mutant of the DEKA locus permeates larger organic cations than the AAAA mutant [32]. Our recent model of the selectivity-filter region
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6. Gating of the activator-bound channel In our model, the activators extend from the selectivity filter to the intracellular parts of the inner helices. The latter region is significantly different between the open and closed K+ channels. Mutations in this region affect the Na+ channel gating [3]. To analyze the effect of VTD on Na+ channel gating, we simulated the closing of the VTD-bound channel using the approach described in [33]. Despite the presence of the bulky VTD, the Na+ channel was able to reach the closed KcsA-like conformation from the open state (Fig. 2F). However, the VTD-channel interaction energy increased from 28.0 kcal/mol in the open state to 16.8 kcal/mol in the closed state. The latter was destabilized by the repulsion between VTD and the gate region. Stabilization of the open Na+ channel by VTD explains why the activator-modified channels open more easily and stay open for a longer time than normal channels. This model is also consistent with the proposal that BTX reaches its receptor through the cytoplasmic end of the openchannel pore and can be trapped in the closed channel [34].
7. Conclusions
Fig. 3. Ion permeation via the ligand-bound Na+ channels. (A) Energy of Na+ in the ligand-bound channels. Each point corresponds to the MC-minimized structure calculated with Na+ constrained to a plane, which is normal to the pore axis. The abscissa shows distance of the Na+-constraining plane from the selectivity filter. The VTD-bound channel imposes only a small energy barrier for Na+ permeation, whereas a high barrier in the pancuronium-bound channel would obstruct the ion permeation. Mutation of Asn784 by Cys results in less favorable interactions of Na+ with the VTD-bound channel. (B and C) Side and cytoplasmic views of 25 superimposed MC-minimized complexes of Na+ with the VTD-bound Na+ channel. At different positions of Na+, the side chain of Phe1750, which strongly interacts with VTD, experiences only small fluctuations. In contrast, the side chain of Asn784 moves to preserve attractive contacts with Na+.
[18] has provided a structural explanation of this effect. Altered conformations of Lys1237 in the activator-bound channel (Fig. 2B) can cause permeation of larger ions, the effect similar to that observed in the DEAA mutant.
Our hypothesis provides the structural explanation for the key aspects of the action of Na+ channel activators. The activators fit in the open pore and interact with all four inner helices in agreement with mutational experiments. Ions can permeate through the activator-bound but not the blockerbound channel. The key determinant of this effect is the hydrophilic lining of the narrow pathway in the agonist-bound channel. Asn784 contributes importantly to this pathway. Obvious distinction of the pathways through the normal and activator-bound channels explains essentially different levels of conductance observed. The extreme case of the zero conductance corresponds to the classical block. Direct interaction of the activators with the selectivity filter could explain altered selectivity of the activator-modified channels. Binding of activators to the inner pore at the gate region stabilizes the open state of the channel. This results in the state-dependent action of the activators and causes easier and longer opening of the activator-modified channels. Predicted interactions between the activators and P-loops open a way for experimental testing of our hypothesis. Mutations of P-loop residues upstream from the DEKA locus should affect the action of activators. In particular, our models suggest that Phe1236 can significantly contribute to the binding of activators (Fig. 2B). Coordinates of the models are available upon request from BSZ. E-mail: [email protected]. Acknowledgments: We thank Boris Khodorov and Ging-Kuo Wang for helpful discussions and Iva Bruhova for reading the manuscript. This work was supported by the grant from the Canadian Institutes of Health Research (CIHR). B.S.Z. is a recipient of the CIHR Senior Scientist award. Computations were performed, in part, at the SHARCNET supercomputer center of McMaster University.
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