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Zipper transition in an alpha-helix: A mechanism for gating of voltage-sensitive ion channels in a biological membrane
J. theor. Biol. (1983) 104,137-158
Zipper Transition in an Alpha-Helix: A Mechanism for Gating of VoItage&nsitive Ion Channels in a Biological Membrane ROGER E. CLAPP Basic Research Associates Incorporated, 19 Copiey Street, Cambridge, Massachusetts 02138, U.S.A. (Received
16 December
1981, and in final form 7 April
1983)
It is suggested that the gating currents which control the ion channels in a biological membrane are comprised of positive charges crossing the membrane along chains of hydrogen bonds. These chains are the sets of hydrogen bonds which hold a-helical protein segments in their rigid conformations. The passage of a positive charge in one direction along such a chain will convert hydrogen bonds from the usual rigid N--C=O.. -H--N form to a flaccid N=C-0-H.. .N form. This “zipper” transition can be reversed by the passage of the positive charge along the return route. A flaccid protein rod can clog an ion channel and thereby close it. When all of the protein rods framing an ion channel are in the rigid conformation, the channel is open. This mechanism is used to explain some of the observed characteristics of calcium ion channels and sodium ion channels.
1. Introduction Proteins control ion channels in biological membranes. How is this control accomplished? The answer often proposed is: through conformational changes induced by voltage changes across the membrane. For example, Llin&, Steinberg & Walton (1976, 1981a,b) have studied the calcium currents in the squid giant synapse and have proposed a model (Fig. 1) in which there are five protein rods, all of which must straighten before the calcium channel which they frame can open to Ca*+ ions. This model is able to account for their measurements, provided that the straightening is a result of a voltage change across the membrane. But Llintis et al. (1976) do not propose a mechanism for this straightening in response to voltage. Protein rods in calcium channels are hypothetical, but protein rods in other membrane proteins are well established. The membrane protein in the photosensitive bacterium Halobacterium halobium is known to contain seven alpha-helical protein rods which span the membrane. This membrane protein has been fully sequenced (Ovchinnikov et al., 1979; Khorana et 0022-5193/83/170137+22$03.00/0
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FIG 1. Schematic representation of hypothetical Ca*+ channel. (a) Side-view showing longitudinal section of channel, which consists of five monomers extending the thickness of the membrane. On the left is a channel at rest with a charge distribution such that each monomer is positive at the inner, and negative at the outer, membrane surface (shown in left monomer). The middle section shows an intermediate state following a change in the electric field across the membrane (membrane polarization). The chargo redistribution in each monomer is assumed to produce a conformational change such that a “trap”, indicated by a deformity of the molecule that obstructs the channel, is removed. Extreme right portion of diagram shows an open channel when the conformational change of all five monomers is accomplished, allowing Ca2+ to how across the membrane. (b) Front view of channel from inward membrane surface. Reproduced by permission from Llinas ef al. (1976).
al., 1979) and its conformation is suggested in Fig. 2, which is based on X-ray measurements. The arrangement of protein rods in Fig. 2, which is taken from Henderson (1977), includes a group of five rods that could be imagined as framing an ion channel. However, an argument will be presented later that suggests that these five rods could not correspond to the five protein rods proposed by Llinas et al. (1976), though they might correspond to the elements needed in a model for sodium-ion channels, the m3h model of Hodgkin & Huxley (1952). Both the calcium-ion and the sodium-ion channels might be explained in terms of a protein transformation illustrated in Fig. 3. This transformation is between proteins which are stiffened alpha-helices and protein rods which have a more relaxed conformation. These latter flaccid rods will be denoted as “primed” cy-helices. The voltage-dependent transition will be called a “zipper” transition, for reasons that will emerge in the following discussion. 2. A Zipper Transition One of the forms that a protein chain can assume is the alpha-helix, described for example in Pauling (1960). This protein helix, which is
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FIG. 2. Drawing showing the arrangement of a-helices in one protein molecule of the purple membrane of Halobacterium halobium, derived from electron microscopy. (Reproduced, with permission, from R. Henderson (1977). Ann. Rev. Biophys. Bioeng. 6, 87. Copyright 1977 by Annual Reviews, Inc.)
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FIG. 3. Two protein rods forming part of the framing for a voltage-sensitive ion channel. (a) Both rods are in a flaccid primed-a-helix conformation, and the channel between them is closed. (b) Both rods are in the rigid a-helix conformation, and the channel between them is open.
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right-handed, contains cross-linking via hydrogen bonds, with the CO group of residue n hydrogen-bonded to the NH group of residue n +4. Figure 4 shows a ten-residue protein a-helix, seen from the outside. If this figure were rolled into a cylinder, with the right-hand ends of the second and third rows, respectively, connecting to the left-hand ends of the first and second rows, then the resulting representation would be very close to the actual alpha-helical protein as found in biological proteins.
FIG. 4. Structure of a ten-residue protein chain, in a right-handed a-helix. Successive turns of the helix are hydrogen-bonded, as shown. The cylindrical structure has been slit and flattened for display in the figure; the actual connections between chain segments are indicated by dashed bandings, connecting the left-hand ends of rows one and two to the right-hand ends of rows two and three, respectively. The R, are the protein side-groups.
From the measured bond lengths, as discussed by Lehninger (1975) and Stryer (1981), it is evident that the CN bond connecting a CO group to an NH group has a partially double-bond character. This can be interpreted as a resonance between the two configurations shown in Fig. 5. The second of these two has an evident affinity for the addition of a proton, to associate with the negatively-charged oxygen atom.
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FIG. 5. The peptide group. The coplanarity of the peptide linkage results from resonance between structures (a) and (b).
While the resonance places only a partial negative charge on the oxygen atom, if a proton were actually added it would draw the full negative charge to the oxygen, leaving a full positive charge on the nitrogen. There are three exposed carbonyl groups at the bottom of the helix in Fig. 4. Any or all of these might attach protons, under appropriate conditions. If we could somehow introduce three such protons, then the bottom row in Fig. 4 would take the altered form shown in Fig. 6, where the positively charged H’ ions can be considered to have bonded to the Oatoms in the ionized participants in the peptide resonances, O- atoms as shown in Fig. 5(b). Now let us consider the sequence shown in Fig. 7, which begins with a detail from Fig. 6 including the leftmost N’ ion in Fig. 6. In Fig. 7 the positive charge on this N’ ion is first transferred to the attached H atom, and the resulting proton (H+) proceeds to move upward along its hydrogen bond to the associated carbonyl group above. The positive charge next shifts to the oxygen atom, which becomes a trivalent O+, then moves on to the carbon atom which becomes a trivalent C+. At this point, Fig. 7(e), the positive charge moves sideways to the upper N atom, as shown in Fig. 7(f). It is now the upper nitrogen that has become a tetravalent N’. The bond configuration between R, and % has now become essentially the same as the earlier bond configuration between RIO and R9. Next we can consider’the positive charge on the N’ located between Rg and Rs in Fig. 6. This positive charge can also transfer to an HC, then to
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FIG. 6. The protein chain of Fig. 4, after the addition of three H+ ions to the three carbonyl groups nearest the carboxylic end of the protein. The three positive charges have migrated to the associated nitrogen atoms, which have accordingly become tetravalent N+ ions.
an 0’, a C’, and finally an N+, where this N’ is now located between Rg and R5. The upward stepping of positive charges can continue. Figure 8 shows the situation after the three positive charges have all stepped upward to the top row of residues. We can see that this stepping has rearranged double bonds. The middle row of Fig. 6 had double bonds in the CO groups, oriented along the axis of the a-helix, though Fig. 5 indicates that these are only partial double bonds. The middle row of Fig. 8, on the other hand, has full double bonds in the CN groupings, oriented transverse to the axis of the helix.
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(b)
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FIG. 7. Valence-bond representation of the steps by which a positive charge is transferred from the nitrogen atom nearest to R 1O,across a hydrogen bond to the nitrogen atom between Ra and R,. The positive charge first moves to the associated proton which carries it across the hydrogen bond. After this the positive charge moves to the carbonyl oxygen, then to the carbonyl carbon, and finally to the adjacent nitrogen atom, the nitrogen between & and R7.
The rearranged bonding has about the same total bond energy as the original bonding. In the new grouping, N=C-O-H, the bond energies are 147 + 84.0 + 110.6 = 341.6 kcal/mole, while the original grouping, H-N-C=O, would give bond energies of 93*4+69.7 + 174 = 337.1 k&/mole if the bonds were the simple single and double bonds indicated in the conventional formula. Actually, the resonance shown in Fig. 5 indicates that there is participation of the ionized grouping, H-N+=C-O-, in the original a-helical structure, which will increase the sum of bond energies beyond 337.1 kcal/mole, probably to a value which
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FIG. 8. The protein chain of Fig. 6, after the three positive charges at the lower end of this chain have migrated stepwise across hydrogen bonds until they have reached the nitrogen atoms at the top of the chain. The a-helix has thereby been transformed into an a”‘- helix.
exceeds 341.6 kcal/mole, making the original structure the more stable in the absence of a membrane voltage. The individual bond energies used above are taken from Pauling (1960), who does not give energy values for the bonds that enter into the peptide grouping. The above bond energies, in any case, can only be approximate, since they will be affected by electrostatic potentials and by steric strains introduced by the configuration of side chains in the actual membrane protein. Such strains could tip the balance toward a bonding which was flexible rather than rigid. (The small hydrogen-bond energies, omitted from the calculations, could also play a role.) Elmore (1968) has considered the barrier to rotation in the peptide bonding, and suggests that the energy involved might amount to perhaps 10 kcal/mole. Actually the barrier energy will be about twice the resonance
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energy, in the general case where the wave-function overlap is attractive for the most stable configuration, repulsive for the top of the barrier. If this is the case, then a barrier height of 10 kcal/mole translates to a resonance energy of about 5 kcal/mole. Adding this resonance energy to the sum of bond energies given earlier for the original a-helical structure, namely 337.1 kcal/mole, gives 342.1 kcal/mole as the energy to be associated with the resonating peptide structure of Fig. 5, the resonating combination of the bond groupings H-N-C=0 and H-N+=C-O-. This is very close to the estimate of 341.6 kcal/mole given above for the new bond grouping of N=C-O-H. From these estimates, it would appear that the new grouping is probably a little less stable than the original a-helical bond grouping. However, the introduction of the characteristic membrane voltage will help to draw electric charges across the membrane, perhaps making the new bonding become the resting state for many of the protein rods spanning the membrane, provided that there is a mobile positive charge available to carry out the requisite zipper transition that converts to the new bond grouping. It is evident that the transition from Fig. 6 to Fig. 8 has altered bonds, and has therefore altered certain of the bond lengths. This would in itself change the a-helix, but not by very much. What is more important is that the basis for the rigidity of the a-helix has been lost in this transition. The rigidity of an a-helix depends on the coplanarity of the HNCO grouping, as shown in the resonance of Fig. 5. The pi-electron delocalization enforces coplanarity over this grouping. It is this coplanarity, combined with the N--C=O.e.H-N hydrogen bonding, that gives the a-helix its stiffness. After the transition from Fig. 6 to Fig. 8, the resulting N=C-0--H...N bonding does not impose any particular directionality upon the molecular grouping that follows the second N atom. The transformed helix, while remaining hydrogen-bonded, will be loosened and somewhat disordered, perhaps like the flaccid rods shown in Fig. 3(a). In particular, if an ion channel was present in a membrane, framed by a set of rigid a-helices such as those in Fig. 2 and Fig. 3(b), then the transformation of some of these stiffened rods into flaccid helices could result in the clogging of this ion channel, particularly if some of the more bulky side chains were pushing the helix away from its rigid a-helical conformation. The transition is accomplished by the stepping of positive charges along the chains of hydrogen bonds. After the stepping transition, all of the hydrogen bonds in each of the three parallel chains have been altered from N-C=O.h.H-N bonds to N=C-0--H.--N bonds. This change of all bonds in a string, resulting from the passage of a single positive charge, resembles the operation of a zipper, except that the change is not between
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“open” and “closed” but between two differing forms of closure, one rigid and the other flaccid. The “zipper” transition is most clearly characterized when there are three positive charges which traverse the length of an a-helix, transforming this stiffened protein rod into a flaccid worm. Driving the three positive charges in the other direction should reverse the transition, stiffen the rod, and open the ion channel which it borders. However, if there is only one mobile positive charge available, rather than three, then there can still be a transition resulting from the travel of this positive charge along one of the three chains of hydrogen bonds in an a-helical protein rod. The alteration of one of the three parallel chains of
FIG. 9. The protein chain of Fig. 4, after the addition of a single proton to one of the lower carbonyl groups, followed by the migration of a single positive charge to the top tier, transforming the a-helix to an a’-helix.
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hydrogen bonds should not give a drastic change in conformation, but the change that it does give could nevertheless be very significant. Figure 9 shows the change in an a-helix that results when a single positive charge, introduced through the attachment at one end of a single proton, moves the length of the protein rod along one of the three chains of hydrogen bonds. It is evident that there is a change in the chemical bonding along this one chain of hydrogen bonds. Some distortion in the physical conformation of the protein rod can be expected to result from this chznge in bonding. The protein rod that results from this single-charge travel will be called an (Y’- helix. Figure 10 shows the corresponding change in an a-helix that results when two positive charges travel along two of the three parallel chains of hydrogen bonds. The loss of rigidity along these two chains, combined with the retention of rigidity along the third chain, could cause a twisting
FIG. 10. The protein chain of Fig. 4, after the addition of two protons, to two of the lower carbonyl groups, followed by the migration of two positive charges from the bottom tier to the top tier, transforming the a-helix to an a”- helix.
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distortion, perhaps a distortion similar to that depicted in Fig. 3(a). The protein rod that results when two positive charges have crossed over will be called an a”- helix. Correspondingly, when all three chains of hydrogen bonds have been primed through the traversal of one positive charge per chain, the flaccid protein rod that results, whose chemical structure is shown in Fig. 8, will be called an a”‘- helix. 3. Ion Channels The model for a calcium ion channel, pictured in Fig. 1, was based on experiments that pointed to the participation of five protein segments, but these experiments went further and also indicated that a single charge crossed the membrane when a single segment was straightened. If we are going to choose from among the primed helices in Figs 8-10, it is clear that we should choose the cu’-helix of Fig. 9 as the candidate structure for a protein segment that frames and clogs a calcium ion channel in the squid giant synapse. That is, each one of the protein segments pictured by Llinls et al. (1976) in their model (Fig. 1) may contain an a-helical structure with a mobile positive charge that can travel along a chain of hydrogen bonds from one side of the membrane to the other. One direction of travel converts the a-helix to an a’- helix which is somewhat distorted, just enough to block the calcium channel. The other direction of travel straightens the distorted (Y’- helix into a rigid a-helix and removes this blockage. When all five protein segments have had their (Y’- helices straightened into cu-helices, the channel opens and the calcium ions flow freely across. The model for a sodium ion channel, introduced by Hodgkin & Huxley (1952), is different in detail but not in character. In the sodium-ion-channel model, there are three segments that must all act together to open the channel, and a fourth segment that acts, somewhat more slowly, to close the channel shortly after it has been opened. All four are voltage-sensitive, as are the five segments in the model for the calcium ion channel. The Hodgkin-Huxley model is given the mathematical expression m3h, where m3 represents the three segments that participate in the opening of a sodium ion channel while h represents the segment that closes the channel. Measurements by Hodgkin & Huxley (1952) indicate that the fast-opening step involves the transfer of six electronic charges across the membrane. Later study of this “gating current” by Armstrong & Bezanilla (1973) has demonstrated that the charge movement carries positive charges from the inside of the membrane to the outside (or negative charges from the outside
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to the inside). The numerical estimate of six charges per opened channel is reinforced by this later work. If there are three protein segments (m 3, participating in the fast opening of a sodium ion channel, and six charges crossing the membrane to accomplish this channel opening, then it is difficult to avoid the conclusion that each segment passes through a transition in which two electronic charges travel a distance which carries them across the thickness of the membrane. In terms of the proposed zipper mechanism, the participating segments would seem to be those in which there are two mobile positive charges per protein segment, and the transition is therefore between a rigid a-helix and the distorted a”- helix of Fig. 10. The countering charge transfer which activates the h-factor and closes the channel would need to introduce a comparable distortion and would probably also involve a pair of charges, though the experiments are not clear on this point. The experiments do tell us that the deactivation is independent of the activation, that h is independent of m3. The pertinent experiments are those examining the effects of neurotoxins. Some of these are very specific in acting upon m3, while others act specifically upon h. Both sets of neurotoxins are discussed in some detail, later in this paper. The observations to date can be compared with the model proposed here, and shown in Fig. 11. It is suggested here that a sodium ion channel is framed by five protein rods, directed alternately across the membrane as in the purple membrane of Fig. 2. The directionality of the alternating proteins distinguishes the five-rod model of Fig. 11 from the five-rod calcium-channel model of Fig. 1. For the calcium channel in Fig. 1, the five protein rods framing the channel need to have the same directionality, while for the sodium channel in Fig. 11 the directionality alternates. In Fig. 11, the three rods labeled m are directed with amino ends facing the inside of the nerve membrane, carboxyl ends facing the outside. To be functional, each of these three m-rods should be carrying a pair of mobile positive charges. In the resting state, the three pairs of positive charges are at the inside (amino) ends of the m-rods and these rods are therefore in the form of flaccid a”- helices, as in Fig. 10. During an action potential, the six charges are driven outward across the membrane, stiffening the helices and opening the sodium-ion channel. The other two protein rods in Fig. 11, labeled g and h, are oriented in the opposite direction, with carboxyl ends on the inside of the membrane, amino ends on the outside. The resting conformation is the conventional a-helix, rigid rather than flaccid. However, at least one of the two, the one labeled h in Fig. 11, is presumed to carry two mobile positive charges which are poised at the carboxyl (inside) end of the protein rod, ready to
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1
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FIG. 11. A model for the sodium-ion channel, comprising five protein rods spanning the membrane.
move across to the amino (outside) end. When this movement takes place, transforming the a-helix to an a”-helix (as in Fig. lo), the h-rod becomes distorted and closes this sodium channel, countering the effect of the m3 transitions. In the proposed model, this h-rod transition provides the h-factor that enters into the m 3h formula of Hodgkin & Huxley (1952). The fifth protein rod in the diagram of Fig. 11 has been designated as a g-protein. It has the directionality of an h-protein, opposite to the m-directionality, but it has not been allocated any mobile positive charges in this proposed protein configuration. It is here pictured as a bystander. However, with mobile charges incorporated it could play the alternative role of an extra h-rod. 4. Induced Ion Channels Recent observations by Baud, Kado & Marcher (1982) provide evidence that sodium channels can be induced in membranes that do not ordinarily contain such channels. The membrane studied was that of the Xenopus faevis oiicyte. The sodium channels appeared after the membrane, with resting potential of about -60 millivolts, was depolarized and held at a
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positive potential of more than about +30 millivolts for a substantial time interval (several minutes). Once formed, these channels responded to voltage in the conventional way, except that there was no built-in mechanism for inactivation. There was an m3 process but no h process. These induced channels opened and closed with membrane potential, following m3 kinetics with a threshold of about -25 millivolts, selectively allowing Na’ ions to pass. The channels remained excitable for a limited time at resting potential, disappearing after about 10 minutes. They could be made to reappear by again depolarizing the membrane and holding it to a positive voltage for a sufficiently long time interval. In terms of the model proposed here for the functioning of voltagesensitive ion channels, the above observations would be explained in the following terms. The proteins in the oocyte membrane are initially in an appropriate configuration to frame sodium channels, but these rods lack the mobile positive charges necessary for the zipper transition. The prolonged reverse polarization to which the membrane proteins are subjected draws protons into the outer interstices of the protein chains and causes proton attachments to exposed carbonyl groups. The membrane is now excitable, with mobile positive charges along chains of hydrogen bonds in a-helices. Zipper transitions can take place under voltage control as long as enough positive charges remain bound to the helices and mobile. As the positive charges pause on protons, and the protons become dislodged and diffuse away, the membrane loses its excitability. Reversing the membrane polarization, once again, can restore the excitability by drawing in protons for reattachment. Since the experimental procedure does not draw protons to the inside surface of the membrane, the h-mechanism for channel inactivation is never given its needed mobile positive charges. 5. Protons and Neurotoxins The addition of protons to carbonyl groups, as in the change from Fig. 4 to Fig. 6, is one way to introduce mobile positive charges into the chains of hydrogen bonds which maintain the helical structure in a protein rod. This direct addition, it has been suggested here, may account for the observations of induced sodium channels reported by Baud et al. (1982). A more protected mechanism having much the same result could utilize sulfhydryl groups in amino acid residues located near the carboxyl ends of the helical rod segments. A study by Albuquerque et al. (1971) provides evidence that there are sulfhydryl groups in close association with the site of action of batrachotoxin (BTX), the potent neurotoxin obtained from the skin of the Colombian
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arrow poison frog Phyllobates aurotaenia. In measurements on the giant axon of the lobster (Homarus americanus) it was found that pretreatment by each of several chemicals, p-chloromercuribenzene sulfonic acid (PCMBS), N-ethylmaleimide (NEM), and dithiothreitol (DTI’), reduced the effects of BTX. Each of these chemicals reacts with sulfhydryl groups, with DTT acting to reduce disulfides. Batrachotoxin is understood to act from inside the nerve membrane to disable the h-process and prolong the sodium current, thereby depolarizing the nerve. BTX is also observed to shift the voltage dependence of the m activation and thereby to induce activation under conditions where there would ordinarily be little or nor activation (no action potentials). Veratridine, aconitine, and several grayanotoxins behave similarly to BTX, and compete for the same site of attachment (Catterall, 1979). Masutani et al. (1981) have examined these toxins and many molecular analogs to determine the location of the biological activity, and have concluded that in each case there is a triangular arrangement of three important reactive groups, two of them proton acceptors and the third a proton donor or acceptor; and that a methyl group nearby plays an important role in almost every case. Scorpion toxin similarly inhibits inactivation (blocks the h-process) but acts from outside the membrane and enhances the effects of BTX and the other toxins that act from inside the membrane, rather than competing with them. In the proposed model, the h-process involves a zipper transition in the protein rod labeled h in Fig. 11. The resting conformation is a rigid o-helix, but with two protons available at two of the three carbonyl groups shown exposed at the carboxyl end of the helix, at the bottom of Fig. 4. These two protons could be made available through the formation of a disulfide bond between two sulfhydryl groups near the carbonyls. The addition of the protons to the carbonyls frees positive charges to move along the chains of hydrogen bonds, as discussed in connection with Figs 6-10. When a molecule of an “inside” toxin approaches, the model suggests that the triangular arrangement of reactive oxygen groups clings by hydrogen bonding to the three exposed carbonyls and draws off the two protons, thereby removing the two mobile positive charges and disabling the zipper process so that the h-rod remains a rigid a-helix. Masutani et al. (1981) suggest that the nearby methyl is a hydrophobic anchoring group that nests in a pocket of the channel protein. In the model proposed here, an alternative possibility is suggested, in which the methyl group aids in holding the two positive charges, at least momentarily, in the configuration (C’H*)H’ with resonance among the three hydrogens.
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FIG. 12. The molecular structure of batrachotoxin (BTX). (Reprinted, with permission, from T. Narahashi, Modulation of nerve membrane sodium channels by neurotoxins, in Advances in Cytopharmacology, vol. 3, edited by B. Ceccarelli and F. Clementi. Copyright 1979 by Raven Press, New York.)
The molecular structure of BTX is shown in Fig. 12, taken from Narahashi (1979). The three reactive oxygen groups are the two OH groups at the left of the figure and the hemiketal oxygen nearby. While there is no obvious methyl group immediately adjacent, Masutani et al. (1981) suggest that a methyl on the pyrrole (at the right of the figure) might fold to be close to the reactive triangle. However, batrachotoxinin A, which does not contain the pyrrole-carboxylate moiety, remains biologically active though at a much reduced level. Measured in terms of the lethal dose, LDso intravenous in mice (Windholz, 1976), batrachotoxin itself is much the most powerful, at two micrograms per kilogram of body weight. Batrachotoxinin A, at 1 mg/kg, is 500 times less lethal, but nearly as powerful biologically as aconitine (0.166 mg/kg intravenous, 0.328 mg/kg intraperitoneal) and veratridine (1.35 mg/kg intraperitoneal). If the role of the methyl group, as suggested here, is to provide a momentary resting place for a pair of positive charges, then in BTX this role could be taken over by the seven-membered ring and its attached methyl, shown at the center of Fig. 12. There are many bondings that can be written down to locate two positive charges at various positions along this moiety. The resulting delocalization of the positive charges would then represent a substantial displacement of these two charges above the end of the h-rod. This displacement could account for the observed shift in the voltage dependence of the m3 activation in the presence of BTX. Furthermore, the substantial displacement of the pair of positive charges, with no
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easy route back to the end of the h-rod, would help to explain the observed irreversibility of the attachment of BTX to its binding site (19). The accessibility of this binding site would be reduced by the sulfhydryl reagents discussed earlier if these reagents blocked direct access to the three exposed carbonyls at the (inside) end of the h-rod. Thus the proposed model would account for many of the observed characteristics of the h-process, as modified by the sulfhydryl reagents and the “inside” neurotoxins such as BTX. It would be appropriate to use this model for the suggestions it contains as to experimental investigations. For example: does BTX bind to the end of a helical protein segment in the sodium channel protein ? Are there sulfhydryl groups nearby? More explicitly, does BTX bridge the h and g protein rods, as suggested in Fig. 13?
FIG. 13. Batrachotoxin bridging a sodium-ion channel.
Another major group of neurotoxins includes tetrodotoxin (TTX) and saxitoxin @TX). These are “outside” toxins that block the sodium-ion current by interfering with the m3-process. It is observed (Kao & Walker, 1982) that STX and TTX compete for the same sites. It is also observed (Catterall, 1979) that there are three saxitoxin sites for each scorpion toxin receptor site. If (in the proposed model) it is the outside end of the h-rod (see Fig. 11) that is the location of the scorpion toxin receptor site, then the threefold count of saxitoxin receptor sites suggests that each STX
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molecule can attach to one of the m-rods at its outside end. Since all three m-rods must stiffen (in the proposed model) for the sodium channel to open, it is evident that disabling any one of the three m-processes will keep the channel closed. The primary active region, in each of these toxins and their analogs, is a guanidinium moiety, a positively-charged group containing three nitrogens in a triangular configuration. Also important to the biological activity are oxygens elsewhere in the molecule, particularly certain OH groups. The triangle of nitrogens parallels the triangle of oxygens in BTX and the other “inside” neurotoxins. The proposed model suggests that this triangle of nitrogens, in STX or TTX, clings to the exposed ends of the three hydrogen-bond chains at the outside end of one of the m-rods, shown in Fig. 11. The clinging could be by hydrogen bonding to the exposed carbonyl groups, two of which will have been converted to COH groups by the earlier addition of protons. After the zipper transition, however, the two positive charges would have crossed to the inside of the membrane, as indicated in Fig. 10, leaving the NCOH groups at the outside end susceptible to the addition of a further proton at each nitrogen. The insertion of protons in this way could evidently interfere with the reverse zipper transition and prevent the flaccid m-rod from stiffening. In addition, the TTX or STX molecule, clinging to the outside end of an m-rod, could protrude into the channel aperture and interfere sterically with the passage of Na’ ions. Sodium-ion channels are further subject to channel block by protons, and there is evidence (Campbell, 1982) that the blockage can take place without the need for entry of the protons into the channel itself. The proposed model for sodium channels would explain proton block as the addition of protons to NCOH groups, as shown in Fig. 10, to produce NH’COH groupings that obstruct the reverse zipper transition. The hydrogen-bond chain, N=C-0--H...N=C-O-H, is disturbed by the insertion of a proton, giving N=C-0-H...NH’=C-O-H in which the protonated nitrogen no longer can accept easily another proton crossing the hydrogen bond from the next peptide group. There is a certain degree of symmetry suggested here in the action of protons and neurotoxins upon the sodium channels. The blockage of sodium currents is carried out through the insertion of protons into the outer ends of m-rods, where the exposed terminations of the hydrogen-bond chains are vulnerable. In particular, TTX’ and STX each contain a triangle of nitrogen atoms which can nest against these three terminations, carrying an easily donated proton as a part of the guanidinium moiety.
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Similarly, the inner end of the h-rod is vulnerable to the attachment of a triangle of oxygen atoms and to the transfer of protons and positive charges from the protein to such toxins as BTX. The charge transfer disables the h-process and prevents the sodium currents from turning off after an action potential. The model in Fig. 11 does not undertake to be a complete picture, but only to suggest mechanisms that could be playing a part in the functioning of the sodium-channel protein. This protein is about ten times as large as the seven-rod protein of Fig. 2, so that the five rods in Fig. 11 can be only a small fraction of the total protein. Even if the proposed mechanism is found to be present and to give a good explanation for the m3h formula of Hodgkin & Huxley (1952), there remains the need to account for the ion selectivity of this protein. The functioning of peptide toxins such as scorpion toxin will also depend strongly upon the form of the remaining protein accompanying the five rods of Fig. 11. . 6. Discussion Enough is known about voltage-dependent ion channels in biological membranes for us to say that the transfer of electrical charges across the membrane must be altering the conformation of certain proteins. It has been shown here that a mechanism for modifying an a-helical protein rod is theoretically available, in which the motion of one or more positive charges, along the chains of hydrogen bonds that are always present, will change the rod from rigid to flaccid, or from flaccid to rigid, depending on the direction of the charge motion with respect to the intrinsic directionality of the protein chain. Is this mechanism actually at work in excitable membranes? If it is, then it would provide an explicit basis for the calcium-channel model of Llinas et al. (1976) and the sodium-channel model of Hodgkin & Huxley (1952). It would also provide an explanation for the observations by Baud et al. (1982) of induced sodium channels. The proposed mechanism is subject to experimental checks. Since it depends on the presence of a-helical protein rods spanning the membrane, any demonstration that a channel protein has no a-helical segments would rule out this mechanism for that channel protein. Another kind of experiment would start with a membrane protein known to contain a-helical segments spanning the membrane, such as the purple mebrane protein of Halobacterium halobium, and undertake to induce ion channels in this protein through the procedure of Baud et al. (1982). It is hoped that this proposal of an explicit mechanism will stimulate experiments that can test for its realm of validity.
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Further theoretical work can also be carried out. In a channel protein as found in an axon membrane, how is a mobile positive charge inserted? Are there sulfhydryl groups nearby that can donate their protons to the carbonyl groups? Current work toward the detailed knowledge of channel proteins may answer this question. We can also note that the movement of positive charges along the chains of hydrogen bonds in an a-helix will involve a sideways stepping at each peptide group (see Fig. 7). This leads to a left-handed helical motion of each positive charge. This is a solenoidal current flow, as illustrated in Fig. 14 for a relatively long protein rod with a mobile positive charge on each of the three chains of hydrogen bonds. A solenoidal current flow will generate a magnetic field. Conversely, a changing magnetic field with its field lines threading a solenoidal current
5’ +& ; .?I, j : . \ :. I .’ !.’ : -\ : : \’ \..’ ‘, f : \ :.. I .’ 1.’ : ,\ j \’ ; 3, ,i : f \:’ \ ,..‘\ I -\
FIG. 14. The helical travel of three positive charges carrying out an a-a”’ zipper transition in a protein rod. The solenoidal trajectories of the three charges are indicated by dashes and dots, along the backbone within the protein rod. The dashes are on the side of the backbone nearer to the viewer, while the dots are on the farther side.
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path will generate an induced voltage, leading to an induced current flow if there are mobile charges available. A zipper transition can thus possibly involve a magnetic field. What experiments might disclose such an involvement? That a chain of hydrogen bonds can provide a path for a positive charge is a central concept of this paper. Part of the charge travel requires the movement of protons across the hydrogen bonds, and this changes the protein conformation. What results from this picture is a proposed answer to two related questions: (1) How do the ion channel gating currents cross from one side of the membrane to the other? (2) How do they change the protein conformation to open or close the channel? REFERENCES ALBUQUERQUE,
234,93.
E. X., SASA,
M., AVNER,
B. P. & DALY,
J. W. (1971).
Na!ure
New Biol.
ARMSTRONG, C. M. Kc BEZANILLA, F. (1973). Nature 242,459. BAUD, C., KADO, R. T. & MARCHER, K. (1982). Proc. natn. Acad. Sci. U.S.A. 79, 3188. CAMPBELL, D. T. (1982). Nature 298, 165. CATIERALL, W. A. (1979). In: Neurotoxins: Tools in Neurobiology (Ceccarelli, B. & Clementi, F. eds), pp. 305-316. Advances in Cytopharrnacology, Vol. 3. New York: Raven Press. ELMORE, D. T. (1968). Peptides and Proteins. Cambridge: Cambridge University Press. HENDERSON, R. (1977). Ann. Rev. biophys. Bioeng. 6,87. HODGKIN, A. L. & HUXLEY, A. F. (1952). J. Physiol. 117,500. KAO, C. Y.&WALKER, S. E. (1982). J. Physiol. 323,619. KHORANA, H. G., GERBER, G. E., HERLIHY, W. C., GRAY, C. P., ANDEREGG, R. J., NIHEI, K. & BIEMANN, K. (1979). Proc. natn. Acad. Sci. U.S.A. 76,5046. LEHNINGER, A. L. (1975). Biochemistry, 2nd edition. New York: Worth Publishers. LLINAS, R., STEINBERG, I. Z. & WALTON, K. (1976). Proc. natn. Acad. Sci. U.S.A. 73,2918. LLINAS, R., STEINBERG, I. Z. & WALTON, K. (1981a). Biophys. Z. 33,289. LLINAS, R., STEINBERG, I. Z. & WALTON, K. (19816). Biophys. J. 33,323. MASUTANI, T., SEYAMA, I., NARAHASHI, T. & IWASA, J. (1981). Z. Pharmac. exp. Therap.
217,812. NARAHASHI, T. (1974). Physiol. Rev. 54, 813. NARAHASHI, T. (1979). In: Neurotoxins: Tools in Neurobiology (Ceccarelli, B. & Clementi, F. eds), pp. 293-303. Advances in Cytopharmacology, Vol. 3. New York: Raven Press. OVCHINNIKOV, Yu. A., ABDULAEV, N. G., FEIGINA, M. Yu., KISELEV, A. V. & LOBANOV, N. A. (1979). FEBSLett. 100,219. PAULING, L. (1960). The Nature of the Chemical Bond, 3rd edition. Ithaca, New York: Cornell University Press. STRYER, L. (1981). Biochemistry, 2nd edition. San Francisco: W. H. Freeman and Company. WINDHOLZ, M., ed. (1976). The Merck Index, 9th edition. Rahway, New Jersey: Merck and Co., Inc.
Zipper Transition in an Alpha-Helix: A Mechanism for Gating of VoItage&nsitive Ion Channels in a Biological Membrane ROGER E. CLAPP Basic Research Associates Incorporated, 19 Copiey Street, Cambridge, Massachusetts 02138, U.S.A. (Received
16 December
1981, and in final form 7 April
1983)
It is suggested that the gating currents which control the ion channels in a biological membrane are comprised of positive charges crossing the membrane along chains of hydrogen bonds. These chains are the sets of hydrogen bonds which hold a-helical protein segments in their rigid conformations. The passage of a positive charge in one direction along such a chain will convert hydrogen bonds from the usual rigid N--C=O.. -H--N form to a flaccid N=C-0-H.. .N form. This “zipper” transition can be reversed by the passage of the positive charge along the return route. A flaccid protein rod can clog an ion channel and thereby close it. When all of the protein rods framing an ion channel are in the rigid conformation, the channel is open. This mechanism is used to explain some of the observed characteristics of calcium ion channels and sodium ion channels.
1. Introduction Proteins control ion channels in biological membranes. How is this control accomplished? The answer often proposed is: through conformational changes induced by voltage changes across the membrane. For example, Llin&, Steinberg & Walton (1976, 1981a,b) have studied the calcium currents in the squid giant synapse and have proposed a model (Fig. 1) in which there are five protein rods, all of which must straighten before the calcium channel which they frame can open to Ca*+ ions. This model is able to account for their measurements, provided that the straightening is a result of a voltage change across the membrane. But Llintis et al. (1976) do not propose a mechanism for this straightening in response to voltage. Protein rods in calcium channels are hypothetical, but protein rods in other membrane proteins are well established. The membrane protein in the photosensitive bacterium Halobacterium halobium is known to contain seven alpha-helical protein rods which span the membrane. This membrane protein has been fully sequenced (Ovchinnikov et al., 1979; Khorana et 0022-5193/83/170137+22$03.00/0
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FIG 1. Schematic representation of hypothetical Ca*+ channel. (a) Side-view showing longitudinal section of channel, which consists of five monomers extending the thickness of the membrane. On the left is a channel at rest with a charge distribution such that each monomer is positive at the inner, and negative at the outer, membrane surface (shown in left monomer). The middle section shows an intermediate state following a change in the electric field across the membrane (membrane polarization). The chargo redistribution in each monomer is assumed to produce a conformational change such that a “trap”, indicated by a deformity of the molecule that obstructs the channel, is removed. Extreme right portion of diagram shows an open channel when the conformational change of all five monomers is accomplished, allowing Ca2+ to how across the membrane. (b) Front view of channel from inward membrane surface. Reproduced by permission from Llinas ef al. (1976).
al., 1979) and its conformation is suggested in Fig. 2, which is based on X-ray measurements. The arrangement of protein rods in Fig. 2, which is taken from Henderson (1977), includes a group of five rods that could be imagined as framing an ion channel. However, an argument will be presented later that suggests that these five rods could not correspond to the five protein rods proposed by Llinas et al. (1976), though they might correspond to the elements needed in a model for sodium-ion channels, the m3h model of Hodgkin & Huxley (1952). Both the calcium-ion and the sodium-ion channels might be explained in terms of a protein transformation illustrated in Fig. 3. This transformation is between proteins which are stiffened alpha-helices and protein rods which have a more relaxed conformation. These latter flaccid rods will be denoted as “primed” cy-helices. The voltage-dependent transition will be called a “zipper” transition, for reasons that will emerge in the following discussion. 2. A Zipper Transition One of the forms that a protein chain can assume is the alpha-helix, described for example in Pauling (1960). This protein helix, which is
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FIG. 2. Drawing showing the arrangement of a-helices in one protein molecule of the purple membrane of Halobacterium halobium, derived from electron microscopy. (Reproduced, with permission, from R. Henderson (1977). Ann. Rev. Biophys. Bioeng. 6, 87. Copyright 1977 by Annual Reviews, Inc.)
(a)
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FIG. 3. Two protein rods forming part of the framing for a voltage-sensitive ion channel. (a) Both rods are in a flaccid primed-a-helix conformation, and the channel between them is closed. (b) Both rods are in the rigid a-helix conformation, and the channel between them is open.
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right-handed, contains cross-linking via hydrogen bonds, with the CO group of residue n hydrogen-bonded to the NH group of residue n +4. Figure 4 shows a ten-residue protein a-helix, seen from the outside. If this figure were rolled into a cylinder, with the right-hand ends of the second and third rows, respectively, connecting to the left-hand ends of the first and second rows, then the resulting representation would be very close to the actual alpha-helical protein as found in biological proteins.
FIG. 4. Structure of a ten-residue protein chain, in a right-handed a-helix. Successive turns of the helix are hydrogen-bonded, as shown. The cylindrical structure has been slit and flattened for display in the figure; the actual connections between chain segments are indicated by dashed bandings, connecting the left-hand ends of rows one and two to the right-hand ends of rows two and three, respectively. The R, are the protein side-groups.
From the measured bond lengths, as discussed by Lehninger (1975) and Stryer (1981), it is evident that the CN bond connecting a CO group to an NH group has a partially double-bond character. This can be interpreted as a resonance between the two configurations shown in Fig. 5. The second of these two has an evident affinity for the addition of a proton, to associate with the negatively-charged oxygen atom.
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FIG. 5. The peptide group. The coplanarity of the peptide linkage results from resonance between structures (a) and (b).
While the resonance places only a partial negative charge on the oxygen atom, if a proton were actually added it would draw the full negative charge to the oxygen, leaving a full positive charge on the nitrogen. There are three exposed carbonyl groups at the bottom of the helix in Fig. 4. Any or all of these might attach protons, under appropriate conditions. If we could somehow introduce three such protons, then the bottom row in Fig. 4 would take the altered form shown in Fig. 6, where the positively charged H’ ions can be considered to have bonded to the Oatoms in the ionized participants in the peptide resonances, O- atoms as shown in Fig. 5(b). Now let us consider the sequence shown in Fig. 7, which begins with a detail from Fig. 6 including the leftmost N’ ion in Fig. 6. In Fig. 7 the positive charge on this N’ ion is first transferred to the attached H atom, and the resulting proton (H+) proceeds to move upward along its hydrogen bond to the associated carbonyl group above. The positive charge next shifts to the oxygen atom, which becomes a trivalent O+, then moves on to the carbon atom which becomes a trivalent C+. At this point, Fig. 7(e), the positive charge moves sideways to the upper N atom, as shown in Fig. 7(f). It is now the upper nitrogen that has become a tetravalent N’. The bond configuration between R, and % has now become essentially the same as the earlier bond configuration between RIO and R9. Next we can consider’the positive charge on the N’ located between Rg and Rs in Fig. 6. This positive charge can also transfer to an HC, then to
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L
FIG. 6. The protein chain of Fig. 4, after the addition of three H+ ions to the three carbonyl groups nearest the carboxylic end of the protein. The three positive charges have migrated to the associated nitrogen atoms, which have accordingly become tetravalent N+ ions.
an 0’, a C’, and finally an N+, where this N’ is now located between Rg and R5. The upward stepping of positive charges can continue. Figure 8 shows the situation after the three positive charges have all stepped upward to the top row of residues. We can see that this stepping has rearranged double bonds. The middle row of Fig. 6 had double bonds in the CO groups, oriented along the axis of the a-helix, though Fig. 5 indicates that these are only partial double bonds. The middle row of Fig. 8, on the other hand, has full double bonds in the CN groupings, oriented transverse to the axis of the helix.
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(b)
(dl
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FIG. 7. Valence-bond representation of the steps by which a positive charge is transferred from the nitrogen atom nearest to R 1O,across a hydrogen bond to the nitrogen atom between Ra and R,. The positive charge first moves to the associated proton which carries it across the hydrogen bond. After this the positive charge moves to the carbonyl oxygen, then to the carbonyl carbon, and finally to the adjacent nitrogen atom, the nitrogen between & and R7.
The rearranged bonding has about the same total bond energy as the original bonding. In the new grouping, N=C-O-H, the bond energies are 147 + 84.0 + 110.6 = 341.6 kcal/mole, while the original grouping, H-N-C=O, would give bond energies of 93*4+69.7 + 174 = 337.1 k&/mole if the bonds were the simple single and double bonds indicated in the conventional formula. Actually, the resonance shown in Fig. 5 indicates that there is participation of the ionized grouping, H-N+=C-O-, in the original a-helical structure, which will increase the sum of bond energies beyond 337.1 kcal/mole, probably to a value which
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FIG. 8. The protein chain of Fig. 6, after the three positive charges at the lower end of this chain have migrated stepwise across hydrogen bonds until they have reached the nitrogen atoms at the top of the chain. The a-helix has thereby been transformed into an a”‘- helix.
exceeds 341.6 kcal/mole, making the original structure the more stable in the absence of a membrane voltage. The individual bond energies used above are taken from Pauling (1960), who does not give energy values for the bonds that enter into the peptide grouping. The above bond energies, in any case, can only be approximate, since they will be affected by electrostatic potentials and by steric strains introduced by the configuration of side chains in the actual membrane protein. Such strains could tip the balance toward a bonding which was flexible rather than rigid. (The small hydrogen-bond energies, omitted from the calculations, could also play a role.) Elmore (1968) has considered the barrier to rotation in the peptide bonding, and suggests that the energy involved might amount to perhaps 10 kcal/mole. Actually the barrier energy will be about twice the resonance
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energy, in the general case where the wave-function overlap is attractive for the most stable configuration, repulsive for the top of the barrier. If this is the case, then a barrier height of 10 kcal/mole translates to a resonance energy of about 5 kcal/mole. Adding this resonance energy to the sum of bond energies given earlier for the original a-helical structure, namely 337.1 kcal/mole, gives 342.1 kcal/mole as the energy to be associated with the resonating peptide structure of Fig. 5, the resonating combination of the bond groupings H-N-C=0 and H-N+=C-O-. This is very close to the estimate of 341.6 kcal/mole given above for the new bond grouping of N=C-O-H. From these estimates, it would appear that the new grouping is probably a little less stable than the original a-helical bond grouping. However, the introduction of the characteristic membrane voltage will help to draw electric charges across the membrane, perhaps making the new bonding become the resting state for many of the protein rods spanning the membrane, provided that there is a mobile positive charge available to carry out the requisite zipper transition that converts to the new bond grouping. It is evident that the transition from Fig. 6 to Fig. 8 has altered bonds, and has therefore altered certain of the bond lengths. This would in itself change the a-helix, but not by very much. What is more important is that the basis for the rigidity of the a-helix has been lost in this transition. The rigidity of an a-helix depends on the coplanarity of the HNCO grouping, as shown in the resonance of Fig. 5. The pi-electron delocalization enforces coplanarity over this grouping. It is this coplanarity, combined with the N--C=O.e.H-N hydrogen bonding, that gives the a-helix its stiffness. After the transition from Fig. 6 to Fig. 8, the resulting N=C-0--H...N bonding does not impose any particular directionality upon the molecular grouping that follows the second N atom. The transformed helix, while remaining hydrogen-bonded, will be loosened and somewhat disordered, perhaps like the flaccid rods shown in Fig. 3(a). In particular, if an ion channel was present in a membrane, framed by a set of rigid a-helices such as those in Fig. 2 and Fig. 3(b), then the transformation of some of these stiffened rods into flaccid helices could result in the clogging of this ion channel, particularly if some of the more bulky side chains were pushing the helix away from its rigid a-helical conformation. The transition is accomplished by the stepping of positive charges along the chains of hydrogen bonds. After the stepping transition, all of the hydrogen bonds in each of the three parallel chains have been altered from N-C=O.h.H-N bonds to N=C-0--H.--N bonds. This change of all bonds in a string, resulting from the passage of a single positive charge, resembles the operation of a zipper, except that the change is not between
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“open” and “closed” but between two differing forms of closure, one rigid and the other flaccid. The “zipper” transition is most clearly characterized when there are three positive charges which traverse the length of an a-helix, transforming this stiffened protein rod into a flaccid worm. Driving the three positive charges in the other direction should reverse the transition, stiffen the rod, and open the ion channel which it borders. However, if there is only one mobile positive charge available, rather than three, then there can still be a transition resulting from the travel of this positive charge along one of the three chains of hydrogen bonds in an a-helical protein rod. The alteration of one of the three parallel chains of
FIG. 9. The protein chain of Fig. 4, after the addition of a single proton to one of the lower carbonyl groups, followed by the migration of a single positive charge to the top tier, transforming the a-helix to an a’-helix.
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hydrogen bonds should not give a drastic change in conformation, but the change that it does give could nevertheless be very significant. Figure 9 shows the change in an a-helix that results when a single positive charge, introduced through the attachment at one end of a single proton, moves the length of the protein rod along one of the three chains of hydrogen bonds. It is evident that there is a change in the chemical bonding along this one chain of hydrogen bonds. Some distortion in the physical conformation of the protein rod can be expected to result from this chznge in bonding. The protein rod that results from this single-charge travel will be called an (Y’- helix. Figure 10 shows the corresponding change in an a-helix that results when two positive charges travel along two of the three parallel chains of hydrogen bonds. The loss of rigidity along these two chains, combined with the retention of rigidity along the third chain, could cause a twisting
FIG. 10. The protein chain of Fig. 4, after the addition of two protons, to two of the lower carbonyl groups, followed by the migration of two positive charges from the bottom tier to the top tier, transforming the a-helix to an a”- helix.
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distortion, perhaps a distortion similar to that depicted in Fig. 3(a). The protein rod that results when two positive charges have crossed over will be called an a”- helix. Correspondingly, when all three chains of hydrogen bonds have been primed through the traversal of one positive charge per chain, the flaccid protein rod that results, whose chemical structure is shown in Fig. 8, will be called an a”‘- helix. 3. Ion Channels The model for a calcium ion channel, pictured in Fig. 1, was based on experiments that pointed to the participation of five protein segments, but these experiments went further and also indicated that a single charge crossed the membrane when a single segment was straightened. If we are going to choose from among the primed helices in Figs 8-10, it is clear that we should choose the cu’-helix of Fig. 9 as the candidate structure for a protein segment that frames and clogs a calcium ion channel in the squid giant synapse. That is, each one of the protein segments pictured by Llinls et al. (1976) in their model (Fig. 1) may contain an a-helical structure with a mobile positive charge that can travel along a chain of hydrogen bonds from one side of the membrane to the other. One direction of travel converts the a-helix to an a’- helix which is somewhat distorted, just enough to block the calcium channel. The other direction of travel straightens the distorted (Y’- helix into a rigid a-helix and removes this blockage. When all five protein segments have had their (Y’- helices straightened into cu-helices, the channel opens and the calcium ions flow freely across. The model for a sodium ion channel, introduced by Hodgkin & Huxley (1952), is different in detail but not in character. In the sodium-ion-channel model, there are three segments that must all act together to open the channel, and a fourth segment that acts, somewhat more slowly, to close the channel shortly after it has been opened. All four are voltage-sensitive, as are the five segments in the model for the calcium ion channel. The Hodgkin-Huxley model is given the mathematical expression m3h, where m3 represents the three segments that participate in the opening of a sodium ion channel while h represents the segment that closes the channel. Measurements by Hodgkin & Huxley (1952) indicate that the fast-opening step involves the transfer of six electronic charges across the membrane. Later study of this “gating current” by Armstrong & Bezanilla (1973) has demonstrated that the charge movement carries positive charges from the inside of the membrane to the outside (or negative charges from the outside
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to the inside). The numerical estimate of six charges per opened channel is reinforced by this later work. If there are three protein segments (m 3, participating in the fast opening of a sodium ion channel, and six charges crossing the membrane to accomplish this channel opening, then it is difficult to avoid the conclusion that each segment passes through a transition in which two electronic charges travel a distance which carries them across the thickness of the membrane. In terms of the proposed zipper mechanism, the participating segments would seem to be those in which there are two mobile positive charges per protein segment, and the transition is therefore between a rigid a-helix and the distorted a”- helix of Fig. 10. The countering charge transfer which activates the h-factor and closes the channel would need to introduce a comparable distortion and would probably also involve a pair of charges, though the experiments are not clear on this point. The experiments do tell us that the deactivation is independent of the activation, that h is independent of m3. The pertinent experiments are those examining the effects of neurotoxins. Some of these are very specific in acting upon m3, while others act specifically upon h. Both sets of neurotoxins are discussed in some detail, later in this paper. The observations to date can be compared with the model proposed here, and shown in Fig. 11. It is suggested here that a sodium ion channel is framed by five protein rods, directed alternately across the membrane as in the purple membrane of Fig. 2. The directionality of the alternating proteins distinguishes the five-rod model of Fig. 11 from the five-rod calcium-channel model of Fig. 1. For the calcium channel in Fig. 1, the five protein rods framing the channel need to have the same directionality, while for the sodium channel in Fig. 11 the directionality alternates. In Fig. 11, the three rods labeled m are directed with amino ends facing the inside of the nerve membrane, carboxyl ends facing the outside. To be functional, each of these three m-rods should be carrying a pair of mobile positive charges. In the resting state, the three pairs of positive charges are at the inside (amino) ends of the m-rods and these rods are therefore in the form of flaccid a”- helices, as in Fig. 10. During an action potential, the six charges are driven outward across the membrane, stiffening the helices and opening the sodium-ion channel. The other two protein rods in Fig. 11, labeled g and h, are oriented in the opposite direction, with carboxyl ends on the inside of the membrane, amino ends on the outside. The resting conformation is the conventional a-helix, rigid rather than flaccid. However, at least one of the two, the one labeled h in Fig. 11, is presumed to carry two mobile positive charges which are poised at the carboxyl (inside) end of the protein rod, ready to
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1
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FIG. 11. A model for the sodium-ion channel, comprising five protein rods spanning the membrane.
move across to the amino (outside) end. When this movement takes place, transforming the a-helix to an a”-helix (as in Fig. lo), the h-rod becomes distorted and closes this sodium channel, countering the effect of the m3 transitions. In the proposed model, this h-rod transition provides the h-factor that enters into the m 3h formula of Hodgkin & Huxley (1952). The fifth protein rod in the diagram of Fig. 11 has been designated as a g-protein. It has the directionality of an h-protein, opposite to the m-directionality, but it has not been allocated any mobile positive charges in this proposed protein configuration. It is here pictured as a bystander. However, with mobile charges incorporated it could play the alternative role of an extra h-rod. 4. Induced Ion Channels Recent observations by Baud, Kado & Marcher (1982) provide evidence that sodium channels can be induced in membranes that do not ordinarily contain such channels. The membrane studied was that of the Xenopus faevis oiicyte. The sodium channels appeared after the membrane, with resting potential of about -60 millivolts, was depolarized and held at a
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positive potential of more than about +30 millivolts for a substantial time interval (several minutes). Once formed, these channels responded to voltage in the conventional way, except that there was no built-in mechanism for inactivation. There was an m3 process but no h process. These induced channels opened and closed with membrane potential, following m3 kinetics with a threshold of about -25 millivolts, selectively allowing Na’ ions to pass. The channels remained excitable for a limited time at resting potential, disappearing after about 10 minutes. They could be made to reappear by again depolarizing the membrane and holding it to a positive voltage for a sufficiently long time interval. In terms of the model proposed here for the functioning of voltagesensitive ion channels, the above observations would be explained in the following terms. The proteins in the oocyte membrane are initially in an appropriate configuration to frame sodium channels, but these rods lack the mobile positive charges necessary for the zipper transition. The prolonged reverse polarization to which the membrane proteins are subjected draws protons into the outer interstices of the protein chains and causes proton attachments to exposed carbonyl groups. The membrane is now excitable, with mobile positive charges along chains of hydrogen bonds in a-helices. Zipper transitions can take place under voltage control as long as enough positive charges remain bound to the helices and mobile. As the positive charges pause on protons, and the protons become dislodged and diffuse away, the membrane loses its excitability. Reversing the membrane polarization, once again, can restore the excitability by drawing in protons for reattachment. Since the experimental procedure does not draw protons to the inside surface of the membrane, the h-mechanism for channel inactivation is never given its needed mobile positive charges. 5. Protons and Neurotoxins The addition of protons to carbonyl groups, as in the change from Fig. 4 to Fig. 6, is one way to introduce mobile positive charges into the chains of hydrogen bonds which maintain the helical structure in a protein rod. This direct addition, it has been suggested here, may account for the observations of induced sodium channels reported by Baud et al. (1982). A more protected mechanism having much the same result could utilize sulfhydryl groups in amino acid residues located near the carboxyl ends of the helical rod segments. A study by Albuquerque et al. (1971) provides evidence that there are sulfhydryl groups in close association with the site of action of batrachotoxin (BTX), the potent neurotoxin obtained from the skin of the Colombian
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arrow poison frog Phyllobates aurotaenia. In measurements on the giant axon of the lobster (Homarus americanus) it was found that pretreatment by each of several chemicals, p-chloromercuribenzene sulfonic acid (PCMBS), N-ethylmaleimide (NEM), and dithiothreitol (DTI’), reduced the effects of BTX. Each of these chemicals reacts with sulfhydryl groups, with DTT acting to reduce disulfides. Batrachotoxin is understood to act from inside the nerve membrane to disable the h-process and prolong the sodium current, thereby depolarizing the nerve. BTX is also observed to shift the voltage dependence of the m activation and thereby to induce activation under conditions where there would ordinarily be little or nor activation (no action potentials). Veratridine, aconitine, and several grayanotoxins behave similarly to BTX, and compete for the same site of attachment (Catterall, 1979). Masutani et al. (1981) have examined these toxins and many molecular analogs to determine the location of the biological activity, and have concluded that in each case there is a triangular arrangement of three important reactive groups, two of them proton acceptors and the third a proton donor or acceptor; and that a methyl group nearby plays an important role in almost every case. Scorpion toxin similarly inhibits inactivation (blocks the h-process) but acts from outside the membrane and enhances the effects of BTX and the other toxins that act from inside the membrane, rather than competing with them. In the proposed model, the h-process involves a zipper transition in the protein rod labeled h in Fig. 11. The resting conformation is a rigid o-helix, but with two protons available at two of the three carbonyl groups shown exposed at the carboxyl end of the helix, at the bottom of Fig. 4. These two protons could be made available through the formation of a disulfide bond between two sulfhydryl groups near the carbonyls. The addition of the protons to the carbonyls frees positive charges to move along the chains of hydrogen bonds, as discussed in connection with Figs 6-10. When a molecule of an “inside” toxin approaches, the model suggests that the triangular arrangement of reactive oxygen groups clings by hydrogen bonding to the three exposed carbonyls and draws off the two protons, thereby removing the two mobile positive charges and disabling the zipper process so that the h-rod remains a rigid a-helix. Masutani et al. (1981) suggest that the nearby methyl is a hydrophobic anchoring group that nests in a pocket of the channel protein. In the model proposed here, an alternative possibility is suggested, in which the methyl group aids in holding the two positive charges, at least momentarily, in the configuration (C’H*)H’ with resonance among the three hydrogens.
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FIG. 12. The molecular structure of batrachotoxin (BTX). (Reprinted, with permission, from T. Narahashi, Modulation of nerve membrane sodium channels by neurotoxins, in Advances in Cytopharmacology, vol. 3, edited by B. Ceccarelli and F. Clementi. Copyright 1979 by Raven Press, New York.)
The molecular structure of BTX is shown in Fig. 12, taken from Narahashi (1979). The three reactive oxygen groups are the two OH groups at the left of the figure and the hemiketal oxygen nearby. While there is no obvious methyl group immediately adjacent, Masutani et al. (1981) suggest that a methyl on the pyrrole (at the right of the figure) might fold to be close to the reactive triangle. However, batrachotoxinin A, which does not contain the pyrrole-carboxylate moiety, remains biologically active though at a much reduced level. Measured in terms of the lethal dose, LDso intravenous in mice (Windholz, 1976), batrachotoxin itself is much the most powerful, at two micrograms per kilogram of body weight. Batrachotoxinin A, at 1 mg/kg, is 500 times less lethal, but nearly as powerful biologically as aconitine (0.166 mg/kg intravenous, 0.328 mg/kg intraperitoneal) and veratridine (1.35 mg/kg intraperitoneal). If the role of the methyl group, as suggested here, is to provide a momentary resting place for a pair of positive charges, then in BTX this role could be taken over by the seven-membered ring and its attached methyl, shown at the center of Fig. 12. There are many bondings that can be written down to locate two positive charges at various positions along this moiety. The resulting delocalization of the positive charges would then represent a substantial displacement of these two charges above the end of the h-rod. This displacement could account for the observed shift in the voltage dependence of the m3 activation in the presence of BTX. Furthermore, the substantial displacement of the pair of positive charges, with no
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easy route back to the end of the h-rod, would help to explain the observed irreversibility of the attachment of BTX to its binding site (19). The accessibility of this binding site would be reduced by the sulfhydryl reagents discussed earlier if these reagents blocked direct access to the three exposed carbonyls at the (inside) end of the h-rod. Thus the proposed model would account for many of the observed characteristics of the h-process, as modified by the sulfhydryl reagents and the “inside” neurotoxins such as BTX. It would be appropriate to use this model for the suggestions it contains as to experimental investigations. For example: does BTX bind to the end of a helical protein segment in the sodium channel protein ? Are there sulfhydryl groups nearby? More explicitly, does BTX bridge the h and g protein rods, as suggested in Fig. 13?
FIG. 13. Batrachotoxin bridging a sodium-ion channel.
Another major group of neurotoxins includes tetrodotoxin (TTX) and saxitoxin @TX). These are “outside” toxins that block the sodium-ion current by interfering with the m3-process. It is observed (Kao & Walker, 1982) that STX and TTX compete for the same sites. It is also observed (Catterall, 1979) that there are three saxitoxin sites for each scorpion toxin receptor site. If (in the proposed model) it is the outside end of the h-rod (see Fig. 11) that is the location of the scorpion toxin receptor site, then the threefold count of saxitoxin receptor sites suggests that each STX
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molecule can attach to one of the m-rods at its outside end. Since all three m-rods must stiffen (in the proposed model) for the sodium channel to open, it is evident that disabling any one of the three m-processes will keep the channel closed. The primary active region, in each of these toxins and their analogs, is a guanidinium moiety, a positively-charged group containing three nitrogens in a triangular configuration. Also important to the biological activity are oxygens elsewhere in the molecule, particularly certain OH groups. The triangle of nitrogens parallels the triangle of oxygens in BTX and the other “inside” neurotoxins. The proposed model suggests that this triangle of nitrogens, in STX or TTX, clings to the exposed ends of the three hydrogen-bond chains at the outside end of one of the m-rods, shown in Fig. 11. The clinging could be by hydrogen bonding to the exposed carbonyl groups, two of which will have been converted to COH groups by the earlier addition of protons. After the zipper transition, however, the two positive charges would have crossed to the inside of the membrane, as indicated in Fig. 10, leaving the NCOH groups at the outside end susceptible to the addition of a further proton at each nitrogen. The insertion of protons in this way could evidently interfere with the reverse zipper transition and prevent the flaccid m-rod from stiffening. In addition, the TTX or STX molecule, clinging to the outside end of an m-rod, could protrude into the channel aperture and interfere sterically with the passage of Na’ ions. Sodium-ion channels are further subject to channel block by protons, and there is evidence (Campbell, 1982) that the blockage can take place without the need for entry of the protons into the channel itself. The proposed model for sodium channels would explain proton block as the addition of protons to NCOH groups, as shown in Fig. 10, to produce NH’COH groupings that obstruct the reverse zipper transition. The hydrogen-bond chain, N=C-0--H...N=C-O-H, is disturbed by the insertion of a proton, giving N=C-0-H...NH’=C-O-H in which the protonated nitrogen no longer can accept easily another proton crossing the hydrogen bond from the next peptide group. There is a certain degree of symmetry suggested here in the action of protons and neurotoxins upon the sodium channels. The blockage of sodium currents is carried out through the insertion of protons into the outer ends of m-rods, where the exposed terminations of the hydrogen-bond chains are vulnerable. In particular, TTX’ and STX each contain a triangle of nitrogen atoms which can nest against these three terminations, carrying an easily donated proton as a part of the guanidinium moiety.
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Similarly, the inner end of the h-rod is vulnerable to the attachment of a triangle of oxygen atoms and to the transfer of protons and positive charges from the protein to such toxins as BTX. The charge transfer disables the h-process and prevents the sodium currents from turning off after an action potential. The model in Fig. 11 does not undertake to be a complete picture, but only to suggest mechanisms that could be playing a part in the functioning of the sodium-channel protein. This protein is about ten times as large as the seven-rod protein of Fig. 2, so that the five rods in Fig. 11 can be only a small fraction of the total protein. Even if the proposed mechanism is found to be present and to give a good explanation for the m3h formula of Hodgkin & Huxley (1952), there remains the need to account for the ion selectivity of this protein. The functioning of peptide toxins such as scorpion toxin will also depend strongly upon the form of the remaining protein accompanying the five rods of Fig. 11. . 6. Discussion Enough is known about voltage-dependent ion channels in biological membranes for us to say that the transfer of electrical charges across the membrane must be altering the conformation of certain proteins. It has been shown here that a mechanism for modifying an a-helical protein rod is theoretically available, in which the motion of one or more positive charges, along the chains of hydrogen bonds that are always present, will change the rod from rigid to flaccid, or from flaccid to rigid, depending on the direction of the charge motion with respect to the intrinsic directionality of the protein chain. Is this mechanism actually at work in excitable membranes? If it is, then it would provide an explicit basis for the calcium-channel model of Llinas et al. (1976) and the sodium-channel model of Hodgkin & Huxley (1952). It would also provide an explanation for the observations by Baud et al. (1982) of induced sodium channels. The proposed mechanism is subject to experimental checks. Since it depends on the presence of a-helical protein rods spanning the membrane, any demonstration that a channel protein has no a-helical segments would rule out this mechanism for that channel protein. Another kind of experiment would start with a membrane protein known to contain a-helical segments spanning the membrane, such as the purple mebrane protein of Halobacterium halobium, and undertake to induce ion channels in this protein through the procedure of Baud et al. (1982). It is hoped that this proposal of an explicit mechanism will stimulate experiments that can test for its realm of validity.
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Further theoretical work can also be carried out. In a channel protein as found in an axon membrane, how is a mobile positive charge inserted? Are there sulfhydryl groups nearby that can donate their protons to the carbonyl groups? Current work toward the detailed knowledge of channel proteins may answer this question. We can also note that the movement of positive charges along the chains of hydrogen bonds in an a-helix will involve a sideways stepping at each peptide group (see Fig. 7). This leads to a left-handed helical motion of each positive charge. This is a solenoidal current flow, as illustrated in Fig. 14 for a relatively long protein rod with a mobile positive charge on each of the three chains of hydrogen bonds. A solenoidal current flow will generate a magnetic field. Conversely, a changing magnetic field with its field lines threading a solenoidal current
5’ +& ; .?I, j : . \ :. I .’ !.’ : -\ : : \’ \..’ ‘, f : \ :.. I .’ 1.’ : ,\ j \’ ; 3, ,i : f \:’ \ ,..‘\ I -\
FIG. 14. The helical travel of three positive charges carrying out an a-a”’ zipper transition in a protein rod. The solenoidal trajectories of the three charges are indicated by dashes and dots, along the backbone within the protein rod. The dashes are on the side of the backbone nearer to the viewer, while the dots are on the farther side.
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path will generate an induced voltage, leading to an induced current flow if there are mobile charges available. A zipper transition can thus possibly involve a magnetic field. What experiments might disclose such an involvement? That a chain of hydrogen bonds can provide a path for a positive charge is a central concept of this paper. Part of the charge travel requires the movement of protons across the hydrogen bonds, and this changes the protein conformation. What results from this picture is a proposed answer to two related questions: (1) How do the ion channel gating currents cross from one side of the membrane to the other? (2) How do they change the protein conformation to open or close the channel? REFERENCES ALBUQUERQUE,
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217,812. NARAHASHI, T. (1974). Physiol. Rev. 54, 813. NARAHASHI, T. (1979). In: Neurotoxins: Tools in Neurobiology (Ceccarelli, B. & Clementi, F. eds), pp. 293-303. Advances in Cytopharmacology, Vol. 3. New York: Raven Press. OVCHINNIKOV, Yu. A., ABDULAEV, N. G., FEIGINA, M. Yu., KISELEV, A. V. & LOBANOV, N. A. (1979). FEBSLett. 100,219. PAULING, L. (1960). The Nature of the Chemical Bond, 3rd edition. Ithaca, New York: Cornell University Press. STRYER, L. (1981). Biochemistry, 2nd edition. San Francisco: W. H. Freeman and Company. WINDHOLZ, M., ed. (1976). The Merck Index, 9th edition. Rahway, New Jersey: Merck and Co., Inc.