The Problem of Aligning S4

Recently, a number of papers have come out that have used sequence homology to generate structural models of human Hv1 (Musset et al,. 2010; Ramsey et al., 2010; Wood et al., 2012). These models have then been used for docking and molecular-dynamics simulations to try to extract some mechanistic insight into the channel function. In this post, I will be discussing the problems that arise when trying to align sequences of the S4 transmembrane helix of voltage-gated cation channels. This has serious implications for the creation of structural models of any voltage-senor domain (VSD). After a brief introduction, I discuss the “register problem” using possible sequence alignments between the Kv1.2-2.1 paddle chimera and human Hv1 as an example. Keep in mind, however, that this problem exists with alignments between any two S4 helix sequences. Next, I discuss a problem that may arise when aligning the S4 helices of VSDs of known structure: it may not be completely straightforward to do these structure-based alignments. The lack of unambiguous methods for assigning the S4 register will result in significant differences in homology models. Researchers should be aware of these problems and carefully discuss their alignment methodology.


Figure 1. Transmembrane helix topology of a 6TM channel domain. Black lines represent the membrane boundaries.

The canonical voltage-gated ion channels, i.e. the voltage-gated potassium (Kv), sodium (Nav), and calcium channels (Cav), are comprised of repeated domains of six transmembrane helices (6TM). In the case of Kv, the channels are formed by four identical 6TM protomers, making the channel a four-fold symmetric tetramer. In the cases of eukaryotic Nav and Cav, the four 6TM subunits are expressed in tandem in a single polypeptide. The transmembrane helices of these channels are referred to as helices S1 through S6 (Fig.1). Helices S1, S2, S3 and S4 form the voltage-sensor domain (VSD, orange in Fig.1) and helices S5 and S6 form the pore domain (brown in Fig.1). The S4 helix is special in that it contains the gating-charge amino acid residues. The positively charged side chains (represented as “+” in Fig.1) of these gating-charge residues are responsible for sensing changes in electrical potential across the membrane (Aggarwal & MacKinnon, 1996).

Hv is different. It also has transmembrane helices S1 through S4, but it does not have helices S5 and S6 (it is only the orange part of Fig.1): Hv is a VSD without a separate pore domain. The gene encoding the Hv channel was discovered only recently by searching genomes for protein-coding sequences that shared homology to the known VSD sequences (Ramsey et al., 2006; Sasaki et al., 2006). One of the most conspicuous features of the VSD sequence is the repeating, positively charged (arginine or lysine) amino acid residues on the S4 helix. These gating-charge residues occur along S4 every three residues (see sequences in Fig. 2). The number of gating-charge residues can vary, but the pattern is strongly conserved. Although this pattern is helpful when it comes to finding putative VSD sequences in the genome, it can lead to ambiguity when trying to properly align those sequences.

The Register Problem

Figure 2. The register problem. Four of several possible alignments for the S4 helices of the Kv1.2-2.1 paddle chimera and human Hv1. The gating charge residues are shown in red. The amino acid residues of the S4 helix from the structure of Kv1.2-2.1 paddle chimera are boxed in orange. The exact boundaries of the human Hv1 S4 helix are unknown.

In Fig.2, four possible alignments between the Kv1.2-2.1 paddle chimera and Hv are shown. I show the Kv1.2-2.1 paddle chimera sequence in Fig.2 because its three-dimensional structure is known and it is commonly used for building homology models of human Hv1. Clearly, the two S4 sequences differ: although they both contain repeated arginine residues, the Kv1.2-2.1 paddle chimera has seven (glutamine (Q) is given position 1 in Fig.2 since it comes from the Kv2.1 sequence, this position is an arginine (R) in native Kv1.2) and human Hv1 only has three. The question then becomes: which three of the seven Kv1.2-2.1 paddle chimera gating charge residues correspond to the three gating charge residues of human Hv1?

Because of these repeated arginine residues, there are several different possible “registers” with which any two S4 sequences can be aligned. By just looking at the sequences, there are no rigorous, unambiguous criteria by which we could say that one of those registers is correct. This is a significant problem since the position of the residue along S4 correlates to the z-position (“depth”) of the residue in the membrane (displacement relative to the plane of the extracellular leaflet). Thus, the different alignments imply significantly different three-dimensional structures for both the open and closed states of the VSD. Clearly, the correct alignment is required to build any plausible homology model based on known structures of VSDs.

Without taking into account additional biochemical data it is not possible to determine which of these alignments is correct. Indeed, since the different conformations of the VSDs between the two channels may vary significantly, it may be impossible to define a correct alignment. As discussed in the conclusions below, using different alignments to build a structural homology model of a VSD will result in very different structures. However, after two structures of VSDs have been experimentally solved it must greatly increase the ease with which we can align their S4 helices, right? Maybe not.

Problems with structural alignments

The structures of only a few VSDs have been solved. First, the structure of the VSD from the archaeal voltage-gated K+ channel KvAP was solved, both in the presence and absence of the pore domain (Jiang et al., 2003; Lee et al., 2005; Butterwick & MacKinnon, 2010). Next, the structure of the eukaryotic Kv1.2 was solved; however, the electron density corresponding to the VSDs was low resolution due to local disorder (Long et al., 2005). This was followed by the structure of the Kv1.2-2.1 paddle chimera, the highest resolution (i.e. most detailed) structure of a eukaryotic VSD to date (Long et al., 2007). Most recently, the structures of the bacterial voltage-gated Na+ channels NavAB and NavRh were solved (Payandeh et al., 2011; Payandeh et al., 2012; Zhang et al., 2012). All of these structures represent depolarized or “up” conformations of the VSD. No structure of a hyperpolarized VSD has been experimentally determined.

Can we get the correct sequence alignment for the S4 helices by comparing the different depolarized VSD structures? Before we can answer this question we need to consider another: Is it possible that different VSDs have different numbers of closed conformations?

Since the first observation by Cole and Moore that long hyperpolarizations lead to slower opening of voltage-gated K+ channels in squid giant axons (the “Cole-Moore effect”), it has been understood that VSDs have multiple closed conformations (Cole & Moore, 1960). It is now thought that these different closed conformations correspond to different gating charge residues occupying a charge transfer center within the VSD (Tao et al., 2010). As the VSD goes from a deactivated conformation to the activated conformation, the gating charge residues pass through this center “one by one” (for additional explanation and discussion, please see my earlier post and watch Jensen et al.’s simulations of the process). Is it possible that different VSDs open the pore after a different number of gating charges have been transferred?

Figure 3. Alternate S4 helix alignments between Kv1.2-2.1 paddle chimera and KvAP. a) Sequence based alignment. b) Structure based alignment. Colors are as in Fig.2.The S4 helical structure box is shown open ended for KvAP because of ambiguity, S4 ends at different residues in different structures.

Fig.3 illustrates this idea with an example. In Fig.3a, the sequence-based alignment of the Kv1.2-2.1 paddle chimera and the archaeal voltage-gated K+ channel KvAP is shown. It is clear that, with the exception of a “gap” at position 4 caused by the insertion of a residue within the S4 sequence of KvAP, the two S4 sequences line up nicely. (The insertion of a residue in the KvAP sequence is very interesting and may have to do with compensation for the fact that the KvAP S4 is an α-helix whereas the Kv1.2-2.1 paddle chimera S4 forms a 310-helix. While the 310-helical geometry allows for each of the gating charge residues to be displayed on the same face of the helix, such an amino acid insertion is necessary for the gating charges to be displayed on the same face of an α-helix.) The structure-based alignment, however, tells a different story. In order to do a structure-based alignment, the easiest question to ask is which gating charge residue is in the charge transfer center. In the structure of the Kv1.2-2.1 paddle chimera, this residue is lysine (K) 5. However, in the structure of KvAP, lysine (K) 6 is in the charge transfer center (using the numbering from Fig.3a). The resulting structure-based alignment requires us to shift the KvAP sequence one register, thus creating a (-1) gating charge position (Fig.3b).

Which of these two alignments is correct? Really, it depends on what we want to use as a reference. If we want to use the structure of the depolarized conformation as a reference, then, clearly, the alignment in Fig.3b is correct. However, if we want to use the sequence-based alignment in Fig.3a as a reference, then we have to conclude that it is possible for Kv1.2-2.1 paddle chimera and KvAP to have a different number of closed conformations. For the Kv1.2-2.1 paddle chimera channel to open, only the gating charge residue in position 5 has to make it into the charge transfer center. For KvAP to open, S4 has to move an additional register, placing gating charge residue 6 in the charge transfer center.

Of course, KvAP is an extreme example since it comes from a hyperthermophilic archaea and its structures don’t clearly delineate the C-terminal boundary of its S4 helix. However, my purpose here was only to use it as an example in the above thought experiment. Obviously, if different VSDs have different numbers of gating-charges they will likely have a different number of closed states. Nevertheless, it certainly seems possible that other eukaryotic voltage-gated cation channels with the same number of gating charge residues may have different numbers of closed states; i.e. that gating charge residues at different register positions occupy the charge transfer center in the activated conformation. As shown above, this can lead to problems with structural alignments.


Because of the repeated pattern of the gating charge residues, there is no straightforward method of correctly aligning the S4 helices of different VSDs. This has profound implications for the construction of accurate homology models of VSDs of unknown structures. A lot of careful thought needs to go into the alignment used. For example, many homology models of human Hv1 have been published (Musset et al,. 2010; Ramsey et al., 2010; Wood et al., 2012). However, in none of these papers have I been able to find any discussion of why the authors chose the particular alignments they employed. To the credit of Wood et al. they build two separate homology models based on two possible S4 helix alignments. They state that the two alignments chosen are the “most probable” but do not say why. I know that a lot of time and thought must have gone into the construction of these models but, because of the lack of disclosure, we cannot know what the authors’ thought-processes were. It would be helpful for the community at large if the justification for these models were shown and discussed. In the absence of such clarification, my default approach to models of VSDs will always be highly skeptical.

It might seem that the problem of structural alignments is really only a problem of definitions. If we all agree that the correct alignment comes from the experimentally determined structures then we can always just define it based on which gating charge residue occupies the charge transfer center. However, the very recent structure of the bacterial voltage-gated Na+ channel NavRh complicates things even more, in that all of its gating charge residues are above the charge transfer center (Zhang et al., 2012). Clearly, nothing is straightforward when thinking about these channels.

Please let me know what you think. Is there a way to reach a definitive alignment of VSD S4 helices that I haven’t thought of? Do you agree that it is possible that different eukaryotic VSDs have different numbers of closed states? What do you think is the proper alignment for homology models of human Hv1 and why? Please leave any comments or questions below.

Works Cited in this Post and Further Reading

Aggarwal, S. K., & Mackinnon, R. (1996). Contribution of the S4 segment to gating charge in the Shaker K+ channel. Neuron, 16(6), 1169–1177.

Butterwick, J. A., & Mackinnon, R. (2010). Solution structure and phospholipid interactions of the isolated voltage-sensor domain from KvAP. Journal of Molecular Biology, 403(4), 591–606. doi:10.1016/j.jmb.2010.09.012

COLE, K. S., & MOORE, J. W. (1960). Potassium ion current in the squid giant axon: dynamic characteristic. Biophysical Journal, 1, 1–14.

Jensen, M. Ø., Jogini, V., Borhani, D. W., Leffler, A. E., Dror, R. O., & Shaw, D. E. (2012). Mechanism of voltage gating in potassium channels. Science, 336(6078), 229–233. doi:10.1126/science.1216533

Jiang, Y., Lee, A., Chen, J., Ruta, V., Cadene, M., Chait, B. T., & Mackinnon, R. (2003). X-ray structure of a voltage-dependent K+ channel. Nature, 423(6935), 33–41. doi:10.1038/nature01580

Lee, S.-Y., Lee, A., Chen, J., & Mackinnon, R. (2005). Structure of the KvAP voltage-dependent K+ channel and its dependence on the lipid membrane. Proceedings of the National Academy of Sciences of the United States of America, 102(43), 15441–15446. doi:10.1073/pnas.0507651102

Long, S. B., Campbell, E. B., & Mackinnon, R. (2005). Crystal structure of a mammalian voltage-dependent Shaker family K+ channel. Science, 309(5736), 897–903. doi:10.1126/science.1116269

Long, S. B., Tao, X., Campbell, E. B., & Mackinnon, R. (2007). Atomic structure of a voltage-dependent K+ channel in a lipid membrane-like environment. Nature, 450(7168), 376–382. doi:10.1038/nature06265

Musset, B., Smith, S. M. E., Rajan, S., Cherny, V. V., Sujai, S., Morgan, D., & DeCoursey, T. E. (2010). Zinc inhibition of monomeric and dimeric proton channels suggests cooperative gating. The Journal of Physiology, 588(Pt 9), 1435–1449. doi:10.1113/jphysiol.2010.188318

Musset, B., Smith, S. M. E., Rajan, S., Cherny, V. V., Morgan, D., & DeCoursey, T. E. (2010). Oligomerization of the voltage-gated proton channel. Channels (Austin, Tex.), 4(4), 260–265.

Payandeh, J., Scheuer, T., Zheng, N., & Catterall, W. A. (2011). The crystal structure of a voltage-gated sodium channel. Nature, 1–7. doi:10.1038/nature10238

Payandeh, J., Gamal El-Din, T. M., Scheuer, T., Zheng, N., Catterall, W. A. (2012). Crystal structure of a voltage-gated sodium channel in two potentially inactivated states. Nature, doi:10.1038/nature11077

Ramsey, I. S., Mokrab, Y., Carvacho, I., Sands, Z. A., Sansom, M. S. P., & Clapham, D. E. (2010). An aqueous H+ permeation pathway in the voltage-gated proton channel Hv1. Nature structural & molecular biology, 1–9. doi:10.1038/nsmb.1826

Ramsey, I. S., Moran, M. M., Chong, J. A., & Clapham, D. E. (2006). A voltage-gated proton-selective channel lacking the pore domain. Nature, 440(7088), 1213–1216. doi:10.1038/nature04700

Sasaki, M., Takagi, M., & Okamura, Y. (2006). A voltage sensor-domain protein is a voltage-gated proton channel. Science, 312(5773), 589–592. doi:10.1126/science.1122352

Wood, M. L., Schow, E. V., Freites, J. A., White, S. H., Tombola, F., & Tobias, D. J. (2012). Water wires in atomistic models of the Hv1 proton channel. Biochimica et biophysica acta, 1818(2), 286–293. doi:10.1016/j.bbamem.2011.07.045

Zhang, X., Wenlin, R., DeCaen, P., Yan, C., Tao, X., Tang, L., Hasegawa, K., Kumasaka, T., He, J., Wang, J., Clapham, D. E., & Yan, N. (2012) Crystal structure of an orthologue of the NaChBac voltage-gated sodium channel. Nature, doi:10.1038/nature11054

4 Responses to “The Problem of Aligning S4”
Check out what others are saying...
  1. […] this post, I will elaborate upon a statement I made in last week’s post. There, I discussed how important a proper alignment of the S4 helices in voltage sensor domains […]

  2. […] provide a better model for aligning S4 helices. As I have discussed previously in my posts “The Problem of Aligning S4” and “Is the S4 Helix of Hv Short?“, any data that help us constrain the limits […]

  3. […] if you need a refresher on VSD architecture and gating charge residues check out my previous post, The Problem of Aligning S4). The formation of this omega pore is state dependent, meaning that the VSD must be in a specific […]

  4. […] shown) is not intended as a definitive alignment. As I have stated previously, (in my post The Problem of Aligning S4) it is very difficult to generate a satisfying alignment for S4 helix sequences. Many possible […]

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