Human Hv1: Architectural Overview

Although voltage-gated proton currents have been measured in cell membranes since the early 1980s (Tomas & Meech, 1982), the genes encoding the voltage-gated proton channels were not discovered until 2006 (Sasaki et al., 2006; Ramsey et al., 2006). What the gene sequence demonstrated was that Hv channels share sequence homology with the voltage-sensor domains (VSDs) of canonical six-transmembrane (6TM) voltage-gated cation channels. However, whereas the 6TM channels contain a VSD (helices S1-S4) and a pore domain (helices S5 and S6), Hv channels are only comprised of four transmembrane helices (S1-S4) forming a lone VSD. Hence, Hv was coined the voltage sensor only protein (VSOP). In this post I will use the protein sequence of human Hv1 as a template to describe the different structural and biochemical features of this protein. The purpose of this post is to set the stage for future discussions that may require some knowledge of Hv1’s general features.

Overall Features

Figure 1. Amino acid sequence of human Hv1. The putative transmembrane helices are shaded in grey and are labeled S1 through S4. The two known phosphorylation sites are highlighted in green. The two histidines responsible for Zn2+ binding are highlighted in blue. The S4 arginines are colored red.

Human Hv1 is comprised of the 273 amino acid residues shown in Fig.1. Both the N– and C-termini of Hv channels reside in the cytoplasm of the cell. Secondary structure prediction indicates that there are four putative transmembrane helices (highlighted in grey in Fig.1 and labeled S1 through S4). Although it is very clear that these four transmembrane helices are present in the structure of the channel, I refer to them as putative in the figure because the exact boundaries (i.e. the amino acid positions at which the helices start and terminate) are unknown.

The first ~90 amino acid residues of human Hv1 are thought to be disordered. The high proportion of negatively-charged [glutamate (E) and aspartate (D)] and proline (P) residues suggest an unstructured random coil. However, some secondary structure prediction algorithms indicate that two short helices may be present: one spanning residues A19-H27, distant from the transmembrane helices in primary sequence, and the other spanning residues D87-S97, directly preceding S1. Short amphipathic helices preceding the first transmembrane helix (S1) have been observed in the structures of other voltage-sensor domains (VSDs) and have been termed S0. Therefore, it is possible that human Hv1 also contains a S0 helix preceding S1.

The C-terminal end of human Hv1 forms a coiled-coil. This feature of the protein is involved in dimerization and will be fully discussed in the next section.

Hv is a Dimer in the Membrane

Figure 2. Human Hv1 dimer model based on inter-subunit cross-linking studies. Transmembrane helices S1-S4 are indicated and the cytoplasmic coiled-coil is shown. The horizontal lines represent the membrane boundaries. Adapted from Lee et al., 2008.

Unlike  6TM channels, which form tetramers from four identical subunits or pseudo-tetramers from tandem 6TM domains, Hv channels form homodimers in cell membranes (Lee et al., 2008; Koch et al., 2008; Tombola et al., 2008). This dimerization is mediated in part by the C-terminal cytoplasmic coiled-coil. It has been shown that cysteine 249 in the coiled-coil readily forms an inter-subunit disulfide bond, covalently cross-linking the dimer (Lee et al., 2008). The structure of the C-terminal coiled-coil of human Hv1 has been determined and it unambiguously shows this disulfide bond (Li et al., 2010). The coiled-coil structure was used in Fig.1 to delineate the exact boundaries of the coiled-coil in the protein sequence (underlined region). It has also been demonstrated that cysteine residues mutated into the top of the S1 transmembrane helix also form inter-subunit disulfides, indicating that the dimer interface extends into the membrane along S1, as shown in Fig.2 (Lee et al., 2008).

It is possible to generate Hv monomers in the membrane by truncating both the N– and C-termini of the channel; these “transmembrane-only” constructs are still able to conduct protons, indicating that each Hv1 subunit contains its own proton conduction pathway (Koch et al., 2008). This has also been demonstrated by mutagenesis studies performed on tandem dimer constructs in which both subunits are expressed as a single polypeptide (Tombola et al., 2008). Although it is clear how the C-terminal coiled-coil promotes dimerization, it is unknown what interactions are formed between the N-terminus and other regions of the channels in the dimer.

Although the monomer is able to conduct protons, it is also clear that its gating behavior is significantly altered (Koch et al., 2008). This is in part because the dimeric protein displays strong co-operativity between the two subunits (Tombola et al., 2010; González et al., 2010; Musset et al., 2010). In order for either Hv subunit in the dimer to conduct protons, both must adopt the open conformation. The mechanism of this co-operativity is still unknown.

Zinc inhibition

The presence of extracellular Zn2+ inhibits Hv channels. The  Zn2+ binding site in human Hv1 bridges two histidine residues, one at position 140 (near the extracellular end of S2) and the other at position 192 (in the voltage-sensor paddle motif; shown highlighted in blue in Fig.1). When either of these two histidines are mutated to alanine, Zn2+ inhibition is alleviated (Ramsey et al., 2006). It is thought that Zn2+ binding to these two histidines stabilizes the closed conformation of the channel, thereby preventing the channel from opening. Although it is clear that the Zn2+ binds to the closed conformation of the channel, an attempt has been made to explain the action of Zn2+ inhibition using a homology model built from the depolarized “open” conformations of the KvAP and Kv1.2 VSDs (Musset et al., 2010). I have already discussed in previous posts why I am very skeptical when it comes to homology models of VSDs, but I have to say that I am extra skeptical about this proposed mechanism, since they are using the wrong conformation of the VSD to build their model.

It is also interesting to note that some recently characterized Hv channels from coccolithophores (a type of single-celled algae, check them out on wikipedia)  remain sensitive to  Zn2+ even though the the two histidine residues identified in the human sequence are not conserved (Taylor et al., 2011). Clearly, another mechanism of inhibition is used in these Hv channels.

Phosphorylation

Two phosphorylation sites have been identified on human Hv1, both of which are located in the N-terminal cytoplasmic portion of the channel (Musset et al., 2010). The two amino acid residues that can be phosphorylated are threonine 29 and serine 97 (highlighted green in Fig.1). It is very interesting that only phosphorylation of the more distant (in primary sequence) threonine 29 has a significant affect on the gating of the channel (Musset et al., 2010). Mutations of serine 97, which is adjacent to the transmembrane helix S1, do not alter the activation of the channel by phosphorylation (Musset et al., 2010). This suggests that, although a significant portion of the N-terminus may be disordered, there could be functionally important interactions between the N-terminal amino acid residues and those of the transmembrane helices. As stated in the dimer section above, the specific interactions between the N-terminus and the rest of the protein are currently unknown. It is also interesting to note that threonine 29 resides adjacent to a putative helix (amino acid residues A19-H27) predicted by secondary structure analysis.

Conclusion

In this post I gave a brief overview of some of the biochemical features of the human Hv1 channel. Using this information, it is possible to build a picture of the general architecture of these channels. Human Hv1 is a dimer in the membrane, it is inhibited by  Zn2+ via the action of two extracellular histidine residues and is regulated by phosphorylation on its cytoplasmic N-terminus.  The interface between the subunits within the dimer is defined by the C-terminal cytoplasmic coiled-coil and continues into the membrane along S1 (Fig.2).  Although there is mounting evidence that some potentially structured features of the N-terminus interact with the rest of the channel, we still do not known the exact nature of this interaction.

Works Cited and Further Reading

González, C., Koch, H. P., & Larsson, H. P. (2010). Strong cooperativity between subunits in voltage-gated proton channels. Nature structural & molecular biology, 17(1), 51–56. doi:10.1038/nsmb.1739

Koch, H. P., Kurokawa, T., Okochi, Y., Sasaki, M., Okamura, Y., & Larsson, H. P. (2008). Multimeric nature of voltage-gated proton channels. Proceedings of the National Academy of Sciences of the United States of America, 105(26), 9111–9116. doi:10.1073/pnas.0801553105

Lee, S.-Y., Letts, J. A., & Mackinnon, R. (2008). Dimeric subunit stoichiometry of the human voltage-dependent proton channel Hv1. Proceedings of the National Academy of Sciences of the United States of America, 105(22), 7692–7695. doi:10.1073/pnas.0803277105

Li, S. J., Zhao, Q., Zhou, Q., & Zhai, Y. (2009). Expression, purification, crystallization and preliminary crystallographic study of the carboxyl-terminal domain of the human voltage-gated proton channel Hv1. Acta Cryst (2009). F65, 279-281 [doi:10.1107/S1744309109003777], 1–3. doi:10.1107/S1744309109003777

Li, S. J., Zhao, Q., Zhou, Q., Unno, H., Zhai, Y., & Sun, F. (2010). The Role and Structure of the Carboxyl-terminal Domain of the Human Voltage-gated Proton Channel Hv1. Journal of Biological Chemistry, 285(16), 12047–12054. doi:10.1074/jbc.M109.040360

Musset, B., Capasso, M., Cherny, V. V., Morgan, D., Bhamrah, M., Dyer, M. J. S., & DeCoursey, T. E. (2010a). Identification of Thr29 as a critical phosphorylation site that activates the human proton channel Hvcn1 in leukocytes. Journal of Biological Chemistry, 285(8), 5117–5121. doi:10.1074/jbc.C109.082727

Musset, B., Smith, S. M. E., Rajan, S., Cherny, V. V., Sujai, S., Morgan, D., & DeCoursey, T. E. (2010b). 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

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

Taylor, A. R., Chrachri, A., Wheeler, G., Goddard, H., & Brownlee, C. (2011). A Voltage-Gated H+ Channel Underlying pH Homeostasis in Calcifying Coccolithophores. (P. G. Falkowski, Ed.)PLoS biology, 9(6), e1001085. doi:10.1371/journal.pbio.1001085.g007

Thomas, R. C., & Meech, R. W. (1982). Hydrogen ion currents and intracellular pH in depolarized voltage-clamped snail neurones. Nature, 299(5886), 826–828.

Tombola, F., Ulbrich, M. H., & Isacoff, E. Y. (2008). The Voltage-Gated Proton Channel Hv1 Has Two Pores, Each Controlled by One Voltage Sensor. Neuron, 58(4), 546–556. doi:10.1016/j.neuron.2008.03.026

Tombola, F., Ulbrich, M. H., Kohout, S. C., & Isacoff, E. Y. (2010). The opening of the two pores of the Hv1 voltage-gated proton channel is tuned by cooperativity. Nature structural & molecular biology, 17(1), 44–50. doi:10.1038/nsmb.1738

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3 Responses to “Human Hv1: Architectural Overview”
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  1. […] residues along the sequence of human Hv1 (underlined residues in Fig.1; see my previous post “Human Hv1: Architectural Overview” for the entire human Hv1 sequence). For example, the structured region of the coiled-coil ends at […]

  2. […] the mechanism of the voltage-gate H+ (Hv) channels. As I have discussed in an introductory post (Human Hv1: Architectural Overview), Hv channels are comprised only of a dimer of VSDs without the associated pore domain seen in […]

  3. […] the N-terminus has a significant impact on the channel gating (Musset et al. 2010; see my post Human Hv1: Architectual Overview to learn more). Specifically, they showed that, upon phosphorylation of Thr29, the opening of the […]



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