Aspartate¹¹² is the Selectivity Filter of the Human Voltage Gated Proton Channel

Abstract

The ion selectivity of pumps and channels is central to their ability to perform a multitude of functions. Here we investigate the mechanism of the extraordinary selectivity of the human voltage gated proton channel¹, hH_V1. This selectivity is essential to its ability to regulate reactive oxygen species production by leukocytes^2–4, histamine secretion by basophils⁵, sperm capacitation⁶, and airway pH⁷. The most selective ion channel known, H_V1 shows no detectable permeability to other ions¹. Opposing classes of selectivity mechanisms postulate that (a) a titratable amino acid residue in the permeation pathway imparts proton selectivity^{1, 8–11}, or (b) water molecules “frozen” in a narrow pore conduct protons while excluding other ions¹². Here we identify Aspartate¹¹² as a crucial component of the selectivity filter of hH_V1. When a neutral amino acid replaced Asp¹¹², the mutant channel lost proton specificity and became anion selective or did not conduct. Only the glutamate mutant remained proton specific. Mutation of the nearby Asp¹⁸⁵ did not impair proton selectivity, suggesting that Asp¹¹² plays a unique role. Although histidine shuttles protons in other proteins, when histidine or lysine replaced Asp¹¹², the mutant channel was still anion permeable. Evidently, the proton specificity of hH_V1 requires an acidic group at the selectivity filter.

Voltage gated proton channels are considered specific (perfectly selective) for protons, because no evidence exists for permeation of anything but H⁺. Specificity, combined with a large deuterium isotope effect⁹ and extraordinarily strong temperature dependence of conduction¹⁰ suggests a permeation pathway more complex than a simple water wire, as exists in gramicidin¹³. All proton conduction appears consistent with a hydrogen-bonded chain (HBC) mechanism¹⁴; a HBC including a titratable group could explain several unique properties of H_V1¹, especially proton selectivity¹⁴. Yet in a recent study, mutation of each titratable amino acid in all four transmembrane (TM) helices of hH_V1 failed to abolish conduction¹². Thus, the mechanism producing proton selectivity remained unknown.

We noticed that a human gene, C15orf27 (of unknown function), contains a predicted voltage sensor domain (VSD) that shares 25% sequence identity and 52% similarity [http://www.ebi.ac.uk/Tools/emboss/align/] with the VSD of hH_V1, and includes three Arg residues in the S4 TM helix that are conserved among all known H_V1 homologues. Phylogenetic analysis of VSD sequences (Fig. S1) reveals that a group comprising H_V1, C15orf27, and voltage sensitive phosphatase (VSP) sequences separated early from the two phylogenetically distinct groups of depolarization activated VSDs described previously (K_V channels and Na_V/Ca_V channels), supporting the modular evolution of VSD-containing proteins¹⁵. Furthermore, H_V1 VSDs occupy a discrete lineage, distinct from those of VSP and C15orf27 orthologs.

When we cloned the C15orf27 gene and expressed the product in HEK-293 or COS-7 cells, the GFP-tagged protein localised at the plasma membrane (Fig. S2), but we detected no currents beyond those in non-transfected cells. We reasoned that substitutions based on sequence elements that differ between hH_V1 and C15orf27 should be structurally tolerated while revealing residues responsible for proton conduction. We therefore mutated residues that are perfectly conserved in 21 H_V1 family members and differ between C15orf27 and H_V1. We replaced five candidate residues in hH_V1 (D112, D185, N214, G215, and S219) (Fig. 1a & 1b) with the corresponding residue in C15orf27. Four mutants exhibited large currents under whole-cell voltage clamp (Fig. 1d). The reversal (zero current) potential, V_rev, measured at several pH_o and pH_i, was close to the Nernst potential for protons, E_H (Fig. 1c), demonstrating proton selectivity. D112V mutants localised to the plasma membrane (Fig. S3), but displayed no convincing current (Fig. 1d). Some D112V transfected HEK-293 or COS-7 cells (and non-transfected cells) exhibited small native proton currents. His140A/H193A double mutants^{16, 17}, in which the two Zn²⁺ binding His residues are neutralized, resemble WT, with similar ΔpH dependent gating¹², and V_rev near E_H (Fig. S4). We expressed mutants in this Zn²⁺ insensitive background (D112x/A/A) to distinguish their currents from native currents that are abolished by 100 μM Zn²⁺ at pH_o 7.0. We tentatively concluded that Asp¹¹² is crucial to proton conduction.

Figure 1. Identification of five key amino acids that differ in hH_V1 and C15orf27, and the currents generated in a heterologous expression system by hH_V1 mutants in which hH_V1 residues were replaced by the corresponding amino acid in the non-conducting C15orf27.

a, Representative subset of multiple sequence alignment of 122 VSDs; only TM helices are shown. Gene families include: H_V1; voltage sensitive phosphatases; C15orf27; Ca²⁺ and Na⁺ channels; and K⁺ channels (cf. Fig. S1). b, Location of the key amino acids in the open hH_V1 channel VSD viewed from the side (membrane), based on a homology model¹⁷. c, V_rev in the four conducting mutants is near E_H (dashed line), indicating proton selectivity. V_rev was measured using tail currents; in G215A V_rev was positive to threshold and was observed directly. d, Voltage-clamp current families in cells expressing hH_V1 mutants. Depolarizing pulses were applied in 10-mV increments from a holding voltage, V_hold = −40 mV (D185M, D112V), −60 mV (G215A, S219P), or −90 mV (N214D), with the most positive pulse labelled. After membrane repolarisation, an inward “tail current” is seen as channels close (see inset for D185M); pH is given as pH_o//pH_i. D112V exhibited no clear current.

The absence of detectable currents in D112V led us to make other D112x substitutions. These mutants (Fig 2a) exhibited slowly activating outward currents upon depolarization that resembled hH_V1 currents. As reported previously¹², Asp¹¹² mutation had little effect on the ΔpH dependence of gating. The proton conductance-voltage, g_H-V, relationship of all D112x mutants shifted roughly −60 mV when pH_o increased from 5.5 to 7.0 (Fig. S5), as in WT channels^{1, 8, 18}. Mutation of Asp¹¹² did influence channel opening and closing kinetics (Table S2).

Figure 2. Currents in Asp112 mutants resemble proton currents, but are not.

a, Currents generated by WT, D112E, D112H, D112K/A/A, D112N/A/A, D112S, D112A/A/A, and D112F/A/A in COS-7 cells (pH_i 5.5) at pH_o 5.5 (column 1) or 7.0 (column 2), during families of pulses in 10 mV increments up to indicated voltages. Tail currents at pH_o 7.0 (column 3) reveal that V_rev deviates from E_H, indicating loss of proton selectivity. At pH_o 5.5 V_hold was −40 mV (−60 mV for WT). At pH_o 7.0 V_hold was −40 mV (K, N, S), −50 mV (F), −60 mV (H, A), −80 mV (E), or −90 mV (WT); V_pre was −65 mV (WT), −40 mV (E), −10 mV (H), +50 mV (K), +40 mV (N, S), or +20 mV (A, F). V_rev (arrows) was determined from the amplitude and direction of tail current decay. For D112N, V_rev was above V_threshold and was evident during pulse families. b, Shift in V_rev when the TMACH₃SO₃ bath solution was changed from pH 5.5 to 7.0. There is no difference between WT and D112E, but the shift in all other mutants is smaller than WT (p<0.001, by one-way ANOVA followed by Tukey’s test, n = 7, 4, 9, 8, 6, 7, 9, and 4. Dashed line shows E_H.

Measurements of V_rev in Asp¹¹² mutants revealed a marked departure from WT hH_V1 properties. At symmetrical pH 5.5, V_rev was near 0 mV (not shown). At pH_o 7.0, pH_i 5.5 (Fig. 2a, column 3), WT channels reversed near E_H (−87 mV), indicating proton selectivity. But for all mutants except D112E, V_rev was substantially positive to E_H (Fig. 2b), ranging from −58 mV (D112H) to −13 mV (D112N). Substitutions at Asp¹¹² eliminated the proton specificity that distinguishes H_V1 from all other ion channels¹. A previous study described currents in D112A and D112N mutants¹² but did not report V_rev.

We expected that loss of proton selectivity would result in nonselective permeation of cations. Surprisingly, V_rev did not change detectably when Na⁺, K⁺, N-methyl-D-glucamine⁺, or TEA⁺ replaced TMA⁺ (Table S4). To test anion vs. cation selectivity, we adopted the classical tactic of replacing a fraction of the bath solution with isotonic sucrose¹⁹. The Nernst equation predicts that dilution of all extracellular ions except H⁺ and OH⁻ (leaving internal ion concentrations unchanged) will shift V_rev negatively for a cation selective channel, but positively for an anion selective one. Despite the 10-fold reduction of buffer concentration, direct measurement confirmed that pH remained constant.

Fig. 3 illustrates determination of V_rev from tail currents in a D112H transfected cell at pH 5.5//5.5 in CH₃SO₃⁻ (Fig. 3a) or Cl⁻ solution (Fig. 3c), and after 90% reduction of external ionic strength (Figs. 3b & 3d). Astonishingly, for all Asp¹¹² mutants except D112K/A/A, sucrose shifted V_rev positively (Fig. S6), indicating anion selectivity both in CH₃SO₃⁻ (Fig. 3e) and Cl⁻ solutions (Fig. 3f). For hH_V1 and D112E, V_rev did not change, reaffirming their proton specificity. Neutralization of a single Asp residue converts a proton channel into a predominantly anion selective channel. Thus, Asp¹¹² mediates charge selectivity as well as proton selectivity.

Figure 3. Dilution of ionic strength by 90% with isotonic sucrose shifted V_rev positively, indicating that most Asp¹¹² mutants are anion selective.

a Measurement of V_rev by tail currents in a cell transfected with D112H at pH 5.5//5.5 and (b) after sucrose. c, V_rev in the same cell in pH 5.5 Cl⁻ solution, and (d) after sucrose. Arrows indicate zero current. V_hold = −40 mV, V_pre = +60 mV. e, Mean shifts of V_rev with decreasing ionic strength in CH₃SO₃⁻ solutions or (f) in Cl⁻ solutions. Each value was determined in 3–6 cells. X = not done. g, Shifts of V_rev when CH₃SO₃⁻ was replaced by Cl⁻. Values for WT and D112E do not differ significantly from 0 mV. For all anion selective mutants except D112N, the difference between shifts at pH 5.5 and 7.0 was significant (p<0.001, one-way ANOVA followed by Tukey’s test; n = 3–8). Error bars in e–g are s.e.

To confirm anion permeability of Asp¹¹² mutants, we replaced the main external anion, methanesulfonate⁻ (CH₃SO₃⁻), with Cl⁻. Consistent with previous studies¹, V_rev in hH_V1 was unchanged. As shown in D112H (Fig. 3a vs. 3c), V_rev shifted negatively in Cl solutions in all mutants (except D112E), indicating that Cl⁻ is more permeant than the larger CH₃SO₃⁻ anion (Fig. 3g). That all conducting non-acidic mutants exhibited Cl^-permeability indicates that Asp¹¹² mediates not only proton selectivity, but also charge selectivity. Currents were smaller than WT in cells expressing some mutants (Fig. S7), suggesting a smaller unitary conductance. Evidently, these channels conduct anions, but not very well.

Although the mutant channels have diminished selectivity, V_rev did shift negatively when pH_o increased from 5.5 to 7.0 (Fig. 2b). Because these solutions differ mainly in buffer species and concentrations of H⁺ and OH⁻, Asp¹¹² mutants must have significant permeability to H⁺ and/or OH⁻. The Goldman-Hodgkin-Katz equation shows how V_rev depends on ion concentrations:

$$V_{\mathit{rev}}=\frac{RT}{zF}log\frac{P_{{Cl}^{-}}{[{Cl}^{-}]}_i+P_{{CH}_3{SO}_3^{-}}{[{CH}_3{SO}_3^{-}]}_i+P_{{OH}^{-}}{[{OH}^{-}]}_i+P_{H^{+}}{[H^{+}]}_o}{P_{{Cl}^{-}}{[{Cl}^{-}]}_o+P_{{CH}_3{SO}_3^{-}}{[{CH}_3{SO}_3^{-}]}_o+P_{{OH}^{-}}{[{OH}^{-}]}_o+P_{H^{+}}{[H^{+}]}_i}.$$ (1)

Ions with greater permeability dominate V_rev. Permeation of H⁺ and OH⁻ are difficult to distinguish because they have the same Nernst potential¹. The data can be interpreted assuming permeation of either (Table S3), but the anion selectivity of Asp¹¹² mutants and the pH dependence of sucrose effects (Figs. 3e & 3f) support OH⁻ permeation. The relative permeability of conducting Asp¹¹² mutants was OH⁻ (or H⁺) > Cl⁻ > CH₃SO₃⁻.

Although Asp¹¹² is essential to selectivity, other acidic groups might participate. We mutated Asp¹⁸⁵, located in the presumed conduction pore (Fig. 1b)^{12, 17}. However, like D185M (Fig. 1b), D185V, D185A, and D185N remained proton selective (Fig. S8). Speaking against additive effects, the double mutant, D112N/D185M did not differ from D112N (Fig. S8).

Consistent with earlier predictions that a titratable amino acid provides the selectivity filter of H_V1^{1, 8–11}, only channels with acidic residues (Glu or Asp) at position 112 manifested proton specificity. Asp¹¹² lies at the constriction of the presumed pore (Fig. 1c), a logical location for a selectivity filter, and just external to the postulated gating charge transfer centre²⁰. Our original prediction envisioned selectivity arising from protonation/deprotonation of a residue during conduction, but other mechanisms are possible. For example, proton selectivity of the influenza A M₂ viral proton channel has been explained by (a) immobilized water²¹, (b) successive proton transfer and release by His³⁷ (refs.^{22, 23}), or (c) delocalization of the proton among His³⁷ and nearby waters²⁴.

The Cl⁻ permeability of D112H was completely unexpected given strong precedents for His imparting proton selectivity to channels. Histidine shuttles protons in K⁺ or Na⁺ channel VSDs with Arg→His mutations^25–27, in carbonic anhydrase²⁸, and in M₂ channels^{22, 23}. However, these molecules are not proton specific^{27, 29}. Evidently, His shuttles protons, but does not guarantee proton selectivity. In hH_V1, Asp¹¹² (or Glu¹¹² in D112E) excludes anions, resulting in proton specific conduction. His may fail to exclude anions because it is cationic when protonated, whereas Glu and Asp are neutral.

The anion selectivity of neutral Asp¹¹² mutants suggests that electrostatic forces due to the charge distribution in the rest of the channel deter cation permeation, and that the cation selectivity of the WT channel is due to the anionic charge of Asp¹¹². Asp¹⁸⁵ does not participate directly in selectivity (Fig. S8). VSP family members possess the equivalent of Asp¹¹² (Fig. 1a), yet conduct no current³⁰, illustrating that Asp¹¹² requires a specific microenvironment to achieve selectivity. Although permeation of Cl⁻ and CH₃SO₃⁻ suggests a wide pore in D112x mutants, local geometry might differ in WT channels due to the presence of anionic Asp¹¹².

Regulation of voltage gating by ΔpH is distinct from permeation. Pathognomonic of H_V1 is a strict correlation between the g_H-V relationship and V_rev, in which V_threshold shifts 40 mV/Unit change in ΔpH⁸. The ΔpH dependence persisted in mutants exhibiting shifted g_H-V relationships ¹². Here we show uncoupling of V_rev and voltage gating. Asp¹¹² mutants retained normal ΔpH dependence (Fig. S5), despite the dissociation of V_rev from ΔpH (Figs. 2 & S9). This uncoupling of pH control of gating from permeation speaks against any mechanism that invokes regulation by local proton concentration in the vicinity of S4 Arg residues¹².

In summary, Asp¹¹² is a critical component of the selectivity filter of hH_V1, crucial to both proton selectivity and charge selectivity. That D112E was proton selective, but D112H conducted anions indicates that this proton channel requires an acid at the selectivity filter. That neutralization of nearby Asp¹⁸⁵ did not affect selectivity suggests that Asp¹¹² plays a unique role.

‘Methods Summary’

The pipette solution (also used externally) contained (mM) 130 TMACH₃SO₃, 2 MgCl₂, 2 EGTA, 80 MES, titrated to pH 5.5 with ~20 TMAOH. In the pH 5.5 TMACl solution, TMACl replaced TMACH₃SO₃. Bath solutions at pH 7.0 had (mM) 90 TMACH₃SO₃ or TMACl, 3 CaCl₂, 1 EGTA, 100 BES, and 36–40 TMAOH. For experiments with Zn²⁺, solutions contained PIPES without EGTA. Experiments were done at 20–25°C. Currents are shown without leak correction. V_rev data were corrected for liquid junctions potentials measured in each solution¹⁹.

‘Methods’

Exhaustive searches to identify H_V1 homologs were performed using protein BLAST and PSI-BLAST. A sample of VSDs from K⁺, Na⁺, and Ca²⁺ channels (that open with depolarization like H_V1, and in addition one that opens with hyperpolarization), along with putative H_V1, VSP, and C15orf27 homologs were chosen. For cation channels, we sampled from the range of subfamilies, from the VSD repeats within Na⁺ and Ca²⁺ channels, and from the range of species. VSD sequences, including crystallized K⁺ channels (PDB ids 1ORS, 2R9R, and 2A79), were aligned using PromalS3D³¹, which incorporates structural information, allowing high confidence identification of VSD boundaries. Sequences were trimmed to the VSD, realigned with PromalS3D, and the resulting alignment was analyzed with PhyML (maximum likelihood)³² and Protpars (maximum parsimony)³³ at the Mobyle portal³⁴. Trees were visualized with TreeDyn³⁵ and iTOL³⁶. Parsimony (not shown) and maximum likelihood trees had similar topology, including H_V1 and C15orf27 families separating into discrete branches. A homology model of the VSD of hH_V1 was constructed as described previously¹⁷.

The C15orf27 clone was PCR amplified from human cerebellum and subcloned into pcDNA3.1(+) expression vector (Invitrogen, Carlsbad, CA). The coding sequence of human H_V1 (HVCN1) was cloned into either pcDNA3.1(−) or pQBI25-fC3 (to make GFP-H_V1) vectors as described previously¹⁶. Site directed mutants were created using the Stratagene Quikchange (Agilent, Santa Clara, CA) procedure according to the manufacturer’s instructions. All the positive clones were sequenced to confirm the presence of the introduced mutation. HEK-293 or, more often COS-7 cells were grown to ~80% confluency in 35 mm cultures dishes, usually by seeding cells 1 d ahead of transfection. Cells were transfected with 0.4–0.5 μg of the appropriate cDNA using Lipofectamine 2000 (Invitrogen). After 6 h at 37oC in 5% CO₂, the cells were trypsinized and re-plated onto glass cover slips at low density for patch clamp recording the following day. We selected green cells under fluorescence for recording. Patch clamp methods were described previously¹⁸.

The main pipette solution (also used externally) contained (in mM) 130 TMACH₃SO₃, 2 MgCl₂, 2 EGTA, 80 MES, titrated to pH 5.5 with ~20 TMAOH. In the pH 5.5 TMACl solution, TMACl replaced TMACH₃SO₃. Bath solutions at pH 7.0 had (mM) 90 TMACH₃SO₃ or TMACl, 3 CaCl₂, 1 EGTA, 100 BES, and 36–40 TMAOH. For experiments with Zn²⁺, solutions contained PIPES buffer³⁷ without EGTA. Experiments were done at 20–25°C. Currents are shown without leak correction. V_rev data were corrected for liquid junctions potentials measured in each solution.