Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011 Oct 23;480(7376):273-7.
doi: 10.1038/nature10557.

Aspartate 112 is the selectivity filter of the human voltage-gated proton channel

Boris Musset  1 , Susan M E Smith, Sindhu Rajan, Deri Morgan, Vladimir V Cherny, Thomas E Decoursey
Affiliations

Aspartate 112 is the selectivity filter of the human voltage-gated proton channel

Boris Musset et al. Nature. .

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 channel, H(V)1 (also known as HVCN1). This selectivity is essential to its ability to regulate reactive oxygen species production by leukocytes, histamine secretion by basophils, sperm capacitation, and airway pH. The most selective ion channel known, H(V)1 shows no detectable permeability to other ions. Opposing classes of selectivity mechanisms postulate that (1) a titratable amino acid residue in the permeation pathway imparts proton selectivity, or (2) water molecules 'frozen' in a narrow pore conduct protons while excluding other ions. Here we identify aspartate 112 as a crucial component of the selectivity filter of H(V)1. When a neutral amino acid replaced Asp 112, 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 Asp 185 did not impair proton selectivity, indicating that Asp 112 has a unique role. Although histidine shuttles protons in other proteins, when histidine or lysine replaced Asp 112, the mutant channel was still anion-permeable. Evidently, the proton specificity of H(V)1 requires an acidic group at the selectivity filter.

PubMed Disclaimer

Conflict of interest statement

COMPETING FINANCIAL INTERESTS

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Identification of five key amino acids that differ in hHV1 and C15orf27, and the currents generated in a heterologous expression system by hHV1 mutants in which hHV1 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: HV1; voltage sensitive phosphatases; C15orf27; Ca2+ and Na+ channels; and K+ channels (cf. Fig. S1). b, Location of the key amino acids in the open hHV1 channel VSD viewed from the side (membrane), based on a homology model. c, Vrev in the four conducting mutants is near EH (dashed line), indicating proton selectivity. Vrev was measured using tail currents; in G215A Vrev was positive to threshold and was observed directly. d, Voltage-clamp current families in cells expressing hHV1 mutants. Depolarizing pulses were applied in 10-mV increments from a holding voltage, Vhold = −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 pHo//pHi. D112V exhibited no clear current.
Figure 2
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 (pHi 5.5) at pHo 5.5 (column 1) or 7.0 (column 2), during families of pulses in 10 mV increments up to indicated voltages. Tail currents at pHo 7.0 (column 3) reveal that Vrev deviates from EH, indicating loss of proton selectivity. At pHo 5.5 Vhold was −40 mV (−60 mV for WT). At pHo 7.0 Vhold was −40 mV (K, N, S), −50 mV (F), −60 mV (H, A), −80 mV (E), or −90 mV (WT); Vpre was −65 mV (WT), −40 mV (E), −10 mV (H), +50 mV (K), +40 mV (N, S), or +20 mV (A, F). Vrev (arrows) was determined from the amplitude and direction of tail current decay. For D112N, Vrev was above Vthreshold and was evident during pulse families. b, Shift in Vrev 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 EH.
Figure 3
Figure 3. Dilution of ionic strength by 90% with isotonic sucrose shifted Vrev positively, indicating that most Asp112 mutants are anion selective
a Measurement of Vrev by tail currents in a cell transfected with D112H at pH 5.5//5.5 and (b) after sucrose. c, Vrev in the same cell in pH 5.5 Cl− solution, and (d) after sucrose. Arrows indicate zero current. Vhold = −40 mV, Vpre = +60 mV. e, Mean shifts of Vrev 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 Vrev 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.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. DeCoursey TE. Voltage-gated proton channels and other proton transfer pathways. Physiol Rev. 2003;83:475–579. - PubMed
    1. Capasso M, et al. HVCN1 modulates BCR signal strength via regulation of BCR-dependent generation of reactive oxygen species. Nat Immunol. 2010;11:265–272. - PMC - PubMed
    1. DeCoursey TE, Morgan D, Cherny VV. The voltage dependence of NADPH oxidase reveals why phagocytes need proton channels. Nature. 2003;422:531–534. - PubMed
    1. Henderson LM, Chappell JB, Jones OTG. The superoxide-generating NADPH oxidase of human neutrophils is electrogenic and associated with an H+ channel. Biochem J. 1987;246:325–329. - PMC - PubMed
    1. Musset B, et al. A pH-stabilizing role of voltage-gated proton channels in IgE-mediated activation of human basophils. Proc Natl Acad Sci USA. 2008;105:11020–11025. - PMC - PubMed

Publication types

MeSH terms