The arachidonic acid activated proton flux was absent in the CHO cell lines expressing the mutations of H111/115L, H111/119L or H115/119L, compared to that observed for wild-type Nox2.. T
Trang 1Investigation of the contribution of histidine 119 to the conduction
of protons through human Nox2
Tosti J Mankelow*, X Wen Hu, Kate Adams and Lydia M Henderson
Department of Biochemistry, School of Medical Sciences, University of Bristol, UK
The conduction of protons through human Nox2 has
pre-viously been shown to be dependent upon His115
Align-ment of sequences for both animal and plant Nox proteins
indicated that histidines 115 and 119 are both highly
con-served, while His111 was conserved among animal
homo-logues of Nox1–4 To investigate the possible role that these
histidine residues might play in the conduction of protons
through Nox2, we have introduced both paired and single
mutations into these histidine residues Each construct was
used to generate a CHO cell line in which the expression of
the mutated Nox2 was assessed Nox2 was expressed in each
of the CHO cell lines generated, however, the level of
expression of H111/115L in CHO cells was lower and that of
H111L very much reduced, compared to that of wild-type
Nox2 The arachidonic acid activated proton flux was
absent in the CHO cell lines expressing the mutations of
H111/115L, H111/119L or H115/119L, compared to that observed for wild-type Nox2 Similarly only a small efflux of protons was observed from CHO cells expressing either H119L or H111L In all cases the expected proton flux was elicited through the addition of the protonophore, carbonyl cyanide m-chlorophenylhydrazone Conclusions regarding the role of His111 in the conduction of protons cannot be drawn due to the reduced expression We can, however, conclude that His119, in addition to His115, is required for the conduction of protons through Nox2 His119 has been identified as a highly conserved residue for which no function has previously been proposed
Keywords: gp91phox; membranes; NADPH oxidase; proton transport
The phagocytic white blood cells engulf, kill and digest
microorganisms within the body These cells possess a
membrane bound enzyme, NADPH oxidase, which
gener-ates superoxide (OÆ2 ) and contributes to the killing of the
engulfed microorganisms within the phagosome The
NADPH oxidase is composed of two membrane subunits,
gp91phoxand p22phox, and three cytosolic proteins, p67phox, p47phox and p40phox The predicted binding sites for the enzyme cofactors, FAD and the two b-type heme, are located within gp91phox[1]
A number of proteins with amino acid sequences similar
to that of the human gp91phox have been described in human [2–4], mouse, rat, Caenorhabditis elegans, Dictyo-stelium, rice [5], tomato and Arabidopsis [6,7] genomes The identification of multiple sequences with similarity to gp91phox within the human genome led to the need to rename and number the family of proteins from phox (phagocyte oxidase) to Nox (NADPH oxidase-like) Of the Nox family members, Nox2 is the originally described human phagocyte protein gp91phox In addition, genes encoding dual oxidases (Duox), which contain both a peroxidase and Nox have been identified in a number of different genomes [8]
The generation of superoxide by the phagocytic NADPH oxidase is associated with an efflux of protons [9] The expression of wild-type Nox2 in CHO cells has previously been shown to be associated with the presence of a characterized arachidonic acid activated proton conduction pathway [10,11] The membrane topology of gp91phox is predicted to consist of 4–6 transmembrane domains located within the N-terminal half of the protein followed by a hydrophilic, cytosolic domain [12] The third predicted transmembrane domain of Nox2 contains a sequence
of three regularly spaced histidine residues, HSAIHT-IAH(111–119) The mutation of His115 to Leu has previously been shown to greatly reduce proton conduction,
Correspondence to L M Henderson, Department of Biochemistry,
School of Medical Sciences, University of Bristol, University Walk,
Bristol, BS8 1TD, UK Fax: +44 117 9288274,
Tel.: +44 117 9289046, E-mail: L.M.Henderson@bris.ac.uk
Abbreviations: BCECF-AM, 2¢,7¢-bis-(2-carboxyethyl)-5-(and
-6-)-carboxyfluorescein, acetoxymethyl ester; cPLA 2 , cytosolic
phospho-lipase A 2 ; CHO91, CHO cell line resulting from the transfection of
CHO cells with full-length wild-type human Nox2; CHO91H111/
115L, CHO/Nox2 cell line with His111 and His115 mutated to Leu;
CHO91H115/119L, CHO/Nox2 cell line with His115 and His119
mutated to Leu; CHO91H111/119L, CHO/Nox2 cell line with His111
and His119 mutated to Leu; CHO91H119L, CHO/Nox2 cell line with
single mutation His119 to Leu; CHO91H111L, CHO/Nox2 cell line
with single mutation His111 to Leu.
*Present address: Bristol Institute for Transfusion Sciences,
Southmead Hospital, Bristol, UK.
Present address: Department of Biochemistry, University of Oxford,
UK.
Note: The data presented in this paper have previously been published
as an abstract for the 56th Harden conference on Biological
electron and proton transfer Plymouth, UK, 2003.
(Received 16 July 2004, accepted 20 August 2004)
Trang 2whether introduced into the full-length Nox2 [13] or only
the N-terminal membrane spanning domains [14] Similarly,
proton conduction was greatly reduced if all three histidine
residues were mutated to leucine [13,14] Therefore the
ability of Nox2 to act as proton conduction pathway has
previously been demonstrated to be dependent upon
His115
An alignment of the amino acid sequences for Nox
and Duox proteins available in the genome databases
demonstrated that His115 and His119 in human Nox2
are both conserved through out the animal and plant
sequences of Nox and Duox proteins (Fig 1A) Only the
sequence of Arabidopsis RbohE appeared to lack the
His119 residue However, the introduction of a four
amino acid gap in the sequence improved the sequence
alignment and suggested that both His115 and 119 are
also conserved in RbohE (Fig 1B) His111 is conserved
in Nox2 sequences from different animal species (Fig 1)
Histidines 101, 115, 209 and 222 have been proposed to
function as the 5th and 6th coordinate ligands for the
two b-type hemes due to similarities in sequence position
and spacing of the histidine residues to those of yeast ferric reductase [15] However, no function has previ-ously been proposed for a conserved histidine residue at position 119
The contribution of His119 and/or His111 to the conduction of protons through Nox2 has not previously been investigated To determine whether the mechanism for the conduction of protons through Nox2 requires His111 and/or His119, we have established a number of CHO cells lines which express human Nox2 containing mutations to each of the possible pair of histidine residues (111 + 115,
115 + 119 and 111 + 119) or to individual histidine residues (111 or 119) The CHO cell lines expressing each
of these mutations failed to show an arachidonic acid activated proton flux However the mutation of His111 to Leu resulted in a reduced level of expression of full-length Nox2 Therefore, we conclude that the conduction of protons through Nox2 requires His119 as well as the previously demonstrated role for His115 The possible role for His111 in proton conduction through Nox2 cannot be established from the current data
Fig 1 Amino acid sequence alignment of Nox
and Duox proteins with the third predicted
transmembrane domain of human Nox2 The
amino acid sequences for 40 Nox and Duox
proteins were obtained from the PubMed
nucleotide and protein databases (A) The
alignment of the amino acids from each Nox
and Duox protein with sequence similarity to
that of residues 100–131 in human Nox2 The
arrow indicates the conserved His residue
(119) in addition to the previously reported
conserved histidine at position 115 The
sequences shown include the Arabidopsis
(Rboh), rice (RbohAOsp), tomato
(Rboh-Tomato) and induced in tomato by whitefly
(RbohTomIn), potato (Strboh) and
Dictyos-telium (DictoNox2) The pig and rabbit
Nox1 are incomplete partial sequences lacking
the N-terminus There are sequences for
human Nox5 with and without the extended
N-terminal containing the EF-hands The
residues numbers for both are shown (B) The
introduction of a gap of four amino acids into
the RbohE sequence (residues 473–503) is
necessary to align the histidine residues with
those of the other Arabidopsis, plant and
human Nox2.
Trang 3Materials and methods
The composition of the salt solutions used are as described
previously [11,13]
Sequence alignment
The PubMed protein and nucleotide databases contain
sequence data for a number of Nox and Duox proteins from
human, mouse, rat, cow, bison, pig, dolphin, rabbit,
guinea-pig, Japanese puffer fish, Dictyostelium, Arabidopsis, rice,
tomato, potato and C elegans Alignment of these sequences
in species groups identified a region of sequence similarity
including and in addition to the previously described
conserved histidine at position 115 in human Nox2 The
amino acids with sequence homology to residues 100–131 of
human Nox2 were identified in each Nox and Duox
sequences and aligned usingGENEDOCandDNASTAR The
histidine residues 115 and 119 (human Nox2) are conserved
throughout the 40 sequences currently available Amino
acids 100–129 of human Nox2 are predicted to form the third
transmembrane domain
Mutagenesis of His111, 115 and/or 119 to Leu
in full-length Nox2
cDNAs encoding full-length Nox2 containing each of the
following mutations: His111 & 115 to Leu (H111/115L);
His115 & 119 to Leu (H115/119L); His111 & 119 to Leu
(H111/119L); His111 to Leu (H111L) and His119 to Leu
(H119L) were generated from two overlapping PCR
products as described previously [14], using the specific
oligonucleotide primers contained in Table 1 The two
fragments were annealed and joined by extension from the
overlap region before final PCR amplification using
Full-length Nox2 fwd and Full-Full-length Nox2 rev oligonucleotide
primers (Table 1) These PCR products were inserted as a
HindIII/BamHI fragment into the multiple cloning site of
the plasmid pMEP4 (Invitrogen) Oligonucleotides were
synthesized by MWG-Biotech AG
Construction and maintenance of transfected CHO cell lines
Stable CHO cell lines expressing each of the mutant forms
of full-length human Nox2 (CHO91) were established following transfection by electroporation (230 V, 975 lF)
of the constructs described above, followed by selection with 100 lgÆmL)1 hygromycin b as described previously [11,14] The following stable cell lines were established: CHO91H111/115L (Nox2 with histidines 111 and 115 mutated to leucine); CHO91H115/119L (Nox2 with histi-dines 115 and 119 mutated to leucine); CHO91H111/119L (Nox2 with histidines 111 and 119 mutated to leucine); CHO91H111L (Nox2 with histidine 111 only mutated to leucine) and CHO91H119L (Nox2 with histidine 119 only mutated to leucine)
All CHO cell lines were maintained in Ham’s F-12 nutrient mixture with GlutaMAX-I, 10% (v/v) fetal bovine serum, 50 UÆmL)1penicillin and 50 lgÆL)1 streptomycin The cells were divided 1 : 2 once a week following trypsinization [11]
Expression of the mutant Nox2 proteins Verification of the expression of each of the mutant Nox2 proteins and determination of their cellular localization were assessed by immunostaining and confocal microscopy
as previously described [11,13] The immunocytochemistry was performed with an antipeptide [PRGVHFIFN KENF(558–570)] polyclonal antibody [11,16] upon each
of the CHO91 mutant cell lines Expression of Nox2 was driven from the inducible metallothionein promoter in pMEP4 through the preincubation of the cells with 10 lM
Cd2+for 16–24 h The CHO cell lines were grown on round cover slips for 40–48 h prior to fixation in 4% (v/v) formaldehyde for 10 min and permeabilization with 0.2% (v/v) Triton in phosphate buffered saline (2 min) Binding of the antibody raised against Nox2 was detected with a fluorescein isothiocyanate-labelled anti-rabbit Ig An image
of an optical plane through the cells was collected with a
Table 1 Sequences of the oligonucleotide primers used for the introduction of mutations in full-length Nox2 Full-length Nox2 fwd contains a HindIII restriction site (underlined) for the directional insertion into pMEP4 and covers the N-terminal initiating Met codon (bold) of Nox2 Full-length Nox2 rev contains a BamHI restriction site (underlined) for the directional insertion into the expression vector, pMEP4 and covers the stop codon
of the C-terminus of Nox2 (bold) Full-length Nox2 mutants were generated from two overlapping PCR products (nucleotide 1–352 and 336–1713) which were annealed and joined by extension before final PCR amplification using Full-length Nox2 fwd and Full-length Nox2 rev Bold underlined single nucleotides are the substitutions in the mutant and groups of three nucleotides underlined show the positions of the histidine residues.
Full-length Nox2 fwd CTCAGCTAAGCTTGCCACCATGGGGAAC All full length constructs Full-length Nox2 rev AGCTGAGGATCCCTACCCACGTACAATTCG All full length constructs His111Leu rev CAATGGTGTGAATCGCAGAGAGAAGTG His111/119Leu and His111Leu His115Leu fwd TGCGATTCTCACCATTGCACATCTATTT His111/115Leu
His115Leu rev CAATGGTGAGAATCGCAGAGTGAAGTGC His115/119Leu
His119Leu fwd TGCGATTCACACCATTGCACTTCTATT His111/119Leu and His119Leu His111/115wt rev CAATGGTGTGAATCGCAGTGAGAAGTG His119Leu
His115/119wt fwd TGCGATTCACACCATTGCACATCTATT His111Leu
His115/119Leu fwd TGCGATTCTCACCATTGCACTTCTATTT His115/119Leu
His111/115Leu rev CAATGGTGAGAATCGCAGAGAGAAGTGC His111/115Leu
Trang 4Bio-Rad MRC 600 inverted confocal microscope and
presented as a Kalman average of five successively collected
scans, with excitation at 488 nm The expression levels of all
the mutants were compared to that observed for wild-type
Nox2 in CHO91 cells (positive control) Nonspecific
binding of the antibodies was assessed in nontransfected
CHO cells ± 10 lMCd2+(negative control)
Transmembrane proton flux in mutant CHO91 cell lines
The conduction of protons through the arachidonate
activated, NADPH oxidase associated H+ channel was
assessed as change in pHiwith
2¢,7¢-bis-(2-carboxyethyl)-5-(and -6-)-carboxyfluorescein, acetoxymethyl ester
(BCECF-AM) for all mutant CHO91 cell lines, as described
previously [11,13,14] The pH of the external solution
was rapidly altered by the addition of HEPES (pH 6.6) or
Tris (pH 8.3) The addition of the potassium ionophore,
valinomycin, equilibrates K+ ions across the plasma
membrane, depolarizing the membrane potential of cells
in a high K+solution and hyperpolarizing the membrane
potential if cells are in a Na+ solution (low K+)
Therefore, the addition of Tris and valinomycin to cells
in a high K+medium establishes a strong electrochemical
gradient for the efflux of protons A gradient favouring
the influx of protons is established following the addition
of HEPES and valinomycin to cells in a Na+solution
(low K+)
The predicted third transmembrane helix for Nox2
A predicted structure for amino acids 98–129 of human
Nox2 was modelled by C Dempsey (Department of
Biochemistry, University of Bristol, UK) based on the
assumption that the amino acids of the third predicted
transmembrane domain adopt an a-helix conformation
Results
The conduction of protons through Nox2 was previously
observed to be greatly reduced in CHO cells expressing
Nox2 in which His115 had been mutated to Leu and when
all three histidines, 111, 115 and 119, were mutated to Leu
[13,14] The alignment of Nox protein sequences indicates
that His119 is highly conserved and His111 is conserved in
Nox2 proteins from different animal species However, the
contribution of histidines 111 and 119 to the conduction of
protons through Nox2 has not previously been investigated
despite their possible location on the same face of an a-helix
as His115 To determine the contribution of histidines 111
and 119 to the conduction of protons through human Nox2,
we have established CHO cells that express Nox2, into
which mutations to either paired or single histidine residues
within the third transmembrane domain have been
intro-duced The expression of the resulting protein and
conduc-tion of protons in response to arachidonic acid was assessed
for each of the cell lines
Expression of Nox2 histidine mutations in CHO cells
Table 1 contains the sequences of the oligonucleotide PCR
primers used to introduce the mutations of Hisfi Leu for
the three histidine residues of interest within the third transmembrane domain of Nox2 Each of the three possible combinations of pairs of histidine residues were mutated to leucine (111/115, 115/119 and 111/119) and used to establish stable CHO cell lines, CHO91H111/115L, CHO91H115/ 119L and CHO91H111/119L Transcription and transla-tion of the Nox2 gene is under the control of an inducible metalothionine promoter (10 lMCd2+for 16 h) Immuno-staining, using an antibody raised against the Nox2 C-terminal, was used to assess the inducible expression of Nox2 protein in each of the cells lines
The immunostaining was observed to be greater for CHO91 (Fig 2A), CHO91H115/119L (Fig 2C), CHO91H111/115L (Fig 2E) and CHO91H111/119L (Fig 2G) when the cells had been grown in the presence of
10 lMCd2+compared to the same CHO cell lines grown in the absence of Cd2+(Figs 2B,D,F,H, respectively) Non-transfected CHO cells showed low levels of immunostaining whether grown in the presence (Fig 2I) or absence (Fig 2J)
of Cd2+ Therefore, each of the CHO cell lines show an induced expression of protein, which was dependent upon transfection with Nox2 cDNA The level of expression observed in CHO91H111/115L cells, in which histidines 111 and 115 were both mutated to leucines (Fig 2E) was lower than that observed for CHO91 with the wild-type Nox2 (Fig 2A), while the expression of Nox2 in each of the remaining mutant CHO cell lines (Fig 2C,G) was compar-able to that observed for CHO91 cells (Fig 2A) as observed previously [11,13,14]
Arachidonic acid activated proton conduction The arachidonic acid activated proton conduction pathway has previously been reported to be greatly reduced by the mutation of His115 to Leu compared to that observed for wild-type Nox2 [13,14] To assess the contribution of histidines 119 and 111 to the conduction of protons through Nox2, the ability of CHO91H111/115L, CHO91H115/119L and CHO91H111/119L cells to conduct protons in response
to stimulation by arachidonic acid was assessed Electro-chemical gradients for either proton influx (Fig 3A–C) or proton efflux (Fig 3D,E) were imposed upon the cells prior
to the addition of arachidonic acid CHO91H115/119L (Fig 3C) cells showed little change in pHi in response
to arachidonic acid compared with the response of CHO91 cells (Fig 3A) expressing human Nox2 A similar low conduction of protons following the addition of sodium arachidonate was observed for CHO91H111/115L (Fig 3E) The observed very low conduction of protons for both CHO91H111/119L and CHO9H115/119L cells is as would be predicted and may be the result solely of the mutation of His115 to Leu in both of these cell lines However, CHO91H111/119L cells failed to exhibit an arachidonic acid activated conduction of protons whether assessed as a proton influx (Fig 3B) or as a proton efflux (Fig 3D) The subsequent addition of the protonophore, carbonyl cyanide m-chlorophenylhydrazone, resulted in the observed pHi change expected for the established proton electrochemical gradient in all cases (Fig 3A–E) CHO91, CHO91H111/115L, CHO91H115/119L and CHO91H111/ 119L all demonstrated a similar pHichange in response to a sodium acetate imposed acid pulse (Fig 3F) indicating that
Trang 5Fig 2 Expression of wild-type and double histidine mutations of Nox2 in CHO cell lines The expression of full-length Nox2 was determined by immunocytochemistry as described in the Materials and methods The inducible expression of Nox2 was determined for CHO91 (A and B), CHO91H115/119L (C and D), CHO91H111/115L (E and F), CHO91H111/119L (G and H) and for nontransfected CHO cells (I and J) The CHO cells were incubated in the presence of 10 l M Cd 2+ for 16 h prior to fixation and staining (A, C, E, G and I) The images are Kalman averages of five successively collected scans The intensity of the fluorescence is represented by a pseudocolour scale where red is highest and blue
is lowest.
Trang 6the cytoplasm in each of these cell lines has a similar
buffering capacity, with that of CHO91H111/119L a little
lower The calibration of BCECF-AM response to pHi
was similar in all four cell lines (Fig 3G) We therefore
conclude that the absence of an arachidonate activated
proton conduction in CHO91H111/119L cells suggests that
His111 and/or His119 are required, in addition to His115,
for the conduction of protons through Nox2
Mutation of individual histidine residues in human Nox2
To investigate the individual roles of His111 and His119, in
the conduction of protons through Nox2, two CHO cells
lines were constructed, CHO91H111L and CHO91H119L,
in which the single Hisfi Leu mutations had been
intro-duced The expression of Nox2 in which the histidine at
position 119 had been mutated to leucine was inducible in
CHO91H119L cells (Fig 4A) and the protein was expressed
at a level comparable with that observed for wild-type
Nox2 (Fig 2A) However, the expression of Nox2 in
CHO91H111L cells, in which the histidine at position 111
had been mutated to leucine, although enhanced by
activation of the inducible promoter, was observed to be
significantly lower than that of wild-type Nox2 (Figs 4B
and 2A)
CHO91H119L cells in the presence of a transmembrane gradient for the efflux of protons exhibited a very small conduction of protons following the addition of arachido-nate (Fig 4E) The efflux of protons was similar to that observed for CHO91H111/119L cells (Fig 3D) The efflux
of protons was only observed following the subsequent addition of carbonyl cyanide m-chlorophenylhydrazone (Fig 4E) The reduction in proton efflux observed for CHO91H119L cells is not due to a reduction in the level of protein expression as a result of the introduction of this mutation into Nox2 Therefore, we can conclude that the reduced proton efflux is as a consequence of the mutation of His119 to Leu, and therefore that His119 is required for the conduction of protons through Nox2
We note that proton efflux was also not observed following the addition of arachidonate to CHO91H111L cells suspended in a K+ medium (not shown) However, due to the reduced level of expression of H111L Nox2 in CHO compared with wild-type and the other mutations described here and reported previously [13,14], no conclu-sions can be draw regarding the role of His111 in the conduction of protons through Nox2
Therefore, we can conclude that the conduction of protons through human Nox2 requires both His119 and His115
Fig 3 Arachidonic acid stimulated proton flux The CHO cell lines were incubated with 10 l M Cd 2+ for 16 h prior to the assay Cells from each of the CHO cell lines were harvested and loaded with BCECF-AM as described in the Materials and methods Following the addition of the potassium ionophore, valinomycin (2.7 l M ), the transmembrane potassium gradient dictates the membrane potential The addition of 5 m M HEPES rapidly decreases the external pH to 6.6, or to pH 8.3 following the addition of 5 m M Tris This established an electrochemical gradient for the influx (A–C)
or efflux (D and E) of protons The influx of protons was assessed for control CHO91 cells expressing wild-type gp91 phox (A), CHO91H111/119L (B) and CHO91H115/119L (C), all suspended in the Na+medium In addition the efflux of protons was assessed in both CHO91H111/119L (D) and CHO91H111/115L (E) with cells suspended in a K + medium (A–E) The opening of the proton conduction pathway and the transport of protons was monitored as an alteration in internal pH, following the addition of 10 l M arachidonic acid (AA) The maximum change in pH i was determined following the addition of the protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP; 66 l M ) where indictated The requirement for a second addition of CCCP in (E), in order to elicit a full pHi change, probably results from a larger number of cells The buffering capacity was assessed following the addition of 26 m M NaAc (F) to CHO91H115/119L, CHO91H111/119L, CHO91, CHO91H111/115L (left to right) and the response of the BCECF-AM to pH was calibrated (G) in CHO91 (j), CHO91H111/119L (m), CHO91H115/119L (d) and CHO91H111/115L (r).
Trang 7It has previously been shown that His115 is required for the
conduction of protons through human Nox2, in either the
full-length protein [13] or the N-terminal membrane domain
alone [14] The third transmembrane domain of Nox2
contains three histidine residues separated from each other
by three amino acid residues [14] In this paper we have
generated CHO cell lines expressing human Nox2 into
which double and single mutations to histidines 111, 115
and 119 had been introduced Mutation of His119 to Leu
severely reduced the arachidonic acid activated conduction
of protons We therefore conclude that His119, as well as
His115, is required for the conduction of protons through
human Nox2 The role of His111 in proton conduction
cannot be firmly established from the current data The
alignment of sequences for Nox proteins indicates that
His119 is a highly conserved residue and yet no role for the His119 has previously been proposed
The M2 protein is an integral membrane, coat protein of the influenza A virus It has previously been reported to function as a proton channel [17–21] The virus enters the cells via endocytosis The M2 protein proton channel is opened by the acidic pH environment of the endosomes, resulting in a fall in pH of the viral particle, leading to dissociation of the viral coat and release of the viral genome [17,22] Amantidine, an anti-influenza drug, acts as
an inhibitor of the influenza A M2 proton channel and hence prevents the uncoating of the viral particle and release of its genome into the cell [18,23] Therefore, the influx of protons through the M2 protein is important in the life cycle of the virus The M2 protein has a histidine residue, His37, located in the transmembrane domain, which is required for the opening of the channel by acidic
pH and for the influx of protons through the channel [22,24] A possible cation–p interaction between the prot-onated imidazole ring of His37 and the indole ring of Trp41 has been identified It has recently been proposed that the Trp41 side chain blocks the pore of the channel and that the interaction between protonated His37 and Trp41 pulls the Trp41 side chain out of the pore of the channel, thereby opening the M2 proton channel [22,25] A tryptophan occurs at position 125 of human Nox2, located within the transmembrane domain that also contains His111, 115, and 119 The model of this region as an a-helix places Trp125 two turns of the helix above His119 and not on the same face of the helix Therefore, the distance and the orientation of His119 to Trp125 is too great to permit a cation–p interaction similar to that described for the pH stimulated gating of the M2 protein
by the Trp41–His37 interaction [22,25]
A role for cytosolic phospholipase A2 (cPLA2) in the opening of the NADPH oxidase associated proton channel has previously been demonstrated through the development and use of a cPLA2deficient PLB-985 cell line (PLB-D cells) [26–28] Gating of the proton channel formed by either full-length human Nox2 [27] or just the N-terminal membrane domain alone [28] have both been reported to be deficient in PLB-D cells The requirement for cPLA2 for both the activation of superoxide generation [26] and the gating of the proton channel [27,28] can be over come by the addition
of arachidonate acid The site of interaction with and the mechanism by which arachidonate results in the activation
of both the NADPH oxidase and the gating of the proton channel is at present unknown
In this paper we have demonstrated a requirement for His119 in the conduction of protons through human Nox2 This, combined with the previously described role for His115 in proton conduction, suggests a mechanism that involves passage of the proton from one histidine residue to the next
Acknowledgements
The authors wish to thank Dr Chris Dempsey, Department of Biochemistry for the modelling of the transmembrane domain as an a-helix The work was supported by Grant No HO604 from the Arthritis Research Campaign and a MRC PhD studentship to T.J.M.
E
D C
Fig 4 Expression of the single mutations, H119L and H111L, in CHO
cells and determination of proton efflux from CHO91H119L cells The
expression of mutant Nox2 in CHO91H119L (A and B) and
CHO91H111L cells (C and D) was assessed as described in the
Materials and methods The CHO91H119L (A) and CHO91H111L
(C) were grown in the presence (A,C) or absence (B,D) of 10 l M Cd 2+
for 16 h prior to the assay The efflux of protons from CHO91H119L
cells preincubated with 10 l M Cd2+(E) was determined as described in
the Materials and methods The CHO91H119L cells were resuspended
in a K + medium and additions of 2.7 l M valinomycin, 5 m M Tris,
10 l M arachidonate (AA) and 66 l M carbonyl cyanide
m-chlorophe-nylhydrazone (CCCP) were made where indicated in the figure (E).
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Supplementary material
The following material is available from http://www blackwellpublishing.com/products/journals/suppmat/ EJB/EJB4340/EJB4340sm.htm
Table S1 The Nox and Duox gene sequences used to construct the alignment in Fig 1
Fig S1 The third predicted transmembrane helix of Nox2