Arabi CLC__JBC_really_final_revision_March3_09_unmarked

17 Pages·2009·0.78 MB·English

JBC Papers in Press. Published on March 4, 2009 as Manuscript M901170200
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M901170200

RESIDUES IMPORTANT FOR NITRATE/PROTON COUPLING IN
PLANT AND MAMMALIAN CLC TRANSPORTERS*
Bergsdorf, Eun-Yeong 1, Anselm A. Zdebik1,2 and Thomas J. Jentsch1
From 1Leibniz-Institut für Molekulare Pharmakologie (FMP) and Max-Delbrück-Centrum für
Molekulare Medizin (MDC), D-13125 Berlin, Germany, and 2 University College London
(UCL), Department of Neuroscience, Physiology and Pharmacology, London Epithelial
Group, Royal Free Campus, London NW3 2PF, United Kingdom
Running head: Nitrate-proton coupling in CLC transporters
Address correspondence to: Thomas J. Jentsch, FMP/MDC, Robert-Rössle-Str. 10, D-13125
Berlin, Germany. Fax: [phone]; E-mail: [email protected]

Members of the CLC gene family either CLC proteins are found in all phyla from bacteria
function as chloride channels or as to men and either mediate electrogenic
anion/proton exchangers. The plant AtClC-a anion/proton exchange or function as chloride
uses the pH gradient across the vacuolar channels (1). In mammals, roles of plasma
membrane to accumulate the nutrient NO - in membrane CLC Cl- channels include
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this organelle. When Arabidopsis thaliana transepithelial transport (2-5) or control muscle
AtClC–a was expressed in Xenopus oocytes, it excitability (6), whereas vesicular CLC D
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mediated NO3-/H+- and less efficiently Cl-/H+- exchangers may facilitate endocytosis (7) and wnlo
exchange. Mutating the ‘gating glutamate’ lysosomal function (8-10) by electrically shunting a
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Replacing the ‘proton glutamate’ E270 b3y isoforms (AtClC-a, -g) (12-15) which may mostly http
alanine abolished currents. These could be reside in intracellular membranes. AtClC-a uses ://w
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restored by the uncoupling E203A mutation. the pH gradient across the vacuolar membrane to w
Whereas mammalian endosomal ClC-4 and transport the nutrient nitrate into that organelle .jb
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ClC-5 mediate stoichiometrically coupled 2Cl- (16). This secondary active transport requires a .org
/H+-exchange, their NO3- transport is largely tightly coupled NO3-/H+-exchange. Astonishingly, by g/
uncoupled from protons. By contrast, the however, mammalian ClC-4 and -5 and bacterial u
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AtClC-a-mediated NO3- accumulation in plant ecClC-1 display tightly coupled Cl-/H+-exchange, st on
vacuoles requires tight NO - to H+-coupling. but anion flux is largely uncoupled from H+ when A
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Comparison of AtClC-a and ClC-5 sequences NO3- is transported (17-21). The lack of ril 9
identified a proline in AtClC-a that is replaced appropriate expression systems for plant CLC , 2
0
by serine in all mammalian CLC isoforms. transporters (12) has so far impeded structure- 19
When this proline was mutated to serine function analysis that may shed light on the
(P160S), Cl-/H+ exchange of AtClC-a ability of AtClC-a to perform efficient NO -/H+-
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proceeded as efficiently as NO -/H+-exchange, exchange. This dearth of data contrasts with
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suggesting a role of this residue in NO -/H+- extensive mutagenesis work performed with
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exchange. Indeed, when the corresponding CLCs from animals and bacteria.
serine of ClC-5 was replaced by proline, this The crystal structure of bacterial CLC
Cl-/H+-exchanger gained efficient NO -/H+- homologues (22,23) and the investigation of
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coupling. When inserted into the model mutants (17,19-21,24-29) have yielded important
Torpedo chloride channel ClC-0, the equivalent insights into their structure and function. CLC
mutation increased nitrate relative to chloride proteins form dimers with two largely
conductance. Hence, proline in the CLC pore independent permeation pathways (22,25,30,31).
signature sequence is important for NO -/H+- Each of the monomers displays two anion binding
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exchange and NO - conductance both in plants sites (22). A third binding site is observed when a
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and mammals. Gating and proton glutamates certain key glutamate residue, which is located
play similar roles in bacterial, plant, and halfway in the permeation pathway of almost all
mammalian CLC anion/proton exchangers. CLC proteins, is mutated to alanine (23).
Mutating this ‘gating’ glutamate in CLC Cl-
channels strongly affects or even completely
1
Copyright 2009 by The American Society for Biochemistry and Molecular Biology, Inc.
suppresses single pore gating (23), whereas CLC were obtained by surgery from deeply
exchangers are transformed by such mutations anaesthetized (0.1 % tricaine (Sigma Aldrich))
into pure anion conductances that are not coupled pigmented or albino Xenopus laevis frogs.
to proton transport (17,19,20). Another key Oocytes were prepared by manual dissection and
glutamate, located at the cytoplasmic surface of collagenase A (Roche Applied Science) digestion.
the CLC monomer, seems to be a hallmark of 23 ng (ClC-5), 46-50 ng (AtClC-a) or 1-3 ng
CLC anion/proton exchangers. Mutating this (ClC-0) cRNA were injected into oocytes.
‘proton glutamate’ to non-titratable amino acids Oocytes were kept in ND96 solution (containing
uncouples anion transport from protons in the (in mM) 96 NaCl, 2 KCl, 1.8 CaCl ., 1 MgCl , 5
2 2
bacterial ecClC-1 protein (27), but seems to HEPES, pH 7.5) at 17 °C for 1-2 days (ClC-0), 3-
abolish transport altogether in mammalian ClC-4 4 days (ClC-5) or for 5-6 days (AtClC-a). Two-
and -5 (21). In those latter proteins, anion electrode voltage-clamp was performed at room
transport could be restored by additionally temperature (20-24 °C) using a TEC10 amplifier
introducing an uncoupling mutation at the ‘gating (npi, Tamm, Germany) and pClamp9 software
glutamate’ (21). (Molecular Devices). The standard bath solution
The functional complementation by AtClC-c contained (in mM): 96 NaCl, 2 K+-gluconate, 5
and –d (12,32) of growth phenotypes of a yeast Ca++ D-gluconate, 1.2 MgSO , 5 HEPES, pH 7.5
4
strain deleted for the single yeast CLC Gef1 (33) (or MES for buffering to pH 5.5 or 6.5; Tris for
suggested that these plant CLCs function in anion pH 8.5). For some experiments, NaCl was
transport, but could not reveal details of their substituted with equal amounts of either NaNO ,
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biophysical properties. We report here the first NaBr, or NaI. Ag/AgCl electrodes and 3M KCl w
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functional expression of a plant CLC in animal agar bridges were used as reference and bath lo
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cells. Expression of WT and mutant AtClC-a in electrodes, respectively. ed
Xenopus oocytes indicate a general role of Measurement of relative intracellular pH changes fro
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‘gating’ and ‘proton’ glutamate residues in using the “Fluorocyte” device - Proton transport h
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anion/proton coupling across different isoforms was measured semi-quantitatively by monitoring ://w
and species. We identified a proline in the CLC intracellular fluorescence signal changes using the w
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signature sequence of AtClC-a that plays a crucial Fluorocyte device (21). Briefly, 23nl of saturated .jb
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role in NO -/H+-exchange. Mutating it to serine, aqueous solution of the pH indicator BCECF .o
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the residue present in mammalian CLCs at this (Molecular Probes) were injected into oocytes 10 b/
y
position, rendered AtClC-a Cl-/H+-exchange as to 30 min before measurements. Oocytes were g
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efficient as NO3-/H+-exchange. Conversely, placed over a hole of 0.8 mm in diameter, through est o
changing the corresponding serine of ClC-5 to which BCECF fluorescence changes were n A
pexroclhinane gecro.n vWerhteedn ipt roinlitnoe arne pelaffciecdie ntth eN Ocr3i-t/iHca+-l mseervaseudr edto inr edruecspe ontshee tpoo spsuiblslee tarcatiinvsa,t iownh icohf pril 9, 2
serine in Torpedo ClC-0, the relative NO - endogenous oocyte currents. Starting from a 01
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conductance of this model Cl- channel was holding V of -60 mV, depolarizing pulse trains
drastically increased and ‘fast’ protopore gating clamped oocytes to +90 mV for 400 msec and -
slowed. 60mV for 100 ms, whereas hyperpolarizing pulse
trains started from -30 mV and clamped to -160
mV for 400 ms and to -30mV for 100 ms. BCECF
EXPERIMENTAL PROCEDURES fluorescence was measured with a photodiode and
digitally Bessel-filtered at 0.3 Hz. These non-
Molecular biology - cDNAs of Arabidopsis ratiometric measurements generally show drifts
thaliana AtClC-a (12), rat ClC-5 (34), and owed to bleaching or intracellular dye distribution
Torpedo marmorata ClC-0 (35) were cloned into (21), but allow for sensitive measurements of pH
the pTLN (36) expression vector. Mutations were changes upon changes in voltage or external ion
generated by recombinant PCR and confirmed by composition.
sequencing. Capped cRNA was transcribed from
linearized plasmids using the Ambion
mMESSAGE mMACHINE kit (SP6 RNA RESULTS
polymerase for pTLN) according to
manufacturer’s instruction. Previous attempts to functionally express plant
Expression in Xenopus oocyte and two-electrode CLC proteins in animal cells proved unsuccessful
voltage clamp (TEVC) studies - Pieces of ovary (12). However, when Xenopus oocytes were
2
measured five or more days after injecting AtClC- NO - failed to influence the rate of acidification
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a cRNA, currents well above background were because proton influx is coupled to an efflux of
observed in two-electrode voltage-clamp anions from the interior of oocytes.
experiments (Fig. 1). These outwardly rectifying In ecClC-1, ClC-4 and -5, a certain ‘gating
currents were roughly 30% larger when glutamate’ is important for coupling chloride
extracellular chloride was replaced by nitrate fluxes to proton countertransport (17,19,20). In
(NO -) (Fig. 1A-C), smaller with extracellular crystals of the bacterial protein, the negatively
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iodide, and nearly unchanged with a replacement charged side chain of this glutamate seems to
by bromide (Fig. 1C). Reversal potentials block the permeation pathway (22,23). Mutating
indicated that the apparent anion permeability was this glutamate to alanine in ClC-4 and -5 not only
larger for NO - than for other anions tested. It leads to flux uncoupling, but also abolishes the
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should be noted that for CLC exchangers the strong outward rectification of either transporter
observed apparent permeabilities and (26). Likewise, currents of the E203A mutant had
conductances represent those of coupled lost their outward rectification (Fig. 2). Rather
anion/proton exchange rather than diffusive anion than being linear, and again similar to the
transport. Contrasting with the slow activation of analogous mutants of ClC-4 and -5 (19,20,26),
currents by depolarization observed in plant currents showed slight outward rectification at
vacuoles (16), heterologously expressed AtClC-a positive and slight inward rectification at negative
currents almost totally lacked time-dependent voltages. Contrasting with moderate changes in
relaxations (Fig. 1A,B). Like currents elicited by ion selectivity observed with such mutations in
D
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ClC-4 or -5 (26), AtClC-a currents were reduced ClC-4 and -5 (26), the E203A mutation w
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by acidic extracellular pH (pH ) (Fig. 1D-F). drastically altered the anion selectivity of AtClC- lo
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current decrease than with Cl- (Fig. 1F). for Cl- (Fig. 2C, inset), its conductance was now h
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We next explored proton transport of reduced rather than increased when extracellular ://w
AtClC-a by measuring semi-quantitatively pH of Cl- was replaced by NO - (Fig. 2A-C, Fig. 4F). w
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voltage-clamped oocytes using the Fluorocyte With either external anion, currents were .jb
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system (21). Net ion transport was elicited by insensitive to changes in pH (Fig. 2D,E) and .o
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strongly depolarizing oocytes to +90 mV. trains of depolarizing pulses failed to change pHi by/
Because prolonged strong depolarization can (Fig. 2F). Hence, the E203A mutant displays g
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elicit endogenous transport processes in oocytes, uncoupled anion transport and drastically reduced st o
trains of depolarizing pulses were used instead conductance in the presence of NO3-. n A
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electrogenic anion/proton exchangers. Pulsing mutations of ecClC-1 (27), ClC-4 and -5 (21) 1
9
AtClC-a expressing oocytes to positive voltages (Fig. 3). Neither currents (Fig. 3A-C), nor
indeed induced intracellular alkalinization (Fig. depolarization-induced H+-transport (Fig. 3G,H)
1G). Importantly, alkalinization was also were different from background with this mutant.
observed when protons must be extruded against However, when being combined with the
their electrochemical potential (at pH =5.5), uncoupling E203A mutation in the ‘gating
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suggesting that their transport is driven by the glutamate’, the AtClC-a(E203A, E270A) double
coupled anion entry (Fig. 1H). Under either mutant gave currents which resembled those of
condition (pH =7.5 or 5.5), alkalinization the single E203A mutant (Fig. 3D-F). Like
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occurred more rapidly with extracellular NO - AtClC-a(E203A), the double mutant failed to
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than with Cl-. This shows that AtClC-a more transport protons in response to depolarization
efficiently mediates NO -/H+- than Cl-/H+- (Fig. 3I).
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exchange, a finding compatible with its role in Two main biophysical differences between
accumulating NO - in plant vacuoles (16,37). AtClC-a and ClC-4 and -5 are (i) the much
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Contrasting with ClC-4 and -5 (26), AtClC-a stronger rectification of the mammalian isoforms
mediates robust currents also at negative voltages and (ii) their partial uncoupling of anion from
(Fig. 1A-E). Accordingly, and in contrast to ClC-5 proton transport in the case of nitrate. We
(Fig. 5H), AtClC-a mediated proton influx when therefore searched for differences in their primary
oocytes were pulsed to negative voltages (-160 sequences that may underlie these differences. A
mV) (Fig. 1I). Substituting extracellular Cl- by salient feature is the presence of a proline in a
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stretch of highly conserved ‘signature’ sequence relaxations when jumping to positive voltages. In
(Fig. 4A). In AtClC-a, this proline replaces a contrast to WT ClC-5 (Fig. 5G), trains of
serine that is found at this position in most CLCs. depolarizing pulses alkalinized the oocyte interior
The importance of that serine was recognized much more rapidly when extracellular Cl- was
early on (25). Even the conservative exchange of replaced by NO - (Fig. 5I). ClC-5(S168P)
3
this serine for threonine (S123T) changed the ion transported H+ efficiently against its
selectivity, single-channel conductance and open- electrochemical gradient (with pH =5.5) using
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channel rectification of the ClC-0 Cl- channel NO - as driving ion (Fig. 5J). When S168 of ClC-
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(25). In the crystal of ecClC-1, the equivalent 5 was replaced by glycine or alanine, only the
serine (S107) participates in the coordination of S168A mutation yielded a moderately higher
Cl- in the central binding site (22). Mutating this I(NO -)/I(Cl-) current ratio (Fig. 5K). This
3
particular proline in AtClC-a to the ‘consensus’ contrasts with AtClC-a, where glycine could
serine (P160S) resulted in less rectifying currents substitute for proline without compromising its
(Fig. 4B-D), indicating that the difference in I(NO -)/I(Cl-) ratio (Fig. 4K) and its efficient NO -
3 3
rectification between AtClC-a and ClC-5 WT /H+-exchange. Hence, a single mutation that
proteins is not determined by the residue at this replaced a serine by proline, which is found at
position. Importantly, both the apparent that position in AtClC-a, strongly increased the
permeability (from reversal potentials) and preference of ClC-5 for NO - and converted it into
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conductance no longer differed measurably an efficient NO -/H+-exchanger.
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between NO - and Cl- solutions (Fig. 4B-D). The uncoupling E211A mutation in the ClC-
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Furthermore, the mutant performed Cl-/H+- and 5 gating glutamate slightly increased its relative ow
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NO -/H+-exchange with similar efficiencies (Fig. nitrate conductance (Fig. 5K), whereas the lo
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4E). When this proline was mutated to glycine equivalent mutation in AtClC-a quite drastically ed
(P160G), the ratio of currents measured in the lowered its nitrate vs. chloride conductance (Fig. fro
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presence of extracellular NO - (I(NO -)) to those 4F). Exchanging serine168 for proline in the h
measured with extracellular C3l- (I(Cl-)3) resembled uncoupled ClC-5 mutant increased relative ttp://w
the ratio I(NO -)/I(Cl-) of WT AtClC-a (Fig. 4F), conductance in the presence of NO - (Fig. 5K), w
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with which it also shares the higher efficiency of whereas a similar exchange in the uncoupled .jb
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NO -/H+-exchange as compared to Cl-/H+- AtClC-a mutant failed to change the I(NO -)/ I(Cl- .o
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exchange (data not shown). When the P160S ) current ratio (Fig. 4F). b/
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mutation was combined with the uncoupling We finally asked whether also CLC Cl- g
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mutation in the gating glutamate, the resulting channels gain higher NO3- conductance with an est o
double mutant AtClC-a(P160S, E203A) displayed equivalent mutation and used the Torpedo Cl- n A
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proton coupling (data not shown). S123P mutant gave robust currents that were, 1
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The above experiments indicate that the however, about 4-fold smaller than those from
presence of proline in the CLC signature sequence WT ClC-0 (Fig. 6A,D). Whereas the WT channel
may be responsible for the efficient NO -/H+- conducts Cl- much better than nitrate (Fig. 6A-C),
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coupling of AtClC-a. We asked next whether NO - conductance was larger than Cl-
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changing the equivalent serine to proline in ClC-5 conductance in the S123P mutant (Fig. 6D-F).
would increase its efficiency of NO -/H+- The selectivity was changed also for other anions,
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coupling. In the presence of extracellular chloride, as evident e.g. in the large increase of Br-
currents from the ClC-5(S168P) (Fig. 5D) were conductance (Fig. 6F). In addition, the mutation
much smaller (~5 to 10-fold) than WT currents drastically slowed the fast protopore gate (as
(Fig. 5A). They increased about 3-fold in the evident from current relaxations after stepping to
presence of extracellular NO - (Fig. 5E), agreeing negative voltages, Fig. 6A,D) and introduced an
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with recent parallel work by Zifarelli and Pusch open-pore outward-rectification (Fig. 6C,F). The
(38). While currents of WT ClC-5 are also larger concomitant change of pore and gating properties
with NO - than with Cl- (Fig. 5A-C), the I(NO - reflects the tight coupling of permeation and
3 3
)/I(Cl-) ratio is markedly increased in the mutant gating in CLC Cl- channels (39).
(Fig. 5D-F,K). The strong rectification of ClC-5
currents is not appreciably affected by the S168P
mutation (Fig. 5A-F), but currents showed
somewhat more pronounced and slower ‘gating’
4
DISCUSSION may be influenced by different lipid compositions
of membranes from oocytes and plant vacuoles,
We have achieved for the first time the functional or AtClC-a might endogenously form complexes
expression of a plant CLC transporter in animal with other proteins. These other proteins may
cells. This enabled us to study currents and proton include structurally unrelated ancillary β-subunits
transport of wild-type and mutant AtClC-a, a (as known for some mammalian CLCs (10,41)),
NO -/H+-exchanger that serves to accumulate the other AtClC isoforms (12) (CLC proteins function
3
plant nutrient NO - in vacuoles (16,37). We have as dimers (22,25,30)), or anchoring proteins.
3
studied the role in AtClC-a of key glutamate The outward rectification of AtClC-a is less
residues that are important for anion/proton strong than that of ClC-4 and -5, both of which do
coupling in bacterial and mammalian CLC not transport measurably at negative voltages
isoforms and have shown that a proline-serine (26). This difference is not owed to the presence
exchange in a highly conserved stretch (GSGIPE, of proline instead of serine in the signature
the ‘CLC signature sequence’) strongly affects sequence of AtClC-a, as revealed by our
nitrate/proton transport in AtClC-a and ClC-5. mutagenesis experiments. The weaker
In our previous study, we were unable to rectification of AtClC-a may be crucial for the
detect plasma membrane currents in Xenopus proton-driven uptake of NO - into vacuoles which
3
oocytes injected with AtClC-a to AtClC-d (12). may have a lumen-positive voltage. The much
Currents reported previously for the oocyte- stronger rectification of ClC-4 and -5 is a priori
expressed tobacco CLC ClC-Nt (40) resemble difficult to reconcile with their presumed role in
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endogenous oocyte currents (12). Hence the electrical compensation of H+-ATPase currents ow
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present study may represent the first functional (11). lo
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characterization of plant CLCs in animal cells. The heterologous expression of AtClC-a ed
This renders AtClC-a accessible to structure- allowed the first structure-function analysis of a fro
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function analysis by mutagenesis. The most plant CLC protein. We began with studying two h
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important technical differences to our previous glutamate residues that are conserved in all ://w
study (12) are the injection of about twice the confirmed CLC exchangers. The ‘gating w
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amount of RNA and a longer time of expression. glutamate’ is important for anion to proton .jb
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Whereas we previously measured oocytes three coupling in CLC exchangers, as well as for gating .o
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days after injection, we now found that AtClC-a in CLC Cl- channels. Its replacement in AtClC-a b/
y
currents rise above background levels only after 5 by alanine uncoupled anion flux from protons like g
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days. Because AtClC-a is physiologically in other CLC transporters (17,21). Similar to ClC- st o
expressed in the plant vacuole (16), the presence 4 and ClC5, this mutation also drastically changed n A
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The currents reported here for oocyte- equivalent mutations in ClC-4 and -5, this 1
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expressed AtClC-a resemble in many aspects uncoupling AtClC-a mutation also drastically
currents from Arabidopsis vacuoles that were changed the I(NO -)/I(Cl-) current ratio. This
3
studied by patch-clamp in the whole-vacuole change did not depend on the presence of a
configuration and that were absent in strains with proline in the signature sequence.
AtClC-a gene deletions (16). Both currents In CLC anion/proton exchangers the
showed similar rectification and proton coupling. pathways of Cl- and H+ diverge at a point
However, there are also conspicuous differences. approximately half-way through the membrane
When studied under asymmetric ionic conditions and reach the cytoplasm at different points (27). A
with NO - in the pipette (vacuole) and Cl- in the ‘proton glutamate’ at the cytoplasmic CLC
3
bath (cytosol), which resembles our oocyte surface is thought to bind protons, handing them
experiments with external NO -, vacuolar currents over to the ‘gating glutamate’ using a poorly
3
showed prominent, depolarization-induced defined path. In both mammalian and E.coli CLC
activation that remained incomplete after several exchangers this glutamate could be replaced by
seconds (16). Such a current relaxations were other titratable amino acids without abolishing
clearly absent from our recordings. Moreover, anion/proton coupling (21,27,42). When mutated
whereas our currents increased with extracellular to non-titratable residues, however, Cl- and H+
NO - only about 1.6-fold, this increase was much transport were below detection levels in ClC-4
3
more drastic with vacuolar currents (16). Several and -5 (21), whereas small, uncoupled anion
explanations may be invoked. Current properties currents were observed with ecClC-1 (42). This
5
can be rationalized by a blockade of anion/proton were reported to complement (12,32) growth
exchange at the ‘gating glutamate’ when the phenotypes of a S. cerevisiae strain deleted for the
supply of protons ceases (21). The uncoupled, but single yeast CLC (ScClC or Gef1(33)). Whether
reduced anion flux in ecClC-1 might be owed to this is related to the fact that AtClC-c and –d, just
‘slippage’ past the central exchange site (42). This like ScClC, carry serine in their signature
model is strongly supported by double mutants in sequence and are hence probably prefer chloride
ClC-4 and -5, in which the uncoupling ‘gating over nitrate, remains unclear.
glutamate’ mutation rescued uncoupled anion flux The interpretation of conductance ratios of
(21). Exactly this situation was found here with ClC-5, and by extension of AtClC-a, is
AtClC-a. complicated by results from noise analysis that
It was puzzling that anion transport of ClC- indicate that ClC-5 switches from transporting to
4, -5, and ecClC-1 is largely uncoupled from non-transporting modes of operation (21). Such a
proton transport with the polyatomic anion NO - ‘gating’ of transport activity probably underlies
3
(21), whereas AtClC-a efficiently couples NO - to the time-dependent current relaxation upon
3
proton countertransport (16) – an essential depolarization of ClC-5 and might also explain
property for its role in accumulating this plant the slow depolarization-induced activation of
nutrient in vacuoles. By sequence comparison, we vacuolar currents observed by de Angeli et al.
identified a proline in the CLC signature sequence (16). Zifarelli and Pusch recently concluded from
as a likely candidate for this difference. In all their noise analysis that the increase in ClC-5
animal CLCs, this position is occupied by serine. currents with NO - is not due to an increased
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Indeed, when P160 in AtClC-a was mutated to ‘unitary conductance’ of the ‘turned on’ w
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serine, the plant transporter performed Cl-/H+- transporter (which would include ‘slippage’ of the lo
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WT. Importantly, when the equivalent serine of that the difference in I(NO -)/I(Cl-) ratios between h
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ClC-5 was mutated to proline, ClC-5 mediated WT and AtClC-a (P160S) is solely due to an ://w
efficient NO -/H+-exchange both in the present effect on ‘gating’. This is because this mutation w
3 w
study as well as in parallel work by Zifarelli and not only modified conductance ratios, but also .jb
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Pusch (38). A novel method (43) to measure changed the apparent permeability (reversal .o
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proton transport allowed these authors to show potentials). We therefore conclude that the b/
y
that ClC-5 (S168P) had gained a NO -/H+ proline-serine exchange affects the ion selectivity g
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coupling ratio of about 2 at voltages between +40 of the exchange process. This conclusion is st o
and +60 mV, indistinguishable from that for Cl- indirectly bolstered by the single channel analysis n A
/mHu+t-aenxtcsh aant gteh is (p3o8s)i.t ioOnu r( Fdiga.t a5 Ko)n laortgheelry CalgCre-5e ochf atnhgee Cs lCi-n0 Si1o2n3 Ts emleucttaivnitt, yw haicnhd deomthoenr strpaoterde pril 9, 2
0
with their results (38), but show interesting properties (25), and by the changed ion selectivity 1
9
differences with data obtained for AtClC-a (Fig. of the ClC-0(S123P) mutant described here.
4F). The substitution of the critical proline by Unfortunately, we cannot draw similar
glycine in AtClC-a did not interfere with its conclusions for ClC-5 because its strong
efficient NO -/H+-exchange, maybe suggesting rectification precludes the determination of
3
that a helix breaker might be sufficient to support reversal potentials.
such an exchange. However, glycine at the In the crystal of ecClC-1, the equivalent
equivalent position in ClC-5 does not increase serine participates in the coordination of a Br- ion
currents in the presence of NO -, nor does it (used as Cl- substitute in crystallography) in the
3
enable efficient NO -/H+-exchange (data not central binding site (22). Several mutations in
3
shown and (38)). Y445, another residue involved in this
Only four out of seven Arabidopsis CLC coordination (22), uncoupled chloride from
proteins have a proline in their signature proton fluxes (44). Such mutations were
sequence, with the remaining three displaying a associated with a reduced presence or complete
serine like all known animal CLCs. This suggests absence of anions at the central binding site (44).
that all these four proline-containing AtClCs Likewise, crystals obtained in the presence of
function as 2NO -/H+ exchangers. A more NO - revealed that this anion, which uncouples Cl-
3 3
definitive assignment of their physiological roles, from H+ transport in ecClC-1, cannot be detected
however, is not yet possible. In this respect, it is at this position (18). Thus, anion/proton coupling
interesting to note that only AtClC-c and AtClC-d seems to require that an anion occupies this site. It
6
was proposed that this anion serves as an changes of proton coupling and rectification that
intermediate binding site for protons on their way that bear close resemblance to results from
from the ‘proton glutamate’ to the ‘gating mammalian endosomal CLCs. However, there
glutamate’, leading to the seemingly outlandish were also significant differences in effects on
proposal of HCl as a proton transport intermediate NO - conductance. We further identified an
3
(45). We suggest that the replacement of serine by important proline residue in the CLC signature
proline in the GSGIPE sequence enables NO - to sequence of AtClC-a that is crucial for its
3
occupy the central anion binding site, thereby efficient NO -/H+-exchange activity and that
3
leading to efficient proton coupling. conferred more efficient NO -/H+-coupling on the
3
In summary, a breakthrough in heterologous mammalian ClC-5 exchanger and an increase in
expression of AtClC-a has allowed us to extend NO - conductance on the Torpedo Cl- channel
3
structure-function analysis of anion/proton ClC-0. Our work provides a basis for future
exchange to plant CLCs. Mutagenesis of critical studies of other plant CLCs and their comparison
‘gating’ and ‘proton’ glutamates resulted in to mammalian counterparts.

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FOOTNOTES

* We thank A. Fast, N. Kroenke, P. Seidler and S. Zillmann for technical assistance. This work was
supported by a grant of the Deutsche Forschungsgemeinschaft to AAZ and TJJ and by the Leibniz
Graduate School of Biophysics.

The abbreviations used are: ClC-n, member n of the CLC family of chloride channel and transporters
(in animals); AtClC-n, member n of the CLC family of Cl- channels and transporters in the plant
Arabidopsis thaliana; ecClC-1, one of the 2 CLC isoforms in E. coli; .HEPES, N-(2-
hydroxyethyl)piperazine-N’-(2-ethansulfonic acid); MES, (2-(N-morpholino)ethanesulfonic acid);
BCECF, 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein; pH, intracellular pH; pH ,
i o
extracellular pH; WT, wild-type; I(NO -) and I(Cl-), current in the presence of NO - and Cl-,
3 3
respectively, which with CLC exchangers also involves an H+-component.
8
FIGURE LEGENDS

Fig. 1. Electrophysiological characterization of AtClC-a in Xenopus oocytes. A,B, Voltage-clamp
traces of oocyte-expressed AtClC-a in either Cl-- (A) or NO -- (B) containing solutions. From a holding
3
potential of -30 mV, the oocyte was clamped in 20 mV steps to voltages between -100 and +100 mV
for 500 msec. C, steady-state I/V curves of AtClC-a with different extracellular anions. Currents were
normalized for individual oocytes to current at +80 mV in Cl- solution (with a mean value of 3.38 ±
0.40 µA). AtClC-a has a NO - > Br- > Cl- > I- conductance sequence (mean values from 13 oocytes
3
from 4 batches; error bars, SEM). Inset, higher magnification of I/V curve to show reversal potentials.
D,E, steady-state I/V curves of AtClC-a at different values of extracellular pH (pH ) in Cl-- (D) or NO -
o 3
- (E) solutions. Voltage-clamp protocol as in (A,B). Currents were normalized for individual oocytes to
the respective current at +80 mV at pH 7.5. (5 oocytes for each data point; error bars, SEM). F,
Currents at +80 mV as a function of pH with extracellular Cl- (□) or NO - (○). For each curve, currents
o 3
were normalized to currents at pH =8.5. G-I, AtClC-a mediated, voltage-driven H+-transport assayed by
o
pH-sensitive BCECF fluorescence using the Fluorocyte system (21). Proton transport was stimulated by
trains (black bars) of 400-msec long voltage-pulses, either to +90 mV (depol., depolarization) in (G,H)
or to -160 mV (hyperpol., hyperpolarization) in (I) in the presence of either extracellular Cl- or NO -
3
(shown in boxes above). Depolarizing pulses elicited intracellular alkalinization both with pH =7.5 (G)
o
and pH =5.5, when proton transport occurs against a gradient (H). Extracellular NO - accelerates
o 3
depolarization-driven alkalinization (G,H), but not hyperpolarization-induced acidification (I: pH =7.5).
o D
o
Only changes in relative fluorescence (rel. fluor.) induced by changes in voltage or external anions are w
n
relevant because of the drift inherent to the Fluorocyte technique (21). Results as in G-I were obtained lo
a
d
in 26, 6, and 7 independent experiments, respectively. ed
fro
m
Fig. 2. Impact of ‘gating glutamate’ mutant E203A on AtClC-a properties. A,B, voltage-clamp traces of h
oocytes expressing the E203A mutant of AtClC-a with extracellular Cl- (A) or NO3- (B) using the pulse ttp://w
protocol of Fig.1A,B. C, steady-state I/V curves of AtClC-a(E203A) with different extracellular anions w
w
reveal a changed conductance sequence of Cl- > Br- > NO3- > I- (mean of 19 oocytes; error bars: SEM). .jbc
Currents were normalized to current in Cl- at + 80mV (with mean current of 3.05 ± 0.22 µA). Inset: .o
rg
Higher magnification of I/V curve to show reversal potentials. D,E currents of AtClC-a(E203A) are b/
y
insensitive to pH both in the presence of extracellular Cl- (D) or NO - (E). Number of oocytes: n=10 g
o 3 u
e
for (D), n=14 for (E); error bars, SEM. F, lack of depolarization-induced alkalinization with the E203A st o
mutant. 25 independent experiments gave similar results. n A
p
Fig. 3. Effect of proton glutamate E270A mutation, alone and in combination with the uncoupling ril 9, 2
0
E203A mutation, on AtClC-a transport. A, B, voltage-clamp traces of oocyte-expressed AtClC-a E270A 1
9
in either Cl- (A) or NO - (B) containing solutions are not different from those from uninjected control
3
oocytes (not shown). C, Steady-state I/V curves of AtClC-a(E270A) are not different from controls. (10
oocytes per data point. Currents were normalized to current at +80mV in Cl-). D, E, Currents of the
AtClC-a(E203A, E270A) double mutant are larger with Cl- (D) than with NO - (E). These results also
3
suggest that the failure to observe AtClC-a(E270A) currents is not due to an absence from the plasma
membrane. F, I/V curves of currents averaged from 9 oocytes from 4 batches. Currents were
normalized to those in Cl- at 80 mV (with mean current of 1.86 ± 0.47 µA). Inset: Higher magnification
of I/V curve to show reversal potentials. G, H, lack of detectable depolarization-induced alkalinization
with AtClC-a(E270A) in the presence of Cl- (G) or NO - (H). Similar negative results were obtained
3
with 9 oocytes (from 4 batches) for G and H. I, lack of depolarization-induced proton transport with
AtClC-a(E203A, E270A). Similar results were obtained with 5 oocytes from 3 batches. Voltage-clamp
protocols identical to those in Fig. 1.

Fig. 4. A critical role of proline residue in AtClC-a in determining anion selectivity. A, sequence
alignment of several CLC proteins in the region of the GSGIPE ‘signature sequence’. E.coli ecClC-1 as
well as all animal CLCs have a serine between the two glycines. It is replaced by proline in plant
AtClC-a and –b, but not in AtClC-c and –d. B,C, typical two-electrode voltage-clamp traces of the
AtClC-a P160S mutant in Xenopus oocytes with external Cl- (B) or NO - (C) (voltage protocol as in Fig.
3
1). D, current-voltage relationship of the mutant appears identical in extracellular Cl- and NO - (mean of
3
9
9 oocytes from 6 batches each). Currents were normalized to those in Cl- at +80mV (where mean
current was 2.33 ± 0.44 µA). E, depolarization-induced H+ transport of AtClC-a (P160S) is similarly
effective with extracellular Cl- or NO -, in stark contrast to the WT (Fig. 1G,H). Similar results were
3
obtained in 14 experiments (14 ooyctes from 6 batches). F, ratio of currents at + 80mV in the presence
of NO - and Cl-, respectively, for AtClC-a and several mutants. Number of oocytes entering mean
3
values: AtClC-a, n=25; AtClC-a(E203A), n=24; AtClC-a(P160S), n=9, AtClC-a(P160G), n=5, AtClC-
a(P160S, E203A), n=4 and AtClC-a(E203A, E270A), n=9.

Fig 5. Effect of the S168P mutation on ClC-5 conductance and proton coupling. A,B, typical two-
electrode voltage-clamp traces of oocyte-expressed ClC-5 in Cl- (A) or NO - (B) containing solutions.
3
C, steady-state I/V curves from such experiments (averaged from 19 oocytes from 7 batches;
normalized for each oocyte to current in Cl- at +80 mV, with mean current of 3.01 ± 0.35 µA). D-F,
current properties of the S168P mutant measured as in (A-C). Data in (F) show means from 23 oocytes
from 5 batches, normalized to I in Cl- at +80mV, with mean I of 0.79 ± 0.17 µA. Voltage-clamp
protocols in A-F as in Fig. 1. G, less efficient depolarization-induced alkalinization by ClC-5 in the
presence of NO - than Cl-. Similar results were obtained in 17 experiments. H, hyperpolarization does
3
not elicit intracellular acidification with ClC-5, in contrast to AtClC-a (Fig. 1I). Similar results were
obtained in 13 experiments I,J, drastically increased NO -/H+-exchange activity with ClC-5 (S168P), as
3
shown by Fluorocyte with pH =7.5 (I) or with pH =5.5 (transport against gradient) (J), resembling in
o o
this respect AtClC-a (Fig. 1G,H). Similar results were obtained in 21 (I) and 8 (J) experiments K, ratio
D
of currents at +80 mV measured with extracellular NO - and Cl-, respectively, for ClC-5 and several ow
3 n
mutants, determined as in Fig. 4F. Number of oocytes used for averages: ClC-5, n=19; ClC-5(S168P), lo
a
d
n=23; ClC-5(S168G), n=17; ClC-5(S168A), n=2; ClC-5(S168P, E211A), n=6; ClC-5(E211A), n=10; ed
ClC-5(E211A, E268A), n=34). Error bars indicate SEM. fro
m
h
Fig 6. Modification of ClC-0 Cl- channel currents by the S123P mutation. A,B, typical voltage-clamp ttp://w
traces of an oocyte expressing WT ClC-0 and measured in Cl- saline (A) and NO - saline (B). After w
3 w
about 8 seconds at -120 mV (to active the slow, hyperpolarization-activated gate of ClC-0), the oocytes .jb
c
were clamped consecutively for 1 sec to -120mV (to keep the ‘slow’ common gate open), 400 ms to + .o
rg
80mV to open the fast protopore gate. At t=0 they were clamped (for 400 ms) to test voltages between b/
y
+80 and -160 mV in steps of 20 mV, followed by a constant pulse to -100 mV for 400 ms. C, g
u
e
normalized I/V curves of WT ClC-0 in presence of different extracellular anions. Values were obtained st o
from exponential fits to t=0 and represent open channel currents because both slow and fast gates were n A
p
obpatecnheeds bwye rhey npoerrm- aalnizde dd efpoor leaariczhin ign dpivreidpuualsle os,o cryestep etoct itvheel yc.u rArevnetr aing eCdl -c autr rVen=t+s 6f0r ommV 5, wohoecryet ems eoafn 2I ril 9, 2
0
was 13.8 ± 2.2 µA. D,E, typical traces from an oocyte expressing the S123P mutant of ClC-0. Voltage- 1
9
clamp protocol similar to (A), but the test voltages were applied for 600 ms, followed by a 500 ms pulse
to -100 mV. F, normalized open-channel I/Vs for the mutant obtained as in (C). Means are from 7
oocytes of 2 batches. Normalized to current in Cl- at +60 mV, where mean I was 4.41 ± 0.90 µA.

10