Calcium-Proton Exchange During Algal Calcification

11 Pages·1991·4.7 MB·English

by  T A Mcconnaughey

Reference: Biol. Bull 180: 185-195. (February,
Calcium-Proton Exchange During Algal Calcification
TED A. McCONNAUGHEY1* AND RICHARD H. FALK2
1Marine Biological Laboratory, Woods Hole, Massachusetts 02543, and2Botany Department,
UniversityofCalifornia, Davis, California 95616
Abstract. Extracellular calcification by the giant celled calcification in characean algae involves active calcium-
alga Chara coralUna may involve active Ca2^ extrusion proton exchange.
from the cell in exchange for protons. The following ev- Characeans calcify as a by-product ofbicarbonate as-
idence is presented: CaCO, incrustations accrete largely similation fromalkalinewaters(Spearetal., 1969; Raven
along the inside, facing the cell, as revealed by X-ray mi- etal.. 1986;Okazaki andTokita, 1988). Theplantsextract
croanalysis using Sr:+ and Mn2+ as tracers for new min- proton equivalents from the medium along parts oftheir
eralization. Inward proton currents are inhibited by the giant cells, forming alkaline patches or bands that may
u
Ca:+ transportantagonistsGd and La34. LowCa2+ con- become heavily calcified. The proton equivalents are ex-
centrations inhibit pH banding and photosynthesis, and trudedelsewhere, formingacidicpatchesorbands. There,
solutionsoflowCa:+activitysupportmorephotosynthesis HCOr is apparently protonated to form CO:, which the
in the presence ofadditional buffered calcium. The ratio plant absorbs (Walker el al., 1980; Smith and Walker,
ofcalcification to photosynthesis in moderately alkaline 1980; Price and Badger, 198?). Pericellular carbonic an-
solutions containing sufficient calcium remains stable at hydrase and complicated invaginations of the plasma
about 1.0 independent of solution Ca24 concentration. membrane within the acid zones may facilitate CO2 gen-
Ion specific microelectrodes placed close to the calcified eration and absorbtion (Price el al., 1985).
surface sometimes detect increases in Ca:+ activity coin- Characeanscanbemorethan halfCaCO3bydryweight,
cident with decreases in proton activity. As the pCa of and as will be shown here, calcification is often stoichio-
solution increases, the maximum pH observed at the al- metric to photosynthesis. Nevertheless, calcification
kaline surface increases, as does the maximum solution physiology hasbeen largely neglected, and calcification is
pH which supports electrochemical currents by the cell. generally assumedtobeindependentofactiveCa2+ trans-
CcaolmcbuilnataetdiotnhseromfoedxtyrnaacemlilculalrimpitHs afonrdApTCaPadprpirvoeanch2Hth+e/ lpeosrsts(ueg.gg.e,stRsatvheantaetctail.v.e1C9a862+).trVaanrsipoourstemviigdhetncbeenienvveorltvhede.-
Ca2+ exchange against the cytosol. First, Ca2+ ATPases apparently catalyze Ca2+ extrusion
from cells in exchange for protons (Niggli et al.. 1982;
Introduction Villalobo and Roufogalis, 1986; Rasi-Caldogno et al.,
Ca2+ ATPase appearsto be associated with calcification 1987; Dixon and Haynes, 1989). Ca2+ ATPase could
in various animals and plants (e.g., Klaveness, 1976; therefore catalyze proton uptake at the site ofcalcification
Okazaki, 1977;OkazakieM/., 1984;KingsleyandWatabe, in Chara. Second, characeans are functionally analogous
1985).Thisreportexploresthepossibilitythatextracellular to coccolithophorid algae, which also calcify in an ap-
proximate ratio of 1:1 to photosynthesis, but do so intra-
Received 3 April 1990;accepted 6 November 1990. cellularly (e.g.. Sikes et al.. 1980). Ca2+ and carbon pre-
*Addresscommunicationsto:Dr.TedMcConnaughey.U.S.Geolog- sumably traverse thecytoplasm to reach the vesicularsite
ical Survey, Box25046 MS413, Lakewood,CO 80225. ofcalcification, and Ca2+ ATPase seems to be involved
Abbreviations: CAPS = 3-(cyclohexylamino)propanesulfbnate; CHES (Klaveness, 1976; Okazaki etal., 1984). Third, molecular
l=ino2lt-pr(riNo-spca(ynhceylsdourhlefooxxnyyalmtaeem;tinhPoyI)lPe)EtmShea=ntehlsy,ul4la-fpmoiinpanetorepa;rzionpMeaOdniPeeSthasnu=el-ts'2oun-la(ftoNen-;amtoerT;pTRhAIoPS-S CduOri2nagppacraelncitfliycaptrioovnidbesymovsartioofustheplparnetcsipiatantdingacnairmbaolns
= tris(hydroxymethyl)aminomethane. (McConnaughey, I989a, b, c), including Chara (Mc-
185
186 T. A. McCONNAUGHEY AND R. H. FALK
Connaughey, in prep). Since HCO3 is more abundant fluxes measured extracellularly then reflect cellular H+
in alkaline solutions, its unimportance in calcification transport most closely.
suggests that the calcifying region can be fairly isolated The energy (E) required for proton uptake under both
from solution. The calcifying cell must therefore supply models is given by:
Ccaal2c+iu+m,CaOnd+reHmovOet=heCparCotOons+ge2nHer+atAedndbythereacCtaio2n+ E = FV(aZCa - bZH) + RT In (Ca,/Cat,)a/(H,/H )b ( 1)
2 2 3 . finally,
is well known to affect characean photosynthesis and The terms on the right represent work done against the
membrane properties associated with pH banding (e.g., membrane electrical potential V, and against the mem-
Lucas, 1976;Wiesenseel and Ruppert, 1977;Luhringand brane chemical gradients. F is the Faraday constant, "a"
Tazawa, 1985; Bisson, 1984; Tazawa el a/.. 1987). and"b"arethe numbersofCa2i and H+ ionstransported
A Ca:+ ATPase model and a more conventional proton per cycle, Z is ionic charge. R is the gas constant, and T
channel model for characean calcification are illustrated is Kelvin temperature. Cytoplasmicandexternal Ca2+ and
in Figure 1. Both modelsareelaboratedtofittheavailable H+ activities are subscripted "i" and "o," respectively.
data. Ca2+ influx intothecell, in the Ca:+ ATPase model, Forthe proton channel model, protonsare drawn into
occurs within the alkaline band (Fig. Ib) to produce the the cell by the membrane electrical potential. The ther-
=
observed electrogenic character of pH banding (Walker modynamic limit forpassive(E 0) proton uptakeoccurs
and Smith, 1977). Figure Ic, eshowsthe useofmolecular when the membrane electrical and chemical gradient
COi from the plant as the major carbon source for cal- energies balance, yielding a proton Nernst equation:
cification (McConnaughey, in prep.) and the accretion of = FV + 2.3 RT(pH - pH,) (2)
extracellular calcium deposits from the inside (demon-
strated here). Figures Idand Ifdepict non-calcifyingcon- Foran illuminated cell inalkalinesolution, the membrane
ditions, such as when Ca2+ or carbon levels are too low potential might be around 200 mV and cytoplasmic
tosustain much CaCO^ precipitation. The non-calcifying pH, might be about 7.5-8.0 (Smith and Raven, 1979;
condition can be experimentally useful, because the H+ Spanswick and Miller, 1977; Mimura and Kirino, 1984;
Ca"
Alkaline 2H
Band
CaCOj
HChannel
CALCIFYING NON-CALCIFYING
Ca"
Ca ATPase
H*Channel 2OH
Figure 1. Modelsofextracellularcalcificationand itscouplingtobicarbonateutilizationinChora. Left:
schematic ofa cell, showing alkaline band at top (with trapezoidal CaCO3 incrustations), and acid band
below(withplasmalemmasomes,participatinginbicarbonateuse)."P"representsphotosynthesis,(a)Proton
channel model. HCO3~diffusestothealkalinesurfaceanddonatesaproton, becomingconvertedtoCO3",
whichprecipitateswithCa2+.(b)Ca2+ ATPasemodel. ATPdriven 2H+/Ca2+exchangealkalimzestheexternal
medium and locally increases its Ca2+ concentration. CO2 diffuses from the cell and reacts with water to
yield the protons needed forexchange with Ca;+, and theCO5" which precipitatesasCaCOj. A 1:1 ration
ofcalcificationtophotosynthesisisshown forboth models. Right:elaborationsonthe H* channelandCa2+
ATPasemodelsforthealkalineband, incorporatinginwardaccretionofCaCO, incrustations, usingCO2as
the carbon source. Proton channel (e, d) and Ca:+ ATPase models (e, I) showingthe alkaline band under
calcifying(c. e)and non-calcifyingconditions(d. f). The ion fluxesdetectableexternally are highlighted.
ALGAL CALCIFICATION 187
Smith. 1984a, b). The maximum pH in the alkaline band maps, line scans, and area scans made with an accelerat-
would then be about 10.9-1 1.4, independent ofsolution ing voltage of 15 KeV revealed distributions of Ca, Sr,
Ca2+ and Mn.
activity.
ForATP-driven 2H+/Ca:+ exchange, theenergyofATP Ratesofcalcificationandphotosynthesiswereestimated
hydrolysis (E) is about -50 to -55 KJ/mol (e.g.. Hashi- from changes in the alkalinity and total dissolved inor-
moto el a/.. 1984). The electrical term in eq. (1) drops ganiccarbon content ofsolution. Alkalinitywasmeasured
out, leaving by acidometric titration using the Gran method (Stumm
= - - - and Morgan, 1970). Heavily calcified plants were incu-
E/2.3RT pCa,) 2(pH pH,) (3) bated in stoppered flasks at 25C under a mixture offlu-
sCSpayantdcoeeprlhsaa,svm1ii9n8cg7)pa.CsaElqo(pu=eat-oilfoon1g/2{3.Cdaaens2+cdr,}idbiisesspalbaaolcuietnde6fi.nr9o((mpMiHtlh(lle,eprcCoaamn,-d^ toairnaeldslyc0e-cn5otn0atanmidAn/eidnCcaa(Cinnld,em,s/clpe//Hnlt)8lNitgoahtH8s.C2f,Oor,ad6j-1u8,stNhe.adCSlwoliut1th,iKNonCas1OiHn.i.2-.
position ofthecytosol, (pH,, pCa,), by4.4to4.8 pH units. Calcification was calculated as halfthechange in alkalin-
The presentexperiments look forevidencethat CaCO3 ity, and photosynthesis was calculated as the change in
incrustationsaccretefrom the inside,aswould beexpected total carbon minus calcification.
iftheplantsuppliesthe precipitatingcalcium andcarbon. In experiments designed to see whether buffered Ca2+
Proton and calcium specific microelectrodes search for stimulated photosynthesis in solutions oflow Ca2+ activ-
regionsofelevatedCa2+ anddepressed FT activitiesalong ity, photosynthesis was monitored using an oxygen elec-
the calcifying surface. The combinations ofcalcium and trode(Orion 97-08). Wide mouthjars(500 ml)containing
proton activities are compared with the thermodynamic about 5 gofalgaewere filled withsolutionsprepared from
constraints ofATPdriven 2H+/Ca2+ exchange against the partially degassed, deionized water, and capped under-
cytosol. Calcium transport antagonistsare used to inhibit waterto excludeair bubbles. Control solutionscontained
proton uptake. And the stoichiometry ofcalcification to (in mA/) CaCl: 0.05, KC1 0.1, NaCl 1, NaHCO, 1.8. and
photosynthesis is examined to see ifcalcium merely dif- Na2CO3 0.2. Test solutions contained an additional 0.8
fusesto thecalcification site. In theend, theCa2' ATPase mA/CaCl: and sodium citrate(1.5 mA/). Thesesolutions
model offers some advantages, but presents some inter- exhibited the same Ca2' activity, using a Ca2+ specific
estingdifficulties. Thediscussiontoucheson howtheplant microelectrode. A relatively high pH (9.1) ensured that
uses calcification as a photosynthetic adaptation. the plants obtained most of their carbon through the
physiologyassociated with pH banding. Halfoftheplants
Materials and Methods had been mostly decalcified before the experiment by
soaking them for 2 days in a solution containing 10 mA/
The present experiments used male plants of Cham MES buffer, initial pH 5.2. Cool white fluorescent lights
corallina from South Australia, provided by Bill Lucas. provided illumination during 2-3 h incubations at about
Plants were maintained in the laboratory, in aquaria ini- 25C.
tially containing "CPW/B" solution (in mA/, CaCl2 0.2, The effect ofcalcium transport antagonists on inward
NaHCO, 1, NaCl 1, KG 0.2) overlying 5-20 cm mud. proton currents were investigated by exposing an illu-
Nutrientsand additional calcium andcarbon were some- minated cell to LaCl3 or GdCl3, while measuring proton
times added to stimulate growth and calcification. Cool uptake with an extracellular vibrating H' specific micro-
white fluorescent lights provided illumination. electrode (Kuhtrieber and Jaffe, 1990). The cell was
Regions of new mineralization were identified by X- mounted in an open Petri dish (solution volume about 3
ray microanalysis. Plants first accumulated CaCO, in a ml) and perfused at a rate of 0.08 ml/s with a solution
medium containing(in mA/)CaCl2 2, NaHCO3 2, CaSO4 containing, in mA/. CaCl: 0.2, KC1 0.2, NaCl 1.0, TRIS
0.2, KC1 0.2, and NaCl 1, and were then transferred to 5, pH adjusted to 8.3 with NaOH. Fiberoptic lights pro-
media containing additional SrCli 1, and MnSO4 0.1. to vided illumination. The proton electrode vibrated per-
label regions ofnew mineralization. Cells showing heavy pendicularly to the cell over an excursion of 10 ji, at a
calcification and good cytoplasmic streaming were rapidly frequency of0.5 Hz, at a distance ofabout 10 ^m from
frozen in liquid nitrogen slush, fractured, andgivenathin the cell. 4 ^Moles of the lanthanide was added to the
coating ofaluminum by vacuum evaporation (Emscope input stream without changing flow rate. The signal here
SP2000) at -196C, to increase surface conductivity. is the voltage difference registered by the electrode as it
Frozen hydrated specimens were transferred under vac- moves back and forth near the cell. A proton gradient of
uum to the cryostage ofa scanning electron microscope one pH unit within the sampled region ideally yields a
(Hitachi S800) equipped with a solid state X-ray spec- signal ofabout 58 mV, although in practice the signal is
trometer (Kevex 8000 series). Secondary electron mode smaller. Fluxesofproton equivalentscarried by H+, OH~,
images provided details of surface morphology. X-ray and protonated TRIS buffer were calculated from Pick's
188 T. A. McCONNAUGHEY AND R. H. FALK.
first law, usingdiffusion coefficients 93, 53, and 7 X 10 6 trical currents were calculated from the electrical con-
cnr/s, respectively, and concentrations calculated from ductivity of solution, using Ohm's law. The example
the measured pH. In the case illustrated, the pH at the shown used a divided chamber, so that opposite halves
electrodewasabout 9 before addingthe lanthanides. The ofthecell wereexposedtodifferentsolutions. Cytoplasmic
voltagefield arising from netcharge uptakebythealkaline streamingbetween thetwohalveswasuninterrupted. The
band introducesonlyasmall biastothe pH signal; relative "control" half was bathed in CPW/B, while the "test"
to background solution, the alkaline band might show a halfwentfromCPW/BtocarbmonM-freesolutionscontaining
voltagedifferential ofabout -4 mV, whilethepH gradient zwitterionic buffers and 20 CaCl2 at progressively
of around 2 units produces a voltage signal of about higher pH.
-120 mV.
Ca2+ and FT activitiesatthealkaline surfaceofthecell Results
were measured using stationary ion specific microelec-
trodes, constructed as described by Borelli et al. (1985). Mineralization patterns
Electrodeswereconnected to a high impedance amplifier Calcified cells exposed to solutions enriched in Sr and
(World Precision Instruments FD223), with output to a Mn accumulate significant Sr and Mn mainly along the
wcfiheearlrdet(sraoebmcoeourttdiem-re.4sAmudsVdeidtrieaolsnaatwlievlelp.ottoTehnbteaicaplkegrrsiecoenulsnlidunl)garbeilealeseccettdrroipdceaHsl deixintswrtaarrcideblusltuuilroafnracsseuigtogefesoCtfasdCemOpeot3saiiltncitrorunas,ntsaaptloitrohtnosfug(rFhoimgdsi.tfh2fe.usc3ie.ol4ln).taoTlhotinhseg
andpCameasurementsbyabout +.07 and0.14pCaunit, the cell wall is also possible. Mn/Sr ratios are spatially
respectively. The cellswere exposed tobuffered solutions variable, suggesting some elemental segregation during
of various calcium concentrations, usually lacking dis- transport or precipitation. This is indicated by variations
solved inorganiccarbon todiscourage calcification. Fiber in the relative intensitiesoftheir X-raypeaksobserved in
optic lights provided illumination. area scans. Some ofthis variability is visible in the X-ray
In experiments comparing pericellular pH against a maps presented in Figure 3.
thermodynamic model for 2H+/Ca2+ exchange, the pH
datarepresentthehighest valuesobservedduringelectrode
scansofthecell surface, andduringobservationsofseveral
minutes duration at particularly alkaline locations. So-
lution pCa was calculated from solution Ca2+ concentra-
tions and ionic strength, using Davies' individual ion ac-
tivitycoefficient (see Stumm and Morgan, 1970), ormea-
sured using an Orion 93-20 electrode for solutions
containing citrate. In experiments comparing simulta-
neousvariationsin pericellularpH and pCa, H+ andCa:+
electrodes were placed close together near the alkaline
surface ofthecell, and the intensity ofpH bandingeither
fluctuated spontaneously or was modulated by turning
the light offand on. In the examples shown, the medium
contained MOPS(5 mA/)andcitrate(2 mM),plusNaOH
and CaCl2 to produce pH 7.98, pCa 3.89.
Extracellularelectrical currentswere measured usinga
vibrating probe electrometer(Jaffe and Nuccitelli, 1974).
The probe vibrated perpendicular to the cell surface, ap-
proximately 30-50 Mm from thecell whilethecell moved
by on a motorized stage. Fiberoptic lights provided illu-
mination. Most experiments used nominally carbon-free
solutions containing (in mAf) KC1 0.2, NaCl 1, and a
zwitterionic buffer (MOPS, PIPES, EPPS, CHES, or
CAPS, 5 mM), pH adjusted to the desired value using
NaOH. At the chosen concentration of CaCl2 (0.1-50
mM), the cell was repeatedly scanned along its length for Figure 2. Scanningelectron micrograph offrozen, hydrated cell la-
eplHectorricaClaa2c+ticvointcyewnhtirlaetisoonluwteiroensaodfdepdr.ogTrehsesicveelllywhaisghaelr- bweiltehdiwniwtahrdSrd:i+mapnldingMnof2+t.hsehcoewllinagndextarpapcaerlleunltarduCpalCicOa,tioinncroufsttahteiocnesl,l
lowed to adjust in each solution forat least 30 min. Elec- wall. Magnification: top 162x, bottom 830x. Scalebar = 30 .
ALGAL CALCIFICATION 189
CALCIFICATION TO PHOTOSYNTHESIS RATIO
1 -
15KV X1.00K 30UM
Figure3. DistributionsofCa(yellow),Sr(blue),and Mn(red)inan
extracellularCaCO3 deposit, visualized by X-ray mappingofa frozen.
hydratedcellexposedtoSr:+andMn:+afterfirstaccumulatingsignificant
CaCO
3.
Sr and Mn accumulations presumably consist of di-
valent metal carbonates and MnO2, the latter inferred
from its dark color. Manganese oxidation, Mn2+ + H O
+ '/2O2 = MnO2 + 2H+, isfavored inthealkaline, oxyge2n-
rich environment ofthe plant surface. More or less pure
Mn
accumulations, based on relative X-ray counts for
120
4 6 10
ENERGY
(KeV)
Figure4. X-rayspectrataken atpoints"A" and "B"ofcell shown
in Figure 2. Spectra correspond to materials deposited after (A) and
before(B)addition ofSr2+ and Mn2+ tothe medium. X-raycountsare
scaled relativetotheCa peak (100%);spectrum A hasbeen shifted up-
wardsby 20% forclarity.
190 T. A. McCONNAUGHEY AND R. H. FALK.
Proton cycling involves calcium
Low Ca2+ concentrations inhibit photosynthesis (Fig. >
5, inset). This inhibition appears to involve Ca2+ fluxes,
because plants incubated at the same low Ca2+ activity oHI
show more photosynthesis ifadditional buffered Ca2+ is
aadnddetdhetostsiomluultaitoinon(TbaybblueffI)e.reTdhCear2a+tearoefgrpehaotteorswyintthhecsails- EHCI
cified than withdecalcified plants(twowayANOVA, both U 3 -
factors and interactions significant at P < 0.05). OUJ
Proton uptake at the alkaline band, measured using a
|.H
vibrating FT specific electrode, is inhibited by the Ca2+ O
transport antagonists Gd3+ and La3+ (Fig. 6). In this ex-
ample, theelectrode was positioned overapoint showing
particularly strong alkalinization. Gd3+ reduced the signal
registered by the vibrating electrode by about half, but
thecellsoon recoveredabout 80-90% ofitsformersignal.
Subsequent treatment with La3+ reduced the signal more
strongly, and H+ uptakedid not recoverforoveran hour.
Beforeaddingthe lanthanides, thevoltagedifferencesignal
mV
registered by the vibrating electrode (about 7 at pH
9) corresponds ideally to a flux of proton equivalents
around 2 nMoles cirT2/s, carried mostly by OH" and
TRIS buffer (calculation. Fig. 6 inset).
The alkaline bands of C/iara can turn on and oft in-
dependently, sometimeswithout obvious provocation,so
reductions in the pH gradient are not necessarily propor-
tional to pathology. The pH gradient is also affected by
CaCO}dissolution atthe plantsurface,andby ion pairing
and precipitation ofthe introduced lanthanides. The ef-
fects here appear to be mostly physiological, however.
Perfusion ofthe chamber should have brought solution
pH back to normal within a few minutes (theoretical di-
lution time about 38 s).
An approximately 2-3 min oscillation in apparent H+
influx is observed in this experiment (Fig. 6). Such oscil-
lations are detected using various techniques (Fisahn et
ai. 1989), andean sometimes be induced by addingCa2+
to the medium.
Electrochemicaldetection ofcalcium efflux
BoththeCa2+ATPaseand proton channel modelspre-
dict Ca2+ diffusion toward the alkaline surface undercal-
Table I
Stimulation ofphotosynthesisbybufferedcalcium, atlowsolution
calciumactivity. Photosynthesisestimatedbyoxygenevolution,
in micromolesO;pergram wet weightperhour,
withstandarddeviation (n = 10)
ALGAL CALCIFICATION 191
Extracellular electricalcurrents
The ion fluxes associated with pH banding create ex-
tracellular current loops which can be measured with a
vibrating probe electrometer. These currents persist until
solution pH israisedaboveacritical value, atwhich point
the currents cease or may reverse with much diminished
amplitude. The solution pH at which current cessation
occurs varies with solution Ca2+ activity in more or less
the same way as the extracellular pH data in Figure 8.
The Ca2+ dependence suggests that proton uptake iscou-
pled to Ca2+ expulsion.
Presumably, as 2H+/Ca:+ exchange becomes impos-
40 60 sible, cytosolic Ca2+ rises and inhibits Ca2+ influx (see
TIME(MIN) Eckert and Chad, 1984). The proton ATPase ofthe acid
Figure 7. Extracellular pH and pCa measured with stationary ion band shuts down as the cytoplasm becomes alkalinized
specific electrodes placed close to the calcined surface of a cell under (due to cessation of proton uptake) and the membrane
pnorno-bcaabllcyifcyaiunsgecdonbdyitiinocnrse.as(eAd)CPaosCiOti,vedicsosrorleultaitioonnobreltewaecehninpgHatalnodwppCHa., potential increases (due to cessation of Ca2+ uptake).
(B)Anticorrelationbetween pHandpCa.suggestingcalcium-protonex- Consequently, even though 2H+/Ca2+ exchange is elec-
change. trically silent, preventing this exchange can stop extra-
cellular electrical activity.
Figure 9 illustrates an experiment in which a cell is
t2hHe+d/eCcar2e+aseexcihnapnegreicmelulsutlaralCsoa2b+eastufhfiicgihenptHt.ocoavuesrecdombey spollaucteidonisncaondtiaviindiendg2c0hammAbIerC,aCaNnd(piCnacr=ea2s.in1g)layreaalpkpalliiende
trheeduccetlliownaslli.n CaCO, dissolution and Ca2+ leaching from tpoHth8e.2,rigphCta(t=est3).8s.ideC.ytTohpelalsefmtic(cosnttrreoalm)isnigdebertewmeaeinnsthaet
twosidesisuninterrupted. Asthetestsideapproachesthe
calculated thermodynamic limits for ATP driven 2H+/
Thermodynamics Ca2+ exchange, itscurrents diminish, but currents on the
The maximum pH observed at the alkaline surface us- control side are unaffected. In the last test solution (pH
ingmicroelectrodesapproachesthethermodynamic limit 10.0, pCa 2.1). banding is strongly suppressed and an ap-
for ATP driven 2H+/Ca:+ exchange, calculated using eq.
3 (Fig. 8). The approach is closest at high solution Ca2+
activities (low pCa). As pCa:+ increases, the maximum 11
pH also increases, although not as much as allowed by
thermodynamics. At pCa >4, higher pH readings are ob-
10-
tained in the presence of the weak Ca2+ buffer citrate,
suggesting that the rate of Ca2+ supply to the cell may
limit proton uptake. All pericellular pH, pCaobservations 9-
fall within thethermodynamic constraints forATPdriven
2H+/Ca2+ exchange, and the Ca2+ dependence for peri- pH
cellular pH provides some support for the Ca2+ ATPase 8 tI CYTOSOL O
model. 4
pHATPLANTSURFACE
The pH and pCa in large culture vessels containing 345--WITHCITRATE
Chamalsoapproach thecalculatedthermodynamic limits CULTURESOLUTIONS
for 2H+/Ca:+ exchange (Fig. 8). The most extreme con- 1
ditions observed (pH 10.78, pCa 4.30) are close to the pCaofsolution
most extremeconditionsobserved atthecell surface with Figure 8. pH observations at the alkaline surface as a function of
microelectrodes. Ratherhigh pericellularpH (about 10.7) solution Ca2+ activity. Diagonal line: calculated thermodynamic limits
is observed transiently in Ca2+ free solutions, but pH for ATP driven 2H+/Ca:+ exchange between the cytosol and external
bquainrdeimnegnetvefonrtubaalnldyincgo.llIanptseesr,nalcoCnsai2s4testnotrewsi,tpherahCapas2+surpe-- csoyobtlsouestriovoleni,dcaactsosmauplmkoaislniitgnieEo(nsA,urTfpPa)Hce==un5d08,eKrpJe/Cxmapoel=r.imS6e.y9nm.tbao(llSsqc:uoan(rdDeisit)iaomnmosan,xdw)iimtahusosmuutmpeciHd-
plemented by CaCO,dissolution and Ca2+ leaching from trate.(+)Same,withcitrate.(A)Combinationsofsolution pHandpCa
the cell wall, may support banding for awhile. observed in large vatcultures.
192 T. A. McCONNAUGHEY AND R. H. FALK.
I
LOU
S3
Si
ao:
2
-1.8
-20 -10
DISTANCE FROM DIVIDER (mm)
Figure9. Extracellularcurrentsmeasured usinga vibratingvoltageprobe. Cellwasplaced in adivided
chamber, and solution on the right side was replaced with solutions having higher Ca2* activity and pro-
gressively higherpH. Duplicatescansare shown in each medium, andcell responsestodifferent mediaare
oSfoflsuettiboyns-c5o0n0ta/inieAd/c(min2.rProuVs/i)tiNvaeCclurIr,enKtsGd0e.n2o.tpelursegtihoensfoolflopwoisintgiavdedcituirornesn:t(ian)flCuaxStOo4ce0l.l2,(aNlakaHliCneOb3a1n.dsp)H.
8.2. (b)CaCI2 20,CHES 5,pH 9.0. (c)CaCl, 20.CAPS 5, pH 9.8. (d)CaCl,20, CAPS 5. pH 10.0).
parent efflux ofpositive charge prevails over the test side 50 mA/) has a minimal effect on the ratio ofcalcification
ofthe cell. The control side does not appear to compen- to photosynthesis, suggesting that diffusion to the calci-
sate, so if real, this current efflux should hyperpolarize fication site can be minimal. Ca2+ transport antagonists
the cell. interferewith H+ uptake. In solutionsoflowCa2+ activity,
additional "buffered" Ca:+ enhances photosynthesis and
Discussion proton uptake. Pericellular Ca2+ activities sometimes in-
crease simultaneously with stronger alkalization. Com-
Antecedents to the Ca2+ ATPase model for Chara ex- binations ofextracellularCa2+ and H+ activities are ther-
tend back at least to 1829, when Bishoff(cited in Pring- modynamically compatible with ATP driven 2H+/Ca2~f
sheim, 1888)suggestedthatcharacean limedepositsgrow exchange, and the maximum pH at the alkaline band
from the inside. Classical works on bicarbonate use also increaseswith pCa, aswouldbeexpected ifCa2+ extrusion
favored calcium and carbon movement through the po- accompanies proton uptake.
larized leaves of calcareous aquatic angiosperms and Mostoftheprecipitatingcarbon alsoappearstobesup-
characeans to reach the site of mineralization (Arens, plied by the cell as CO2 (McConnaughey, in prep.). This
1933, 1938, 1939). Kishimoto etal. (1984)suggested that further implies that the calcifying region can become iso-
proton uptake in Chara might occur through an electro- lated from bulk solution. Consequently, the cell must
neutral proton cotransport or countertransport system. supply Ca2+ and remove protons in 1:2 stoichiometry,
Many aspects ofthe Ca2+ ATPase model have therefore asindicated,bythe reaction Ca2+ + CO2 + H2O = CaCO3
been discussed. + 2H+
.
The Ca2+ ATPase model correctly predicts the data The data are less supportive of the proton channel
presented here. Sr2+ and Mn2+ accumulate largely along model, which offers no explanation for the Ca2+ depen-
the inner surface ofCaCO3 incrustations, facing the cell. dence ofphotosynthesis, or the elevations ofpericellular
Increasing the Ca2+ concentration in solution (from 2 to Ca2+coincidentwith H+ depletion. Thediffusion pathway
ALGAL CALCIFICATION 193
to the site of calcification creates additional conceptual the alkaline band of Chara. 45Ca2+ exchange rates are
problems. Why won't it accept OH" and CO?=, which generally less than 3 pMol cm"2 s"1, measured over the
should diffuse away from the cell, reducing the ratio of whole cell (Spanswick and Williams, 1965, Hayama et
calcification to photosynthesis to values below 1.0. and at. 1979; MacRobbie and Banfield, 1988). Some exper-
making it dependent on theCa2+ concentration, orphos- iments employed conditions unfavorable to pH banding,
phate, which fails to precipitate where Sr and Mn do but the disparity between inferred and published steady
(McConnaughey, in prep.)? state45Ca2+ fluxesneverthelessrequiresfurtherstudy. Low
Most of the published data appears compatible with Ca2+ exchange rates may be caused by containment of
theCa2+ ATPase model. Both modelsattribute the extra- fluxestothecortical cytoplasm ofthealkaline band, with
cellularcurrent influx in thealkalineband largely toCa:+ little exchange into majorcellularCa2+ reservoirssuch as
undercalcifyingconditions, and to H+ equivalents under the vacuole, chloroplasts, or mitochondria. Theavailable
non-calcifying conditions (Fig. 1). Membrane hyperpo- evidence supports this possibility. Extracellular electrical
larizationsarecaused byelectrogenic H+ extrusion in the currents presumably reflect Ca2+ uptake mainly within
acid band undereither model. Increased membrane con- the alkaline bands. When cells are placed in a divided
ductivity at high pH (Bisson and Walker, 1980, 1982) chamber with Mn:+ on one side, Mn precipitation is vis-
might result from more favorable thermodynamics for ibleonlyon that side, suggestingminimal transportalong
the proton ATPase oftheacid band, reversibility of2H+/ thecell. 45Ca2+ fluxes measured duringrepeatedelectrical
Ca2+ exchange, and perhaps opening of additional ion stimulation yield values around 60 pMol cm"2 s"1, mea-
channels (e.g.. Kikuyama et a/., 1984). sured overthewholecell, at 1 mAlexternalCa2+ (Hayama
el a!.. 1979). The metabolic machinery needed for large
Calcium transport fluxes therefore appears to be present.
Certain caveats apply to the thermodynamic analysis An analogy to coccolithophorid algae is instructive.
ofCa:+ transport attempted here. Pericellular pCa varies Calcification in these algae occurs within intracellular
locally, and depressions relative to solution values are vesicles, soboth calcium and carbon presumablytraverse
likely at high pCa (Fig. 7). Cytoplasmic pH and pCa may thecytoplasm toreach thecalcification site. Ca2+ ATPase
also vary. Extracellular and intracellular activity scales apparently participates in calcification (Klaveness, 1976;
may be offset with respect to each other. The slope ofthe Okazaki el a!., 1984). Sufficient dataaresometimesavail-
extracellular pH data is closer to 1/3 than 1/2, but pH is abletoestimateCa2+ fluxes. Forexample, Emilianiahux-
too high forATPdriven 3H+/Ca2+ exchange. 4H+/2Ca:+ leyicalcifies at a rateofaround 5-7 X 10"18 moles/s, and
exchange is likewise excluded. Several factors may con- the surface area ofthe finished coccolith is around 1-1.5
Ctrai2b+uteextcohatnhegefaaltl-hofifghfrpoCma.thAeslinmoittesdcaalbcouvlea,teCdaf2o+re2xtHru+-/ X198100)".7Tchmer2e(fPoaraes,chteh,e t1r96a4n;s-Kmleavmebnreasnse, C1a9726+;fSliukxemsaetyabt.e
sion may locally depress pCa below ambient values. Ca2+ 30-50 pMol cm"2 s"1. This is ofthe same magnitude as
diffusion toward the cell may limit the rate ofCa2+ and estimated for Chara.
Htdeionf+sofyrcunymstciohloeuintsnigloc,yfraaasatslsekpsuaeglorgibienctsieattliyelndueflbdaryrowmptihtHehthhibeniucgfprhfleeeaarrnseptedsHCs(uasvrea2fel+au.cFeiFesgi.naian6nlcldiryne,psaehtstohe)-es, sTfooutrmBamaellbcyalCuyarsai2et+nhvcecoyroltnvolsceooeCwln,iatcr2la+"atfrrirgioeecnehs"tvreCiasnnaisc2-cl+cehesac,lrolvaunaclccaeeuranontlCreaacsty,2i+trooenfptslilucaxauserlmsea,upaenrtniecd--.
so ifthe proton flux remains constant, diffusion should vacuoles is in the millimolar range (Okihara and Kiyo-
reduce pericellularpH moststronglyathigh pCa. In sum- sawa, 1988). To the extent that cytosolic free ion concen-
mary, there are many reasons why the data might fall trations pose atransport problem, the issue may be more
short ofthe thermodynamic limit, even ifthe plant op- acute with protons. The proton fluxes are presumably
erates close to the limit. The more interesting feature is twice as large and involve longer distances.
thatthe plant apparently approachesthe thermodynamic Applicability to other organisms
limit. IfCa2+ ATPase underlies extracellular calcification in
Whyareextracellular H+ fluxessomucheasiertodetect Chara and intracellularcalcification in coccolithophorids,
thanCa2+ fluxes?Thedifference, presumably, isthatCa2+ it might contribute similarlyelsewhere. Forexample, large
influx andeffluxoccurclosetogether, while proton fluxes Ca2+ dependent proton influxes, sensitive to lanthanides,
must be separated to create the acid bands needed for also occur at the calcified rhizoid of the siphonaceous
bicarbonate assimilation. Proton electrodesare alsotwice marine alga Acetabularia (McConnaughey, in prep.).
as sensitive as Ca2+ electrodes, and proton fluxes should
be twice as large. Couplingcalcification tophotosynthesis
The Ca2+ ATPase model postulates high rates ofCa2+ Although aphotosynthetic organism may looseCO2to
cyclingthrough thecell, around 100pMol cm"2 s"1 within calcification, it gainstwo protons foreach carbon lost. In
194 T. A. McCONNAUGHEY AND R. H. FALK
mildly alkaline waters, these 2H+ potentially enable it to Arens, K. 1939. Physiologische Multipolantat der zelle von Nitella
cfoornvpehrotto2syHnCthOes3ist.o 2AnCOa2p,pyrioexlidmiantgealynet1:g1airnatoifooonfecaClcOi-2 Barnweash,reD.ndJ.d,earnPdhoDt.osLy.ntTahyelsoer..P1r9o7l3o.plaIsnmasi3t3u:s2tu9d5i-e3s00of.calcification
andphotosyntheticcarbonfixationinthecoralMonlastreaanniilaris.
fication to photosynthesis is observed not only in Chara, llelgol. li'iss. Meeresunlers. 24: 284-291.
but sometimes also in coccolithophorid algae (Paasche, Bisson,M.A. 1984. Calciumeffectsonelectrogenicpumpandpassive
1964; Sikes el a/., 1980), calcareous seaweeds (Pentecost, permeability ofthe plasma membrane ofChara corallina. J Mem-
1978), and invertebrate-algae symbioses (Goreau, 1963; braneBiol 81: 59-67.
BarnesandTaylor, 1973; DuguayandTaylor, 1978; Kuile Bisson, M. A.,and N. A. Walker. 1980. The Chara plasmalemmaat
et ai, 1989). In the symbioses, the animal calcifies while ohifgHh+pHo.rOElHe'ct.riJcalMmeemabsruarenmeeBnitosl.s5h6o:w1r-a7p.idspecificpassive uniport
the algae use the CO;.. Rapid and massive calcification Bisson,M.A.,and N.A.\\alker. 1982. Controlofpassivepermeability
may exceed structural or defensive uses for CaCO3, and in the Chara plasmalemma. J E\p Hot 33: 520-532.
coccolithophorids, for example, discard excess scales to Borelli,M.M.,W.G.Carlini,W.C.Dewey,andR.R.Ransom. 1985. A
remain suspended in thewater. Coralsbuild uphugeskel- simple method tormakingion-selectivemicroelectrodessuitablefor
etal moundsbut occupy onlythetop few millimeters. No intracellular recording in vertebrate cells. / Neurosci. Methods 15:
structural ordefensive use ofCaCO3 isobvious in Chara. Dixo1n4,1D-.15A4..,andD.H.Haynes. 1989. Ca:+pumpingATPaseofcardiac
eraPtreopteorniceclylcullianrgCtOhe2orceotnicceanltlryaatliloonwssweolrlgaanbiosvmesamtobigeennt- sanadrcCorlegmrmaadiiesntisnsbeuntsirtievqeuitroesmaesmoburraceneofpcootuenntteira-ltrparnosdpuocretdablbeyHKT.+
(Walker et ai. 1980). Photosynthesis generally saturates J Membr Biol 112: 169-183.
altibCrOiu2mcovnacleunetr(ea.tgi..onSsmhiitghhearntdhaWnatlhkeera,tmo1s98p0h)e,riacndequfia-r Dugcuiafyi,catLi.onE.b,yatnhdeDs.oriLt.idTafyolroarm.in1i9f7e8r.anAPrrcihmaatrsyapnrgondluacttuiso(nFiacnhdtelcal&-
higher than present in many natuCraOl waters subjected to EckeMrotl,l)R..,J.anPrdolJo.ioEo.l.Ch2a5d:.351968-43.61.Inactivation ofCa channels. Prog.
strong photosynthesis. Elevating 2 concentrations (by Biophys Molec. Biol. 44: 215-267.
HCO
protonating 3 ) therefore increases carboxylation Fisahn,J.,T. McConnaughey,and \V.J. Lucas. 1989. Oscillationsin
rate. Many aquatic plants and invertebrate-algae appar- extracellularcurrent,externalpH,andmembranepotentialandcon-
ently promote photosynthesis through proton cycling. In ductanceinthealkalinebandsofNitellaandChara.J. E.\p. Bol. 40:
this context, protons, rather than CaCO3, may be the Garre1l1s8,5-R1.19M3.., A. Lerman, and F. T. Mackenzie. 1976. Controlsof
priFnrciopmleaprgoedoucchtemoifccaallcpiefriscapteicotni.ve, biologically precipi- a3t15m.osphericCO2andO2:past,present,andfuture.Am.Sci.64:306-
tated carbonates comprise one of the more abundant Goreau, T. F. 1963. Calcium carbonate deposition by corallinealgae
crustal materials, and represent the principle biogeo- andcoralsinrelationtotheirrolesasreef-builders.Ann. N. Y Acad
chemical reservoir for carbon (Garrels el ai. 1976). Be- Sci. 107: 127-167.
cause organisms often calcify much faster than the am- Hayama,T.,T.Shimmen,andM.Tazawa. 1979. ParticipationofCa:+
bient media in which they live, biological calcification tianticoenssiantiCohnaorfacceyateopilnatsemrincodsatlrceealmlsi.ngPrionldoupcleadsmbay9m9:em3b05r-a3n2e1.exci-
may provide an important brake on the photosynthetic Jaffe, L. F.,and R. Nuccitelli. 1974. An ultrasensitivevibratingprobe
alkalinization ofnatural waters, and thereby affect such formeasuringextracellularcurrents.J. Cell. Biol. 63: 614-628.
processes as the partitioning ofCO2 between the oceans Kingsley, R. J., and N. \\atabe. 1985. Ca-ATPase localization and
and the atmosphere. inhibition in the gorgonian Leplogorgia virgulata (Lamarck) (Coe-
lenterata: Gorgonacea). J Exp. Mar. Biol. Ecol 93: 157-167.
Kishimoto, U., N. Kami-iki, Y. Takeuchi, and T. Ohdawa. 1984. A
Acknowledgments kinetic analysisofthe electrogenic pump ofChara corallina: I. In-
hibition ofthepumpbyDCCD.J. MembraneBiol. 80: 175-183.
Primary fundingwasprovided through NSFfellowship Klaveness, D. 1976. EmilianiaIntxleyi(Lohman) Hayand Mohler. 3.
DCB-8807613 to Ted McConnaughey. W. J. Lucas and Mineral deposition and the origin ofthe matrix during coccolith
L. F. Jaffe contributed laboratory facilities. The authors formation. Protistologica 12: 217-224.
are grateful to J. Fisahn, W. Kiihtreiber, A. Miller, and Kuhtreiber, \V. M., and L. F. Jaffe. 1990. Detection ofextracellular
A. Shipley, for their assistance, and to D. McCorkle, A. calcium gradientswithacalcium specificvibratingelectrode.J. Cell
Biol 110: 1565-1573.
Kuzerian, C. Barr, and others for their enthusiasm and Kuile, B.ter,J. Erez,and E. Padan. 1989. Mechanismsfortheuptake
helpful comments. ofinorganiccarbonbytwospeciesofsymbiont-beanngforaminifera.
Mar. Biol 103: 241-251.
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