effect of external nh4,na, transmembrane electropotential ... · effect of external k, nh4,na,ca,...

Effect of External K, NH4, Na, Ca, Mg, and H Ions on theCell Transmembrane Electropotential of Avena Coleoptile

N. Higinbotham, Bud Etherton3, and R. J. Foster4Department of Botany, Washington State University, Pullman

A number of studies have shown that an electro-potential difference, PD, of about 100 mv (interiornegative), is present across plant cell membranes(2, 3, 7, 8, 10, 11, 13, 21), similar to that acrossanimal celtl membranes. Thus, if minerals movethrough cell membranes as ions, they are subjectnot only to concentration gradients but also to elec-trical fields.

For precise evaluation of the energy relationshipsin ion accumulation and active ion transport by cellsthe electropotential gradient as well as the chemicalgradient must be known (4, 5). Active ion trans-port, requiring metabolic energy, is defined as trans-port against the electrochemical activity gradlient(4 5). Cases are known in which ion accumulationtakes place without the expenditure of metabolicenergy (5, 18).

The use of the Nernst equation in elucidating thefundamental relationships of electropotential differ-ences to ionic concentration gradients across plant cellmemiibranes lhas been discussed recently in detail byDainty (5) and by Briggs, Hope, an(l Robertson (4).The results of similar studlies of aninmal cells (12, 16,19) and of giant cells of algae (2, 11, 13, 21) showthat the resting electropotentials have an approximlatequantitative relationship to the concentration gra(lientat equilibrium of the ions K+, Na+, and Cl-. Thisrelationship is mlore closely approached when consid-eration is given to the relative permeability coefficientsof these ions as in Goldmlan's modification (9) of theNernst equation. This suggests that the resting po-tential is diffusional in origin, that it nmay be primar-ily a function of the relatively fewv more rapidly per-mleatiing ions, andl that K+, in particular, may have apre(lomlinant effect.

Previous reports have shown that oat, corn, andpea see(lling tissue cells, exhibit a transmembrane PDwhich is influenced by external K+ concentration inthe direction predicted by the Nernst equation (8).HoNever, the effect on these plant cell PD's of ionsother than K+ has not been evaluate(l. The present

1 Received revised manuscript June 28, 1963.2 This research was supported by a Grant (G-9560)

from the National Science Foundation and also, in part,by funds provided for biological and medical research bythe State of Washington Initiative No. 171.

3 Present address: Department of Plant Science, Vas-sar College, Poughkeepsie, N.Y.

4 Department of Agricultural Chemistry and Depart-ment of Chemistry.

report deals wvith a limilited survey of the effect onPD of manipulating the ion coIcenltration graClieints1w altering the external concentration. This papergives details of the resuilts mlientioedI in aIn earlierbrief report (10).

Materials and Methods

The mletlho(d for measuiring plant cell transmiiemii-branie potentials witth glass capillary miiicroelectro(leshias been described elsewhere (7, 8) anid is essentiallythe same as the teclhniiqtie used extenisively withanimiial cells (1, 17). In brief, the excise(d tissue wasmiouinted in a special1y constructe(d Ilcite perfusioncell nmounted on a microscope stage So that an elec-tro(le controlled by a micromanipulator couilc be in-serted inlto the enId( wvall of a cell. Electrodes usedfor insertionl into the cell wvere fille(d with 3-xt KC1solution by boiling whereas the independent electro(lewas fille(d wvith agar ima(le xwith 3 z\ KCl solution.The iniserte(l electro(le was coinniectedi to a cathodefollower amplifier haxvinlg 10,000 iiiegohitn impe(ldancein series xvith a suitable electrometer-e.g., anI elec-tronic galvanometer (Kinl Tel \Model 204A) or oscil-loscope (Tektroni.x Type 532 with Type D plug-inampllifier) and theince to -round(I (ind(lepend(lenit elec-tro(le in perfusing solution)i. The junictioIns wvith thecal)illary electro(les xx-ere via sil-er-silver clhlori(lexWi res.

The oat grains, Aveia .%oti.v( var. \ictory, weresoaked for 2 hours in tal) wvater tlheni planlte(d invermiculite and( allowed to groxlfor 90 to 96 hoturs at250. They xere expose(d to 2 to 4 hours of weak redlight after about 70 hours. The xerniiiculite was wellxatere(d xith nutrienit soltutioni of the folloxing coIll-position in nimiloles/ KCI, 1.0: Ca(N03)2 4H2O,1.0; MIgSO4 7H20., 0.25 NaH.P0,4 * H 0 plusNa.,HPO4 2H.,O to gixe an Na. concentration of1.00 and a pH of 5.5. Tlis nutrienit solutioni at theabove concentration is refer-re(d to as 1 X and XX lientise(l at threefold concentration as 3 X, etc.

To prepare tissue for an experiment the upper51 inni of the coleoptile xas remox-ed and a section ofahout 1 cim was excise(l. Four sections were mlouinte(din each lucite perfusioln cell and place(d itn a beaker ofthe experimental solution to be shaken until PD nieas-urements could be ma(le, usually 3 to 7 hours.

7Thleory. Accor(ling to the Nernst equation ap-pliedl to the case of tissue in KC1 solution the rela-tionship betwveen a, (liffusioInal electropotential and

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HIGINBOTHAM ET AL.-TRANSMEMBRANE ELECTROPOTENTIAL

the concentration gradient at equilibrium and at 25°is (with E in mv):

E = -59 log [KI]1inIn this case the cell interior is electrically negative tothe exterior providing that [K+]10 exceeds [K+1011tand C1- is restrained. If the external concentrationis changed then:

E' = -59 log [K+]'in II

However, isotopic tracer studies have shown thatafter an initial wall space exchange, K+ influx is ata rate such that many hours are required before equili-bration takes place. Thus it may be assumed for thepurposes of the present tests that [K+ ] in remainsconstant since changes in external concentration weregenerally 10-fold or more. Then, subtracting equa-tion II from equation I:

E - E' = -59 log [K+]'011t III[K+10ut

This equation is not valid since it assumes that [K+]inis in equilibrium at 2 quite different external conceni-trations which is obviously not true under the usualphysiological conditions. However, it can be usedto approximate limiting values for the change of PDwith changes of external concentration. Thus themaximum depolarization on increasing external KClconcentration 10-fold would be 59 mv; appreciableinflux of C1- would result in lesser PD changes.Ions actively transported or having low permeabilitycoefficients would have little or no effect. For a givenion, e.g. K+, the PD for a 10-fold change in externalconcentration is expressed as AlOEK.

ResultsThe data presented in table I show a marked de-

polarizing effect of both NaCl and KCl with increas-ing concentration. For NaCl the maximum changeof PD was attained between solutions 1 and 2; thisamounts to 20.5 mv for a 10-fold concentrationchange, i.e., AlOEN-a = 20.5 mv.

For KCl the maximum depression of cell PD was25 mv between solutions 1 and 3; this gives a AlOEKvalue of 25 mv. However, the results indicate thatthe effect on PD of either Na+ or K+ concentrationis in part a function of the absolute concentrationsince A1oE between solutions 2 and 4 (treated for ashorter time) was 13 mv.

Unfortunately, the accuracy of the A values is nothigh owing to variability between coleoptiles (tableI). For this reason, measurements were made ofthe change in PD of single cells while changing theNaCl or KCl concentrations. Under these conditionsthe AE maximum for a 10-fold concentration changeof NaCl was 14 mv, and, for KCl, 27 mv.

Despite the uncertainty of the AE values it seemedclear from these early tests that K+ had a greatereffect on PD than Na+, and that in either case, theremust be some effect of other ions since neither yieldedthe maximum potential slope of 59 mv. Conse-quently, additional experiments were performed inwhich coleoptile segments were treated for 4 to 6hours in KCl or in NaCl solutions at the concentra-tions in which PD's were measured. This is be-lieved to displace ions other than those in the ex-ternal solution from the apparent free space of thetissue. Under these experimental conditions the re-lationship of PD to concentration of external solu-tions of KCl and NaCl was measured (fig 1). In

~E140

100~~~~~~~~~~~20~~~~~~~~~~~~~~

~~~~6O~ ~ ~ CC

40o Theor.-\ K CI_

20~~~~ ~ ~ ~ ~ ~ ~~~~~~2

KCI + me/

1.0 10.0 100.0 0.1 0.3 1 3 10 30 100K Cl or Na CI m eq/i. ex1. sol n. KCI or CaCI2 meq/l. ext. solin.

FIG. 1. (left) The relationship of cell PD, interior negative, to KCl and NaCl concentration in the externalsolution. The theoretical curve is in accord with the Nernst relation using measured PD at 1 meq/liter as the startingpoint and assuming no Cl- effect.

FIG. 2. (right) The effect of cell PD of concentration of CaCl2 (data of Table II), and of KCl alone (curve basedon means of 2 experiments previously reported (8)) and of KC1 in the presence of 3x nutrient solution containing6 meq/liter Ca(N03)2-

197

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Table I

Effec-t on Cc/I Elcctropotential of I 'trilouis Exrtcrol/ Soluitionis

Cell PD, mv

External Hours in No. of Total range of avgsolution No. of coleoptile means of coleoptilesolutioni (approx) coleoptiles cells and range rneans*

of sn's*

Experiment 11. nutrient solution 3X

(pH 5.5)2. nutrient solution 3X plus

NaCl 100 meq/l (pH 5.5)3. nutrient solution 3X plus

KC1 27 meq/1 (pH 5.5)4. nutrient solution 3X plus

NaCl 100 meq/1 andKCl 27 meq/1 (pH 5.5)

Experiment 25. nutrient lOX miniusMgSO4 (pH 5.4)

6. MgCl2, 20 meq/'1(pH 5.5)

7. CaC12, 20 meq/1(pH 5.6)

5

1

6

5

.3

4

4

4

4

3

20

22

18

25

15

3

4 20

102-109(SD, 4-5)74-86

(SD, 2-10)74-88

(SD, 2-7)64-69

(SD, 2-6)

99-108(SD, 2-5)12-140

137-149(SD, 4-7)

107

80

82

67

104

80*

143

* A mean cell PD value was obtainied for each coleoptile. The average given in column 6 (far right) is the averageof the coleoptile meanis. The range of coleoptile means and the range of standard deviations is giveni in columni 5.

** PD's were unistable and cells appeared to have lost normal turgor.

these experimiienits there was a greater (lepolarizingeffect of both KCl an(d NaCl, particularly at the higherconcentration range. However, in the range fron1 to 10 nmeq/liter the AlOEKvalue was 39 nmv. Forthis same change in external concentration NaClcause(l a PD change of only 5 ImyV although a much

greater PD dIrop wvas apparent between external coIn-centrations of 10 and 100 meq/liter. (The A10E be-tween 1 andl 10 meq/liter is less for Na+ in this par-

ticular experimlent than in others not reported here).The several experinments comlparing K+ anld Na+

concentration effects are in agreement in showingthat up to 10 nmeq/liter external concentration K+has the greater depolarizing effect. The increase inthe depolarizing effect of KCl and( NaCl followingpretreatment at the higher concentrations yields AEvalues approaching those found for muscle cells and

algae.Comlparison of effects on PD of CaCl2 and M\IgCl,:

The data of sonme experiments on the effects of MIg+ +

and Ca+ + are shown in table I. Whereas increasingthe concentration of Na+ and K+ resulted in a de-crease of cell PD (in the absence of a complete nu-

trient solution externally) it is apparent that Ca+ +increased the polarization (relative to that in thecomplete nutrient). Omission of MNgSO4 from thenutrient solution had no effect on cell PD. However,the cell electropotentials were unstable in tissuesexposed to a solution of MgCl2 only (table I). Thecells apparently lost turgor, and the nmeasurementsappeare(l to be unreliable. In general there was (le-

polarization. This was also true ot tissues treatedonly a few minutes in MIgCI2 solution.

Additional experiments with Ca+ + concentra-tions confirm the fact that this cation has a uniqueeffect by causing polarization. This is showni graph-ically in figure 2 in whiclh the effects of CaCl. con-centration may be conmpared over a rather wide rangeof concentration. Increasing KCl concentrationi inthe presence of Ca(NO,)2 again caused depolariza-tion; however, the cell PD wN-as at a greater level,an(l this level apparently was largely determinie(d bythe external concentration of Ca±++.

This action of Cac+ imiglht appear to resemblean anion effect; that is, increasing CaCl2 concentra-tion externally may result in increasing anion influxrelative to cation influx thus yielding greater separa-tion of charge across the mlembrane and a higher P1).Such an effect should be largely nullified un(ler con-ditions in whiclh Ca"+ is varied while Cl- is keptconstant, providing that relative permeabilities re-main unchanged. The result of one such experimentkeeping Cl- constant antl substituting Ca"+ forTris is shown in table II. It is readily apparent thatthe Ca+ + effect is essentially unchanged, giving anincrease in PD of 57 mv between 0.1 meq/liter to20 meq/liter. In another experinment (table III)the effect of CaCl2 was tested with similar results atconcentrations of 0.1, 1.0, and 10.0 nmeq/liter made upin 100 meq/liter NaCl. Under these conditions Cl-concentration effects should be negligible.

An additional test of CaCI2 at 20, 70, and 130

198S PLANT PHYSIOLOGY




Table IIEffect of Calcium antd Tris Chloride Solutions on Cell Electropotential

Cell PD, myExternal Hours in No. of Total range of avg

solution No. of coleoptile means of coleoptilesolution (approx) cells and range meansof SD'S*

1. CaCl2 0.1 meq/l 5 4 20 77-91 81Tris Cl 21 meq/l (SD, 5-21)(pH 6.1)

3 4 33 72-84 78(SD, 8-15)

2. CaC12 2.0 meq/l 7 4 26 97-123 110Tris Cl 20 meq/l (SD, 3-11)(pH) 6.0)

3. CaCl2 20.0 meq/l 3 4 34 133-140 137Tris Cl 1 meq/l (SD, 7-14)(pH 6.0)

* See footnote under table I.

meq/liter was made in the absence of other electro-lytes. The data of this experiment, given in tableIII, and of others not reported here, suggest thatthere may be no leveling off of the increase in cellPD at higher Ca+ + concentrations.

Comiparison of Anions Accomnpanying Ca+ +. Asan additional test of whether the Ca"+ effect on PDcould be a function of the accompanying anion solu-tions of CaCl2, CaSO4, and CaH2PO4 were used toperfuse the tissue. The data (table IV) indicaterather clearly that any anion effect per se is quitesmall relative to the Ca+ + effect. None of the anions

caused any appreciable reduction in cell PD such asthat found by use of K+ or Na+.

Influence of NH4+. Experiments withNH4H2PO4 solutions in the presence and absence ofCaCl2 revealed that this salt induces a sharp drop inPD. It seems apparent that NH4+ caused markeddepolarization of coleoptile cell membranes; thechange in potential was about that found for K+.Since NH4+ is believed to permeate cells readily, thedepolarizing effect of NH4+ is in accord with theconcept that cation diffusion is involved in cell po-tentials.

Table III

Effect of CaC12 Co0tcentrationt ont Cell Electropotential in the Presence and Absence of NaCl at 100 nieq/liter

Cell PD, mv

External Hours in No. of Total range of avgsolution solution coleoptiles No. of coleoptile means of coleoptile(approx) cells and range means*

of sn's*

Experiment 11. CaCl, 0.1 meq/1

NaCf 100 meq/l (pH 5.6)2. CaCl, 1.0 meq/l

NaCl 100 meq/l (pH 5.6)3. CaCl2 10.0 meq/l

NaCl 100 meq/l (pH 5.5)Experiment 24. CaCl2 20 meq/l

(pH 5.9)changed to CaCl,130 meq/l (pH 6.6)

5. CaCI2 70 meq/l(pH 6.1)

6. CaCl2 130 meq/l(pH 6.6)

* See footnote unider table I.

6

4

7

3

1

6

5

4

4

4

4

1

4

4

20

21

19

20

5

20

26

35-47(SD, 2-4)57-73

(SD. 3-4)78-98

(SD, 1-6)

120-134(sD, 0-4)

162(SD, 9)144-156(SD, 3-6)143-161

(SD, 6-11)

41

62

88

128

162

151

156

199



PLANT PHYSIOLOGY

Table INCcll PD's Comiiparcd int CaCI2, Ca (NO3) 2' CdSO4, and Ca (H2PO4) 2 Solultions

Externialsolutioni

Hours insolutioni(approx)

Experiment 11. CaCl2 20 meq/l

(pH 5.6)2. Ca (NO )2 20 meq/l

(pH 5.7)Experiment 23. CaSO4 10 meq/l

(pH 5.6)changed to Ca (NO3) 210 meq/l (pH 5.7)

4. Ca(NO3)2 10 meq/l(pH 5.7)chainged to CaSO410 meq/l (pH 5.6)

5. CaH2PO4 10 meq/l(pH 5.4)

6

5

5

6

1.5

5

No. ofcoleoptiles

4

4

4

4

4

3

5

TotalNo. ofcells

20

22

29

27

28

16

25

Cell PD, myrange of

coleoptile meanisaind rangeOf SD's*

129-138(sD, 2-5 )135-151

(SD, 2-13)

104-119(sD, 4-11)114-127

(SD, 8-14)125-142

(SD, 2-14)123-129(SD, 3-8)115-130(so, 3-4)


Influence of H+. H+ and HCO-, may be con-si(lered to be of unique importance since they areconstantly being producecl by metabolisnm and each hasbeen involvedl in theories of ion accumulation or iontransport. Consequently to test the effect of H +several experimiients were performedl at different pHvalues. The effect of a nutrient solution adjusted topH 3.6 with HCl in comparison with the normal nu-trient of pH 5.5 is shoNwn in table V. On the basisof the assumlptions miia(le above (e.g., for equation

III) increases in the externcal concentration of anycation passively (listributed slhoulcl cause a depolariza-tion, providing the permeability of aniions (loes notchange. However, increasing H + ion resulted in asmall but definite increase in PD. This was truealso for the comparison of pH 5.6 andcl pH 7.0 (tableV). For the H + ion the AE -alue amounted to amaximum of about 8 mv for a 10-fold concentrationchange. Such a result is consistent with hypothesesthat H+ ions may be actively pumlped outw-ard but

Ccll Electropotential intTable V

3X Nutrient Soliutiont at Varioius pH l alucs

Externalsolutioni

Hours insolution(approx)

Experiment 11. pH 5.5

2. pH 3.6

Experimenlt 23. pH 5.6

chaniged to 1H 7.0

4. pH 7.0

changed to 1)H 5.6

10

4

4

8

1

No. ofcoleoptiles

4

4

4

3

4

4

TotalNo. ofcells

20

20

21

16

27

25

Cell PD, myrange of

coleoptile meanisand rangeof SD'S*

101-107(SD, 1-4)109-123(SD, 4-5)

113-120(SD, 3-10)100-111(sD, 2-6)95-114

(SD, 2-10)109-123(so, 1-3)


avgo(_f coleoptile

means*

133

142

111

12")

132

1 25

122

avg(f coleoptile

means*

104

118

115.5

103

108.5

117

200




does not exclude the interpretation that lower pHvalues selectively favor inward anion transport.

As a further test of whether or not increasing H+ion concentration might under some conditions de-polarize cell PD, experiments were performed insingle salt solutions to compare the effect with thatin complete nutrient. In brief exposures to 10 mMt

KCl solution at pH 3.5 no change in PD relative topH 5.3 was noted. However, prolonged exposure

(4 hours) in KCl solution at pH 3.5 caused a markedreduction in cell PD. Since this was associated withnoticeable deterioration of the tissue it is believednot to be of great significance with respect to normalcell PD and ion transport. It does serve to show thatcell PD, as other properties of healthy cells (e.g.,turgor), becomes abnormal with prolonged treat-ment at low pH.

In a test of pH effect in a solution mixture of10 mAi CaCl2 and 0.1 mAi KCl it was found that Ca+ +greatly protects the tissue from the deleterious effectof low pH as indicated by cell PD's. Again, higherH+ concentration (comparing pH 5.5 and 4.1) gave

a slight increase (11 mv) in cell PD. This was notnoted in tissue transferred briefly to pH 3.1, whichshowed a lowering of the PD (12 mv).

Effect of HCOy-. In addition to the H+ ion,CO2 is constantly being liberated from cells. If theCO2 is not liberated fronm the protoplast as such butrather passes through the membrane as HCO3- thenthis process could influence the electropotential grad-ient. A high rate of HC03- efflux, relative to ca-

tion efflux, would be expected to reduce the cellelectropotential; raising the external HC03- con-

centration might then increase the potential by in-creasing the influx rate. In a test of the effect ofexternal HCO3- ion no significant difference was

found at a level of 0.5 mmoles/liter of NaHCO3 atpH 7.2.

The Effect of Tris, Chloride, anid Glntamiate Ions.In several experiments it was found expedient to sub-stitute Tr,is or glutamate for mineral ions. The rela-tive effect of these ions may be seen in table VI, inwhich 20 meq/liter of Tris chloride is compared withTris glutamate and CaCl. In this experiment a

change of 200-fold in Cl- concentration, accomplishedby substituting glutamate for Cl-, resulted in a de-polarization of 22 mv. Such a depolarization wouldbe expected if the flux of Cl- is somewhat less thanthat of glutamate ion. Transfer to CaCl2 solutionresulted in a rapid stronig increase in cell PD showingclearly that neither glutamate nor Tris has an irre-versible effect. Calculation of the change of cellPD for a 10-fold change of Cl- to glutamate concen-

tration (table VI) gives a value less than 10 mv.Thus the effect of external concentration of theseanions, as with others, appears small compared to theeffect of extreme cation concentrations.

In another test in xvhich the Ca+ + concentra-tion was essentially constant (at 20.1-22 meq/liter)glutamate was substituted for chloride (table VI).Under these conditions Cl- gave a slight reduction inPD relative to glutamate.

Discussion

The changes of cell transmembrane potenitialswith increasing external KCl concentration are in thedirection predicted by the Nernst and Goldman equa-

tions (9) if K+ is the ion with the faster diffusionrate. Thus cells of higher plants appear to behavemuch as in the giant coenocytes of the algae Nitella,Chara, and Halicystis, and in animal muscle fibers

Table VI

Inifluence of Chloride and Glutamnate Coniceittrations, as Tris or Ca Salts, oil Cell Electropotenitial

Cell PD, mv

External Hours in No. of Total range of avg

solution solution coleoptiles No. of coleoptile meanis of coleoptile(approx) cells and range means*

of SD'S*

Experiment 11. Tris Cl 0.1 meq/l 5 2 10 50-56 53

Tris glutamate 20 meq/l (SD, 8-1 1)2. Tris Cl 20 meq/1 5 3 17 29-35 31

Tris glutamate 0.1 meq/l (sD, 6-10)changed to CaCl2 0.5 1 7 123 12320 meq/l (SD, 20)

Experiment 23. CaCl2 0.1 meq/l 6 4 22 145-155 150

Ca glutamate 20 meq/l (SD, 4-10)4. CaCl2 2.0 meq/l 5 4 27 135-147 142

Ca glutamate 20 meq/1 (SD, 3-9)5. CaC12 20 meq/l 8 4 21 130-154 141

Ca glutamate 0.1 meq/l (SD, 4-6)


201



PLANT PHYSIOLOGY

and nerves. From a quantitative viewpoint the ex-

perimental (lesign was such that conclusions fromthe data must be rather limited. It has been demon-strate(l that the slope of cell PD may approach thetheoretical limit of 59 nmv for a 10-foldl change ofexternal concentration. A pretreatment of tissue inKCl at 100 meq/liter yielded a change of 51 mv per

10-foldl change in external concentration. Since thisapproaches closely the maximum possible value inthe Nernst equation it suggests that Cl- diffusion maybe small relative to that of K+

The effect of NaCl solution as similar exceptthat the present data indicate that NaCl gives lessdepolarization in the lower concentration range

(1.0-30 meq/liter). Between 10 andl 100 meq/liter,however, external NaCl concentration yielded a PDchange amounting to as much as 61 mv per 10-foldNa+ concentration difference which is close to thetheoretical maximum value.

The closest approaches to the maximum depolar-ization effect of both K+ and Na+ were under con-

ditions of several hours pretreatment at higher con-

centrations; presumably these ions essentially satu-rated the wall space at the expense of Ca+". Thiswas the condition sought by Hope and XValker (13)in their work on Chara autstralis in which they foundthat cell PD conformed to the Goldman equation with2 provisions. These were that the permeability co-

efficient of Na+ was about 0.06 that of K+; andl Cl-permeation was negligible (relative to K+ and Na+).

Especially interesting is the finding that increasesof external CaCl2 concentration increases cell polar-ization. Of the other salts tested-including the ionsMg+ +, NH4+, Cl-, NO3-, SO4=, HQPO4-, andTri,-only H+ and glutamate showed a similar, butlesser, effect. It has been noted that increased ex-

ternal concentration of the basal nutrient solutiolnfailed to depolarize the cell PD over a range of

100-folcl (0.1 X to 1XO) despite the ide range ofK+ concentration involved (7). This may now beattributed to the presence of Ca++ which causes a

polarization approximately equal to the (lepolariza-tion effect of K+ and( other cations.

Previous workers have noted that Ca++ may in-duce greater resting PD's in nerve and muscle cells(19). This effect appears to reach a greater miagni-tude in oat coleoptile cells thani any previously re-

ported for other tissues. According to the Nernstequation, for a divalent cation the PD may have thefollowing relationship:

RT Cl,clE =-ln ~29 loo IV

2F C1 ( I

where C1 an(d C2 represent different concentrationsof CaCI2. A 10-fold change in external concentra-

tion may result in a maximum change in PD of 29 mv.However, an influx of Ca++ as an ion wxouldl be ex-

pected to cause depolarization as other cations do.What accounts for the increase in cell PD inducedby increasing external Ca+ + ? The following are

possible hypotheses; they are not nmutually exclusive:1) Ca+ + may increase the mobility of K+ relative

to Cl-. The polarizing effect of Ca+ + is nearlyequal and opposite to the depolarizing effect of K+(under conditions in which Ca++ is present or hasnot been displace(l from the apparent free space). 2)Ca+ + may be enhancing a catioIn efflux puimp, e.g.,for Na+ (7) or for H+, or, perhaps, for Ca+ +

itself. 3) Ca+ + nmay enhance an anion influx puImlp.4) Ca"+ may be affecting some ioIn not conlsi(lere(here.

The data at hand do not allow an unambiguouschoice between these hypotheses. The idlea that theCa+ + effect is a result of increasing a passive K+escaping tendency would appear not to lhold un(lerthe conditions in whiclh the PD excee(ls the valuepredicted from the K+ gradient as shown by Etherton(7). It seems unlikely, also, on the ground thatCa+ + generally induces a greater K+ accumulation(6, 15) and thus is unlikely to increase K+ effluxmlarkedly, if at all.

Evidence for a sodium efflux pump in oat and peatissues has been recently reported (7). The presentdata slhowing greater PD's with increasiing Ca+ +and H+ concentrations are consistent with the idleathat these ions also nmay be actively extrude(l. (How-ever, it does not constitute p)ositive evidence thatthey are actively extruded). Simlilarly, for thethird hypothesis, there is considerable evidence boththat active accumulation of anions occurs and thatanion accumulation is increased in the presence ofcalciuml. In hypotheses 2 and( 3 there is the implica-tion that the ion pumps w\ould be electrogenic, that is.would generate a PD. Dainty lhas recently statedthat according to present evidence ion pUnips are notelectrogenic (5). Nevertheless, the relatively rapid(lecrease in PD of oat coleoptile cells following 2.4-dinitrophenol adldition (8) suggests thlat a miieta-bolic electrogenic punmp may be involve(l.

Hypothesis 4 is purely speculative but is men-tione(d because of the general occurrence of signifi-cant amounts of organic acids in planit cells. Theseconceivably could play a major role.

It is well known that calciumii affects cell perme-ability giving increased selectivity and, under appro-priate conditions, enhances the uptake of certainother ions (6,14,15). Sinice calcium increases tlletransmembrane electropotential gradient, this, inturn, wVould constitute a nonispecific force tending todrive cations into the cell. This force wouldl notnecessarily affect an anlioin acculmulation process or aprocess in which cationis are pullpedl outwardl stuclas a sodium pumlp (7).

The influence of the H+ ionl is anotlher extremelyinteresting feature of the results. In the presence ofCa+ +-in a comlplete nutrient solutioln or in a CaClIplus KCI mixture-increasing H + concentrationi ex-ternally gave small but apparently significant in-creases in cell IPD. This is the result to be expectedif H+ is actively transporte(l. Otherwise increas-ing H+ concentration mliglht be expected to cause adepolarizatioln as in the case of K+ or Na+ provid-inig H+ is quite highly permeable relative to anionis.The polarizing effect of H + wvas reversed un(ler

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longer treatments or at higher H + concentration ofpH 3.1, that is, conditions in which H+ might be ex-pected to partially displace Ca"+ at the cell surface.

The substitution of Tris for some cations appar-ently caused depolarization much as K+ and NH+4.This may be considered surprising in view of thefact that Tris has been reported to increase K+ in-flux rates (20). Both Tris and glutamate appar-ently affect cell PD much as inorganic electrolytes.For this reason the use of these substances must bemade with care in ion uptake studies.

Summary

The electropotential differences in the coleoptilecells of Avena sativa L. var. Victory were measuredwith glass capillary microelectrodes. The coleoptileswere perfused with solutions of nutrient salts invarious concentrations and combinations in a surveyof the effect of various ions on the transmembranepotential of cells in this tissue.

Increasing the external concentrationis of K+,NH4+, and Na+ ions lowered the cell electropoten-tial difference. This was also true for increasingconcentration of Tris.

Increasing the external concentration of Ca+ +caused a marked increase in cell electropotential dif-ference. Increasing the concentration of either H+ions or glutamate ions had a similar but lesser effect.

The kind of anion present in the external solutioninfluenced the PD slightly under the conditionstested; for example, the electropotential differenceappeared to be slightly higher in NO3- than in Cl-,H2PO4j, or S04= solutions.

The present data on cells of higher plants are con-sistent with the idea that the resting electropotentialdifference is particularly related to K+ diffusion.

Acknowledgments

The authors wish to thank Dr. L. B. Kirschner, Dr.A. B. Hope, Dr. M. G. Pitman, and Professor R. N.Robertson for reading the manuscript prior to revisionand for many stimulating discussions (with the seniorauthor). Technical assistance was given by Mr. JamesSugg and Mr. Michael Williams.

Literature Cited

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2. BLINKS, L. R. 1949. The source of the bioelectricpotentials in large plant cells. Proc. Natl. Acad.Sci. 35: 56675.

3. BLOUNT, R. W. AND B. H. LEVEDAHL. 1960. Activesodium and chloride transport in the single celledmarine alga Halicystis ovalis. Acta Physiol.Scand. 49: 1-9.

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10. HIGINBOTHAM, N., B. ETHERTON AN-D R. J. FOSTER.1961. The source and significance of th-e electro-potential of higher plant cells. Plant Physiol.36: xxxv.

11. HILL, J. E. AND W. J. V. OSTERHOUIT. 1938. Cal-culations of bioelectric potentials. If. The con-centration potential of KCl in Nitella. J. Gen.Physiol. 21: 541-56.

12. HODGKIN, A. L. AND P. HOROWICZ. 1959. The in-fluence of potassium and chloride ions on themembrane potential of single muscle fibres. J.Physiol. (London) 148: 127-60.

13. HOPE, A. B. AND N. A. WALKER. 1961. Ionic rela-tions of cells of Chara autstralis R. Br. IV. Mem-brane potential differences and resistances. Aus-tralian J. Biol. Sci. 14: 26-44.

14. JACOBSON, L., D. P. MOORE, AND R. J. HANNAPEL.1960. Role of calcium in absorption of monoval-ent cations. Plant Physiol. 35: 352-58.

15. JACOBSON, L., R. J. HANNAPEL, D. P. MOORE ANDM. SCHAEDLE. 1961. Influence of calcium onselectivity of ion absorption process. PlantPhysiol. 36: 58-61.

16. JENNERICK, H. P. AND R. W. GERARD. 1953. Mem-brane potential and threshold of single musclefibers. J. Cellular and Comp. Physiol. 42: 79-102.

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19. SHANES, A. M. 1958. Electrochemical aspects ofphysiological and pharmacological action in excit-able cells. Pharmacological Rev. 10: 59-164.

20. VAN STEVENINCK, R. F. M. 1962. Potassiumfluxes in red beet tissue during its "lag phase."Physiol. Plantarum 15: 211-15.

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effect of external nh4,na, transmembrane electropotential ... · effect of external k, nh4,na,ca,...

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