plant cell physiol 1997 shimmen 691 7

Plant Cell Physiol. 38(6): 691-697 (1997)JSPP © 1997

Studies on Mechano-Perception in Characeae: Effects of External Ca2+ andC\~

Teruo ShimmenDepartment of Life Science, Faculty of Science, Himeji Institute of Technology, Harima Science Park City, Hyogo, 678-12 Japan

The effects of modification of extracellular concentra-tions of Ca2+ and Cl~ on mechano-perception were studiedin internodal cells of Chara corallina. Cells were stimulatedby dropping a piece of glass tubing on them, and the re-sulting receptor potentials and action potentials were ana-lyzed. When the Ca2+ concentration was extremely loweredby adding EGTA, the amplitudes of both receptor poten-tials and action potentials were attenuated, suggesting theinvolvement of Ca2+ channels. However, the possibility re-mained that attenuation of the amplitude of the receptorpotential was caused by modification of membrane charac-teristics by extreme lowering of [Ca2+]o. When the plasmamembrane was depolarized to about 0 mV by adding 100mM KC1, responses in the negative direction were inducedupon mechanical stimulation. When the plasma membranewas depolarized by adding 50 mM K2SO4, responses in thepositive direction were induced. Thus, Cl" channels maybe involved in responses induced by mechanical stimula-tion under K+-induced depolarization.

Key words: Action potential — Ca2+ channel — Chara —Cl" channel — Mechano-perception — Receptor potential.

Some plants, such as Mimosa and carnivorous plants,respond to mechanical stimuli with dynamic motile re-sponses (Sibaoka 1991). What is now becoming evident isthat non-motile plants also seem to perceive mechanicalstimuli (Turgeon and Webb 1971, Jaffe 1973, Jones andMitchell 1989, Braam and Davies 1990, Knight et al. 1991).

Electrical analysis of mechano-perception has beenmainly studied with carnivorous plants, Dionea and Aldoro-vanda. These plants are equipped with sensory hairs con-taining sensory cells responsible for mechanoperception.

Abbreviations: APW, artificial pond water; [Ca2+], Ca2 con-centration; [Ca2+]c> [Ca2+] in the cytoplasm; [Ca2+]0, [Ca2+] inthe external medium; [Cl~], Cl" concentration; [Cl~]c, [Cl~] inthe cytoplasm; [C\~]o, [Cl~] in the external medium; EGTA, ethyl-eneglycol-bis-09-aminoethylether)M-/v',A'',A''tetraacetic acid; EQ,,equilibrium potential for Ca2+ across the plasma membrane; E a ,equilibrium potential for Cl~ across the plasma membrane; EK,equilibrium potential for K+ across the plasma membrane; Em,membrane potential; (En,)a, Em at the peak of the action potential}(Em)r, resting membrane potential; H, height from which a pieceof glass tubing was dropped for stimulation.

Upon mechanical stimulation, these cells generate recep-tor potentials, which are graded according to the intensityof the stimuli (Jacobson 1965, Benolken and Jacobson1970, Iijima 1991). When the amplitude of a receptor poten-tial reaches a threshold, an action potential is generated,which is transmitted to the motile tissue. The ionic mechan-ism for the receptor potential upon mechanical stimulationhas not been studied in these plants.

Kishimoto (1968) reported that internodal cells ofChara generate receptor potentials upon mechanical stimu-lation. Although Characeae has no differentiated sensorycells for mechanoperception, the potential change generat-ed satisfies the criteria of a receptor potential, i.e. summa-tion and a graded nature according to stimulus intensity(Kishimoto 1968, Shimmen 1996). When the membranepotential (Em) reaches a threshold upon generation of a re-ceptor potential, an action potential is generated, which istransmitted along the longitudinal direction of the cell(Kishimoto 1968, Staves and Wayne 1993, Shimmen 1996).

The generation of receptor potentials is a mechanosen-sitive process, and the subsequent action potential is a volt-age-sensitive process. Therefore, to elucidate the first stepof mechanoperception, the receptor potential must be ana-lyzed. The ionic mechanism of the receptor potential formechanoperception in plant cells was first challenged byShimmen (1997). Shimmen (1996) developed a simple ap-paratus for electrophysiological analysis of the mechano-perception of characean cells. This apparatus was used tostudy the effects of inhibitors of the proton pump and ionchannels (Shimmen 1997). The results showed that the elec-trogenic proton pump does not play an essential role ingenerating the receptor potential. It was also found that in-hibitors of Ca2+ and Cl" channels do not affect the recep-tor potential even at high concentrations (Shimmen 1997).On the other hand, channel inhibitors significantly in-hibited mechanically induced action potential (Staves andWayne 1993, Shimmen 1997). Although the receptor poten-tial was insensitive to the above channel inhibitors, Shim-men (1997) suggested that Ca2+ and/or Cl" channels areinvolved in generating receptor potentials, based on theequilibrium potential of these ions across the plasma mem-brane. However, this conclusion is still tentative. To findion channels involved in generating receptor potentials,this study was done to analyze the effects of modifyingCa2+ and Cl" concentrations in the external medium onthe amplitude of the receptor potentials.

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692 Mechano-perception in Characeae

Materials and Methods

Chara corallina was cultured as reported by Mimura andShimmen (1994). Internodal cells were isolated from neighboringcells and their lengths were adjusted to about 4 cm by ligation withpolyester thread and cutting. Cells thus prepared were kept in ar-tificial pond water (APW) containing 0.1 mM KC1, 0.1 mM CaCl2

and 1 mM NaCl.Mechanical stimulation and electrical measurement were car-

ried out using the apparatus as reported previously (Fig. 1 in Shim-men (1996)). A cell was partitioned into two halves using a cham-ber composed of two pools. One pool (B) was filled with 100 mMKC1 solution and other pool (A) with APW supplemented with180mM sorbitol, and the potential difference between the twopools was measured. The pH values of both APW supplementedwith sorbitol and KC1 solution were adjusted to 7.0 with 5 mMHEPES-Tris. Hereafter, the APW (pH 7.0) supplemented with180 mM sorbitol is simply called APW. Under such conditions,the membrane potential (Em) of the cell part bathed in pool A fill-ed with APW can be measured without inserting a microelectrode(K-anesthesia method, Shimmen et al. 1976). The Em was recordedusing an amplifier (Nihon Kohden MEZ7101) and a pen-writingrecorder (National, VP-6521A or VP-654A). Mechanical stimuliwere applied to the cell part in pool A by dropping onto it a pieceof glass tubing of 1.3 g. The intensity of the stimulus was controll-ed by changing the height {H) from which the tubing was dropped(Shimmen 1996). Stimuli were applied at about 30-s interval.When the cell generated an action potential, the subsequent stimu-lation was carried out after 7-10 min.

The effects of changing ion concentrations on mechanosensi-tivity were evaluated by comparing the amplitudes of receptorpotentials generated by stimuli of same intensity. Since the intensi-ty of the stimulus adopted to evaluate the sensitivity was differentamong the cells, statistical analysis was difficult. Therefore, theresults are described qualitatively. Experiments were carried out atroom temperature (22-26°C).

Results

Effects of decrease in the Ca2* concentration—Theequilibrium potential for Ca2+ across the plasma mem-brane (ECa) is significantly positive inside (Williamson andAshley 1982) and the Ca2+ channel plays a central role ingenerating action potentials (Lunevsky et al. 1983, Shiinaand Tazawa 1987a, Tsutsui et al. 1987, Tsutsui and Oh-kawa 1993). Presence of mechanosensory Ca2+-selectivecation channel and stretch-activated Ca2+ channel in plantmembrane has been reported (Ding and Pickard 1993, Gar-ril et al. 1992). To examine possible involvement of Ca2+

channel in receptor potential, the effect of lowering the Ca2+

concentration of the external medium ([Ca2+]o) was exam-ined. To decrease [Ca2+]0, EGTA at 1 mM was added toAPW and the pH was adjusted with Tris. To prepare a me-dium of pCa 3, 2 mM CaCl2 was added to APW containing1 mM EGTA. Media of pCa 6 and 7 were prepared usingthe association constant between EGTA and Ca2+, 4.83 x106M~' (Jewell and Ruegg 1966). The resting membranepotential ( ( E ^ ) in APW of pCa 3 averaged -234 mV(Table 1). By decreasing [Ca2+]o to pCa 6, (EJ , slight-

PCa3 pCa6 pCa70 -

-50 -

5- -100 -E

-150 -

-200 -

-250 - 1

20 sec

Fig. 1 Effects of [Ca2+]0 on receptor potentials and action poten-tial. The cell was stimulated in APW of pCa 3, 6 and 7. An actionpotential was generated by stimulus from H of 3, 2 and 2 cm atpCa 3, 6, and 7, respectively. Numbers below the Em trace show H(in cm) from which a piece of glass tubing was dropped. The effectof [Ca2+]o on mechanosensitivity was evaluated by comparing theamplitude of receptor potentials generated by stimuli from HoiXcm.

ly changed in the negative direction (—240 mV). When[Ca2+]o was further decreased to pCa 7, (E^, slightlychanged in the positive direction ( — 203 mV). By decreas-ing [Ca2+]0, the membrane potential at the peak of the ac-tion potential ((EjJa) significantly changed in the negativedirection, resulting in attenuation of the amplitude of theaction potential (Table 1, Fig. 1).

The effect of lowering [Ca2+]0 on the receptor poten-tial was examined by comparing the amplitude of the recep-tor potential generated by stimuli of the same intensity. InFig. 1, the amplitudes of receptor potentials generated bystimuli from H of 1 cm were compared. When [Ca2+]o de-creased to pCa 7, the amplitude of the receptor potential in-creased. However, this was not a general tendency. Theeffect of pCa 6 and 7 varied among the cells. By decreasing[Ca2+]o, the amplitude of the receptor potentials increasedin five cells, decreased in five cells, and did not change inthree cells. Thus, the amplitude of the receptor potentialdoes not seem to be affected by decreasing [Ca2+]0 to pCa 6and 7.

To extremely reduce [Ca2+]o, the medium was pre-pared by adding 1 mM EGTA to APW lacking CaCl2

(APW(-CaCl2)). When APW(-CaCl2) supplementedwith 1 mM EGTA was added to the external medium, (Em)r

changed in the positive direction and action potentials wererepetitively generated. Only in rare cases (3 cells), was theaction potential not generated for some period. In the cellshown in Fig. 2, mechanical stimulation in the presence of1 mM EGTA was possible. By adding EGTA, (E J a signifi-cantly changed in the negative direction, resulting in at-tenuation of the amplitude of the action potential. Theamplitude of the receptor potential generated by stimulifrom H of 0.5 cm decreased when EGTA was added.


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Mechano-perception in Characeae 693

Table 1 Effect of [Ca2+]o on the resting membrane potential ((Em)r) (mV) and the membrane potential at the peak of ac-tion potentials ((EJa) (mV)

Average

SE

(EJr

-234

2

pCa3(EJa

- 8

2

(EJr

-240

2

pCa6(EJa

- 3 7

2

(EJr

-203

4

pCa7

(EJa

- 6 5

2

Average values of 13 cells are shown with standard errors.

Effect of increase in the Cl concentration—The equi-librium potential for Cl~ across the plasma membrane(Ea) is also positive inside (Tazawa et al. 1974) and the Cl"channel is involved in generating action potentials (Lunev-sky et al. 1983, Shiina and Tazawa 1987b, 1988, Tsutsui etal. 1986). Presence of stretch-activated anion channel inplant membrane has been reported (Falke et al. 1988). Toexamine possible involvement of CP channel in generationof receptor potential, the effect of increasing [Cl~]o wasexamined. To increase [Cl~]0, 50 mM choline chloridewas added to APW, and the osmolarity was adjusted bydecreasing the sorbitol concentration. By increasing [Cl~]o,(EJ r slightly changed in the positive direction. (EJ a didnot change (Table 2). The amplitude of the receptor poten-tial generated by stimuli from H of 2 cm was not affectedby increasing [Cl~]0 (Fig. 3). By increasing [C\~]o, the am-

pCa3

0 -

-50 -

-100 -

EGTA

5-150 -

-200 - 0.5

Fig. 2 Effects of EGTA on receptor potential and action poten-tial. The cell was first stimulated in APW of pCa 3. An actionpotential was generated by a stimulus from H of 2 cm. Next, theexternal medium was changed to APW(—CaCl2) supplementedwith 1 mM EGTA. An action potential was generated by astimulus from / / o f 1 cm. Numbers below the Em trace show //( incm) from which a piece of glass tubing was dropped. The effect ofEGTA on mechanosensitivity was evaluated by comparing theamplitude of receptor potentials generated by stimuli from H of0.5 cm.

plitude of the receptor potential did not change in sixcells, increased in one cell and slightly decreased in one cell.Thus, the amplitude of the receptor potential does notseem to change when [Cl~]0 is increased to 50 mM.

Effect of both decreasing [Ca2+Jo and increasing[Crj0—To examine the effect of both decreasing [Ca2+]0

and increasing [Cl~]0, 1 mM EGTA and 50 mM cholinechloride were added to APW( — CaCl2). The osmolaritywas adjusted by decreasing the sorbitol concentration.Upon addition of the medium, (EJ r significantly changedin the positive direction (Fig. 4, Table 3). In this case,repetitive action potentials were not generated. It is sug-gested that decrease in the concentration of divalent cation(addition of EGTA) and increase in the concentrationof monovalent cation (addition of choline) caused drastic

APW

£ -100uf

1 min

Fig. 3 Effect of choline chloride on receptor potential and ac-tion potential. The cell was first stimulated in APW and an actionpotential was generated by a stimulus from H of 3 cm. Next, theexternal medium was changed to APW supplemented with 50 mMcholine chloride. Stimulation was carried out every 10 min. Ac-tion potentials were generated by stimuli from / / of 3, 2 and 2 cmat 10, 20 and 30 min, respectively. Numbers below the Em traceshow H (in cm) from which a piece of glass tubing was dropped.The effect of choline chloride on mechanosensitivity was evaluatedby comparing the amplitude of receptor potentials generated bystimuli from H of 2 cm.


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Table 2 Effects of 50 mM choline chloride on the restingmembrane potential ((E J r ) (mV) and the membrane poten-tial at the peak of action potentials ((E J J (mV)

Average

SE

APW

(EJr

-222

2

(EJa

- 7

2

50 mM Cholinechloride

(EJ,

-205

3

(EJa

- 1 1

2

Average values of 8 cells are shown with standard errors.

membrane depolarization (Shimmen et al. 1976).Even under large depolarization, the cell showed

responses upon mechanical stimulation. Two types ofresponses were observed,"" one in the negative direction(Fig. 4A) and the other in the positive direction (Fig. 4B).The direction of the response was dependent on (EJ r .When (EJ r was more negative than — 30 mV, the responsewas in the positive direction (Table 3, Cell No. 3-9). When(E J r was more positive, the response was in the negative di-rection (Table 3, Cell No. 1 and 2).

Response under K-induced depolarization—Theabove experiments (Fig. 4, Table 3) showed that the re-sponse in the negative direction was induced under large de-polarization. However, it was difficult to control (EJ r us-ing the medium containing EGTA and choline chloride.The level of (E J r was dependent on the cells used (Table3). Since (EJ r is strongly dependent on the concentrationof K+ in the external medium (Shimmen and Tazawa

Table 3 Response direction in relation to resting poten-tial ((EJr) (mV) in the presence of both 1 mM EGTA and50 mM choline chloride

Cell No. (EJrDirection of

response

123456789

- 22

- 4 5- 3 5- 3 5- 4 0- 3 5- 3 0- 4 5

negativenegativepositivepositivepositivepositivepositivepositivepositive

1977), (Em)r could be brought to about 0 mV by increasingthe K+ concentration. KC1 at 100 mM was added to APWlacking sorbitol. In some cells, (E J r changed to about 0 mVjust after the addition of 100 mM KC1. In most cells, how-ever, (E J r remained at levels more negative than —100 mVafter addition of 100 mM KC1 (Fig. 5). In the presence of100 mM KC1, (EJ r became unstable. By mechanical stimu-lation from H of 0.5 cm, a response in the positive direc-tion was observed. Upon stimulation from H of 1 cm, thecell generated an action potential. After small repolariza-tion, Era again changed in the positive direction and re-mained at about 0 mV.

After 5 min of depolarization, the cells were stimulat-ed at about 30-s intervals. As expected, responses in thenegative direction were induced by mechanical stimulation.

0 -

2 5 -UJ

-50 -

£. -50 -

u/ -75 -

-100 -

20 sec

Fig. 4 Response of Em in the presence of EGTA and cholinechloride. Cells were stimulated in APW( — CaCh) supplementedwith 1 mM EGTA and 50 mM choline chloride. Numbers belowthe Em trace show H (in cm) from which a piece of glass tubingwas dropped. In cell A, responses in the negative direction were ob-served. In cell B, responses in the positive direction were observed.

-100 -

I-150 -

-200 -

30 sec

Fig. 5 Depolarization in the presence of 100 mM KC1. Em wasfirst measured in APW. At the time shown by the arrowhead, 100mM KC1 was added. (Em)r became unstable. When the cell wasstimulated from Hof 0.5 cm, an receptor potential was generated.The stimulus from / / o f 1 cm caused an action potential. After abrief repolarization, the membrane again depolarized and remain-ed at the depolarized level. Numbers below the Em trace show H(in cm) from which a piece of glass tubing was dropped.


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I

0 -

-5 -

100 KCl

-10 -

-15 -

30 sec

Fig. 6 Responses in APW supplemented with 100 mM KCl.Upon stimulation, a cusp in the positive direction and a subse-quent response in the negative direction were induced. Stimulifrom / / o f 1, 2 and 3 cm induced a small cusp. However, no cuspwas observed upon stimulation from H of 4 and 5 cm. Numbersbelow the Em trace show H (in cm) from which a piece of glass tub-ing was dropped. A cusp generated by the stimulus from H of 3cm is shown by an arrowhead.

5 -

0 -

50 KCI/25KJSO, 30 KCI/3S KjSO4

-V

Fig. 8 Dependence of response direction on [Cl ]o. The cell wasstimulated in APW supplemented with 100 mM KCl, in APW sup-plemented with 50 mM KCl and 25 mM K2SO4 and then in APWsupplemented with 30 mM KCl and 35 mM K2SO4. Numbersbelow the Em trace show H (in cm) from which a piece of glass tub-ing was dropped. For further explanation, see the text.

The amplitude of the response was dependent on the inten-sity of the stimuli (Fig. 6). In most cases, however, theamplitude of the responses decreased when the stimuli wererepeated, presumably due to damage by stimulation underunusual ionic conditions. In the present study, the direc-tion and not the amplitude of the response was analyzed, asthe amplitude varied. In addition, the amplitude of the re-sponses decreased with time, even when cells were keptwithout stimulation. Therefore, experiments were alwayscarried out within 20 min after addition of K+ of high con-centrations.

Before a large response in the negative direction, asmall and rapid response in the positive direction was ob-

served (Fig. 6 arrowhead). This small response, observed inmany cases, hereafter, referred to as the "cusp". In thepresent study, the slow and large response was analyzedbut not the cusp. The direction of the response was depend-ent on [Cl"]0 (Fig. 7). First, responses in the negative direc-tion were observed in APW supplemented with 100 mMKCl. Next, the external medium was changed to APW sup-plemented with 50 mM K2SO4 and 65 mM sorbitol todecrease [Cl~]o to 1.3 mM. The cell was stimulated after 5min of exchange of the medium. In this case, the directionof the responses was positive. No cusp was observed, pre-sumably due to masking by a large response in the positivedirection. When the external medium was again changed to

100 KCl 50IC,SO4 100 KCl

20 -

-20 -

30s

Fig. 7 Change of direction of responses by exchange of KClwith K2SO4. The cell was stimulated in APW supplemented with100 mM KCl, APW supplemented with 50 mM K2SO4 and thenAPW supplemented with 100 mM KCl. In APW supplementedwith 100 mM KCl, a cusp and a subsequent response in thenegative direction were induced. In APW supplemented with 50mM K2SO4, a response in the positive direction was induced.Numbers below the Em trace show //(in cm) from which a piece ofglass tubing was dropped.

Table 4 Effect of [Cl ]o on response-direction

No.

123456789

1011

(KC1)O (mM)

100 50 30

ND

NDND

+ : response in positive direction, —: response in negative direc-tion, ND: not determined. ?: direction of response could not bejudged. [K+]o was adjusted at 100 mM by adding K2SO4.


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100 mM KC1, a cusp and a subsequent response in thenegative direction were observed. (Em)r in the presence of100 mM KC1 was -0 .3+0 .5 mV (n = 33) and that in thepresence of 50 mM K2SO4 was - 4 . 5 + 1.5 mV (n=19).

The dependence of the direction of responses on[Cl~]o was further analyzed. The KC1 concentrations werechanged in the order of 100, 50 and 30 mM (Fig. 8). K+ con-centration and osmolarity were adjusted with K2SO4 andsorbitol, respectively. In the presence of 100 mM KC1, acusp and a subsequent slow response in the negative direc-tion were observed. By decreasing [Cl~]o to 50 mM, theamplitude of the cusp slightly increased and that of theslow response in the negative direction significantly de-creased. By decreasing [Cl~]o to 30 mM, the amplitude ofthe response in the negative direction became very small.

The dependence of the direction of the slow responseon [Cl~]0 is summarized in Table 4. At 100 mM [Cl~]o, allcells showed responses in the negative direction. At 50 mM[Cl~]0, five cells showed responses in the negative directionand three cells in the positive direction. In one cell, the di-rection of the response could not be judged. At 30 mM[Cl~]0, the responses in the positive direction were ob-served in most cells. In one cell, small response in thenegative direction was observed (Cell No. 11).

Discussion

This is the first report analyzing the effects of externalCa2+ and Cl~ on receptor potentials induced by mechani-cal stimulation in plant cells. Although Characeae has nosensory cells, it is very suitable for analyzing the receptorpotential because of its advantages as a tool for electrophys-iological study (Shimmen et al. 1994).

When [Ca2+]o was lowered to pCa 6 or 7, the am-plitude of the receptor potential was not affected. Thisresult is reasonable, considering the equilibrium potentialfor Ca2+ across the plasma membrane (ECa), which is calcu-lated with the following equation:

(1)

where [Ca2+]o and [Ca2+]c represent [Ca2+] in the externalmedium and that in the cytoplasm, respectively. William-son and Ashley (1982) reported that [Ca2+]c at the restingstate is 0.22 fiM in Chara. Using this value, EQ, is calculat-ed to be 106, 19 and - 9 . 9 mV at pCa 3, 6 and 7, respective-ly- (Em)r at pCa 3, 6 and 7 was -234, -240 and -203 mV,respectively (Table 1). Thus, (E,n)r is significantly morenegative than EQ, at all [Ca2+]o. When 1 mM EGTA is add-ed to APW from which CaCl2 has been removed, Eo,should significantly change in the negative direction. Undersuch conditions, the amplitude of the receptor potential isdecreased (Fig. 2). From this result, it is tentatively conclud-ed that the Ca2+ channel might be involved in generating

the receptor potential. However, the possibility remainsthat attenuation of the amplitude of the receptor potentialwas caused by modification of membrane characteristics byextreme lowering of [Ca2+]o, but not by change of ECa.

To examine the possible involvement of Cl~ channels,[C\~]o was increased to 50 mM (Fig. 3, Table 2). ECi was cal-culated with the following equation:

Ecl = 581og (2)

where [Cl ]c and [Cl ]o represent [Cl ] in the cytoplasmand that in the external medium, respectively. Tazawa et al.(1974) reported [C\~]c to be 21 mM in Chara. Using thisvalue, ECI for cells bathed in APW supplemented with 50mM choline chloride is calculated to be — 22 mV, whichis significantly less negative than (Em)r ( — 205 mV). Evenwhen 100 mM choline chloride was added, E a was calculat-ed to be — 39 mV. Further increase in the concentrationof choline chloride is not technically possible. Thus, it isdifficult to make ECI more negative than (Era)r by addingcholine chloride.

To carry out analysis based on equilibrium potentialfor ions in cells bathed in physiological medium, voltageclamping experiments are recommended. However, in theK-anesthesia method, a large electrical resistance betweentwo pools (series resistance) cannot be avoided (Shimmenet al. 1976). Therefore, it is difficult to clamp the membranepotential at the desired level. To carry out a voltage clamp-ing experiments, a measuring apparatus without a largeseries resistance must be developed. In the present study,however, analysis of Cl~ channel based on the equilibriumpotential became possible by depolarizing the membranewith 100 mM K+ (Fig. 7, Table 4).

In the presence of 100 mM KC1, the response was inthe positive direction before the depolarization (Fig. 5) andwas in the negative direction after depolarization (Fig. 6).The response in the negative direction was also induced inthe absence of K+ at high concentration, when the mem-brane was significantly depolarized (Table 3 Cell No. 1 and2). Thus, it is concluded that the response in the negative di-rection was induced by depolarization but not by an in-crease in KC1 concentration.

K+ concentration in the cytoplasm has been reportedto be 112 mM (Tazawa et al. 1974). In the presence of 100mM K+ in the external medium, the equilibrium potentialfor K+ across the plasma membrane is calculated to be 2.8mV. Thus, (Em)r in the presence of 100 mM KC1 ( -0 .3mV) is close to EK. E a in the presence of 100 mM KC1 inthe external medium is calculated to be — 39 mV, whichis more negative than (E,n)r. Reflecting the situation, Em

changed in the negative direction upon mechanical stimula-tion in the presence of 100 mM KC1 (Fig. 6,7). When the ex-ternal medium was changed to APW supplemented with50 mM K2SO4, [Cr]0 was 1.3 mM and E a was calculated


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to be 70 mV. (EJ r in APW supplemented with 50 mMK2SO4 was -4 .5 mV. Reflecting the situation, Em changedin the positive direction upon mechanical stimulation in thepresence of 50 mM K2SO4.

Tazawa et al. (1974) reported [Cl~]c to be 21 mM inChara, and the direction of the response was suggested tochange at 21 mM [Cl~]o. However, the direction of theresponse changed at [Cl"]0 between 50 and 30 mM. InNitella, Tazawa et al. (1974) reported [CP]C to be 31 mM.In the cells used in the present study, [CP],. might range be-tween 30-50 mM, especially in the presence of high KC1concentration in the external medium.

The present study showed that both Ca+ and CP chan-nels might be involved in generating the receptor potential.The response in the presence of K+ at a high concentrationis not necessarily the same as the receptor potential generat-ed in APW. However, the response in the presence of ahigh K+-concentration may reflect some aspects of me-chanosensitivity of characean cells. What is needed is ameasuring system without large series resistance to enablevoltage clamping experiment during mechanical stimula-tion.

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(Received January 16, 1996; Accepted March 25, 1997)


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