The Plant Journal (1895) 8{6), 811-825 Inward and outward K*-selective currents in the plasma membrane of protoplasts from maize root cortex and stele ‘Stephen K. Roberts* and Mark Tester Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge CB2 3EA, UK ‘Summary In an attempt to understand the processes mediating ion ‘transport within the root, the patch clamp technique was applied to protoplasts isolated from the cortex and stele of maize roots and their plasma membrane conductances investigated. In the whole-cell configuration, membrane hyperpolarization induced a slowly activating inwardly rectifying conductance in most protoplasts isolated from the root cortex. In contrast, most protoplasts isolated from the stele contained a slowly activating outwardly rectifying conductance upon plasma membrane depolarization. The reversal potential of the inward current indicated that it was primarily due to the movement of K*; the outwardly rectifying conductance was compara- tively less selective for K*. Membrane hyperpolarization beyond a threshold of about -70 mV induced inward currents. When E was set negative of this threshold, inward currents activated negative of & and no outward currents were observed positive of Ex. Outward currents in the stelar protoplasts activated at potentials positive of -85 mV. However, when Ex was set positive of -85 mV a small inward current was also observed at potentials negative (and slightly positive) of the equilibrium potential for K+. Inwardly and outwardly rectifying K* channels were observed in outside-out patches from the plasma ‘membrane of cortical and stelar cells, respectively. Charac- terization of these channels showed that they were likely tobe responsible for the macroscopic ‘whole-cell’ currents. Inward and outward currents were affected differently by various K* channel blockers (TEA‘, Ba?* and Cs*). In addition, Ca?* above 1 mM partially blocked the inward ‘current ina voltage-dependent manner but had little effect ‘on the outward current. itis suggested that the inwardly rectifying conductance identified in protoplasts isolated from the cortex probably represents an important com- Ponent of the low-affinity K* uptake mechanism (mechan- ism Il identified in intact roots. The outwardly rectifying ‘conductance identified in protoplasts isolated from the stele could play a role in the release of cations into the xylem vessels for transport to the shoot. Received 11 May 195; rvisud 13 July 1986; acepted 14 Soptember 1885, ‘For comesponsence fax +48 01223 33396). Introduetion Roots of higher plants are responsible for the transport of ions from the soil to the xylem vessels (and hence to the shoot). The roots are a complex organ containing various specialized cell types. In general, the epidermal and cortical cells are the site of ion entry or uptake from the soil into the root (Clarkson, 1993}. In contrast, the stelar cells, which ‘are surrounded by the cortex, regulate the release of ions to the xylem vessels for transport to the shoot (Clarkson, 1993). Separating the cortex from the stele is the endo: dermis, which forms a barrier to the apoplastic movement of ions. Thus, for ions to reach the xylem, they must enter the symplasm, in the epidermis or cortex and then be released into the stele apoplast. The properties of the plasma membrane of individual root cells are likely to regulate the transport of ions from the soil to the shoot, but little is known of the molecular mechanisms involved. The patch clamp technique has demonstrated the presence of cation- and anion-selective channels in the plasma membrane of various higher plant root cells (Findlay et al., 1984; Gassmann and Schroeder, 1994; Ketchum etal, 1989; Schachtman et al, 1991; Skerret ‘and Tyerman, 1994; Vogelzang and Prins, 1992, 1994; Wegner and Raschke, 1994; White and LemtiriChlieh, 1995). However, in most of these studies the whole root ‘was used to isolate protoplasts and thus the origin of the protoplasts within the root (for example, cortex, stele or epidermis) is unknown. Hence itis difficult to interpret the role of these channels in the transport of ions within the intact root. Exceptions to this are two recent reports in which protoplasts of known origin have been isolated from higher plant roots. Wegner and Raschke (1994) isolated protoplasts from xylem parenchyma cells from the stele of barley roots. These cells border the xylem vessels and ‘re thought to control ion release into, and their re- absorption from, the xylem vessels. A patch clamp study revealed both K*- and cation-selective outwardly rectifying conductances and anion channels inthe plasma membrane of these protoplasts which appear likely to control the release of ions into the xylem vessels. A K*-selective inwardly rectifying conductance was also observed in these protoplasts, which may allow the re-absorption of cations from the xylem vessels. In a separate study, Gassmann and Schroeder (1994) isolated protoplasts from wheat root epidermal cells. A K*-selective inwardly rectifying conductance was most commonly seen in the plasma ‘membrane of these protoplasts and is likely to represent the pathway for the low-affinity uptake of K* from the In the present study, the patch clamp technique was an812 Stephen K. Roberts and Mark Tester ‘employed to compare the ion transport properties of proto- plasts from the stele and cortex of maize roots. This study represents the first attempt to isolate plasma membrane {on currents from maize roots and compares currents from different cell types within the same root. The results of this study reveal K*-selective conductances which may function ‘a8 major components of the cation transport mechanisms ‘operating in intact roots of higher plants. Results Morphology Protoplasts isolated from the cortex of maize roots were typically 50-75 um in diameter and contained a large vacuole and relatively little cytoplasm (Figure 1a). These ‘were easily distinguishable from protoplasts isolated from which were typically 25-35 um in diameter and ‘were rich in cytoplasm (Figure 1b). It is probable that fextrastelar protoplasts were from the root cortex rather than the epidermis because: (i) the number of cortical cells is much greater than the number of epidermal cell (id) protoplasts had an internal morphology corresponding to intact cortical cells; and (ii the epidermal cells of maize roots have been reported to be more resistant than cortical cells to enzymatic digestion and remain intact (Kochian and Lucas, 1983), an observation also made in the present study. Whole-cell currents Standard bath solution was initially used to elucidate the types of plasma membrane currents present and to investigate their activation and deactivation kinetics. Upon. plasma membrane hyperpolarization, a large time- and voltage-dependent inward current was seen in 20 out of 21 protoplasts isolated from the cortex (Figure 2a and b). Mean current density for this current at ~160 mV was 51.7 + 9.7 mam? (n=13). The activation of this current was best fitted (using the least-squares fit method) by the ‘sum of two exponential components using the following equation (Figure 2c): expt-tty) + hg expl-tta) +h a where 4 is a constant leak and fa; and fz are the steady- state currents of the two components after activation. The time constants (r) and t) showed no apparent voltage- dependence between -200 and ~120 mV (Figure 2d). The inward current showed no inactivation for at least 6 sec (data not shown). The deactivation of the current upon depolarization to -80 mV followed a single exponential (data not shown), Figure 1 Protoplasts Isolated from (a) the comtex and {b the stle of 8 young mize rot “Tha batch pipette i inicated by the arrow Seale ba is 100m In protoplasts isolated from the stele, membrane hyper- polarization induced an inward current in only five out of 24 cells measured. This inward current had similar activa- tion and deactivation kinetics (data not shown) to that measured for the inward current in cortical protoplasts. Plasma membrane depolarization elicited a time- and voltage-dependent outward current in 19 out of 24 stelar protoplasts (Figure 3a and b). Mean current density for this current at +60 mV was 249 + 83 mAm (n = 16). This current activated and deactivated more slowly than the inward current. The outward current activated moidal time course, and could therefore be fitted by @ Hogkin-Huxley-type model (Hodgkin and Huxley, 1952), ‘according to the equation: = ht helt exp (-tP 2) where p is the number of independent membrane-bound 4gating particles that control the opening of the channel, j, is a constant leak and J, is the steady-state current after ion. Currents were usually best fitted (using the least-squares fit method) when p was set to 2 (Figure 3c); in three cases the best fit was obtained when p was set to 3. The time constant (2) decreased slightly as the voltage increased from 0 to +80 mV (Figure 3d). Deactivation of the current after hyperpolarization to -100 mV followed a single exponential (data not shownl. A time-dependent ‘outwardly rectifying conductance was observed in only two out of 21 protoplasts isolated from the cortex. ThisCation currents in maize root cortex and stele 813 “oo +80mV_ c io zo Som : z +0 10 mv § 400 120m 3 ae é i, ; “ 8 ~ -180 mV -t0onv -1900. 200 mv j eo 1000 To re) fear x o fo = © ze ztu se o © © MM & “1200 Figure 2. Whole-cell currents messured 200-180-160 -140-120 Votiage (mv) the plasma morbrane of protoplasts isolated from th cortex of mala rots Standard bath solution wes used. (a) Plasma membrane curerts resulting fom voltage pulses ranging from +80 to -180 rn (in TO. steps at intervals of 7 aes, Fr clatity,only current responses from votage pulsts ranging from +80 to -160 mV Gin 20 mi tape are shown, Holding potential was -60 rn. The ‘splayed currents have had leak current subtracted llebk conductance was 200 pS). The dlameter ofthe cll was 8 nm (6) Curer-votage (1 relationship ofthe time-dependent (@) and instantaneous (6) component ofthe currents shown in (a. (c} Examples of last-squares feof equation (1) tthe activation of the inward current. Holding pote! was -60 mV. (a Votape-dependence ofthe time coatans of activation ofthe inward cuant the rum of feplestes i indicated In brackets foreach point; err bars ‘denote he SEM ‘outward current had similar activation and deactivation kinetics (data not shown) to those measured for the out: ward current in stelar protoplasts. These results are sum- marized in Table 1 ‘A second type of outward current which activated and deactivated rapidly (‘instantaneously’) was also observed upon membrane depolarization in approximately 80% of protoplasts isolated from the cortex and 50% of protoplasts isolated from the stele (Figures 2a and 3a). This current was not characterized in the present study; only the time- dependent conductances are investigated in the present study, The magnitudes of the time-dependent outward currents presented in current-voltage curves were calcu- lated by subtraction of instantaneous currents from steady- state currents. The time-dependent inward currents wore calculated after subtraction of the linear ‘leak’ current (that is, the current resulting from the voltage-independent whole-cell membrane and seal resistances calculated by applying a small voltage to the pipette) from the pseudo- steady-state current measured 650 msec after the start of the activating voltage pulse. This time was chosen to minimize overestimation of the time-dependent inward current due to the activation of a small, vary slowly activa ing inward current (which has also been shown to exist in many other cells by Blom-Zandstra and Vogelzang (com- ‘municated at Plant Transport Group Workshop, Newcastle upon Tyne, UK, 12-14 September 1994) Identification of the time-dependent inward and outward currents Selectivity of whole-cell currents. Tailcurrent protocols were used to determine the major ion responsible for the time-dependent inward current. Inward currents were activated by a hyperpolarizing prepulse, then were followed by steps back to more positive potentials, giving rise to deactivation (tail) currents (Figure 4a and b). The reversal potentials (Ey) of the tail currents were determined as described in Figure 4. The reversal potential ofthe inward current for protoplast in standard bath solution was -61 =814 Stephen K. Roberts and Mark Tester 1300 1280 ssomv 2 1000. ‘200 b zg conv 750 - i sso mv & s0 3 500 oe : j Tiomv Ee a0 L—™ © 400 o 200 oT a Time (s) d 1500 1200 100 E 10 3 z ° £ ws 2 250 ° Votage (mv) Figure 3. Whole call currnts measured aoe the pasma membrane of protoplasts ioated from tho stale of maze roots. ‘Standard bath solution was used. a) Plasma rembranecutrents resulting from voltage pulses fom +10 to 150 rn (in 10 mV stops at intervals o 0 see For cary, currents responses te voltage pula ranging from +80 to -80 mi fin 20 mV eps are shown. Holding potenti! was =100 ml. The diameter of the cell was 35 ym, (b) LV calationship ofthe ime-depandant component of the currents shown in (cl Examples of east squares ts of equation 2 to te stvation ofthe outward current Holding potential was 100 mW (ch Votioge-sependence of the tims constants of activation ofthe outward cutent aor bars denote SEM: = 10) Table 1. Frequency of occurrence and current density of the time-dependent inward and outward currents in th membrane of protoplasts isolated from the stele and cortex of maize roots (mean current density is shown and where appropriate the standard error of the mean is also presented) Conex Stele Current density at Current density at Current density at Current density at Number of ~160 mV +60 mV Number of ~160 mV 60 mV ells (onder?) (nae?) cals (nde Areva?) Inward current 9 577295 (13) = 3 m 2 : Outward current 1 = 27 ” = 249-83 (15) Both currents 1 7 161 2 3732) 22712) No currents ° e - 2 . s Total number of cals 21 2 2mV (m5). This was much closer to the reversal potential _(Figure 4d). This suggests that K* is the major ion respons- for K* in the solution (E = ~48 mV) than to the reversal _ible for the inward current. Consistent with this, the may Potential for CI” (Ec = ~31 mV). When extracellular K* tude of the inward current increased with increasing was varied over a wide range of concentrations, the E,g, extracellular K* concentration and saturation of the inward of the inward currents followed closely the changes in Ex current with voltage occurred with decreasing extracellular200 -30my Curtent (pA 1000 00 ‘Time (ms) Cation currents in maize root cortex and stele 815 7501 C +57mV J 500. 1000 ~140" 100-60” -20 E, (mv) Figure 4, Measurement of reversal potentials (Ey) ofthe inward and outward currents. (a) Tal currents fom a cortical protoplast resulting from 8 voltage step to 158 mV followed by steps back to potentials ranging from -30 to 78 mi dn ‘nV steps at itera of 7 sel. Fr clr only cutent esponses o 8 rn steps are shown. Holding potential was -63 mV. Standard bath solution was used. The alameter ofthe cell was 60 um. (0) Enlargement ofthe boxed aa in (ah. The rverel ofthe til currontsoccurrad at -50 ml, The reversal ofthe tal cutee (Eq) wae determined follows: the amplitude ofthe ti current was cleuatedimmadatly after the decay ofthe capacitance curent and when curens reached seedy state (yh. Currant at xwessubrectod from that at yand plotted against vltago. The potrtia at which s-y = O (thats, Ey) was determined from near gression. (6) Tal euronts rom 2 solar protoplast resting trom 2 voltage stp to +57 mV folowed by steps back o potentials ranging from 47 mi to -77 mV (in 5 mV stop at intervals of 20 sec. Holding potetal was -67 mV. ESB bath slition wae used. The tail curants versed a 60 The carta of the call ‘wae 35 um. The reversal ofthe al currons (Ey! ws determined a8 Io 1). (dl Ege plod a8 function ofthe K* equilbium potential (forthe inwacd (and outward (@)curonts. Ez was varied by perusing the bath wth solutions of varying K”concentaions;solutons used were ESA (= —115 mV), ESB (= -73 mV, standord extvacalilr solution (Ex = ~48 mV) and ESC (G = -28 mV). The data represent the means of saverl experiments (the number of replicates I incested in bracets foreach point: errr bars dente the SEM). The line represents values whon Ey e9Uals Ey K* concentration (Figure 5). Membrane hyperpolarization beyond -71 + 2.8 mV (n = 12) induced an inward current in standard bath solution. If Ex was sat negative of this threshold, tho inward ourrent only activated at potentials nagative of Ex (Figure §) (that is, the activation potential of the inward current followed 6). No outward current was observed at potentials postive of Ex indicating that the channels which passed the inward current allowed no " release. Dependence of the activation potential of the inward current on E has also been observed in other cell types, including wheat root epidermal cells (Gassmann and Schroeder, 1994), guard cells (Schroeder and Fang, 1991) and cultured Arabidopsis ces (Cerana and Colombo, 1983). Assuming that the whole-cell currents inthe present study reflected the activity of one channel type, the sonsi- tivity of the channel gating to [K"leq was examined by fiting @ Boltzmann function to the relationship between the chord conductance of the inward current and voltage (Figure 5c, insert). In 55, 15 and 35.5 mM [K"loq the Voltage at which half-maximal conductance occurred (Va) was -130 * 4.6 mV, ~137 + 3.8 mV and-132 + 35 mV (n= 4), respectively. This indicated that the voltage-dependent gating of these channels was insensitive to changes in Ex or IK Teq- However, from Figure 5(), the activation poten- til of the inward current was always negative of Ex and no outward currents were observed at potentials positive of Ex. The most likely explanation for this observation is, that these channels can activate at potentials positive of Ex but K* efflux is prevented due to blocking by an intracellular cation (Hille, 1992, p.481). Taik-current experiments were also used to elucidate the major ion responsible for the outward current (Figure 4c). The reversal potential of the outward current for protoplasts in standard bath solution was -30 + 3 mV (n = 5). Tall current experiments were repeated in different bething solutions in which extracellulor K* was varied. As extra- cellular K* was altered, Ey changed by the same amount 128 Ex, However, En was always positive of Ex by approxim. ately 20 mV (Figure 4d), © much greater voltage than found for any measured junction potentials (Experimental816 Stephen K. Roberts and Mark Tester procedures) This is consistent with some other ion with a more positive equilibrium potential contributing to the conductance. In ESC bath solution, the equilibrium poten- tial for Mg2* and C- was such that a passive Mg? or CI flux could not account for the postive value of the Eu relative to Ex. However, @ C2? influx could account for the positive values of Ea, relative to the calculated Ex. A Ca?* influx is also thought to explain a similar deviation of Evy from Ex for the outward current in cultured cells from tobacco (van Duijn etal, 1983). ‘At potentials postive of-86 mV both inward and outward currents were observed in protoplasts isolated from the stele, depending on Ex. At potentials negative (and slightly Positive) of Ex small inward currents were observed; but at voltages more positive of & an outward current dominated ie 400) 5.5mMKy = 400) 35.5 mM K" ao So i “sgn § "szamv & 400 “BBinv E00 8 é Cs mn mane sean 1200. +1200, {202m 080 "1600 Osho "1000 ‘Time (ms) ‘Time (ms) A ISS mM K 9355 mK 250 -200 -150 -100 -60 Vp (OV) (Figure 6). If Ex was set at least 20 mV negative of 85 mV only an outward current was observed. The out ‘ward current may be mediated by a channel which activates at -85 mV and passes small inward currents at potentials Positive of Ex due to the influx of cations (for example, Ca) Alternatively, more than one channel type may be responsible for these currents. The small inward current was not the result of @ passive Cl flux since it was most conspicuous at voltages positive of the equilibrium potential for CI (Figure Ge and d). The voltage-gating of these currents was not investigated by fiting @ Boltzmann, function to the relationship between the chord conductance because it was not clear how many channel ted to the whole-cell currents. Single-channel recordings. Single-channel activity such as that shown in Figure 7(a) was observed in five out of six ‘outside-out patches from cortical cell protoplasts. The reversal potential of the single-channel currents followed predicted changes in E (Figure 7b), as observed for whole- cell inward currents in cortical protoplasts (Figure 4d). The unitary conductance of this channel was calculated to be 11 pS in ESD bath solution (15.5: 121 mM K™, outside: cytosol) and 24 pS in ESE bath solution (100: 121 mM K+, outside: cytosol) (Figure 7b). The steady-state open probability (P,) of the channel (as derived from amplitude histograms) increased with negative going potentials (Figure 7c). The relationship between P, and voltage could be fitted by a Boltzmann function and was similar to that observed for whole-cell inward currents (compare Figures 5c and 7c, confirming that these channels are most likely to be responsible for the inward currents observed in the whole-cell patch configuration. Furthermore, no outward currents were observed at potentials positive of Ex in Fgura 5. The efets of changes in exracelularK* concentration on the urensfrom protoplasts fom the corox (o} Currants in 55 mM K° (ESB) Holding potertial was -64 mV and voltage pulses ranged from 44 to -204 mv tin 20 mV stape at intervals of 7 sc) (b) Currnts in 35. mM K* (ESC Helding potrtia wae ~62 mV and vokage pulses ranged from 42 to -202 mv Vn 20 nV stops a intervals of 7 sec. The displayed currents have had lek current subtractad (ese conductance was 125 pS). The diameter (©) Cunrent-vot a range of ‘exaeral K" concentrations. Calculated K” equilour potentials () for ‘ach solution are incieated by symbols and artows above the wax inset the relationship between steedy-stte chord conductance of the inward current and votage. Chord conductance (G) was eleulated a8 lynx) where ly i6 steady currant ata tst voltage (Vp). Data ware fod to Botzmarin equations ofthe form G = Gnas +0epiVq-Vos HSI where G Is the chord conductance a oetvolage Vn Goa conductance, Vis ls the maximal chord ‘which the chord conductance ig hat valent to RTE where the minimal and Fhave their usual meanings Data were obtained id 355 mM IK" For the deta peesarted, at 85 mM K>, Sous = 28 08 ond Vyg = ~136 mV; at 155 mM Ky Gay = 48 nS and Voe'= 196 mv a 38. MOM K", Gag = 9 and Vag 2131 mV. For all curves, 5 = 2,patches containing the inwardly rectifying K* channels, demonstrating that these channels do not allow K° efflux. This is also consistent with the whole-cell currents measured in cortical call protoplasts. ‘Single-channel activity such as that shown in Figure 8a) was observed in three out of four outside-out patches from Bis nme Dassen 1000 samy zm & 52m zo 12m 3 seo ze ‘Time (s) 100, Z nw di e Curent oA) Fee ‘20 8 anv “zim 100 wis Times) etm Brees a ts3mMKC o3nMiKe Votage (mv) 120" ‘120 t ttt & © wae Figure 6, The affects of changes in extracellular K* concentration onthe currents from protoplasts fom the stl {a} Curent in 1 mM K” (ESA. Holding pototial was 106 mV and votage ules ranged rom +88 to -106 mi in 10 mV stop a interval of 30200, For cary, onl current responses from voltage putes ranging Irom +88 10-86 mV (a 20 mV step) are shown (b) Currmts Ia 35. mM K" (ESC). Holding potemal was -96 mV and voltage pulses ranged from +102 to -88 mV in 10 mV steps at intervals 030 se) For cary, only current responses rom vokage pulses ranging from +82 to-88 mV (n 20 step} are shown. The dlareter of he call was 25 um {6} Det ofthe smal inward current associated with the setvatin of the ‘oumard current. Data asin (b), But only current traces resulting from ++12,-8, and -28 mV pulees ar shown (Ey = -28mnV, Eg = -54 mV) varying K* 250 Cation currents in maize root cortex and stele 817 stelar cal protoplasts. The reversal potential ofthe single- channel currents followed predicted changes in Ex but remained more than 20 mV positive of & (Figure 8b) as observed for whole-cell outward currents in stelar cell protoplasts (Figure 4d). The unitary conductance of this channel was 22 pS in ESF bath solution (1: 121 mM K', ‘outside: cytosol) and increased with increasing external K* Summing single-channel recordings showed that the ‘outward K' channels had similar time-dependence of activation to the whole-cell outward currents (compare Figures 3 and 9), confirming that these outward K* chan- nels are responsible for the macroscopic outward currents observed in stelar cell protoplasts. The steady-state P, of ‘the channel increased with positive going potentials (Figure 8). This relationship between P, and voltage could be fitted by Boltzmann functions; increasing external K* con- centration shifted the voltage at which P, was half-maximal (Vas) more positive (Figure 8), indicating thatthe voltage dependent gating ofthis channel was sensitive to changes in external K*. No inward currents were observed at potentials negative of Ex (up to -80 and -40 mV in ESD and ESE, respectivoly) in patches containing the outwardly rectifying K* channels This suggests that the macroscopic outward and inward currents observed in stelar cell protoplasts (see Figure 6 and related text) resulted from the activity of different channel types. Sensitivity to inhibitors. Extracellular TEA' (10 mM) reduced the inward current by approximately 50% and only a small further reduction of the inward current was achieved with 20 mM extracellular TEA*. This inhibition could be fully reversed after washing for approximately 2 ‘min (Figure 10). Be?* (3M) and Cs* (2mM) in the bathing solution were more effective blockers; both completely ‘and reversibly blocked the inward current (data not shown). The sensitivity of the inward current to extracellular Ca?* was also investigated. A voltage- and dose-dependent block of the inward current was observed with increasing extracellular Ca?” (Figure 11). At 40 mM Ca2* the inward current was reduced by approximately 50% relative to the currents observed with 1 mM Ca?*. The time-dependent outward current had a distinct pharmacology. Extracellular TEA” (10 mM) reduced the time-dependent outward current by approximately 70% and increasing TEA’ to 20 mM induced only @ small ‘additional reduction (Figure 12). This block could only be fully reversed after washing for at least 10 min, It i noteworthy that the small inward current which was ‘observed at potentials negative of the activation of the outwardly rectifying conductance was also blocked by TEA", suggesting that the channel mediating this inward current is also sensitive to TEA". Extracellular Ba?” (5 mM) largely inhibited the time-dependent outward current818 Stephen K. Roberts and Mark Tester mere emery 100 sel Wade “Wal WVU 29 4 as 155 mMK" +100 mM kK" b (va) wauno, c 075, 80 : How Bows ° +200 -150-100° 500 ‘Voltage (mv) Figure 7. Properties of inwaray recivng K° channels fom conical cell prtoplss. 2) Recordings of inwardly rectifying K' channel activity from outside- ‘ut patches. Bat solution was ESD. The holding voltage (eyoplasm wih respect to outs ch recording s shown onthe ight. Downward deflections represent channel openings. Curent traces were selected paricully to ilustate current amplitude and recoding resclution rather than probability of opening (se {b)Singlechanne! curent as a function of voltage. Data shown ‘rom five patches in ESD (15 mM K") and ESE (10 mM), with diffrent symbols fr each call The unitary conductance (6) and reversal potent (Ey were dotrmined by fiting a linear regression to the deta. In ESD (Eq = -88 mV = -28 mV, yy was -B3 mV and G was 1! pS: in ESE Uf = ~4 mV; Ez = “82 nV), Ey Was 4 nV and G was 24. (6 Voltage dependence ofthe steady-state single-channel opon probabil in ESD (soe Experimental procedures for calulton of open probabilities! Dato (trom the same patch shown in (Data were ited toa Bormann equation of the frm P, = PsN exD1Vn~ agNSI where." isthe maximal ‘bed othor symbol area desorbed in Figure 5. P= O68, Vas = -120 Vand whereas Cs* was an ineffective blocker at concentrations of 2-10 mM. Also, up to 40 mM extracellular Ca?* had no significant effect on the outward current (data not shown). These results are summarized in Table 2. Discussion Frequency and distribution of whole-cell currents ‘Aclear pattern emerged in the types of plasma membrane conductances observed in protoplests from the cortex and stele of maize roots. The most frequently observed conduct- {ance in protoplasts from the root cortex was a time-depend- tent K*-solective inwardly rectifying conductance. A tim dependent outwardly rectifying conductance was rarely ‘observed in these protoplasts. In contrast, the most com- monly observed plasma membrane conductance in proto- plasts isolated from the stele was a K"-selective tims dependent outwardly rectifying conductance; only one-fifth had atime-dependent inwardly rectifying conductance. This distribution of the plasma membrane conductances is unlikely to be due to the protoplast isolation technique as both sets of protoplasts were isolated using the same proto- col. The different plasma membrane transport properties probably reflect distinct functions of the cortical and stelar Cells. is noteworthy that the most common plasma mem- brane conductance in protoplasts from cultured maize root cells was a K*-selective outwardly rectifying conductance (Ketchum et al, 1989) and thus they may have originated {from the stele. Alternatively, these cells may have de-differ- entiated and may not have represented any particular root cell type. In the present study when protoplasts were isol- ated from whole roots using the same techniques described here, the morphological and electrophysiological character- istics were the same as those seen in protoplasts isolated ‘from the stele. This suggests that, contrary to immediateintuition, rather than cortical protoplasts. ‘The only other plant in which comparisons can be made e I / els y “100° 60 0 50100 Voltage (mv) Cation currents in maize root cortex and stele 819 between different cell types within the root is in wheat. This can only be done by comparing data from two different studies (Findlay et al, 1994; Gassmann and Schroeder, 1994), A K*-selective inwardly rectifying conductance isthe dominant plasma membrane conductance in protoplasts {from the epidermis (Gassmann and Schroeder, 1994). How- i omv Vr Vpaia = -60 mV Figure 9. Time-dopendencs of the sctvation of outwardly reciying K channels rom stele protoplasts. (a) Outward ecifyingK* channel city in an aoatad autside-ou patch in ESDbath solution mV ater moving fom ahoding potential o-60 mV. (b) Least-squares fit of equation (2) to the sum of 28 isolated patch “The time constant (wes Figure 8. Properis of outwardly retiying K° channos from stelar call protoplasts (a) Rocordings of outwardly ectiyingK* channel ctv from outside-out patches. Bath soliton wae ESF. The holding voltage or eae recording it shown on the ight Upward dflcions represent channel openings Current vaces were selected particulary to ilustate current amplitude recording resolution rather than probably of opening {se {b) Single-channel current ae function of voltage. Data shown from tee patches in ESF (1 mM K'),ESD (155 aM K") and ESE (100 rm"), with ‘floret symbole foreach call. The unitary conductance (G) and reversal potential Ee) were determined by fing a near regression tothe data In ESF (By ~-115 mV! = +24 mV}, Eg, was 20 nV 9nd G was 22S: in ESD (Ey = 48 mV; Ee) ~ -28 mV), Ey was -26 mV and G was 30S: {and in ESE (E, = 4 Ea ~ 82 mV, Ey was +20 mi and Gas 80 9S, {c)Valiage dependence ofthe steady state single channel open probably 1 diferent external K° concentrations (see Experimental procedures for ‘alulaton of open probabilities. Data worefited the Bottmann equation ‘asin Figure 7c). Data were obtained in 1 mM" (WM), 155 mM K” (8) and 100 mM K° (8) 2,79" = 0.75 forall K" concanzaions and 5 = 18. In “mM K*, Veg = #10 min TBS mM K°, Vog = +48 mV in 100 mM K Vos #99 mv820 Stephen K. Roberts and Mark Tester gma > 40a ‘oo,D 2° | eearet — 8 | toma ren ; . . : vow ° ‘naw BO 3 Lome °° 400 -teamy °-400 caer 3 amv B ~123mV eo 5 com 7 ay seam Suo0 Time ns) ° ame ie ae 800 ieicokod c c Votan nn ey ° oie 2 3 ‘4 20mM Ca mw 10mM Co” @ Pretreatment et mM Ca scarce er 2 em 3 5 3 es fea ee eee eee cmnen eeseecnager ence Figure 10. The eect of exracllulr TEA* on the inward current from topless rom the cortex Standard bath solution was usod. Holding potental wes -63 mV and voltage pulses range from ~43 10-208 mV in 20 mV saps at intervals of Yraec, The deplayed currents have had lesk current subtracted (lak conductance was 125 pS). The diameter ofthe cll was 78 um. lal Before addition of TEA’ and Ib) 4 min ater adclng 10 mid ceeraeluae TEA’ [c) Curert-voltage relationship of time-dependent curents la varying concentations of extraceller TEA and ater washing out TEA" ever, a K*-solective inwardly rectifying conductance wa rarely observed in other protoplasts isolated from the whole root (Findlay et al, 1994; Schachtman et al, 1991). In these protoplasts (which were most likely to be from the cortex: Findlay et al, 1994), a K*-selective outwardly rectifying conductance dominated the plasma membrane conductance. Thus, wheat root cortical cells may have different ion transport properties to those found in maize ‘oct cortical celts, which could reflect anatomical, and thus functional differences between corresponding cell types in maize and wheat roots. Alternatively, the protoplasts isolated from the whole wheat root may not have pre- dominately originated from the cortex. Standard bath solution was used. Holding potnial wae -62 mV and ules ranged from ~43 0-208 mV Gin 20 mV stops at intervals of Teec, Exvacalluar Ca?” wae varied between 1 and 40 mM but only ‘cutrnts (a) | mM Ca2* and (b) 40 mM Cer” are shown. The displayed turants have hed leak currant subtrarod (leak conductance was 125 pS) ‘The dismater of the cll was 75 pr. (c)Cutton-votage felatenship of time-dependent currents in varying extracalisar Ca¥ concentrations, Inward rectifier and K* uptake from the soil The K*-selective inward current from the cortex of maize roots has counterparts in other plant cells including root cells (Findlay et al, 1994; Gassmann and Schroeder, 1994; ‘Schroeder et al,, 1994; Wegner and Raschke, 1994; White ‘and Lemtiri-Chlieh, 1996). Its activation kinetics are similar to those described in rye roots (White and Lemtir-Chii 1995) and wheat roots (Findlay et al, 1994) in which inward currents were also best fitted using the sum of two exponential components. However, in wheat roots the activation time constants of the inward current were approximately an order of magnitude slower than those described in the present study and in rye roots. The presence of two exponential time constants in the activa- tion kinetics of the inward current could indicate either two closed states of a channel or two populations of channel