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Excitability in Plant Cells Author(s): Randy Wayne Source: American Scientist, Vol. 81, No. 2 (March-April 1993), pp. 140-151 Published by: Sigma Xi, The Scientific Research Society Stable URL: http://www.jstor.org/stable/29774870 Accessed: 06-04-2016 10:15 UTC

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This content downloaded from 130.223.51.163 on Wed, 06 Apr 2016 10:15:49 UTC All use subject to http://about.jstor.org/terms Excitability in Plant Cells

An external stimulus to a plant, such as touch, can trigger a cellular mechanism that generates a defensive response

Randy Wayne

cells, in fact, are hotbeds of electrical ac? ber of ions crossing the membrane. As a duck a pond, paddles it nips alongat the topsthe ofedge under? of tivity, and plant studies have provided They concluded that ions carry the cur? water vegetation. When one nip catches much of the foundation of what is rents that create the action potential. a shoot of Cham, a relative of the green known generally about electrical activi? Although plants are no longer the algae, it sends a spectacular system into ty in cells. Cham has been important in leading organisms used in research on action. The force of the duck's bite trig? those studies and continues to be. the basis of electrical excitability, a num? gers an electrical mechanism in the Physiological studies of electrical ac? ber of investigators have significantly plant, and ionic current rushes across tivity began in the 19th century, and advanced our knowledge of both the the membrane of the nibbled cell. Then since then animal and plant physiolo? mechanisms and the effects of electricity the fluid inside the cell, the protoplasm, gists have worked in parallel. In order in plants. Modern techniques common stops its normal flow around the pe? to study the activity where it happens, to neurophysiology have been applied riphery. The protoplasm quickly jells, at the cellular level, investigators had to to a variety of plants, and the results preventing any leakage that could arise find organisms in which the activity show that electrical physiology in plants from the duck's attack. could be studied in isolation from the is as complex as the systems found in Cham is hardly the only plant that re? whole plant or animal. They also need? animals. Moreover, a variety of plants sponds to external stimuli. All plants re? ed to find cells large enough that they use electricity to initiate action; exam? spond to as they grow, and could be probed with electrodes. In ani? ples are the closing of the leaves of a plants can have various responses to mal studies, the search led to the long Venus flytrap and the touch-driven Hght. Some follow a 24-hour cycle, ad? nerve cells of squids, in which axons, drooping of the leaves of some Mimosa justing the orientation of their leaves for the fibers carrying messages from the species. Nevertheless, the most detailed the maximum absorption of light dur? cell body, are so large that they were information exists for characean algal ing the day. Some plants respond with originally thought to be blood vessels. cells, which I shall examine here. The movement when they are touched by Plant physiologists, on the other hand, electrical activity in these algae is worth predators. selected species of algae that have large examining not only for its importance in What may be less obvious is how cells, such as the characean algae Cham plant biology, but also because studies plants respond to stimuli. Although and Nitella. of plant excitability may help us under? most people know that electrical signals In 1898 Georg H?rmann, a German stand the evolution of the human ner? mediate the responses of an animal's physiologist, observed that big differ? vous system. nervous system, it is less widely known ences in voltage measurements could that plant behavior, too, is governed by develop across cell membranes of Characteristics of Characeans complex electrical mechanisms. Plant Nitella. When such differences are re? Characean algae have been used in generative they are called action poten? much of the work on plant excitability. Randy Wayne received a Ph.D. in botany at the tials, because the regeneration implies They are stoneworts, with a fossil University of at Amherst under the action?the passing of an impulse. By record stretching back to the Devonian guidance of Peter Hepler. Wayne's doctoral work the 1930s, characean algal cells were so period, which began about 400 million considered the contribution of calcium to the phy well known that many investigators years ago, and they are the ancestors of tochrome-mediated signal transduction chain that studied them. For example, K. S. Cole all higher plants. Extant stoneworts be? leads to -spore . He continued his and Howard Curtis of the National In? long to a single family, Characeae, work on phytochrome as a postdoctoral fellow at the stitutes of Health, who later became which is composed of six genera in? University of Texas with Stan Roux. Wayne began known as pioneers in the electrical ex? cluding Cham and Nitella. The majority his work in membrane biology with Masashi Tazawa citability of squid neurons, began of the extant species inhabit the bottom at the University of Tokyo. He is currently an assis? tant professor of plant biology at , studying excitability in Nitella. These in? of clear freshwater ponds, where they where he tries constantly in his teaching and vestigations showed that an action po? live entirely submerged. research to repay the debt he owes to his teachers. tential in Nitella is accompanied by a As I have noted, the primary attrac? Address: Section of Plant Biology, Cornell 200-fold increase in the 's tion of characean algae as an object of University, Ithaca, NY 14853. conductance, as measured by the num study is the size of their cells. In Chara,

140 American Scientist, Volume 81

This content downloaded from 130.223.51.163 on Wed, 06 Apr 2016 10:15:49 UTC All use subject to http://about.jstor.org/terms Figure 1. Chara, an alga, responds to environmental stimuli, as do many plants. A variety of factors, including mechanical stimulation, can generate an action potential?a transient change in voltage across a cellular membrane?that causes some of this alga's internal fluid to jell, preventing it from leaking through small holes or tears in the plasma membrane. Large cells make Chara an appealing organism for electrical physiology. The plant's shoot is composed of long internodal cells separated by smaller nodal cells, seen here at the tip of a plant (lower right) and supporting reproductive structures (top). Each internodal cell is about six centimeters long and half a millimeter wide; like all plant cells it has three distinct partitions. The outer surface is the cell wall, which is composed of cellulose. Underneath the cell wall is the plasma membrane, which is formed from two layers of lipids. Much of the inside of the cell is taken up by a vacuole, which is bound by the vacuolar membrane. (Photograph at right courtesy of the author.)

the plant body is composed of long in? spersed with proteins. Beneath the plas? ternodal cells separated by smaller ma membrane there is a layer of chloro nodal cells. A single internode may be plasts, the sites of photosynthetic six centimeters long and half a millime? processes. Most of the interior of the cell ter wide, or about half as long as a is a vacuole, a sac filled largely with wa? toothpick and half as wide. The inter? ter and bounded by another membrane. nal structure of an internodal cell is un? The area between the vacuolar mem? like that of an animal cell. Like all plant brane and the plasma membrane is cells, the external border is a cell wall, filled with protoplasm; here are found which is composed of cellulose fibers the cell nucleus and the cytoplasm, a that provide rigidity to the cell but are viscous fluid that contains the cell's or permeable to the extracellular fluid. Just ganelles such as mitochondria and ri beneath the cell wall is a semipermeable bosomes. plasma membrane, which is composed The protoplasm of characean cells of two layers of lipids that are inter moves constantly around the periphery

1993 March-April 141 This content downloaded from 130.223.51.163 on Wed, 06 Apr 2016 10:15:49 UTC All use subject to http://about.jstor.org/terms has more positively charged ions and the other side has more negatively charged ions, then there is a potential, or voltage, across the membrane. Here I shall discuss four potentials: membrane potential, resting potential, receptor potential and action potential. A membrane potential is the voltage across a membrane, or a measurement time of the distribution of ions. The resting Figure 2. Two components form an action potential in Chara. The voltages are recorded by potential is the membrane potential inserting microcapillary electrodes into the cell, and the electrical output appears as a when the cell is not being stimulated. change in voltage across a membrane over time. With a reference electrode placed outside Both a receptor potential and an action the cell and a microcapillary electrode inserted into the vacuole (right), the two-component, potential change the membrane poten? whole-cell action potential is recorded (left) after stimulation. The action potential begins tial. A receptor potential arises when a with a sharp spike followed by a more gentle hump. With the electrode arrangement shown receptor in a membrane, such as a mol? above, the action potential makes the measurement less negative, or decreases the voltage ecular mechanoreceptor, is stimulated. across the cell. (Data from Shimmen and Nishikawa 1988.) The stimulation generates an ionic cur? rent that changes the membrane po? tential, but the receptor potential de? creases in magnitude with distance from the stimulated receptor. An action potential is a large, transient change in the membrane potential that is self perpetuating, or regenerative, and it can travel the length of the cell without decreasing in magnitude. Characean algae generate action po? time tentials when subjected to a variety of Figure 3. Plasma membrane's action potential generates the sharp spike in an action stimuli, including a sudden change in potential of Chara. If a microcapillary electrode is inserted just through the plasma temperature, ultraviolet radiation, odor membrane (right), the plasma membrane's action potential is recorded separately (left). ants and mechanical action. These stim? (Data from Shimmen and Nishikawa 1988.) uli first cause the plant to produce a re? ceptor potential. For example, a small mechanical stimulus is converted by a receptor into electrical energy that is proportional to the magnitude of the stimulus. In a resting characean cell, there is a negative voltage inside the plasma membrane relative to the out? side of the cell. In other words, there are more negatively charged ions inside the membrane and more positively charged time ions outside the membrane. The recep? Figure 4. Vacuolar membrane's action potential produces the gentle hump in an action tor potential generates depolarization, potential of Chara. If one microcapillary electrode is inserted through the vacuolar a decrease in the voltage difference be? tween the inside and the outside of the membrane and a second microcapillary electrode is inserted through the plasma membrane (right), the vacuolar membrane's action potential is recorded (left). The recording of the cell. This potential generally lasts as vacuolar membrane becomes more negative, or hyperpolarizes, during its action potential long as the stimulus is present, and it is because more negative charges accumulate in the protoplasm. (Data from Shimmen and essentially an electrical replica of the Nishikawa 1988.) stimulus. If the stimulus depolarizes the cell to a specific threshold level, an ac? of the cell, just beneath the chloroplasts. cells if diffusion were the only mecha? tion potential is generated. The rotating belt of protoplasm travels nism available. An action potential in one area of a at a speed of about 100 microns per sec? A fundamental concept in electrical characean cell causes protoplasmic ond. Its movement, visible through a physiology is defined by the term po? streaming to stop throughout the cell. microscope, is called protoplasmic tential. A potential is a voltage across a As I shall explain below, the action po? streaming or cyclosis. The streaming membrane, which is created by the sep? tential causes external calcium to move process is driven by the same interac? aration of positive charges from nega? into the protoplasm. The increased cal? tions between actin and myosin that tive charges. In biology, charges are car? cium concentration activates a protein create contraction in muscles. The ried by ions. Positive charges are carried kinase that adds a phosphorus group to movement of the protoplasm mixes and by cations such as sodium, and nega? myosin and thereby innibits its interac? transports molecules through the cell, tive charges are carried by anions such tion with actin, which stops the driving which would take too long in such large as chloride. If one side of a membrane force behind protoplasmic streaming.

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This content downloaded from 130.223.51.163 on Wed, 06 Apr 2016 10:15:49 UTC All use subject to http://about.jstor.org/terms The cell may also become isolated from With no external stimuli, the voltage called a Siemens (the reciprocal of resis? neighboring cells because the streaming difference across a cellular membrane is tance or ohm-1, sometimes called a usually enhances the passage of sub? called the resting potential. In characean mho) per square meter. At rest, the spe? stances between cells via small tubes cells, the average resting potential is cific conductance is 0.83 Siemens per known as plasmodesmata. When the -180 miUivolts across the plasma mem? square meter for the plasma membrane action potential abates, calcium is brane and -10 millivolts across the vac and 9.1 Siemens per square meter for pumped from the protoplasm, and uolar membrane. (The negative sign in? the vacuolar membrane. The specific streaming resumes. dicates that the protoplasmic side is conductance changes during the action negative with respect to the other side potential, and the peak specific conduc? Probing the Potential of the membrane. That is, the plasma tance is 30 Siemens per square meter for It is generally an intricate task to record membrane is negative on the inside and the plasma membrane and 15 Siemens the precise electrical activity of any cell. positive on the outside, and the vacuo per square meter for the vacuolar mem? Such measurements are best made in lar membrane is negative on the outside brane. This result reveals that an in? tracellularly?recording the voltage dif? and positive on the inside.) During an crease in ionic conductance accompa? ference between the outside and the in? action potential, the plasma membrane nies an action potential, but it does not side of the cell. This is done by placing a depolarizes to about zero millivolts, indicate which ions are crossing the reference electrode outside the cell and making the inside and the outside of membranes and carrying the currents a recording electrode inside the cell. In the cell about equal in charge; the vac that create an action potential. many neurons, such recording requires uolar membrane hyperpolarizes (mean? the use of an air table to isolate the ing that it becomes more negative) to Particular Permeabilities preparation from the slightest move? about -50 millivolts. The electrical potential across a mem? ment, a microscope with magnification The changes in membrane potential brane is largely determined by the dif? of as much as 250 times, and a micro that develop during an action potential ferences in ionic concentrations on the manipulator, a mechanical device that arise from ionic currents that flow as a inside and the outside of the mem? controls small, precise movements of consequence of a change in a mem? brane. The ions of interest in most or? the electrode. In Chara, however, intra brane's permeability to specific ions. ganisms are calcium, chloride, sodium cellular recording is easy; it is even pos? The changes in permeability that devel? and potassium. Since characean cells in? sible to do it with a naked eye guiding op can be measured as the specific con? clude two membranes, there are three the movements and a relatively steady ductance of the membrane. This is a fluids of interest: the extracellular fluid hand holding the electrode, although measurement of the membrane's per? (the fluid outside the cell), the proto? investigators employ a low-magnifica? meability to all ions; it is given in a unit plasm (the fluid between the plasma tion microscope and a micromanipula tor to further simplify the task. command The characean action potential voltage voltage-clamp moves away from the receptor in both amplifier directions along the cell at a speed of 0.01 to 0.4 meters per second. This is much slower than the so-called con? cell wall duction velocity of action potentials in nerves, which is between 0.4 and 42 meters per second depending on the specific nerve and organism. When an animal's muscle is stimulated, it pro? duces an event that has been called E-C coupling, or excitation-contraction cou? pling, because the electrical excitation causes the muscle to contract. In characean cells, electrical stimulation produces a different kind of E-C cou? pling, excitation-cessation coupling. In algae this refers to the fact that electri? cal stimulation causes the cessation of protoplasmic streaming. Figure 5. Voltage clamping reveals ionic currents that move across a membrane. A voltage clamp When an electrode is inserted into a holds, or clamps, the voltage across a membrane at a value called the command voltage. An characean vacuole and a reference elec? electrical circuit compares the command voltage with the actual membrane potential and injects trode is placed outside the cell, an action the appropriate current to mmimize the difference. The current passed by a voltage clamp is measured, and it is effectively a mirror image of the ionic current flowing across the cell's potential can be observed after stimula? membrane. When the membrane is clamped at the resting potential, no current is passed by the tion (Figure 2). The response appears to voltage clamp because the membrane is in equilibrium. If the membrane is clamped at voltages contain two components: a fast compo? other than the resting potential, the voltage clamp passes current to offset the current that flows nent and a slow component. In fact, it is across the cell's membrane. Here the plasma membrane is clamped by two microcapillary two separate action potentials. The fast electrodes. The electrode on the right records the voltage across the plasma membrane. A potential is across the plasma membrane voltage-clamp amplifier compares the plasma-membrane voltage to the command voltage and (Figure 3), and the slow one is across the then injects current through the microcapillary electrode on the left. An extracellular electrode vacuolar membrane (Figure 4). monitors the current flowing across the membrane.

1993 March-April 143

This content downloaded from 130.223.51.163 on Wed, 06 Apr 2016 10:15:49 UTC All use subject to http://about.jstor.org/terms membrane and the vacuolar mem? is temperature in degrees Kelvin, z is brane) and the vacuolar fluid (the fluid the ion's valence, F is the Faraday con? inside the vacuole). The concentrations stant (9.65 x 104 Coulombs per mole), C0 of ions can be given in the ratio of extra? is the ion's concentration on the outside cellular concentration to protoplasmic of the membrane and Q is the ion's con? concentration to vacuolar concentra? centration on the inside of the mem? tion, because only the relative values are brane. By assuming a temperature of 20 significant to this discussion. The aver? degrees Celsius or 293 degrees Kelvin, age ionic-concentration ratios are which is approximately room tempera? 100:1:12,000 for calcium, 1:55:405 for ture, the equation can be simplified to: chloride, 1:50:340 for sodium and E = (58/z)log(C0/Q) 1:1,100:1,030 for potassium. In other words, the concentration of calcium is This equation gives E in millivolts. Con? low in the protoplasm and high in the sidering the Nernst potential for sodi? vacuole; the chloride concentration is um across the plasma membrane, the higher in the protoplasm than in the ex? equation would be: tracellular fluid, and higher still in the vacuole; the distribution of sodium is E = (58) log (1/50) = -98.5 similar to that of chloride; and potas? (For sodium, z = 1.) This means that time sium has a higher concentration in both sodium would be in equilibrium across the protoplasm and the vacuole. the plasma membrane at a potential of Figure 6. Two components of the plasma The location of an ion is determined -98.5 millivolts. membrane's action potential appear in a voltage by a chemical force and an electrical Each ion has a Nernst potential. In damp experiment Hie recordings of ionic characean cells, the average Nernst po? current result from clamping the plasma force (Figure 8). In response to the chem? membrane at the voltages listed at right The ical force, an ion tends to go from an tentials for the major ions across the quick, transient current develops first, and it area of higher concentration to an area plasma membrane are 59 millivolts for appears as a sharp, downward drop in the current of lower concentration. The electrical calcium, 103 millivolts for chloride, -100 recording. This current arises from calcium ions force pulls an ion toward an area of op? millivolts for sodium and -180 milli? moving into the protoplasm. The slow, transient posite charge, so that a cation, or posi? volts for potassium. current follows. When the plasma membrane is tively charged ion, is drawn toward a The resting membrane potential aris? depolarized to -30 millivolts, the slow, transient negative area. Consider a potassium ion es from the combined equilibrium po? current appears as an upward deflection, or an in the protoplasm. Potassium is more tentials of all of the ions. You may have outward current In this experiment concentrated in the protoplasm than in noticed, however, that both the resting depolarizations between -40 and -50 millivolts cause the slow, transient current to reverse and the extracellular fluid, and thus the potential of the characean plasma mem? move inward. This current is carried by chloride chemical force tends to pull potassium brane and the Nernst potential for ions. (Data from Lunevsky et aL 1983.) out of the cell. The resting potential of potassium are -180 millivolts. This is the plasma membrane, however, is neg? not merely a coincidence. It has been ative in the protoplasm relative to the shown that in resting characean cells, as extracellular fluid. This creates an elec? well as in most resting animal nerves, trical force that pulls potassium, a the membrane is largely impermeable cation, from the extracellular fluid into to calcium, chloride and sodium, but it the protoplasm. At equilibrium, the is readily permeable to potassium. This chemical and electrical forces balance, means that the resting potential is large? and there is no net movement of ions, ly determined by the passive diffusion or charge. Therefore, an uneven distri? of potassium. I)uring an action poten? bution of ions can create a stable mem? tial, the membrane's permeability to brane potential. specific ions changes. The membrane acts like a capacitor, a Ions in Action time component that separates electrical charge. By knowing the difference in an Ionic movement generates action po? Figure 7. Changes in the flow of ions drive ion's concentration across a membrane, action potentials. In the plasma membrane of tentials in animal, plant and fungal Chora, an action potential arises from it is possible to calculate the voltage dif? cells. In 1949 Alan Hodgkin and movements of calcium, chloride and potassium ference, or potential, at which the chem? Bernard Katz, both then at Cambridge ions across the membrane in response to a ical and electrical forces will be bal? University, showed that external sodi? stimulus. Calcium ions (Ca2+) moving from the anced for that ion. This potential is um is necessary for an action potential extracellular fluid into the protoplasm initiate called the equilibrium potential or the in a squid nerve. Through a series of ex? depolarization of the membrane. This is Nernst potential, after Walter Nernst, periments, they developed the sodium followed by an increased conductance of the German physical chemist who de hypothesis, which states that the mas? chloride ions (CD, which move out of the rived it, and the equation follows: sive depolarization of an action poten? protoplasm, further depolarizing the tial results from sodium rushing into a membrane. Finally, the membrane becomes E = (RT/zF)\n(C0/Q cell. It was later shown that tetrodotoxin permeable to potassium ions (K+), which also move out of the protoplasm, returning the In this equation, E is the Nernst poten? (the deadly poison found in the Japan? membrane to its resting voltage and its tial, R is the universal gas constant (8.31 ese puffer fish and removed before the polarized equilibrium state. joules per mole per degree in Kelvin), T fish is eaten as sashimi) prevents an ac

144 American Scientist, Volume 81

This content downloaded from 130.223.51.163 on Wed, 06 Apr 2016 10:15:49 UTC All use subject to http://about.jstor.org/terms ' ?ts> ^i?i :^^mm;'

Figure 8. Membrane potential, or voltage, depends on the relative strength of a chemical force and an electrical force. This can be seen in the movement of potassium ions (K+) across the plasma membrane of Chara. Potassium ions are about 1,000 times more concentrated in the protoplasm inside the membrane than in the extracellular fluid outside. The ions tend to move from areas of higher concentration to areas of lower concentration (left). This chemical force drives potassium ions out of the protoplasm into the extracellular fluid. The movement of positive ions out of the cell causes the plasma membrane to have a positive charge on its extracellular side and a negative charge on its protoplasmic side. Since opposite charges attract, an electrical force pulls positively charged potassium ions from the extracellular fluid into the protoplasm (right). At a membrane voltage called the equilibrium potential or the Nernst potential, the chemical force and the electrical force balance so that there is no net movement of potassium ions.

tion potential by specifically blocking current of the action potential. Never? proposed that calcium simply activates sodium currents in squid nerves. Only theless, when they employed radioac? a mechanism that causes chloride to higher animals, beginning with the coe tive calcium as a tracer, they were un? move out of the cell. It was shown that lenterates, have sodium-based, able to detect any calcium moving into the level of chloride leaving the cell in? tetrodotoxm-inhibited action potentials. the cell. At the same time, Lorin creases 100 fold during an action poten? The more evolutionarily ancient and Mullins of the University of Maryland tial. This amount of chloride moving ubiquitous action potentials are calci? um-driven. The Nernst potential for sodium in characean algae is not large enough to create the large depolarization that de? velops at the plasma membrane during an action potential. The Nernst poten? tial for sodium drives it into the cell, but once the cell depolarizes from its resting potential of -180 millivolts to sodium's Nernst potential of about -100 milli? volts, sodium no longer moves into the cell. Nevertheless, a characean action potential depolarizes the plasma mem? brane to about zero millivolts. This sug? gests that calcium or chloride con? tributes to the depolarization. Calcium (a cation with two positive charges) could depolarize the cell by moving in, and chloride (an anion with one nega? tive charge) could depolarize the cell by moving out. Potassium is not a candi? date because the depolarization during an action potential moves away from potassium's Nernst potential. Disagreement arose about the impor? tance of calcium and chloride. In the

early 1960s Geoff Findlay and Alex Figure 9. Patch-clamp techniques record the current through a single channel or opening in a Hope of the University of Sydney in membrane. A microcapillary electrode is placed against a membrane, and then suction is Australia showed that the magnitude of applied through the electrode. The suction removes a small piece (about one square micrometer) depolarization depends on the concen? of membrane, which becomes sealed across the opening at the end of the electrode (above). The tration of the external calcium, and small piece of membrane may contain a single channel, and currents through the channel can be they believed that calcium carries the recorded. (Photograph courtesy of Owen Hamill and Don McBride.)

1993 March-April 145

This content downloaded from 130.223.51.163 on Wed, 06 Apr 2016 10:15:49 UTC All use subject to http://about.jstor.org/terms for the quick, transient current is be? tween -60 and -20 millivolts. Studies have shown that this current arises from calcium ions moving across the plasma membrane. The reversal potential of this channel, however, is not equal to the Nernst potential for calcium because the channel is permeable to a number of cations. The slow, transient current appears when the plasma membrane is depolar? ized to between -90 and -120 millivolts. Its reversal potential depends on the ex? ternal concentration of chloride ions. If the external chloride concentration is changed, the reversal potential of the 0.1 1.0 10 slow, transient current changes accord? calcium concentration (micromolar) ing to the Nernst potential for chloride. In addition, the slow, transient current Figure 10. Chloride currents create the plasma membrane's action potential. The entry of calcium can be blocked with ethacrynic acid and ions into the protoplasm opens the channel, allowing chloride ions to move out of the protoplasm into the extracellular fluid and further depolarize the membrane. Current through this channel can anthracene-9-carboxylic acid?well known chloride-current blockers. The be recorded with the patch-clamp technique (see Figure 9). If the level of calcium ions in the protoplasm is too low (left column), no current flows. If the calcium is increased (middle column), slow, transient current, then, is a chlo? current flows through the channel, and it appears as small square waves or the combination of a ride current. number of such waves. A single square wave indicates one opening of a single channel. The These voltage-clamp experiments chloride current depends on the voltage across the membrane as well as the presence of calcium confirm that the depolarization of the ions. More current flows through the channel when the voltage across the membrane is between -80 plasma membrane develops as a result and -160 millivolts. If the calcium level is increased further (right column), current decreases because of the movement of calcium into the cell the channel closes. (Data from Okihara et al. 1991.) (the quick, transient current) and chlo? ride out of the cell (the slow, transient out of the cell is more than enough to damp because the membrane is in equi? current). After these two currents stop, depolarize the plasma membrane to librium. If the membrane is clamped at the steady-state current returns the plas? zero millivolts. less negative voltages, a condition simi? ma membrane to its resting potential The above work and other studies lar to the depolarization that develops through the flow of potassium out of suggested that it is the exodus of chlo? during an action potential, the voltage the cell. Figure 7 shows the temporal re? ride from the cell that is responsible for clamp passes current to offset the cur? lationship between these ionic flows. the massive depolarization. After the rent that flows across the cell's mem? Lunevsky and his colleagues postulat? action potential ends, the plasma mem? brane. An important piece of data that ed that a chloride channel, which is spe? brane returns to its resting potential be? can be determined for a specific current cific for the permeability of this ion, is ac? cause potassium moves out of the cell, is its reversal potential, the potential at tivated by an increase in the protoplasmic again making the inside more negative which the current does not flow into or concentration of calcium, which arises than the outside. out of the cell. This potential is the same from the quick, transient current. The as the Nernst potential for the ion that calcium channel is initially activated by Clamping and Currents carries the current. the receptor potential that is generated In the above experiments, a current was In the early 1980s V. Lunevsky and his by a stimulus. If the calcium channel is applied to a cell and then the change in colleagues at the Institute of Biological blocked, the chloride current does not the membrane potential was measured. Physics in the former U.S.S.R. used a appear, suggesting a causal relationship Another way of measuring electrical ac? voltage clamp to study the action poten? between the two currents. tivity in cells uses the voltage clamp, tial across the plasma membrane of Nitel which was developed by K. S. Cole in lopsis, a characean alga. They placed one Checking Out a Channel 1949. A voltage clamp holds, or clamps, electrode in the protoplasm and one in A current flows through channels that the membrane potential at a set value the extracellular fluid to clamp the plas? are composed of protein molecules em? (Figure 5). This is accomplished through ma membrane. By clamping the plasma bedded in the membrane. These mole? an electrical circuit that continually membrane at a series of different poten? cules create little pores through which compares the desired clamp potential tials, they discovered that there were ions pass. The current through a single with the actual membrane potential, three kinds of ionic currents: a quick, channel can be recorded with the patch and injects the appropriate current to transient current; a slow, transient cur? clamp technique. In this technique, a minimize the difference. The current rent; and a steady-state current. glass microcapillary electrode is placed passed by a voltage clamp can be mea? The quick, transient current lasts for against the surface of the membrane. sured, and it is effectively a mirror im? several hundred milliseconds. When the Suction is applied through the elec? age of the ionic current flowing across plasma membrane is depolarized to -50 trode, and a tiny piece of the plasma the cell's membrane. When the mem? millivolts this current flows inward. At membrane (about one square micron) brane is clamped at the resting poten? more depolarized potentials, the current is pulled away, sealed to the electrode tial, no current is passed by the voltage moves outward. The reversal potential like the head on a drum. If the patch is

146 American Scientist, Volume 81

This content downloaded from 130.223.51.163 on Wed, 06 Apr 2016 10:15:49 UTC All use subject to http://about.jstor.org/terms small enough, and in the right place, it may contain a single channel. The iso? lated membrane patch does not have a natural membrane potential, but a volt? age clamp can be used to set the poten? tial to any value. Typically, a channel opens at a specific potential, and the flow of ions can be measured as current, usually in the range of picoamperes, or trillionths of an ampere. When the channel is closed, no current flows. Kiyoshi Okihara and his colleagues at Osaka University applied patch-clamp techniques to the plasma membrane of a characean cell, and they identified the calcium-activated chloride channel re? a sponsible for the plasma membrane's ac? tion potential. Current passes through this channel only when the calcium con? centration in the protoplasm is about 1 micromolar, approximately 10 times its normal concentration. If the protoplas? mic calcium concentration is higher or

Figure 11. Characean action potential arises from a cascade of processes. At rest, both the plasma membrane and the vacuolar membrane are in electrical equilibrium, and protoplasm streams through the space between the two membranes. An external stimulus, such as touch, generates a depolarization across the plasma membrane called a receptor potential. b No one knows which ion causes the receptor potential. The receptor potential causes calcium ions to move from the extracellular fluid into the protoplasm (a). Some of the calcium ions activate chloride channels in the plasma membrane, allowing chloride ions to move from the protoplasm to the extracellular fluid (b). This outward chloride current depolarizes the membrane, which is negative on the inside at rest, and the current generates an action potential across the plasma membrane. The action potential moves along the cell because the depolarized membrane opens more calcium channels, which open more chloride channels. Calcium ions continue diffusing through the protoplasm, and some of them open calcium-activated chloride channels on the vacuolar membrane, allowing chloride c ions to move from the vacuolar fluid to the protoplasm (c). This chloride current generates a hyperpolarization of the vacuolar membrane because chloride ions have a negative charge and the vacuolar membrane is already negative on the protoplasmic side. The calcium ions in the protoplasm stop protoplasmic streaming by inhibiting the actin-myosin system that drives the streaming. At about the same time, potassium ions flow from the protoplasm to the extracellular fluid, which stops the plasma membrane's action potential by returning the membrane to its polarized state. Finally, potassium ions flow from the vacuolar fluid to the protoplasm, which stops the vacuolar membrane's action potential, and calcium ions are pumped from the protoplasm, allowing streaming to resume (d). d

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This content downloaded from 130.223.51.163 on Wed, 06 Apr 2016 10:15:49 UTC All use subject to http://about.jstor.org/terms system. This is the reason that Cole and Curtis began studying the squid axon rather than continuing with characean cells. The vacuolar membrane of a characean cell adds a second component to the action potential. At rest, the vacuolar membrane po? tential is about -10 millivolts. In this case, the membrane is negative outside the vacuole, which is the reverse of the plasma membrane. During an action potential, the vacuolar membrane hyper polarizes (becomes more negative out? side the vacuole) to about -50 millivolts. The average Nernst potentials at the vacuolar membrane are 121 millivolts for calcium, -51 millivolts for chloride, 49 millivolts for sodium and -2 milli? volts for potassium. These numbers in? dicate that chloride is the only ion capa? ble of carrying the vacuolar membrane potential to -50 millivolts. Chloride moves out of the vacuole to make the membrane potential more negative. In a characean cell, the plasma mem? brane and the vacuolar membrane in? teract. The vacuolar membrane, howev? er, can be studied separately by making the plasma membrane permeable to all ions?essentially, by making it disap? pear. This is done by placing a cell in a calcium-free solution that contains EGTA (ethyleneglycol-b/s-tetraacetic acid)?a calcium chelator?and is at 4 degrees Celsius. This treatment stops Figure 12. Venus flytrap captures insects through an electrical process. The plant's lobes are protoplasmic streaming because ATP covered with trigger hairs. If an insect steps on two hairs or the same hair twice, a cellular escapes from the cell. Streaming can be action potential is generated. The action potential triggers a mechanical system that causes reactivated by adding ATP to the the lobes to close on the insect, which is then digested. bathing solution. In a so-called plasma-membrane-per lower than normal, the current de? the molecule would swing around, meabilized cell, a vacuolar-membrane creases. The channel apparently needs which might uncover or open the chan? action potential can be initiated by in? calcium to open, but too much calcium nel. The calcium-activated chloride creasing the calcium concentration of causes it to close. No one knows how a channel opens when the membrane is the bathing solution from zero to one high calcium concentration inhibits the depolarized to potentials less than -160 micromolar. If the chloride concentra? channel. millivolts. This reveals that a membrane tion is increased in the protoplasm, the The reversal potential of the calcium depolarization, such as a receptor po? calcium-induced action potential de? activated chloride current depends on tential, is necessary to open the calcium creases as predicted by the Nernst equa? the chloride concentration as predicted activated chloride channel, but depolar? tion, if chloride is assumed to be the by the Nernst potential for this ion. ization is not enough. Current will flow only current-carrying ion. Anthracene Moreover, the channel is voltage-de? through the channel only if the mem? 9-carboxylic acid, a chloride-channel pendent. In other words, the channel brane is sufficiently depolarized and if blocker, completely eliminates the po? will open only at specific plasma-mem? both calcium and chloride are present. tential. Mmehiro Kikuyama of the Uni? brane potentials. In a simple form, a versity of the Air in Japan measured the voltage-dependent channel could be Inside Action movement of chloride from the vacuole created from a membrane-bound mole? As I mentioned earlier, a characean cell to the protoplasm and found that an in? cule with a large dipole moment, one has three compartments that bathe two crease in protoplasmic calcium creates end positive and the other negative. The excitable membranes: the plasma mem? an increase in chloride moving out of positive end would swing toward the brane and the vacuolar membrane. An the vacuole. If the fluid in the vacuole is negative side of the membrane, and this animal cell has only one membrane, and replaced with a chloride-free solution, movement could place the molecule this makes an axon much simpler than a there is no increase in protoplasmic across an opening in the membrane, characean cell. An axon is geometrically chloride, indicating that the chloride closing the channel. If the potential similar to an electrical cable, and thus ca? does come from the vacuole. across the membrane were to switch, ble theory can be easily applied to such a In intact cells, the vacuolar mem

148 American Scientist, Volume 81

This content downloaded from 130.223.51.163 on Wed, 06 Apr 2016 10:15:49 UTC All use subject to http://about.jstor.org/terms brane's action potential develops only plasm, it depolarizes the plasma mem? others, noted that many plants respond after the plasma membrane has been ex? brane enough to open the calcium-acti? to mechanical stimulation. Darwin be? cited. This indicates that some mecha? vated chloride channels, generating ad? came interested in carnivorous plants nism couples the plasma membrane's ditional depolarization as chloride such as the Venus flytrap, which he action potential to the vacuolar mem? passes out through the plasma mem? called "one of the most wonderful in brane's action potential. Calcium is as? brane. The calcium diffuses across the the world," and he was the first to show sumed to be the coupling agent for sev? five to 20 microns of protoplasm to the that the plant digests captured insects. eral reasons. First, a characean action vacuolar membrane at a speed of about At rest, the lobes of a Venus flytrap sit potential increases the concentration of one micron per second. At the vacuolar passively open. Each lobe secretes a calcium in the protoplasm. Second, re? membrane, the calcium activates chlo? type of nectar that attracts insects, and moving calcium from a cell's external so? ride channels on the membrane, allow? so-called trigger hairs are embedded in lution does not affect the plasma mem? ing chloride to move from the vacuole the inner surface of each lobe. If an in? brane's action potential (as long as the into the protoplasm, which hyperpolar sect steps on a lobe and either hits two calcium is replaced with a similar ion, izes the second membrane. The plasma trigger hairs or hits the same hair twice, such as barium), but this does inhibit the membrane's action potential ends as the mechanical stimulation generates an vacuolar membrane's action potential. potassium leaves the protoplasm, and action potential, and the lobes close, And finally, a microinjection of calcium the vacuolar membrane's action poten? capturing the insect. into the protoplasm generates an action tial ends as potassium moves from the The sundew gets its name from its potential in the vacuolar membrane. vacuole to the protoplasm. appearance. This plant shines as if coat? It is now possible to describe the fun? ed with dewdrops because it is covered damental steps in a characean action Other Excitable Plants with sticky hairs that can capture in? potential (Figure 11). An external stimu? Other plants employ electrical signals sects. Once an insect is captured, it be? lus causes a depolarization of the plas? to elicit behaviors and physiological gins to struggle, and the mechanical ma membrane (a receptor potential). processes. Although the mechanistic ex? stimulation to the plant induces action The receptor potential arises from the planations for many plant responses potentials that cause the hair to wrap movement of calcium through the plas? have only recently emerged, the re? around the insect. Neighboring hair ma membrane and into the protoplasm. sponses themselves have been known cells also produce action potentials, and If enough calcium moves into the proto for some time. Charles Darwin, among they, too, wrap around the insect, thus

Figure 13. Sundew entangles insects in the plant's sticky hairs, which adhere to an insect's feet. When the insect tries to escape, the mechanical stimulus to the plant generates an action potential that causes the hair to wrap around the insect. Neighboring hairs also produce action potentials, and these hairs, too, wrap around the insect. Secretory cells then release enzymes that digest the insect.

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This content downloaded from 130.223.51.163 on Wed, 06 Apr 2016 10:15:49 UTC All use subject to http://about.jstor.org/terms providing a secure trap. Then nearby secretory cells exude enzymes, forming a little stomach that digests the insect. One of the best-known examples of plant behavior comes from Mimosa pu dica, often called the sensitive plant. When the leaves of the plant are touched, they bend over and appear dead. The drooping arises from a me? chanically driven action potential. More? over, an action potential propagates from the stimulated region throughout the plant. This causes drooping in the rest of the plant, a defense mechanism apparently designed to make the whole plant look unappealing. Not all plant action potentials, how? ever, cause obvious responses. In Luffa?the plant whose gourd or fruit is used for "loofah" sponges?action po? tentials cause a transient inhibition of growth. And in a variety of flowers, landing on the stigma generates an action potential, which may be in? volved in subsequent pollination or the maturation process. In tomato seed? lings, a mechanical wound induces electrical activity that causes the accu? mulation of proteins that limit further damage to the plant. Electrical phenomena control many responses in plants. In a characean alga, we understand many of the details of the mechanism that leads from a duck's nip on the plant to the cessation of pro? toplasmic streaming. But we are just be? ginning to address the similarities be? tween the electrical excitability in characean algae and higher plants, let alone animals. In any case, it is apparent that plants can perform long-distance communication through electrical sig? nals, such as the passing of information from a mechanical stimulus from one Mimosa stem to another. Many biolo? gists continue to describe electrical ex? citability as part of the animal world. In the future, we should think of plants as excitable too.

Acknowledgments Thanks to Drs. Atsushi Furuno, Owen Hamill, Roger Spanswick, Mark Staves, Robert Turgeon and Scott Wayne for their comments on this manuscript.

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This content downloaded from 130.223.51.163 on Wed, 06 Apr 2016 10:15:49 UTC All use subject to http://about.jstor.org/terms eral Physiology 20:229-265. gy of Giant Alga; Cells. Cambridge: Cambridge Shiina, T., R. Wayne, H. Y. L. Tung and M. Taza Briggs, G. E., and A. B. Hope. 1958. Electrical po? University Press. wa. 1988. Possible involvement of protein tential differences and the Donnan equilibri? Kikuyama, M. 1986. Tonoplast action potential of phosphorylation/dephosphorylation in the modulation of Ca2+ channels in tonoplast-free um in plant tissues. Journal of Experimental Characeae. Plant and Cell Physiology 27:1461-1468. cells of Nitellopsis. Journal of Membrane Biology Botany 9:365-371. 102:255-264. Cole, K. S., and Howard J. Curtis. 1938. Electrical Lunevsky, V. Z., O. M. Zherelova, I. Y. Vostrikov Shirnmen, T., and S. Nishikawa. 1988. Studies on impedance of Nitella during activity. Journal and G. N. Berestovsky. 1983. Excitation of Characeae cell membranes as a result of acti? the tonoplast action potential of Nitella felx-. of General Physiology 22:37-64. vation of calcium and chloride channels. Jour? His. Journal of Membrane Biology 101:133-140. Dainty, J. 1962. Ion transport and electrical po? nal of Membrane Biology 72:43-58. Smyukhin, A. M, and E. A. Britikov. 1967. Ac? tentials in plant cells. Annual Review of Plant tion potentials in the reproductive system of Physiology 13:379-402. Mullins, L. J. 1962. Efflux of chloride ions during action potential of Nitelk. Nature 196:986-987. plants. Nature 215:1278-1280. Ewart, A. J. 1903. On the Physics and Physiology of Qkihara, K, T. Ohkawa, I. Tsutsui and M. Kasai. Staves, M. P., and R. Wayne. In press. The touch Protoplasmic Streaming in Plants. Oxford: Clarendon Press. 1991. A Ca2+- and voltage-dependent Cl-sen induced action potential in Ohara: Inquiry into sitive anion channel in the Chora plasmalem the ionic basis and the mechanoreceptor. Aus? Hodick, D., and A. Sievers. 1989. On the mecha? ma: A patch-clamp study. Plant and Cell Phys tralian Journal of Plant Physiology. nism of trap closure of Venus flytrap (Dionaea iobgy 32:593-601. Tominaga, Y, R. Wayne, H. Y L. Tung and M. muscipula Ellis). Planta 179:32-42. Shiina, T., and M. Tazawa. 1986. Action potential Tazawa. 1986. Phosphorylation-dephospho Hope, A. B., and G. P. Findlay. 1964. The action in Luffa cylindlica and its effects on elonga rylation is involved in Ca2+ controlled cyto potential in Cham. Nature 191:811-812. N tion growth. Plant and Cell Physiology plasmic streaining of characean cells. Proto? Hope, A. B., and A. B. Walker. 1975. The Physiolo 27:1081-1089. plasm 136:161-169.

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