Corap, Biochera, Physiol. 1976, Vol. 54A, pp. 323 to 329. Pergamon Press, Printed in Great Britain

THE EFFECT OF S O D I U M O N "PARANO!AC"~A MEMBRANE M U T A N T OF P A R A M E C I U M

YOUKO SATOW, HELEN G. HANSMA* AND CHING KUNG Laboratory of Molecular Biology and Department of Genetics, University of Wisconsin,

Madison, Wl 53706, U.S.A.

(Received 27 October 1975)

Abslraet--l. A behaviorally "paranoiac" strain of Paramecium is studied electrophysiologically and . biochemieally. When bathed in Na + solutions, the mutant shows prol0figed excitation up to 60see.

2. T2.~: ~t,,ation and plateau level of the prolonged depolarization are proportional to the l'Na+]out. 3. Tes~ ~.,~ ,,es show that the membrane is highly conductive during the depolarization. 4. Flame ~hotometry shows up to 70~o loss of internal K +. This loss, no t observed in wild type,

is proportional to [Na+]o,~. 5. Z2Na analyses show a large gain of Na ÷ by the mutant proportional to l'Na+]o~,. 6. We conclude that the mutation-prolonged depolarization represents an otherwise normal excited

state where conductances of K + and Na + are high.

I N T R O D U C T I O N

Understanding the molecular mechanism of mem- brane excitation presents a challenge to neurobiology. To approach this problem, we have begun a multidis- ciplinary study of the excitable membrane of Parame- cium. In this study, we have isolated and characterized over 300 lines of behavioral mutants in Paramecium aurelia, most of which e a s y membrane defects (Kung, 1971 ; Kung et al., 1975). Among them are many Ca. gate mutants, which can no longer generate the Ca- action potentials (Kung & Eckert, 1972; Satow et aL, 1974), and two K-channel mutants, which show differ- ent anomalies in membrane potential and resistance (Satow & Kung, 1976a, b). We describe here the abnormalities of another membrane mutant, "Para- noiac".

"Paranoiac" was among the first group of behav- ioral mutants discovered (Kung, 1971a, b). It spon- taneously gives prolonged avoiding reactions-- swimming backward continuously fo r up to 60see. This "paranoiac" behavior can only be observed when Na + is present. Since Na + exists in millimolar con- centrations in culture medium and the "paramecium saline" (Dryl, 1959), this abnormality is observable in all these solutions. In contrast, the normal parame- cia generate only the transient avoiding reactiocts, lasting less than a second, i n these solutions. An avoiding reaction consists of a period of backward swimming due to the reversal of the ciliary beating direction (Jennings, 1906). Ciliary reversal has been shown to result from the transient increase in Ca 2+ concentration in the cell due to the Ca-action poten- tial (Eckert, 1972). T h e Ca-action potential can be evoked chemically, mechanically and electrically (Nai- toh & Eckert, 1974).

Recording intraeellularly, we have shown the mem- brane potential Changes tfiat accompany the avoiding

reactions of normal and paranoiac paramecia. The normal (wild type)parameehim merhbrane responds actively to the Na ÷ in the bath (Satow & Kung, 1974). I n contrast, the membrane stayed quiet when bathed with solutions containing Ca 2+ and K +. When a solution of Na + and Ca z÷ appeared in the bath, a series of depolarizations was initiated. Each episode lasted about 0.5 see. It was often led by a Ca-spike but always reached a plateau, lower than the peak of the spike. Recording from the mutant, we discovered the physiological correlate cf the "paranoiac" behavior: depolarizations to a plateau lasting up to 60 sec were registered in and only in Ca - N a solutions (Satow & Kung, 1974). These depo- larizations correspond exactly to the periods of back- ward swimming.

In" this paper we study the me mbr a ne - o f this mutant in detail. Current was injected during intracel- lular recording from paramecia bathed in solutions of different Na + concentrations. Sodium influx was measured with 22Na + and the cellular K content was monitored by flame-photometry.

MATERIALS A N D ML-'IT'., I O D S

Paramecium stocks and cultures The two strains used were of species 4 of P. aurelia.

They are the wild-type stock 51s (non-kappa bearing) and stock d4-90, the paranoiac mutant with the gen'otype PaA PaA. The behavioral phenotype of this mutant has been described (Kung, !971a). ! t is a single-gene mutant with a mutation unlinked to other known mutations affecting membrane pr0perti6s (Kung; 1971; Van Houten et al, 1976). Cells were cultured in the Cerophyl medium bacter- ized with £nterobaeter aerogenes (Sonnebom, 1970).

Intracellular recordinc 3 T h e m e t h ~ s for intracellular recording from Parame,

Cium were those of Naitoh & Eckert (1974). Modh'icati0n~ Of the referende electrode and the use of continuous per- fusion have been described by Satow & :Kung (1976a)~ Both the stimulating electrode and therecording electrode

*Present address:" Department of Biological Sciences, UniverSity of California, Santa Barbara, CA 93106, U.S.A.

323

324 YotJKO SATOW, Ht~I~N G. HANSMA AND CHING KDNG

were lilled with 500raM KC! and had resistances of 70-130 MR. All solutions used to bathe the specimens dur- ing recording contained I mM Ca(OH)z, I mM citric acid, buffered to pH 7.15-7.25 with 1.0-1.2 mM Tris [tris (hyd- roxymethyl) aminomethane'l. In addition, the K-solution contained 4 mM KC[, the Na-solutions contained various concentrations of NaCi, as specified, a~nd the Ca.solution contained an additional 2 mM of CaCI 2. Recording began with the paramecium in the K-solution, in which it had no spontaneous activity. Other solutions were perfused into the bath to replace the K-solution as the experiment progressed. Experiments were carried out at room tem- perature with paramecia in log phase growth.

Atudysis of K ÷ content Cells from a culture were concentrated and washed into

! mM Ca ~÷, 1.2mM Tris, I mM citrate, l mM KCI, pH 7.2. After 15-30 rain, the cells were centrifuged and resus- pended at a density of 1-5 × 105 cells/mL One ml aliquots were added to 100ml of different incubation solutions (l mM Ca 2+, 1.2mM Tris, l mM citrate, pH 7.2 plus various concentrations of NaCI). After 30-50 rain of incu- bation at room temperature, the samples were centrifuged and the supernatant thorough)y removed. The Cell pellet was suspended in l0 ml water, and aliquots of this suspen- sion were taken for protein determinations (Lowry, 1951). Inorganic ions were extracted with 99-100% efficiency (Dunham & Child, 1961; Hansma, 1974). The cooled extract was analyzed on an Eppendorf flame photometer with parallel standards of known K + concentrations. The results were expressed as /orioles K+/g cellular protein (Hansma, 1974).

ZZNa uptake Cellswere concentrated and washed into an adaptation

solution (1 mM Tris, I m M HEPES, l mM Ca z+, I mM citrate pH 7.2). After" 10--20 min, the cells were centrifuged into a pellet.Aliquots of the pellet were transferred to vials containing the incubation solution (adaptation solution ~with ~ZNa and NaC! added in various concentrations). After 30-45 rain incubation, aliquots of the cell suspension were layered over a nonradioactive wa ~h solution in centri- fuge tubes made from Pasteur pipetts (Hansma, 1974; Hansma & Kung, 1976). The wash solution was non- radioactive, having the same .omposition as the incubation solution with the addition of 1~'~ sucrose to raise the den- sity. After centrifugation, the tip of the tube containing the cell pellet was broken off and its radioactivity was measured in a gamma counter (Nuclear Chicago). The total cell suspension was also counted to determine the specific activity ofextraecllular sodium. Protein determina- tions were made, and the results were expressed as pmole sodium taken up in 30 min per gram of cellular protein. The incubated and washed cells for both the K + content and Na ÷ influx study were examined and counted under a stereomicroscope. There was no damage of cells or loss of cell count.

R E S U L T S

The Ca-action potentials

Naitoh & Eckert (1974) showed tha t P. caudatum generates graded action potentials. The depolarizing current is carried by Ca2+, which is coupled to a mechanical response of the ciliary microtubules (cili- ary reversal). Ca-action potentials are also generated by P. aurelia. Figure 1 shows these action potentials Upo n current injection in wild type and in the "par- anoiac" mutant . The kinetics of potential r ise and fall, a s well a s the peak height, are essentially the Same in t h e wild type and the mutan t in both

C a

wild type Psrenoiec

Fig. 1. Potential responses to injected currents in wild type (left), and the "paranoiac" mutant (right) of P. aurelia, showing no essential differences between the responses of the two strains in a Ca-solution (upper frames) and in a

• K-solution (lower). In each frame the upper trace is the membrane potentia ! led by a 10msec, 10mV calibration pulse (reference level as marked); the lower trace marks the 100msec, 10-gamp outward current injected. The slight differences between the potential responses of the two strains are within the variations encountered in differ- ent cells of the same strain, See Materials and Methods for the techniques of intraecllular recording and the com-

positions of the solutions.

solutions. Slight differences are no more than those encountered when recording from different specimens of the same strain. The upper frames of Fig. 1 show responses when paramecia are bathed in the Ca-solu- tion in which Ca 2+ is the only cation added to the buffer. The lower frames of Fig. I show the Ca-action potentials recorded from paramecia in the K-solution, conta ining both K + and Ca 2+. Thus, unlike the "pawn" mutants (Kung & Eckert, 1972; Satow et al., 1974) and the "TEA-insensitive" mutant (Satow & Kung, 1976b), the Ca-channel and the K-channel im- portant in excitation are not affected by the "'para- noiac" mutation.

Spontaneous activities in Na-solution

We have studied the behavioral responses of wild- type and paranoiac P. aurelia to Na-solution (Kung, 1971; Satow & Kung, 1974). We have also shown that the action potentials spontaneously generated by the wild type ha a Na-solution are different from the al l-or-none action potentials recorded in Ba2+-con - tainh~g solutions (Satow & Kung, 1974). Typically, the ~pontaneous electrical activity in the Na-solution has a leading spike, a lower plateau and then a sudden r e p o l a d ~ t i o n after about 0.5 see. The pattern of the spontaneous electrical activity is not uniform a n d the spike component is of ten ,obscure . A train of these depolarizations is observed when Na 'solut ion is let into the bath to replace t h e K-solution (Fig, 2 top). These membrane depolarizations correspond to the series of transient avoiding reactions observed in Na-solution.

T h e : pattern of response to the Na-solutions is greatly altered i n the "paranoiac" mutan t (Fig. 2 bot- tom). Instead of the 0.~5 sec depolarizatiofi episode, the depolarization is nmintained from 10 to over 60see. The upstrokes of these prolonged depolar izat ion events are variable in their rate of rise, as in the case

Membrane mutant 325

wild type

11~ t' It Ill | | I | lll IIFIIIwLrl IP~l ' ¢ l l ~ " ' t = = ' " r r ~ r ~ " T - - ' ' I - - ' ~ I ~ ~" " ~ I t a l y U~ I r " ~ " r

Psrsnoiec

~ 2 S t ,c

Fig. 2. Membrane potentials recorded intracellularly from a wild type (top) and a "paranoiac" mutant (bottom) in a 4 mM Na ÷ solution, showing differences in the spontaneous membrane activities upon encountering Na ÷. Cells were first penetrated in the K-solution. Arrows mark the time when the Na-solution begins to replace the K-solution. Note that wild type responds to Na ÷ with irregular and transient depolarization episodes. The mutant responds with greatly prolonged depolarization with plateaus greater than 30see. Reference level is .marked by the broken lines. Calibration: 25sec, 40mV. See Materials and Methods for the techniques of perfusion and the complete composition

of solutions.

of the wild type. The down-stroke kinetics are totally different from those of the wild type. Repolarizat ion from the pla teaus takes 2-10see. Al though not seen in Fig. 2, the mutan t sometimes generates short depo- larizat ion events similar to those of the wild type. The 10 see depolarizat ions correspond to the 10 see continuous backward swimming of the mutant in Na- ~olution. Both the spontaneous membrane depolari- zation and the backward swimming increase in durat ion when the external N a ÷ concentrat ion in- creases, al though variat ion from Cell to cell can be observed.

After the Paranoiac mutant i s bathed in the Na- solution for several minutes, its resting potential often becomes more depolarized (Figs. 2, 3). The decrease in resting potent ial by 10 to 20mV may be due to an ion imbalance resulting from the large K- and Na- fluxes (see below).

Electrically-triggered response in Na-solution The peculiar pat tern of prolonged depolar izat ion

of the mutant can be triggered by current injected through t h e membrane. Figure 3 ( r ight ) shows an event triggered by an outward current pulse of a 100msec, 1 0 - g e t u p Note that the mutant ' s leading action potential, the steady-state depolar iza t ion by in-

jected current, and even the first par t of the repolari- zation after the injected current are very similar to those of the wild type, responding to the same current injection. The difference appears after the 100reset injected current. Since prolonged depolar izat ions of the paranoiac membrane can occur spontaneously (Fig. 2), very little current is needed to trigger the event. The stimulus durat ion is also not critical, since pulses as short as 5msec at 2 x 1 0 - g a m p are~sui'fi- cient. These prlonged depolar izat ion events can even be triggered as the "off-response" to a hyperpolarizing current. In such case, the upstrokes are continuous with the depolar izat ion that is caused by shutting off the injected current. The durat ion and the ampl i tude of the plateau are not correlated with the strength, durat ion or direction of the triggering current. Like the spontaneous depolar izat ion plateaus, the triggered plateaus -vary in durat ion from cell to cell.

The ~otent ial level and the dura t ion o f the plateau are directly related to the concentration of Na ÷ in the solution. Figure 4 shows that the same triggering current leads to progressively longer plateaus when the same mutan t cell is bathed in a series of solutions with increasing concentrat ions of N a ÷. Although this effect has been observed in all specimens examined, quantif ication is limited by the small sampling s ize

wild type O ~

I

Paranoiac

o

, . , ,d - . i

l O O m e c

Fig. 3. Potential responses to injected current in wild type (left) and the "paranoiac" mutant (right) in a 4mM Na* solution. Note the similar potential response (upper traces) during the t00msec, 10-gamp injected current (lower traces) and the different response after the injected current. After the G,lrrent, the paranoiac mutant membrane is suspended at a depolarized level for 17see, part of which is showrl here. Similar responses of the mutant are shown at slower sweep speed in: Fig. 4. Note also that the mutant membrane rests at a less polarized level than wi ld type. Reference level

as marked. Calibration: 30 mV, 100 msee,

326 YOUKO SATOW, HELEN G. HANSMA AND CHING KUNG

P l r e n o l a c ( r a m N i t )

2 - - - . ' ~ " - - l ~ - ,t_, ,__ ,,, ,,,

~IIQC

8 ~ ~,~,~. . ,~. . .~ '

l,

Fig. 4. Sustained depolarization of the same "paranoiac" mutant cell bathed in a series of solutions from 2raM to 16raM Na ÷ (top to bottom). These depolarization are triggered by the same current (10 -~ amp, 100 msee as marked beneath the potential traces). Note that the duration of the depolariza- tion plateau lengthens as the concentration of external Na ~ increases. The marks on the bottom trace indicate the deletion of 13.2see of the plateau from the record to save space. Note also that the plateau potential.as well as the resting potential becomes progressively more depolarized as the concentration of external Na + increases. Solid straight line with each of the potential trace marks

the reference level. Calibration: 2 see, 30 mV.

pract ical in electrophysiology and by the variat ion f rom specimen to specimen (e.g. second trace of Fig. 4 vs Fig. 5). Comparab le da ta has been obtained, however, rby the technically simpler measurements of the durat ion of cont inuous backward swimming of large numbers of cells under similar conditions. They clearly show that there is a Statistically very signifi- cant, a lmost linear increase in the dura t ion of back- ward swimming as the concentrat ion of Na + in- creases up to 2 0 m M (Kung, 1971a).

Figure 4 also shows that both the resting potential and the p la teau levels of the paranoiac become m o r e depolarized in higher concentrat ions of Na *, The resting potential level of the wild type also becomes more depolarized in higher concentrat ions .of Na +, a l though wild !ype never shows a depolor izat ion pla- teau even after current , inject ions in high Na + solu- t ions

The abil i ty of the m u t a n t to show the: prolonged depolarizat ion plateau depends on the resting poten- tial level. Hyperpolar iz ing the mutan t membrane in 4 r a M Na + by 5 - 1 0 m V with a "condit ioning" d.c. pulse prevents the plateau from occurring., even with s trong triggers. Condi t ioning depolar izat ion often appears to lengthen the plateau, a l though insufficient quanti tat ive measurements have been made to be cer- tain o f this, Condi t ioning depolar izat ion of mutant

Pmr&notmc N a 4ram

membrane in K- or Ca-solut ion does not generate depolar izat ion plateaus after the proper trigger. Con- di t ioning depolar izat ion or hyperpolar~.ation of the wild-type membrane up to 20 mV also does n o t gen- erate such a plateau.

Membrane resistance at rest vs at plateau

F r o m the behavioral observations, we suspected that the depolar izat ion at the plateau might represent a prolonged excited state of the membrane. Therefore, we tested the membrane resistance during the plateau and compared it with the resistance at rest. In Fig. 5, a 100msec inward current o f 1 0 - ~ ° a m p was in- jected every 1 sec through the membrane of a mutant. The potential displacement by the injected current is much smaller at the plateau than at the resting level. Thus, the membrane resistance is lower at the plateau than a t the resting potential . I t is a l s o interesting to note that the membrane resistance gradually increases as the plateau continues. Most , if not all, of the resist- a n t e is regained before the membrane repolarizes.

Sodium f l u x e s Presumably there are larger ion f luxes during the

p la teau state, where the membrane resistance is low. Using 22Na as a tracer, the influx of N a + into wild- type and mutant paramecia is examined. Figure 6

1 $eC

Fig. 5: Reduction:of membrane resistance at t he depolarization plateau 0f th~ "paranoiac" mutant,

before and after the plateau:The plateau is triggered by'a~ i o o m ~ qo--9 amp depolarizing current as in Fig. :4i'The'cell is bathed in: a 4 mM :Na + solUfi0n: T h e ~ r k ~ ~ indicdtc a deletion o f 11,~ Scc

of the plateau from the record.Zero reference le£'cl as indicated.:Calibration: I sec, 30mY.

160

c ~0o ~ ~ paronoiae / 1

0 5 IO 15 20 Extetnol No + Concenttofion (raM)

FiB. 6. Ha+ uptake by paramecia 30 rain incubation in solutions of different Na +conocntrations containing nNa. Note that the mutant (triangles) accumulates more Ha + than wild type (circles). Means and standard deviations are shown; N ---- 4, for both wild type and mutant. Ab- scissa: concentration of Na + in the incubation solution in raM; ordinate:/~mole Na + accumulated per gram cellu-

lar protein in 30 rain.

shows that there is a large influx of N a + into the mutants when they are transferred into Na-solutions. The comparab le Na-flux into wild type is much smaller. The N a + influx into the paranoiac mutant is propor t ional to the external N a + concentration, as are the dura t ion of plateau (Fig. 4) and the dura t ion of backward swimming (Kung, 1971). In a 20 raM N a + solution, up to 120/~mole of Na + is taken up per g ram protein in 30 min by the mutant.

The specific radioactivi ty of the wash solution above the cell pellet is only 10--20°,/o of the specific activity o f the original incubation solution. Thus, the procedure for washing cells is sufficient to remove 80-90% of the small amount of N a + t rapped between the cells in the pellet. For the same reason, one can assume that 8 0 - 9 0 ~ of the readily exchangeable Na + bound to the cell surface is removed b y the washing procedure. Therefore, the radioact ivi ty captured b y the cell pellet represents largely the inf lux o f N a +.

Similar results to those in Fig. 6 were obtained when the total N a ~ content of the calls is measured by flame photometry , al though the flame photometer is much less sensitive to sodium, so that these measurements are less precise.

Potassium fluxes

When, the m u t a n t s a re t ransferred i n t o solutions containing N a +, they also show large decreases in total cellular K + (Fig. 7). In a solut ion containing 20 raM N a ÷, these cells lose more than 70% of their total K + in 30 rain. In contrast , the cellular potassium content of wild-type paramecia remains essentially constant a s the external N a + concentrat ion varies from 0 to 20 mM, except for a small and unexplained d ip at ~1 raM Na÷~

The K + loss by the mutan t is o f t h e same magni- tude as its N a + gain. The difference in K+ content between wi ld type and mutant is larger, however, than the difference in N a ÷ inf lux between wild type a n d mutant. Thus, i t is poss ib l e tha t - there a re other ion

|40

" wild (ypll ' ~ 120

.j

l =

;~8o x

E

4O

20

Membrane mutant 327

l • ' , t ! • t , , t . . . . ! . . • , t

O 5 tO • 1 5 20 Exlernol No + Coneentroflon (raM)

Fig. 7. K ÷ content of paramecia measured by flame photo- metryafter 30 to 35 rain incubation in solutions of different Na + concentrations. Note that the mutant(triangle) loses K + in Na + solution and wild type (circles) does not. Means and standard deviations are shown; N = 5 for wild type; N = 4 for the mutant. Abscissa: concentration of Na + in the incubation solution in mM; ordi,ate: /anole

K+/g cellular protein.

fluxes in addi t ion to the Na + and K + fluxes reported here. We have thus far been unable to detect either a chloride efflux o r a significant difference in calcihm

inf lux , however (Hansma, 1974).

D I S C U S S I O N

Formal genetic analyses have shown that the "para- noiac" mutant has a single-gene al terat ion (Kung, 1971b). This single gene m u t a t i o n causes a dramat ic and very specific change in the behavior of the para- no i ac mutant , which has led to the discovery of its biochemical and electrophysiological abnormalit ies. Both the active and passive propert ies of the mem- brane e lee t rophys io logy appear n o r m a l in Ca- or K-solu t ion ' (Fig. 1). A group of related abnormal phenotypes are observed, however,.when the mutants encounter Na ÷ in the solution.

1. They show repeated prolonged cil iary reversals, and hence backward swimming, in N a + solutions (Kung , 1971a).

: 2. T h e r e are sustained membrane depolarizations, at a plateau, corresponding to the prolonged back- w a r d swimming (Fig. 2, 3). • :3. The du ra t ion o f backward swimming and the dura t ion o f the pla teau increase as the external con-

cen t ra t ion o f N a + increases ( K u n g , 1971a): (Fig. 4). 4. The repolarizat ion from the plateau is very slow

(Fig. 2, 4). 5. The plateau potential bve l and the r e s t i n g

potent ial level both become more depolarized with the increase of external N a ~ (Fig. 4); this is also t r ue for wild type.

6. The membrane is much less resistive at the p l a - teau than a t rest (Fig. 5); this may be true for wild type as well.

7. There is a much larger N a * influx in the mutant t h a n i n wild type in the N a + soh t /ons (Fig. 6).

328 YOUKO SA1OW. HELEN G. HANSMA AND CIIING KUNG

8. There is a concomitant loss of internal K + in these solutions in the mutant but not in the wild type (Fig. 7).

9. The large K + loss and Na ~ gain of the mutant are directly correlated with the external concentration of Na* (Fig, 6, 7).

In Na-solutions, the wild-type membrane typical ly shows a series of depolarizations, each with a -,-0.5 sec "plateau." Satow & Kung (1974) postulated that the "paranioac" pattern of prolonged depolariza- tion was the result of mutational blockage of the nor- mal repolarization process. This postulate explains the prolonge21 depolarization to a plateau and the abnormally slow repolarization kinetics of the mutant.

The resistance measurements also suggest that the depolarization plateau in paranoiacs is a pro longa- tion of the normally transient "excited" state where the membrane is highly permeable. Prolonging this state by the mutat ion results in large fluxes of ions in the mutant. Alternatively, the large K + and Na + fluxes could occur While the mutant membrane is at rest. This alternative should be considered seriously since after ~ 10rain i n the Na-sohit ion the mutant membrane often rests at a less polarized level than wild type. However, large fluxes o f ions are not observed when resting potential is changed. Depolari- zation of the resting wild-type membrane by increas- ing external Na +- concentration does not increase the K + efflux (Fig. 7). Also,. the mutant does not have a K + elitux when bathed, in a solution of high K + concentra t ion also known to depolarize the mem- brane (Hansma, 1974; Hansma & Kung, 1976). We view the decrease of the mutant resting potential in Na-solution as the result and not the cause of the high intracellular N a concentration. The mutant loses i t s membrane potential completely and eventually dies much sooner than wi ld type, presumably due to the ion redistribution.

A typical wild4ype depolarization in Na + solution begins with a Ca-action potential. Some wild-type depolarizations lack the Ca 2+ spike , however, and show only the 0.5 see "humps". The spikeless "humps'" a r e also ~ seen: in the " P a w n " m u t a n t , .whose Ca 2 + spike has been eliminated by its m u t a t i o n (Kung & Eckert , 1972; Satow et al., 1974). These observations suggest thepresence of a Na+-sensi t ivedepolar izat ion mechanism independent o f t h e Ca-ac t ion potential . This mechanism could be a N a + binding s i te affecting the permeation of other, ions or a channel specific f o r N a + permeation. The large N a + flu~.es measured in this study suggest the presence of a Na 2 channel. One possible: model for t h e N a +-related depolariza- tion episode is the following: In the presence of N a +, a Na + Channel opens, influx of Na + occurs, the: mere. brahe depolarizes (rise of"hump"), a Ca-action poten- t i a l (spike) is triggered, and the membrane remains in a high conductance s tate for ~0.5 see (plateau) un- til a t ime-dependent repolarization mechanism closes the Na + channel (shut=off). Such a channel is, quite different from the well-known Na + channel in nerves, in~ the: manner of i t s Opening and the slower and longer electrogenesis.

All the mutational effects of the paranoiac mutant can only be seen in Na-solution. Therefore, the mutat ion blocking the repolarization must be affect-

ing a membrane structure which binds o r channels Na +. It is possible that the time-dependent inactiva- tion mechanism of the Na-channel, postulated above, has become defective in this paranoiac mutant. A Na- channel that does not shut properly accounts for the sustained depolarization at the plateau. At the pla- teau, the membrane is highly conductive allowing Na ÷ and K + to flow down their electrochemical gra- dients. Aside from being prolonged, this high conduc- tance state is not necessarily abnormal. The short ('--0.5 sec) plateau observed in wild type presumably represents the same state of the membrane. When the wild-type membrane is depolarized by long d.c. pulses to levels comparable to the plateaus it is also less resistive. The abnormali ty of the "paranoiac" mutant is clearly at the shut-off mechanism of this conductive state. The repolarization is at least ten times slower in the mutant than in the wild type. This repolariza- tion would be more difficult against a larger Na + gradient. Thus, the higher the external Na + concen- tration, the more defective the mutant appears, in terms of the duration of plateau, the durat ion of back- ward swimming, the K + loss and Na + gain.

Alternative explanations of the mutant defect in repolarization are less attractive in this case. The repolarization kinetics after the Ca-action potential are apparent ly normal in this mutant (Fig. l). A weaker K + efflux could explain the slow repolariza- tion after the mutant plateau, but this contradicts the observation that there is a huge K + efflux in the mutant (Fig. 7). However, the possible involvement of N a +, when present, in Ca-inactivation or K- delayed rectification in Paramecimn has not been fully investigated.

Four other independent mutants having similar but not identical "paranoiac" behavior have been dis- covered and examined behaviorally, genetically, and biochemically (Van Houten et al., 1976; Hansma & Kung, 1976). The postulate of a mutational lesion on a Na ÷ channel, ,given here to explain ~his paranoiac mutant, may not apply to the other paranoiac mutants. Of special interest is the finding that two different mutations on the same gene can give a "paranoiac" phenotype and a "fast-2" phenotype. The analysis of the "fast-2" mutant leads to the conclusion that it has a relative increase in resting K-permeabi- lity (Satow & Kung, A976). Preliminary results show that wild-type paramecia bathed in Na ÷ solution exhibit prolonged depolarization plateaux both spon- taneously and after triggering pulses, i f TEA+ is applied to the cells externally or internally. This find- ing furl~her supports the idea that a defective K-chan- nel can also l e a d to the "paranoiac" phenotype.

SUMMARY

The membrane of a "paranoiac" mutant of Parame- cium aurelia was s tudied b y intrace!lular recording, measurement of 22Na uptake, and flame-photometric analysis of K + content. ~

This mutant shows no abnormali ty in its active or passive membrane: propert ies ~ when, bathed in :solu- tions containing Ca z+ and K ~, but its active response to Na* is;abnormal. Normal paramecia r e spond t o external N a +, e.g. 4mM,. with a series o f active d~- polarizations, each with a Na-related ~upstrokeoften

Membrane mutant 329

lead ing to a Ca-spike, a ~ 500 msee plateau and a 10-30 msec repolar izat ion. Pa rano iac mutan t s give a t ra in o f act ive depolar iza t ions , each with a p ro longed p la teau lasting 10 to 60sec fol lowed by a 1-5 sec gra- d u a l repolar iza t ion to t h e rest ing level. This p ro- longed depolar iza t ion can be t r iggered by a small cur- rent del ivered through a s t imula t ing electrode. The d u r a t i o n and potent ia l level o f this p ro longed p la teau re la ted direct ly to the external concen t r a t ion o f N a +. Artif icial hyperpo la r iza t ion by a d.c. pulse abol ishes this plateau. Resis tance measu remen t s wi th shor t pulses show that the m e m b r a n e is highly conduc t ive a t the plateau.

T h e pa rano iac mutan t a lso suffers a large loss of K + a n d a large influx o f Na + when bathed in N a solut ions. These a b n o r m a l fluxes are p ropor t iona l to the external Na + concent ra t ion . U p to 70% of the in ternal K + is lost in a 2 0 r a m N a + solut ion, a n d there is a c o m p a r a b l e ga in of N a +.

T h e possibili ty that the m u t a t i o n affects a m e c h a n i s m of repolar iza t ion such as in inac t iva t ion o f a N a channe l is dis~'~Jssed. T h e failure o f this mechan i sm would suspend the m e m b r a n e a t a depo- lar ized excited state (the plateau). At this state, the m e m b r a n e is highly conduc t ive and is unable to pre- vent K + efflux or Na + influx.

Acknowledgements--We thank Dr. Donata Oertel for her helpful criticisms. This work was supported by NSF Grant BMS 75-10433 and FHS Grant GM 22714-01 to C.K.

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