The electric properties of Paramecium have gained interest because of the emerging role of membrane potential and conductance in the control of ciliary activity (Kinosita, 1954; Okajima, 1953; Naitoh, 1958; Kinosita, Murakami & Yasuda, 1965; Naitoh & Eckert, 1969a, b; Eckert & Naitoh, 1970). Recent evidence indicates that ciliary responses are coupled to membrane potential by membrane-regulated calcium fluxes (Eckert, 1972). Membrane potential changes occur in response to mechanical stimuli (Naitoh & Eckert, 1969 a, b), radiant energy (Hildebrand, 1970), and changes in extracellular electrolyte concentration (Kamada, 1934; Naitoh & Eckert, 1968a). Further interest comes from the isolation of behavioural mutant of Paramecium (Kung, 1971) which has lost the property of regenerative depolarization characteristic of the wild-type membrane (Kung & Eckert, 1972). The cell membrane of Paramecium normally produces a graded, spike-like, regenerative response to applied outward current (Eckert & Naitoh, 1969). This is followed closely in time by a re-orientation of the cilia so that the direction of the effective stroke (and, hence, the direction of swimming) is reversed (Eckert & Naitoh, 1970). A similar graded response is produced by the depolarization in response to mechanical stimulation of the anterior surface of Paramecium (Eckert, Naitoh & Friedman, 1972). In this paper we present evidence that the graded response is dependent on extracellular Ca*, and propose that the membrane of Paramecium undergoes an increase in calcium permeability in response to depolarization, which permits Ca"" to flow inward down its electrochemical gradient. The regenerative response in Paramecium is similar in this and several other respects to the graded, non-propagated responses produced by crustacean muscle (Fatt & Katz, 1953; Fart & Ginsborg, 1958; Werman & Grundfest, 1961).

Die präsynaptische Freisetzung von Noradrenalin bewirkt durch einen Anstieg des zytosolischen Ca2+-Spiegels die kavernöse Muskelkontraktion. Das Ca2+ kann dabei aus dem Extrazellulärraum aber auch aus intrazellulären sarkoplasmatischen Speichern stammen. Die Beteiligung dieser Ca2+-Speicher wurde in einer Organbadstudie an Gewebsstreifen von Kaninchen untersucht, wobei die Kontraktion durch Phenylephrin oder elektrische Stimulation ausgelöst wurden. Nach Inkubation in Ca2+-freier Lösung wie auch nach Gabe von Nifedipin trat keine vollständige Kontraktionshemmung ein. Ryanodin, ein bekannter Blocker intrazellulärer Ca2+-Kanäle, konnte die adrenerge Kontraktion signifikant inhibieren, Caffein verstärkte diesen Effekt. Die Ergebnisse zeigen, daß für die glattmuskuläre Schwellkörperkontraktion sowohl ein Ca2+-Einstrom aus dem Extrazellulärraum, wie auch aus intrazellulären Speichern von Bedeutung ist.

Outward tail currents measured inAplysia neurones after termination of depolarizing voltage-clamp pulses consist of rapidly decaying voltage-dependent K currents and slow tail currents of much slower time course. The rapidly decaying voltage-dependent tail currents were blocked with aminopyridines, and measurements of the slow tail currents were made following decay of any residual rapid tail currents. The slow tail current exhibited two components of differing sensitivity to externally applied tetraethylammonium (TEA) ions. In some neurones of the abdominal ganglion (L-2, L-4), virtually all of the slow tail current was resistant to blockage by TEA, while in others (L-3, L-6) 80% or more of the slow tail current was blocked by low TEA concentrations (KD<1 mM), the remaining slow tail current being resistant to TEA. This TEA-resistant slow tail current was identified as a K current because it reversed near the K equilibrium potential (EK), the reversal potential was shifted by changes in the external K concentration, and it could be blocked by injection of Cs+. It was abolished by replacement of external Ca2+ by Co2+ or Ba2+, by addition of Cd2+, or by injection of EGTA, and thus determined to be a Ca-dependent current. Intracellular injection of TEA or external application of aminopyridine or apamine had little or no effect on the TEA-resistant slow tail current. Quinidine reduced the TEA-sensitive, but not the TEA-resistant current. Both the TEA-sensitive and the TEA-resistant components of the slow tail current exhibited similar time courses of decay. Thus, neurones ofAplysia appear to contain different proportions of two classes of Ca-dependent K channels that differ in their sensitivity to certain channel blocking agents.

The effect on the Ca-dependent potassium current,IK(Ca), of procedures that increase intracellular cAMP levels was studied inAplysia neurons using three different pharmacological approaches.

1. The Ca current seen in response to depolarization was investigated in Paramecium caudatum under voltage clamp. Inactivation of the current was measured with the double pulse method; a fixed test pulse of an amplitude sufficient to evoke maximal inward current was preceded by a conditioning pulse of variable amplitude (0‐120 mV). 2. The amplitude of the current recorded during the test pulse was related to the potential of the conditioning pulse. Reduction of test pulse current was taken as an index of Ca current inactivation. The current recorded during a test pulse showed a progressive decrease to a minimum as the potential of the conditioning pulse approached +10 to +30 mV. Further increase in conditioning pulse amplitude was accompanied by a progressive restoration of the test pulse current. Conditioning pulses near the calcium equilibrium potential had only a slight inactivating effect on the test pulse current. 3. Injection of a mixture of Cs and TEA which blocked late outward current had essentially no effect on the inward current or its inactivation. 4. Elevation of external Ca from 0·5 to 5 m M was accompanied by increased inactivation of the test pulse current. The enhanced inactivation of the test pulse current was approximately proportional to the increase in current recorded during the conditioning pulse. 5. Following injection of the Ca chelating agent, EGTA, the inactivation of the test pulse current was diminished; in addition, the transient inward current relaxed slightly more slowly, and the transient was followed by a steady net inward current. 6. The time course of recovery from inactivation in the double pulse experiment approximated a single exponential having a time constant of 80‐110 msec. Injection of EGTA shortened the time constant by as much as 50%. 7. It is concluded that interference with the entry of Ca or enhanced removal of intracellular free Ca 2+ interferes with the process of Ca current inactivation, while enhanced entry of Ca promotes the process of inactivation. While the mechanism of inactivation is unknown, arguments are presented that the accumulation of intracellular Ca influences the Ca channel conductance.

An androgen-dependent sexual dimorphism of the immune system was demonstrated in mice. The ration of T/B lymphocytes in the spleen was found to be significantly higher in adult females than in adult males. Furthermore, neonatal androgen administration in females gave rise to a permanent significant decrease of the T/B ratio, while neonatal castration in males resulted in a permanent increase of this ratio.

Simultaneous voltage clamping and microcinematography were used to examine the behavior of cilia in response to prolonged hyperpolarizing and depolarizing steps in the membrane potential ofParamecium caudatum. In the absence of stimulation the cilia beat at less than 20 cycles per second with the power stroke directed toward the posterior and somewhat to the right (i.e. 4 o'clock) of the cell.

A behavioral deficiency producedby a single genemutationinParameciumaurelia wastraced toimpairedelectric excitability ofthecellmembrane. Evidence ispresented thatthemutantmembranedoesnot exhibit thenormaldepolarization-activated increasein cal- ciumconductance responsible forregenerative depolariza- tioninthewildtype.Otherelectric properties character- istic ofthewild-type membraneremainnormalinthe mutant. A major unsolved problem inneurobiology isthemechanism ofnegative resistance inelectrically excitable membranes. After asudden depolarization, themembrane exhibits atran- sient increased conductance toanion, usually Na+orCa++, whichthencarries depolarizing charge intothecell. Thead- ditional depolarization results inafurther increase incon- ductance, andthustheprocess becomes regenerative, driving themembrane voltage toward theequilibrium potential of theion(1, 2).Thisregenerative behavior, whichwill betermed electric excitability, isresponsible fortheupstroke ofanac- tion potential. Although theelectrical properties ofexcitable membranes havebeenstudied intensively andhavebeende- scribed elegantly bytheionic hypothesis, virtually nothing is knownaboutmechanisms ofmembraneexcitation atthe molecular levels ofmembrane structure andchemistry. One successful approach tothemechanism ofbiological functions hasbeentomodify ordelete amolecule responsible forone partofacomplex function, andthentoobserve theeffect on thefunction. Manipulations bygenemutation havebeen applied tostudies ofthenervous systembyBenzer (3)in Drosophila melanogaster. Thisrationale isalsobehind the workofKung(4, 5),whichisdirected atthegenetic dissection oftheexcitable membrane ofaciliate, Paramecium aurelia. Paramecium isadmirably suited foragenetic approach to thestudyofmembraneexcitation. Thiscell iselectrically excitable (6-8) andlends itself tointracellular recording and stimulating techniques (9,10). Thelocomotor behavior of thisciliate hasbeenshowntocorrelate withtheelectrical activity ofthemembrane (7, 11,12). Behavioral correlates of membrane activity greatly simplify theproblem ofrecognizing altered membrane function inmutants (4, 5).Since these uni- cells havenosynapses orneural organization other thanthe cell membrane, analysis ofaltered membrane function isfur- thersimplified. Pureclones ofvarious genotypes canbe grownaxenically (13) forbiochemical analysis andcompari- sonsandforimmunological studies. Thedevelopment ofbe- havioral genetics inthis organism rests onanextensive foun- dation ofParamecium genetics (15), andexploits theadvan- tageofautogamy, aprocess ofnuclear reorganization inP. aurelia thatmakesdetection ofmutantsnearly aseasyasin haploid organisms. Finally, a large fraction ofthesurface membrane ofciliates covers thecilia, andthuscanberou- tinely harvested forfractionation (14). Thispaperreports electrophysiological studies onamutantofP.aurelia, first selected for, andrecognized through, itsaltered locomotor behavior (4,5),inwhichthesurface membrane haslost a single function essential forelectric excitation. Theelectric properties ofthewild-type membraneof Paramecium caudatum aresimilar inbasic principles ofthe ionic hypotheses (1)tothose ofother excitable membranes found innerveandmuscle. Theelectric excitability ofPara- meciummostclosely resembles thatofcrustacean muscle membrane (8,16-19). Whenitisdepolarized byinjected cur- rent, Paramecium produces agraded regenerative response in whichthemaximumamplitude varies withtheextracellular concentration bynearly theamountpredicted bythe Nernst relation forapurecalcium electrode (6). This, to- gether withother evidence (19), indicates that theresponse in Paramecium results fromatransient increase incalcium con- ductance evokedbydepolarization. Theincrease inCa++ conductance inresponse todepolarization willbetermed calcium activation after theanalogous Naactivation exhibited bynervemembrane (1).

The amount of Ca++ which is bound to the surface of Paramecium can be controlled by the relative concentrations of cations, and determines, in part, the ciliary response to cationic stimuli (Naitoh and Yasumasu, 1967; Naitoh, 1968). Experiments were performed to examine the relationship between cationic concentration and binding on the one hand, and electrical behavior of the cell on the other. Solutions of Ca++, and solutions of calcium plus Mg++, Ba++, K+, Na+ or Rb+were tested at concentrations of 0.06 to 16 mM. In calcium solutions the I-V characteristics were relatively independent of [Ca], while the resting potential dropped with a slope of 20 mV per decade increase in [Ca]. In mixtures of Ca plus other cations I-V characteristics were also relatively independent of ionic concentrations, provided the amount of bound calcium was held constant. The slope resistance in the region of zero current was also dependent on bound calcium, but was somewhat complicated by specific properties of some cationic species. All the cations tested lowered the resting potential, and hence the membrane appears to have poor cation permselectivity. Cationic permeability was largely a function of the degree of saturation of the anionic binding sites with Ca++; with a decrease in bound Ca the conductance to any cation was increased.

Specimens of Paramecium immersed in solutions of CaCl2 show graded electrogenesis in response to imposed transmembrane current. However, when BaCl2 in a final concentration of 0.25 mM is added to a 1 mM CaCl2 solution, an outward current pulse of 10-10 amp or greater elicits an all-or-none transient reversal in membrane potential having a duration of about 40 msec. An increase of [Ba++] results in (a) lower resting potential, (b) positive shift in critical firing level, (c) increased overshoot of the action potential, (d) decreased hyperpolarizing afterpotential, and (e) increased duration of the action potential (a.p.). If [Ca++] is increased along with [Ba++] so as to keep the ratio √[Ba++]/√[Ca++] constant, the same results are obtained except that the duration of the a. p. remains unaltered. Thus, effects a-d appear to be related to [Ba++] and not to [Ca++] or [Cl-]. The degree of overshoot in 1 mM Ca is linearly related to log [Ba++] with a slope of approximately 22 mv. With the ratio [Ba++]/[Ca++] constant, the slope closely approaches the ideal value of 29 mv. The evidence indicates that prolongation of the action potential is due to a delayed onset of Ba inactivation, and that this in turn is a function of surface-bound Ba. Other features of the action potential are absolute refractoriness during its rising and plateau phases, relative refractoriness lasting several seconds, and repetitive firing in response to steady current depolarization. The response is unaffected by TTX and TEA. Mn prolongs the action potential. Sr has an action similar to Ba, whereas the addition of K, Na, Rb, or Mg to the basic calcium medium is unaccompanied by all-or-none electrogenesis.

Specimens of immersed in solutions of CaCl show graded electrogenesis in response to imposed transmembrane current. However, when BaCl in a final concentration of 0.25 mM is added to a 1 mM CaCl solution, an outward current pulse of 10 amp or greater elicits an all-or-none transient reversal in membrane potential having a duration of about 40 msec. An increase of [Ba] results in (a) lower resting potential, (b) positive shift in critical firing level, (c) increased overshoot of the action potential, (d) decreased hyperpolarizing afterpotential, and (e) increased duration of the action potential (a.p.). If [Ca] is increased along with [Ba] so as to keep the ratio √[Ba]/√[Ca] constant, the same results are obtained except that the duration of the a. p. remains unaltered. Thus, effects a-d appear to be related to [Ba] and not to [Ca] or [Cl]. The degree of overshoot in 1 mM Ca is linearly related to log [Ba] with a slope of approximately 22 mv. With the ratio [Ba]/[Ca] constant, the slope closely approaches the ideal value of 29 mv. The evidence indicates that prolongation of the action potential is due to a delayed onset of Ba inactivation, and that this in turn is a function of surface-bound Ba. Other features of the action potential are absolute refractoriness during its rising and plateau phases, relative refractoriness lasting several seconds, and repetitive firing in response to steady current depolarization. The response is unaffected by TTX and TEA. Mn prolongs the action potential. Sr has an action similar to Ba, whereas the addition of K, Na, Rb, or Mg to the basic calcium medium is unaccompanied by all-or-none electrogenesis.

The amount of Ca which is bound to the surface of can be controlled by the relative concentrations of cations, and determines, in part, the ciliary response to cationic stimuli ( and , 1967; , 1968). Experiments were performed to examine the relationship between cationic concentration and binding on the one hand, and electrical behavior of the cell on the other. Solutions of Ca, and solutions of calcium plus Mg, Ba, K, Na or Rbwere tested at concentrations of 0.06 to 16 mM. In calcium solutions the I-V characteristics were relatively independent of [Ca], while the resting potential dropped with a slope of 20 mV per decade increase in [Ca]. In mixtures of Ca plus other cations I-V characteristics were also relatively independent of ionic concentrations, provided the amount of bound calcium was held constant. The slope resistance in the region of zero current was also dependent on bound calcium, but was somewhat complicated by specific properties of some cationic species. All the cations tested lowered the resting potential, and hence the membrane appears to have poor cation permselectivity. Cationic permeability was largely a function of the degree of saturation of the anionic binding sites with Ca; with a decrease in bound Ca the conductance to any cation was increased.