by n. akaike*, am brown, g. dahlt, h. higashit, g

J. Phy8iol. (1983), 334, pp. 309-324 309With 8 text-figures and 2 plates

Printed in Great Britain

VOLTAGE-DEPENDENT ACTIVATION OF POTASSIUM CURRENT INHELIX NEURONES BY ENDOGENOUS CELLULAR CALCItJM

BY N. AKAIKE*, A. M. BROWN, G. DAHLt, H. HIGASHIt, G. IS1ENBERG§,Y. TSUDA AND A. YATANI

From the Department of Physiology and Biophysics, University of TexasMedical Branch, Galveston, TX 77550, U.S.A.

(Received 6 October 1981)

SUMMARY

1. The effect of endogenous Ca on potential-dependent K current, IKD, wasexamined in identifiable neurones of Helix aspersa. The suction pipette method ofinternal perfusion was used along with a combined voltage-clamp method in whichthe membrane potential was measured by a separate glass micro-electrode and thecurrent was passed by the suction pipette. Activation of the potential-dependent Acurrent, IA, was prevented by using holding potentials of -40 mV where IA isinactivated and by the addition ofthe A-current blocker 4-aminopyridine. ActivationofK currents by transmembrane Ca current, IKCa, was suppressed by Co substitutionfor Ca ion extracellularly.

2. Under these conditions, IKD rose to a peak value and then subsided to a steadylevel. The current-voltage (I-V) relationship for peak IKD had an upward bump atabout + 50 mV that gave it an S-shape. The I-V curve for steady IKD rosecontinuously. Peak and steady IKD were reduced by perfusing with EGTA or F ionsintracellularly. The EGTA effect occurred at intracellular Ca activity levels below10-7 M. Increases in the concentration of EGTA1 at constant Cai had no additionaleffect; however, recovery experiments do not allow us to rule out some direct actionof EGTA on IKD'

3. Prolonged extracellular perfusion with Co-substituted solutions also reducedIKD and the effects occurred more quickly when the solutions were made hypertonicor caffeine was added to them. The peak transient was abolished, and the smallremaining steady IKD (about 5-10% of normal peak IKD) was blocked by tetra-ethylammonium. IKD could be restored by the temporary reintroduction ofCa in theextracellular solution.

4. The S-shape of the peak I-V relationship for IKD may be due to Ca releasedfrom an endogenous site by membrane depolarization. The reduction of steady and

*Department of Physiology, Faculty of Medicine, Kyushu University, Fukuoka, Japan.t Department of Physiology and Biophysics, University of Miami Medical School, Miami,

FL, U.S.A.$ Department of Physiology, School of Medicine, Kurume University, Kurume, Japan.§ Heisenberg Fellow of the Deutsche Forschungsgemeinschaft. Permanent address: II Physio-

logisches Institut, Universitat des Saarlandes, Homburg, F.R.G.

N. AKAIKE AND OTHERS

peak IKD to very low values by Ca chelators or prolonged perfusion with Ca-treesolutions indicates that Cai is important for activation of these K channels.

5. Three cellular structures were identified in electron micrographs of freeze-fractured neurones that could be involved in potential-dependent endogenous Carelease. These were a restricted extracellularly space, an intracellular membranesystem of endoplasmic reticulum that may be fused to the internal face of the plasmamembrane (the subsurface cisterns of Henkart & Nelson, 1979), and intracellularvesicles that also may be fused to the plasma membrane.

INTRODUCTION

In 1979, Aldrich, Getting & Thompson reported that the transient peak of thedelayed K current of Ani8odoris neurones had an N-shaped relationship withmembrane potential when Co ion was substituted for Ca extracellularly. The N-shapehad been shown earlier (Meech & Standen, 1975) to result from activation of Kchannels by transmembrane Ca current, ICa' and since in voltage-clamp experimentsICa is not measurable following Co substitution (Brown, Morimoto, Tsuda & Wilson,1981) another explanation for the result of Aldrich et al. (1979) seems to be required.An obvious one would be a voltage-dependent Ca-activated K current, IK, thatpersists in the absence of measurable Ca current. The latter could arise if Ca enteredthe cell from endogenous structures such as those reported by Rosenbluth (1962),Henkart, Landis & Reese (1976) and Henkart & Nelson (1979) which might beconnected in a limited way to the extracellular space. The present experiments weredone to test this possibility.We separated the delayedK current from Na, Ca and fast transient K or IA (Connor

& Stevens, 1971) currents and blocked Ca currents by substituting Co for Ca in theextracellular perfusion. The substantial K current that remained had an upwardbump or inflexion in the relationship between peak current and membrane potentialthat gave it an S-shape as Aldrich et al. (1979) reported. Intracellular perfusion withthe Ca chelators EGTA or F ions greatly suppressed the peak level of IKD andattenuated the N-shape, Steady IKD was also markedly reduced. The results wereindependent ofEGTA concentrations between 1 and 5 mm at constant Cai activitiesof5 x 10-8 M, suggesting that direct effects ofEGTAi on IK were not major. However,recovery Of IKD from EGTAi was not complete and a direct action cannot beexcluded. Prolonged external perfusion with Co-substituted solutions also greatlyreduced the endogenous Ca-activatedK current. The latter was restored quickly whenCa was reintroduced.The morphology ofHelix neurones was examined for the presence of structures that

might serve as Ca stores. Three types of structures were found which could act assources for Ca release when the plasma membrane is depolarized. These structuresmight also form a source of slowly depletable cellular Ca.

METHODS

The experiments weredone on single, identifiable nerve cell bodies isolated from the suboesophagealganglia of Helix a8per8a- usually E1,2,5 and F1,2,76 and 77 of Kerkut, Lambert, Gayton, Loker &Walker (1975). These cells have diameters of about 80-200 ,um. The basic suction pipette method of

310

K CURRENT AND ENDOGENOUS Ca, VOLTAGE CLAMP 311

internal perfusion was used (Akaike, Lee & Brown, 1978; Lee, Akaike & Brown, 1980) and thevoltage clamp was a combined method in which a separate glass micro-electrode, filled with 3 M-KCIand having a resistance of 1-2 MCI, was used to record membrane potential while the suction pipettewas used to pass current. The membrane potential was summed with the command step duringvoltage clamp (Brown et al. 1981).The series resistance, R., calculated from admittance measurements made with sine waves of

current step was about 5 kfl, which could lead to a maximum error of 5 mV in our estimate of truemembrane potential.

TABLE 1. Experimental solutions (mM)NaCl Tris-Cl KCl CaCl, CoCl2 MgCl2 K-asp. K-fluoride Glucose

ExternalNormal 85 5 5 10 15 - 5.5Tris-Ca 90 5 10 15 5.5Tris-Co - 90 5 10 15 - 5-5

InternalK-asp. - 135K-fluoride 135

Ionic currents were monitored on a storage oscilloscope (Tektronix, 5111), and recorded bothon paper using a fibre optics oscilloscope (TECA Instagraph 4) and on a 12-bit signal averager(Nicolet 1170) at sample rates of 10 pjs to 10 is. The digitized data were stored on a digitaltape-recorder (Kennedy, 9700) for subsequent analysis.The delayed outward K current was separated by suppression of Na, Ca and IA currents. Na

current was suppressed by substitution of Tris hydroxymethyl aminomethane for Na. Ca currentwas abolished by replacing Ca ion with Co ion in the extracellular solution. The IA current wasgreatly suppressed at the usual holding potential of -40 mV (Connor & Stevens, 1971 ; Neher, 1971)and in two experiments 4-aminopyridine (4-AP) was added to the external solution for extrameasure. Leakage currents were determined from the current responses remaining after tetraethyl-ammonium (TEA) at 50 mm (substituted for Tris) was applied.

Solutions used for external and internal perfusion are shown in Table 1. Intracellular andextracellular solutions were buffered to pH 7-2 and 7 4, respectively. The amounts of Ca requiredfor varying EGTA at constant Ca concentration were calculated using the association constant forCaEGTA of 107.13 at pH 7-4 and 20 OC (Bjerrum, Schwarzenbach & Sillen, 1975). Ca activity wascorrected for a calculated single ion activity coefficient of 0-38. Experiments were done at roomtemperature of 21-22 OC. Flow rate of the external solutions was about 5ml/min and the solutioncould be changed completely within 30 s.

Preparations were fixed for freeze-fracture and electron microscopy as follows. Parietal gangliawere fixed at room temperature in a solution composed ofglutaraldehyde (2 %), the osmotic pressureof which was adjusted with Na cacodylate buffer (50 mM) to 260 mosmol. The influence ofhypertonic solution on the ultrastructure was investigated by perfusing the ganglia for 40 min withnormal external solution containing an additional 240 mM-sucrose or 120 mM-NaCl; the sameadditives were used in the fixative. After 24 h of fixation the specimens were immersed in 30%glycerol for 30 min. Freeze-fracturing was performed according to standard techniques in a BalzersBAF 300 unit equipped with an electron beam gun for Pt/C (450) and C evaporation. All specimenswere prepared at a vacuum of 6 x 10-7 torr, and a specimen stage temperature of -100 'C; Pt/Cshadowing (2 nm) was started immediately after fracturing.

Electron photomicrographs were obtained by a Phillips EM300. Nomenclature for the surfacesrevealed by membrane fracture follows the convention of Branton, Bullivant, Gilula, Kasnovsik,Moor, Muchlethaler, Northcote, Packer, Satir, Satir Speth & Weinstein (1975).


RESULTS

The outward K currents in neurones may be separated into three components(Thompson, 1977; Aldrich et al. 1979), one of which, the delayed current, called 'KD,was the focus ofthe present experiments. The fast transientK current, IA, was largelyinactivated at the usual holding potential, VH, of -40 mV. The customary restingpotential in these neurones in the solutions used was -60 mV. Clamping to -40mV

Tris-CoTris-Ca Tris-Co EGTA,

+10 mV 120a_ | ~~~~~~~~~~200 nA+50 mV U

400 ms

Fig. 1. Membrane K current in response to 400 ms pulses and the effects ofCo substitutionfor Ca extracellularly (10 mM) followed by internal perfusion with a solution to which10 mM-EGTA was added. Membrane potential (Vm), +10 mV and +50 mV in upperand lower rows respectively; holding potential (VH), -40 mV. Note that the currentcalibration is different in upper and lower rows. An inward Ca current precedes the Kcurrent in the extreme left trace of the upper row.

required from 2 to 5 nA ofoutward current. In two experiments the A-current blocker4-AP (Thompson, 1977) was applied externally and the results were unchanged. Thereremain two outward K currents - IKD and the K current activated by transmembraneCa-current, 1KCa (Meech & Standen, 1975; Heyer & Lux, 1976; Thompson, 1977) - andseparation of these currents is described in the next section.

Separatio' of IKD from 1KCaOutward K currents during a voltage-clamp step of 400 ms duration are shown in

Fig. 1. The IK rises to a peak which is attained within 50-100 ms and then declinesslowly to a steady level over the next several seconds. A small inward Ca currentprecedes IK at lower potentials shown at slow speed in the upper left-hand trace ofthe Figure and at fast speed in the inset of Fig. 2A. Plots of peak currents againstpotentials (I-V relationships) show inflexions that give them an N-shape (Fig. 2A),as found in previous work (Aldrich et al. (1979). In the presence ofCa extracellularly,IK has two components: the Ca-current-activated 1K' IKCa' and the voltage-dependentIK called IKD by Aldrich et al. (1979). IKca is generally taken as the component that

312

K CURRENT AND ENDOGENOUS Ca, VOLTAGE CLAMP

disappears in Ca-free Co solution (Meech, 1978), although the mechanism by whichCa current activates the K current is not agreed upon (compare Meech, 1978, andHeyer & Lux, 1976). Therefore, we replaced extracellular Ca with Co and assumedthat the remaining IK was equivalent to the 'KD of Aldrich et al. (1979).

In Co-substituted solution the peak components of the outward current weresuppressed as shown in Fig. 1 and Fig. 2A, the effects occurring within 5 min. The

A nA B1200.

nA500 1000

300 - 600

-50 0 50 100 -50 0 50 100 150Membrane potential (mV) Membrane potential (mV)

Fig. 2. A, Relationship ofpeak currents to potential (I-V curves) in 10 mM-Cao (O), 10 minafter in 10 mM-Co. (@) and 20 min after washing in control solution (A). Inset Figuresshow the current and voltage traces in 10 mM-Cao and 10 mM-Coo. Vertical and horizontalbars are calibrated for 100 nA and 20 ms, respectively. The inward Ca current is moreobvious at these sweep speeds and it disappeared in Coo. Vm, +20 mV; VH, -40 mV.B, The I- V relationships for RKD at the peak (0) and at the end of a 5 s depolarizing pulse(@). The cell was larger than the one used in A. Ca current was blocked by Co substitutionfor Ca extracellularly, thereby eliminating IKCa. Note that S-shaped peak I-V is presentin Ca-free Co solution in small (A) and large (B) cells whose current amplitude differ bya factor of 3-4. The steady I-V in B was not inflected although in the control solution(not shown) it was inflected.

I-V relationships for peak and steady components, the latter measured at the endof the 5 s depolarizing pulse in CoCl2 solution, are compared in Fig. 2B. Althoughno membrane Ca current is now apparent (Fig. 2A inset and Fig. 8), peak IKamplitude does not rise steadily with potential, an upward concavity, bump orinflexion occurring in the range of + 35 to + 100 mV. This gives the I-V curve asigmoidal appearance similar to that shown in Fig. 8A of Aldrich et al. (1979) butwithout the distinct N-shape due to a negative slope conductance shown in theirFig. 8B. In fact a definite region of negative slope conductance was not observed inCo-substituted solutions. Any inflexion in the steady-state I-V curve, if present, isless marked than in the peak I-V curve. These results are seen in small and largecells despite three- to four-fold differences in current amplitudes.The effects of brief exposures to Co ions were reversible within 15 min after return

to the control solution, as Fig. 2A shows. During Co substitution the amount ofoutward current required to hold the neurones at -40 mV was unchanged. Thisindicates that leakage current was not affected by the substitution. Furthermore, thecurrent response to 10 and 20 mV hyperpolarizing steps was also unchanged. Theeffects of Co on the A current were also examined. We found that Co reduced this

313


current and that, as for IKD, the effects were reversible. We have not studied theactions of Co on the Na current.One possibility for the inflexion of the peak I-V curve observed in Co solutions

might be incomplete blockage of ICa. Brown et al. (1981) could not detect anyvoltage-dependent inward current when Co was substituted for Ca extracellularly ashere (see Fig. 8). Mg also does not appear to permeate the Ca channel (Akaike et al.1978), but as a further precaution we substituted Co for both Mg and Ca giving alevel of 25 mM-Co extracellularly. Under these conditions the inflected I-V curvepersisted, and as we were concerned with the possibility that Ca might be releasedin small amounts from endogenous stores connected to the extracellular space(Henkart et al. 1976), we attempted to block changes in Ca1 by perfusing intracellularlywith EGTA and F ions. The results are described in the next section.

Effects of internal perfusion with Ca chelatorsPerfusion with internal solutions containing EGTA ions at concentrations of 1

to 10 mm markedly reduced IK (Figs. 3 and 4). Neither the holding current nor thecurrents produced by hyperpolarizing steps of 10 and 20 mV were altered. In adifferent set of experiments to be reported elsewhere we found that activation of theA current was shifted to hyperpolarized potentials (N. Akiake, A. M. Brown, Y. Tsuda& A. Yatani, unpublished data). In eight cells, perfusion with 10 mM-EGTA reducedpeak current at + 50 mV by more than 50% within 25 min. The effect on theamplitudes ofpeak currents was especially marked between + 35 to + 100 mV so thatthe N-shape of the peak I-V plots was attenuated and in some cases abolished. Theamplitude of the current measured at 500 ms from the onset of the clamp step wasalso reduced by a factor of 43 + 10-4% at + 40 mV in eight cells, and the slopeconductance fell as well. The reduction appeared to occur in a dose-dependent mannerbecoming progressively greater at EGTA1 values of0 5, 1-0, 3 0, 5-0 and 10 mm. Therewas, however, no further suppression on increasing the concentration ofEGTA overthe range of 10-20 mm, possibly because Ca1 activities may not be changing (seebelow). The effects appeared steady after about 30 min of perfusion. The amount ofrecovery that occurred over the subsequent 2-3 h of perfusion with EGTA-freeinternal solutions was variable. When 1-0 mM-EGTA was perfused for 30 min untilthe effect was steady, recovery was only partial (Fig. 3C), and was less at morepositive potentials where 'Ca is becoming smaller. Recovery from 5.0 mM-EGTA1was much less than recovery from 1-0 mm-EGTAi.

In each of five cells, the 5 and 10 mm doses of EGTA1 were more effective insuppressing K currents than short-term (10-15 min) extracellular perfusion with Cosolution (Fig. 3A). Moreover, in four cells substitution of Co for Ca extracellularlywhen preceded by the intracellular perfusion of 10 mM-EGTA produced no additionalsuppression of 'K at short times of 10-15 min (Fig. 3B).One possibility for the EGTA1 effect is that EGTA1 has a direct action on IKD

independent of its Ca buffering action. This incomplete recovery from EGTA1 maybe explained this way, In addition, Kostyuk & Krishtal (1977) found a greatersuppression of IK by 10 mM-EGTAi compared with 1 mM-EGTAi at constant Caiactivity of 5.9 x 10-9 M. However, Marty (1981) has reported that single K channelsin chromaffin cells are not affected directly by EGTA1, so we checked this point again.

314


The effects of 1-5 mM-EGTAi were examined at a constant Ca activity of 5 x 10-8 Min five cells. This Ca activity was used because it is below threshold for IKca (Brown,Brodwick & Eaton, 1977; Meech, 1978). Lowering Ca1 from 10-7 M to 5 x 10-8 M wasaccompanied by a reduction in IKD, but the reduction showed no significantdependence on the concentrations of EGTA1 used (Fig. 3D).

A B

/K (nA) /K (nA)

1000 ~~~~~~~1000-

500 ~~~~~~~~500

-50 0 50 100 -50 0 50 100Membrane potential (mV) Membrane potential (mV)

C D/K (nA) /K (nA)

1000 101000

500 -500-

VH VH


Fig. 3. A, peak I-V relationships in 10 mM-Cao (0), 10 mM-Coo (El) and after additionof 5 mM-EGTA (A) to the intracellular solution. B, peak I-V relationships in 10 mM-Ca.(0), 5 mM-EGTA applied intracellularly (I0) before Co substitution for Cao, and followingCo substitution (A). C, peak IK-V relationship in 10 mM-Cao (0), in 1 mM-EGTA perfusedintracellularly for 30 min (-), and recovery after 30 min of washing with EGTA-freeinternal solution (A\). D, effects of intracellular Ca and EGTA.on peak IK. Lowering Caifive-fold from 1.1 x 10-7 M (0) to 5-5 x 10-1 M (-, A) reduced peak K. However, atconstant Cap, increasing EGTA1 five-fold (@, 1 mM; A, 5 mM) had no further effect.

We also examined the effects of substituting fluoride anion (F-) for aspartate anionintracellularly in four neurones. This produced a large suppression ofpeak and steadycurrents (Fig. 4), although the N-shaped peak was still present in the peak I-V curve.The effects of 135 mM-Fi were greater than 10 mM-EGTAi, for F ions produced furthersuppression of IKD in the presence of EGTA1 (Fig. 4). On the other hand additionof EGTA to F-containing solutions had no further suppressive effect.The data to this point show that after suppression ofCa currents by Co substitution

315


for Ca ion extracellularly, intracellular Ca chelators can reduce K currents. Thepersistent inflexion in the peak I-V curve raises the possibility that Ca may bereleased from membrane sites that are not readily exchanged when Ca is replacedby Co in the extracellular solution. The next section deals with possible cellstructures that could subserve the Ca releasing function.

/K (nA)

1500 A B

/K (nA)

1000 ~~~~~~~~~1000-

VH VH

-50 0 50 100 150-50 0 50 100 150Membrane potential (mV) Membrane potential (mV)

Fig. 4. Effects of the Ca chelating agents EGTA and F ions on peak IKD currents (A) and'KD currents at the end of a 1 s depolarizing pulse (B). ICa was blocked by substitutionof COC12 for CaCl2 (10 mM) extracellularly. Intracellular solutions: 0, K aspartate; *, Kaspartate plus 10 mM-EGTA; A, KF plus 10 mM-EGTA.

Ultrastructural correlates for the release of CaThree structures have been identified that could be involved in the effects that have

been described.(1) Restricted extracellular space. A typical large neurone and its surroundings are

shown in freeze-fracture electron micrograph of P1. 1. In several areas, the externalleaflet of the plasma membrane ('E-face', labelled El) is exposed. The E-face of theneurone's plasma membrane is closely adjacent to the plasma membrane of aneighbouring glial cell (protoplasmic leaflet of P-face, labelled P*). Thus, the twoplasma membranes narrow the extracellular space to a cleft (restricted extracellularspace, r.e.s.). The diameter of the r.e.s. measured at 255 locations was 24+6 nm.

Often, two adjacent neurones interdigitate; those interdigitations (labelled I)enlarge the surface membrane area beyond the one calculated assuming a sphericalouter dimension. Usually, a glial cell is interposed between the two neurones.The morphological data suggest that a restricted and lengthened extracellular

pathway may delay and hinder the washout of Ca ions from the extracellular surfaceof the plasma membrane. It has previously been shown (Isenberg & Dahl, 1977) thatexposure of cardiac Purkinje fibres to hypertonic media can enlarge the diameterof the r.e.s. several-fold and reduce the effects of hindered diffusion. We tested thispossibility by bathing the ganglia for 30 min in media containing an additional240 mM-sucrose which doubled the osmolarity. We found that doubling the osmolarity

316


enlarged the diameter of the r.e.s. to 49+ 9 nm in 282 measurements, a result thatis similar to earlier reports in cardiac muscle (Isenberg & Dahl, 1977).

(2) Secretory vehicles. The neurones have numerous Golgi apparatuses (P1. 1:labelled G.a.) and spherical secretary vesicles (V, P1. 1 and 2A). The vesicles maybe concentrated in packages at one pole of the neurone and have a diameter of153 + 36 nm (n = 874). Doubling the osmolarity reduced the diameter of the vesiclesto 91+29 nm (n = 254), indicating that these intracellular compartments shrinktogether with the cytosol. the vesicles make contact with and appear to fuse to theplasma membrane, thereby enlarging the surface area (V* in P1. 2A).

(3) Subsurface cisterns. The endoplasmic reticulum e.r. of vertebrate and inverte-brate neurones has been reported to form cisterns closely apposed to the plasmamembrane (subsurface cisterns or s.s.c.s) (Rosenbluth, 1962; Henkart et al. 1976). InHelix aspersa ganglia, s.s.c.s were observed in the neurones but not in the glial cells.In the freeze-fracture electron micrographs, s.s.c.s appeared as flat sacs when theplane of cleavage transacted their surfaces (PI. 2A) and had a tubular configurationwhen cleaved perpendicularly (P1. 2B).The s.s.c.s face the plasma membrane and in thirty-three cases the apposition

occurred over a length of 1-15+0-73 nm. The gap between the two membranes is20+ 5 nm (n = 12) after correcting for the thickness of the two membrane lamellae,which was 8 nm each. Henkart et al. (1976) showed aggregations of particlesmeasuring 9 nm in diameter on the P-face of the plasma membrane and the luminalface of the s.s.c. membrane. They suggested that the particles might form junctionswhich could couple the surface and the outer e.r. membrane electrically. In thepresent study, we see particles of this diameter at the s.s.c. and the opposing plasmamembrane regularly, but aggregations were only found occassionally. A new findingis the appearance of continuity between the plasma membrane and the membraneof the s.s.c. The continuity illustrated in P1. 2A is typical and shows a narrow regionor 'neck' of 87+11 nm (n = 47) outer diameter. Whether the continuity betweens.s.c. and plasma membrane is transient or permanent could not be determined.The junctions between s.s.c. and plasma membrane may connect the lumen of the

s.s.c. with the extracellular space (e.c.s.). In two neurones, exposure to solutionsof double the normal osmolarity caused the cell body to shrink and the s.s.c. to swell.The amount of swelling was about two-fold to 153 + 52 nm (n = 68) for the neck. Thelumen of the s.s.c. also appeared to swell to about twice its original diameter (eightobservations in four specimens).Our electron micrographs suggest that the r.e.s., the s.s.c. and the fused secretary

vesicles might act as Ca stores connecting intra- and extracellular spaces. This leadsto the prediction that bathing the neurones with Ca-free Co solutions for periods muchlonger than 10-15 min might reduce the Ca content of those compartments. Anotherprediction is that Ca depletion might occur more quickly when the washout isperformed in media with increased osmolarity. The results oftesting these predictionsare presented in the following sections.

Effects of prolonged exposure to Ca-free mediaNeurones were perfused with Co-substituted Ca-free extracellular solutions for

periods of 2 h. Figs. 5A and 6 indicate that the outward peak currents become smallerand the reduction continued for 90 min. After this period the effects are maintained.

317


After 90-120 min in this solution the current tracings lost their time-dependence; thecurrent amplitude measured 100 ms after the start of the clamp step was the sameas the amplitude measured 1900 ms later. The S-shaped inflexion of the peak I-Vcurve (Fig. 5) was greatly attenuated. During this time the holding current wasunchanged and the inward currents produced by 10 and 20 mV hyperpolarizing pulses

nA

1400

A 1200

1000

nA /800 //700~~~~~~~~~~~~~~~~~~600-


Figure 5. A, gradual reduction of peak outward currents by prolonged perfusion withCo-substituted extracellular solution. 0, 10 mM-Ca; A, [. *, 10 mM-Co after 30, 60 and90 min respectively. After 90 min the N-shape of the curve was almost abolished and theeffect remained steady. B, effects of hypertonic solutions (240 mM-sucrose added) on thetime course of decline ofpeak outward currents. 0, 10 mM-Ca; A, 10 mM-Ca plus sucrose;[O. , 10 mM-Co plus sucrose after 20 and 40 iiin respectively. An initial reduction in Casolutions occurred within 10 min. The decline in Co-substituted solutions isnow much fasterthan occurred in A, reaching a new steady level within 20 min. Application of5 mM-EGTAintracellularly did not produce any further depression (cf. Fig. 3A).

were not significantly altered. In two experiments we tested for recovery of the Kcurrents by adding Ca to the solution containing 10 mm-Co. Reintroduction ofCa wasin steps of 2, 5 and 10 mM-Ca to avoid lethal Ca loading as occurs in cardiac muscleafter prolonged exposure to zero Ca solution. The K currents were restored to about70% in 10 mM-Cao and the transient peak of the K current reappeared (Fig. 7). Theeffects occurred quickly, restoration of IKD beginning within 1 min following thereintroduction of Ca to the bathing solution. There were no inward currents evidentduring reintroduction of Ca but they may have been masked by the much larger Kcurrents that reappeared. We examined this point in more detail by studying Cacurrents in mixtures of Cao and Coo. The Ca currents were isolated by blocking Naand K currents according to the method of Brown et al. (1981). Fig. 8 shows thatwith 10 mM-Co and 2-5 or 5-0 mM-Ca in the bath solution no inward Ca current isdetectable. At 10 mM-Coo and 10 mM-Cao a very small inward current flows. The Caflow through Na and K channels is ordinarily so low that we may neglect it (Meves& Vogel, 1973; Inoue, 1980). Hence we may conclude that recovery currents of the

318


type shown in Fig. 7 are hKD currents according to our definition and are notaccompanied by detectable inward Ca currents except at Ca. concentrations of10 mM.

Effects of perfusion with hypertonic extracellular solutionsWe superfused two neurones with media of doubled osmolarity. Figs. 5B and 6A

indicate that reduction of IKD occurs more quickly and 'KD drops within about10 min to a very low level.

min min0 20 40 60 80 0 20 40 60 80

0 r-~~~ISucrose 240m~m 0Caffeine 10mm

L21 CoC12 10MM Ca COC12 10mM

Fig. 6. Comparison of the effects of perfusion with hypertonic solutions (A) or caffeine (B)on the decline of peak K current in Ca-free CoCl2 solution. Note that in B addition of50 mm-TEA extracellularly produced a very small depression. Vm, +50 mV. The 100%value was |100 nA in A and 1200 nA in B.

When the I-V curves obtained 20 min after exposure were evaluated (Fig. 5B),the current amplitudes were strongly depressed and the inflected N-shape was absent.The depressant effect seemed to be maintained because the I-V curve obtained 40 minlater coincided with the previous curve. Also, internal perfusion with 5 mM-EGTAj(20 min) did not produce any additional changes (not shown).The current tracings recorded after 20 min of exposure to doubled osmolarity in

Co solution do not show any time-dependence and are similar in this regard to theresults oLtained following 90-120 min of exposure to Co-substituted extracellularsolution of normal osmolarity.

Effects of caffeineIn skeletal muscle caffeine causes the release of Ca from sarcoplasmic reticulum

as well as preventing its uptake by sarcoplasmic reticulum (Endo, 1977). We werecurious as to whether caffeine might enhance Ca depletion, possibly from the s.s.c,in these neurones. It appears (Fig. 7) that caffeine increased the rate at which IKDfell in Co-substituted solutions.

The current remaining after Ca depletionThe outward currents that remained after prolonged exposure to solutions that

deplete Ca (Ca0, hypertonic sucrose, caffeine) have little or no time-dependence and

319


min0 30 60 90 120 150 180 210

(b4 (C)(a)

ICa CoC12 10.mM

a~~Caffeine 10mm

Fig. 7. Recovery of peak IKD by addition of CaCI2 to the bathing solution after prolongedperfusion with CoCl2 and 10 mM-caffeine. In the right-hand panel a, b and c show actualcurrent (upper trace) and voltage (lower trace) tracings recorded at each phase. Vm,+50 mV; VH -40 mV.

10 Ca

0 Ca

10 Co, 0 Ca1 _in

5 Ca10 Co

10 Ca

[ 100 nA

[50 nA

Fig. 8. Ca currents in the absence (O Co) or presence (10 Co) of 10 mM-Co. Ca currents wereisolated according to Brown et al. (1981). Co substitution for Ca resulted in completeblockage of Ica within 5 min. Addition of 5 mM-CaCI2 (5 Ca) produced no detectable Icabut 10 mM-CaCl2 (10 Ca) produced a small inward current. Vm, + 10 mV; VH,-50mV;40 ms pulse.

320

1000

800

c

I I I I I- IT

I CaC12


are not strongly voltage-dependent. They constitute about 10% of the net outwardcurrent and can be blocked by TEA (Fig. 6B).

DISCUSSION

The present experiments showed as expected that outward K currents are reducedwhen membrane Ca current is blocked. This is due to blockage of the Ca-activatedK current, called IKCa in the nomenclature of Thompson (1977). The new finding isthat further substantial suppression of the remaining K current, IKD, occurs whenCo-substituted solutions, hyperosmotic solutions or caffeine are perfused extracellu-larly. The effect is not general since neither holding nor leakage currents were changedand the effect is reversible. The actions of EGTAi are less clear. It was difficult toexamine the reversibility of the effects on IKD alone. This is because completelyreversible isolation of IKD required Co application extracellularly for 15 min or lessand the effects of internal perfusion with EGTA required about 30 min to reach asteady level. Therefore, the effects of EGTAi were examined usually in the presenceof Cao and 1KCa* The results at constant Caj and different levels of EGTA suggestthat a direct action of EGTA is not a major effect at least at potentials of 60 mVor less (Fig. 3C). However, the partial recovery particularly at potentials positiveto + 60 mV (Fig. 3C), where ICa and consequently IKCa are becoming smaller,suggests some irreversible effect on 'KD- Perhaps the differences between the resultsof Kostyuk & Krishtal (1977) and Marty (1981) may also be explained on this basis.It is also possible that the reduction in existing Cai may be involved in the suppressionofIKD. The greater effects at more positive potentials (see Fig. 3B) may be explainedby the fact that Ca-activation ofK channels at submicromolar concentrations of Cais voltage-dependent (Pallota, Magleby & Barrett, 1981; Lux, Neher & Marty, 1981;Marty, 1981).The effects of EGTAi were not general. For example, leakage and holding currents

were not changed. However, activation of the A current was shifted to more negativepotentials. Intracellular perfusion with F anion had a greater effect than intracellularperfusion with EGTA. The Cai levels were lower for Fi probably for two reasons: thegreater affinity ofF ions for Ca and the higher concentration ofF ions that was used.The effects ofEGTAi are probably not due to a surface charge action since we have

found no evidence for an internal surface charge that affects channel gating. Cacurrent-dependent inactivation cannot be explained on this basis (Wilson, Tsuda,Morimoto & Brown, 1982) and internal perfusion with divalent cations that affectextracellular surface charge is also without effect on Ca channel gating (Brown, Tsuda,Morimoto & Wilson, 1982) and Na and K channel gating (Begenisich & Lynch, 1975).We may also exclude significant action of extracellular Co on surface charge sincewe have found little evidence that Co can affect Ca channel gating (Wilson et al. 1982).F ions are known to have complicated actions on cellular enzymes and we cannotexclude such actions as causes of the F ions effects we have observed.The inflected N-shape of the peak IKD membrane-potential (Em) curve may be

related to Ca entry that in turn depends on a driving force related to Em-EC. in muchthe same way that IKCa depends on Em-ECa. If this is so, the source for the Ca must

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11 PHiY 334


include the extracellular space and could involve the subsurface cisterns and vesiclesthat are fused to the plasma membrane. Some support for this possibility comes fromthe results of experiments using prolonged perfusion with Co-substituted solutions,hyperosmotic solutions and caffeine. The rapid restorationofIKD following reintro-duction of Ca into the bathing solution also supports this idea. The source is not likelyto be exclusively intracellular because in skeletal muscle intracellular Ca release doesnot diminish at positive potentials (Miledi, Parker & Schlow, 1977). The results arenot inconsistent with the absence of a change in arsenazo absorbance followingdepolarizations in Co-substituted (Gorman & Thomas, 1980) or EGTA solutions(Ahmed & Connor, 1979), since the periods of exposure were not specified in theseexperiments and local changes might not have been detected. The Ca currents-responsiblefor our results must be less than 500 pA since larger currents would havebeen detected by our methods. Ca-activated K currents are much larger as a resultof the fact that only a few Ca ions may be required to activate a K channel thatconducts millions of ions.The results raise the question as to whether voltage-dependent K current is present

at all after Ca stores have been depleted. The cannot be readily explained as theconsequence of removal of K channels from the membrane since K currents wereabout 80 % restored within1 min after reintroduction of Ca. following prolonged Coperfusion (Fig. 7) and within a few hours following perfusion with EGTA1. If anindependent K current exists it is very small, on the basis of the TEA results shownin Fig. 6B. Moreover, the residual K currents could have been maintained by a smallamount of cellular Ca that was not yet depleted. Hofmeier & Lux (1981) haveidentified Helix neurones in which nearly all of the K current is Ca-activated. SinceIKD is largely Ca1-dependent, this raises difficulties with nomenclature. We suggest,however, that the distinction between K currents activated by measurable trans-membrane Ca current and K currents activated by endogenous Ca from slowlydepletable stores continue to be made, while recognizing that since both currents areCa-dependent there may be no fundamental difference between them.

This work was supported by NIH grants NS1 1453 and HL25145.

REFERENCES

AHMED, Z. CONNOR, J. A. (1979). Measurement of calcium influx under voltage clamp in molluscanneurones using the metallachromic dye arsenazo III. J. Phy8iol. 287, 61-82.

AKAIKE, N., LEE, K. S. & BROWN, A. M. (1978). The calcium current ofHelix neuron. J. gen. Phy8iol.71, 509-531.

ALDRICH, R. W., GETTING, P. A. & THOMPSON, S. H. (1979). Inactivation of delayed outwardcurrent in molluscan neurone somata. J. Physiol. 291, 507-530.

BEGENISICH, T. & LYNCH, C. (1975). Effects of internal divalent cations on voltage-clamped squidaxon. J. gen. Phy8iol. 62, 675-689.

BJERRUM, J., SCHWARZENBACH, G. & SILLEN, L. G. (1975). Stability Condtant, part 1, OrganicLigand8, pp. 76 and 90. London: The Chemical Society.

BRANTON, D., BULLIVANT, S., GILULA, N., KASNOVSIK, M., MOOR, H., MUCHLETHALER, K.,NORTHCOTE, N., PACKER, L., SATIR, B., SATIR, P., SPETH, V. & WEINSTEIN, R. (1975).Freeze-etching nomenclature. Science, N. Y. 190, 54.

BROWN, A. M., BRODWICK, M. S. & EATON, D. C. (1977). Intracellular calcium and extraretinalphotoreception in Aply8ia giant neurons. J. Neurobiol. 8, 1-18.

BROWN, A. M., MORIMOTO, K., TSUDA, Y. & WILSON, D. (1981). Mechanisms of inactivation of thecalcium current. J. Physiol. 320, 193-218.

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BROWN, A. M., TSUDA, Y., MORIMOTO, K. & WILSON, D. L. (1982). Actions of calcium ion on gatingand permeation of the calcium channel. In The Mechanism of Gated Calcium Transport acrossBiological Membranes, pp. 53-62. New York & London: Academic Press.

CONNOR, J. A. & STEVENS, C. F. (1971). Voltage clamp studies of a transient outward membranecurrent in gastropod neural somata. J. Physiol. 213, 21-30.

ENDO, M. (1977). Calcium release from the sarcoplasmic reticulum. Physiol. Rev. 57, 71-108.GORMAN, A. L. F. & THOMAS, N. V. (1980). Extracellular calcium accumulation during depolar-

ization in a molluscan neurone. J. Physiol. 308, 259-285.HENKART, M. P., LANDIS, D. & REESE, T. S. (1976). Similarity of junctions between plasmamembranes and endoplasmic reticulum in muscle and neurones. J. Cell Biol. 70, 338-347.

HENKART, M. P. & NELSON, P. G. (1979). Evidence for an intracellular calcium store releasableby surface stimuli in fibroblasts (L cells). J. gen. Physiol. 73, 655-673.

HEYER, C. B. & Lux, H. D. (1976). Control of the delayed outward potassium currents in burstingpace-maker neurones of the snail, Helix pomatia. J. Physiol. 262, 349-382.

HOFMEIER, G. & Lux, H. D. (1981). Three distinct effects mediated by calcium ions on electricalmembrane properties of Helix neurones. Arch. Physiol. Sci. 4, 115-126.

INOUE, I. (1980). Separation of the action potential into a Na-channel spike and a K-channel spikeby tetrodotoxin and by tetraethylammonium ion in squid giant axons internally perused withdilute Na-salt solution. J. gen. Physiol. 76, 337-354.

ISENBERG, G. & DAHL, G. (1977). Sheep Purkinje fibre: differerre between reversal potential (EK2)of the pacemaker current and the potassium equilibrium potential (EK) is reduced by hypertonicTyrode solution. Pfluiger8 Arch. 368, R3.

ISENBERG, G. & DAHL, G. (1980). Decoupling of heart muscle cells: correlation with increasedcytoplasmic calcium activity and with changes of nexus ultrastructure. J. Membrane Biol. 53,63-75.

KERKUT, G. A., LAMBERT, J. D. C., GAYTON, R. J., LOKER, J. E. & WALKER, R. J. (1975). Mappingof nerve cells in the subesophageal ganglia of Helix aspersa. Comp. Biochem. Physiol. 50A, 1-25.

KOSTYUK, P. G. & KRISHTAL, 0. A. (1977), Effects of calcium and calcium-chelating agents on theinward and outward currents in the membrane of mollusc neurones. J. Physiol. 270, 569-580.

LEE, K. S., AKAIKE, N. & BROWN, A. M. (1980). The suction pipette method for internal perfusionand voltage clamp of small excitable cells. J. Neurosci. Methods 2, 51-78.

Lux, H. D., NEHER, E. & MARTY, A. (1981). Single channel activity associated with the calciumdependent outward current in Helix pomatia. Pfluigers Arch. 389, 293-295.

MARTY, A. (1981). Ca-dependent K channels with large unitary conductance in chromaffin cellmembranes. Nature, Lond. 291, 497-500.

MEECH, R. W. (1978). Calcium-dependent potassium activation in nervous tissues. Ann. Rev.Biophys. Bioeng. 7, 1-18.

MEECH, R. W. & STANDEN, N. B. (1975). Potassium activation in Helix aspersa neurones undervoltage clamp: a component mediated by calcium influx. J. Physiol. 249, 211-239.

MEECH, R. W. THOMAS, R. C. (1980). Effects of measured calcium chloride injections on themembrane potential and internal pH of snail neurones. J. Phyaiol. 298, 111-129.

MEvES, H. & VOGEL, W. (1973). Calcium inward currents in internally perfused giant axons. J.Physiol. 235, 225-265.

MILEDI, R., PARKER, I. & SCHALOW, G. (1977). Measurement of changes in intracellular calciumin frog skeletal muscles fibres using arzenazo II. J. Physiol. 269, 11P.

NEHER, E. (1971). Two fast transient current components during voltage clamp on snail neurones.J. gen. Physiol. 58, 36-53.

PAGE, E. & UPSHAW-EARLEY, J. (1977). Volume changes in sarcoplasmic reticulum of rat heartsperfused with hypertonic solutions. Circulation Res. 40, 355-366.

PALLOTA, B. S., MAGLEBY, K. L. & BARRETT, J. N. (1981). Single channel recordings ofCa2+-activated K+ currents in rat muscle cell culture. Nature, Lond. 293, 471-474.

ROSENBLUTH, J. (1962). Subsurface cisterns and their relationship to the neuronal plasmamembrane. J. Cell Biol. 13, 405-421.

THOMPSON, S. H. (1977). Three pharmacologically distinct potassium channels in molluscanneurones. J. Physiol. 265, 465-588.

WILSON, D. L., TSUDA, Y., MORIMOTO, K. & BROWN, A. M. (1982). Interaction between calciumions and surface charge as it relates to calcium currents. J. Membrane Biol. (In the Press.)

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324 N. AKAIKE AND OTHERS

EXPLANATION OF PLATES

PLATE 1

Freeze-fracture electron micrograph of a large neurone in the parietal ganglion of Helix aspersa.In the cytoplasm, the labels indicate Golgi apparatus (g.a.), secretary vesicles (V) and endoplasmicreticulum (e.r.). The neurone is surrounded by a restricted extracellular space (r.e.s.). Numerousinterdigitations (I) between the neurone and neighbouring cells further complicate the structureof the r.e.s. The central neurone at the left displays an exoplasmic leaflet of the plasma membrane(El) and the neighboring neurone shows its protoplasmic leaflet (P2). Between the two neurones athin glial cell (GI) is interposed, and the protoplasmic (P*) and the exoplasmic (E*) leaflet of itsmembrane are shown.

PLATE 2

A, organelles of the intracellular space (i.c.s.) (V, secretary vesicles; e.r., endoplasmic reticulum)may contact the membrane, possibly forming connexions with the extracellular space (e.c.s.)(Henkart et al. 1976). V* labels a fused vesicle and e.r.* connected e.r. Numerous particles are seen.The largest particles group about a diameter of roughly 9 nm.

B, In this plane of cleavage the subsurface cisterns (s.s.c.s) appear as long tubules closely apposedto the plasma membrane (P). The membranes of the s.s.c.s expose both the intraluminal (I.1.) andthe protoplasmic leaflet (P*). Numerous particles are seen. The class of the largest particles groupsaround a diameter of roughly 9 nm.

The Journal of Physiology, Vol. 334

,~ ~~ ~ ~ ~ ~ ~ ~ ~~~~~~~~x~'

Plate 1

N. AKAIKE AND OTHERS (Facing p. 324)

The Journal of Physiology, Vol. 334

.S.S.C.


Plate 2

by n. akaike*, am brown, g. dahlt, h. higashit, g

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