321

Journal of Physiology (1992), 448, pp. 321-337 With 9 figures Printed in Great Britain

ELECTRICAL COUPLING BETWEEN CELLS OF THE INSECT AEDES ALBOPICTUS BY FELIKSAS BUKAUSKAS*, CHRISTOPH KEMPFt AND ROBERT WEINGART From the Department of Physiology, University of Bern, Buhlplatz 5, 3012 Bern, Switzerland and the tDepartment of Biochemistry and Central Laboratory, Blood Transfusion Service, Swiss Red Cross, University of Bern, Freiestrasse 3, 3012 Bern, Switzerland

(Received 10 April 1991) SUMMARY

1. Cell pairs of an insect cell line (Aedes albopictus, clone C6/36) were used to study the electrical properties of intercellular junctions. A double voltage-clamp approach was adopted to control the voltage gradient between the cells and measure the intracellular current flow. 2. Determinations of junctional conductance (gj) revealed two types of intercellular contacts, gap junctions and cytoplasmic bridges. Identification occurred by means of functional criteria, i.e. the dependency of gj on (i) junctional membrane potential, (ii) non-junctional membrane potential, and (iii) heptanol. 3. In cell pairs with putative gap junctions, gj was dependent on the junctional membrane potential (Vj). When determined at the beginning of voltage pulses, gj was insensitive to Vj; when determined at the end of 15 s pulses, it depended on Vj in a bellshaped manner (70 % decrease for a change in Vj of + 75 mV). 4. These cell pairs also showed a dependency of gj on the non-junctional membrane potential (Vm). When determined immediately after changing the non-junctional membrane potential in both cells, gj was not affected by Vm; when determined 30 s later, gj was modified by Vm in a S-shaped fashion (100% decrease when Vm was depolarized to + 50 mV). 5. Exposure to 3 mM-heptanol gave rise to complete and reversible block of gj in cell pairs with putative gap junctions. 6. Cell pairs susceptible to uncoupling by heptanol revealed junctional currents indicative of the operation of gap junction channels. The single-channel conductance, determined at a Vm of -50 to -70 mV, was 133 pS. 7. In the case of putative cytoplasmic bridges, gj was insensitive to the junctional and non-junctional membrane potential. In addition, it was not affected by 3 mMheptanol. 8. WAhile most cell pairs showed functional properties characteristic of gap junctions or cytoplasmic bridges, few cell pairs exhibited junctional currents compatible with the co-existence of both junctional structures. *

Present address: Kaunas Medical

Academy,

Mickeviciaus 9, 233000

Kaunas,

Lithuania.

MIS 9293 11-2

322

F. BUKAUSKAS, C. KEMPF AND R. WEINGART INTRODUCTION

Salivary glands from insects, e.g. those from Chironomus larvae, have been used extensively to study intercellular communication (for review, see Loewenstein, 1981; Weingart, 1987). Diffusion and electrical measurements allow the characterization of insect gap junctions in terms of biophysical properties. A comparison with vertebrate gap junctions reveals considerable functional differences (e.g. Spray & Bennett, 1985) reflecting differences in connexon and connexin structure. With respect to diffusional properties, insect gap junctions have pores of larger diameter. Using peptides, Simpson, Rose & Loewenstein (1977) established a diffusion limit of 1200-1900 Da in Chironomus salivary glands. Later on, Schwarzmann, Wiegandt, Rose, Zimmerman, Ben-Haim & Loewenstein (1981) reported the transfer of glycopeptides up to 2450 Da, corresponding to a pore size of 2-3 nm. More recently, examining diffusion profiles, Zimmerman & Rose (1985) estimated an effective pore diameter of 2-9 nm. In contrast, gap junction channels in vertebrate tissues are more restrictive. In cardiac tissue, the cut-off limit for intercellular permeation is 900-1000 Da, equivalent to a pore size of 1-2 nm (Weingart, 1981; Imanaga, 1987). A similar value was derived from primary cultures and cell lines of mammalian origin (Flagg-Newton, Simpson & Loewenstein, 1979). This implies that gap junction channels of vertebrates are narrower, due to bulkier or more polar groups lining the channel pore. With respect to electrical properties, arthropod gap junctions possess a set of interesting features. For example, in insect salivary glands the conductance of gap junctions, gj, strongly depends on the non-junctional membrane potential, Vm (Obaid, Socolar & Rose, 1983; Verselis, Bennett & Bargiello, 1991). Hyperpolarization of both cells of a cell pair leads to an increase in gj, depolarization to a decrease. A weak dependency of gj on Vm was found in squid blastomeres (see Spray, White, Campos de Carvalho, Harris & Bennett, 1984), but not in other cells examined. In insect salivary glands, gj also depends on the junctional membrane potential, Vj (Obaid et al. 1983; Verselis et al. 1991). This behaviour has also been seen in giant axons of crayfish (Jaslove & Brink, 1986) and embryonic cells of squid, amphibian and fish (for references, see Spray & Bennett, 1985). Recent studies have shown that mammalian gap junctions are sensitive to 1j as well (Rook, Jongsma & van Ginneken, 1988; Veenstra, 1990; Moreno, Campos de Carvalho, Verselis, Eghbali & Spray, 1991). The work presented in this paper was initiated to study the electrical properties of insect gap junctions using a double voltage-clamp approach in conjunction with patch pipettes. The classical preparation, salivary glands of Chironomus larvae, seemed inappropriate because of the large cell size (diameter 80-150,m) and membrane coating. Obviously, small cells grown in culture would be more suitable. Hence, we resorted to an established cell line derived from Aedes albopictus larvae (Igarashi, 1978), a mosquito belonging to the diptera. The experiments were performed on pairs of cells spontaneously formed in culture. This approach allows examination of both macroscopic and microscopic currents in the same preparation. An abstract presenting preliminary data has already been published (Bukauskas, Kempf & Weingart, 1991).

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METHODS

Cells An established insect cell line, Aedes albopictus, clone C6/36 (code of the American type culture collection, ATCC: CRL 1660; Igarashi, 1978), was used in this study. The cells were derived from larvae of the mosquito Aedes albopictus (family, culicidae; order, diptera). The cells were grown at 28 0C in culture medium (RPMI 1640; GIBCO, Paisley, UK), containing 20 % fetal calf serum and supplemented with 100,ug/ml streptomycin and 100 U/ml of penicillin (code 2212; Biochrom, Berlin, Germany). The cells were passaged weekly and diluted 1:10. For the experiments, confluent monolayers of cells (- 4 x 105 cells/cm2) were harvested and resuspended in RPMI 1640 containing 20 % fetal calf serum at a cell density of 0 2-1 x 106 cells/ml. The cells were then seeded at a density of 104 cells/cm2 onto glass cover-slips placed in multiwell culture dishes. The cover-slips were previously washed with ethanol and sterilized by flaming. This procedure led to an abundant formation of cell pairs within 24 h. Electrophysiological measurements were performed 1-3 days after cell plating. _

Solutions The electrical measurements were performed in Krebs-Ringer solution containing (mM): NaCl, 140; KCl, 4; CaC12, 2; MgCl2, 1; glucose, 5; pyruvate, 2; HEPES, 5 (pH 7 4). The composition of the pipette solution was as follows (mM): potassium aspartate, 120, NaCl, 10; MgATP, 3; MgCl2, 1; CaCl2, 1; EGTA, 10 (pCa 8); HEPES, 5 (pH 7T2); filtered through 0-22 ,um pores. 1Heptanol (Fluka, Buchs, Switzerland) was dissolved directly in Krebs-Ringer solution (3 mM).

Electrical measurements Cover-slips with adherent cells were transferred from the culture dishes to the experimental chamber and superfused with Krebs-Ringer solution at room temperature (20-24 0C). The chamber, consisting of a Perspex frame with an attached glass bottom, was mounted on the stage of an inverted microscope equipped with phase-contrast optics (Diaphot-TMD, Nikon; Nippon Kogaku, Tokyo, Japan). The bath solution was exchanged by gravity (bath volume 1 ml; exchange rate 2 ml/min). Patch pipettes were pulled from glass capillaries (100,dl; Bardram, Birker0d, Denmark) with a horizontal puller (BB-CH, Mecanex, Geneva, Switzerland). The pipettes had DC resistances of 3-5 MCI (tip size approximately 1 ,zm). The experimental set-up used has been described in detail before (Weingart, 1986; Riidisiili & Weingart, 1989). Electrical measurements on single cells and cell pairs were performed in the whole-cell, tight-seal recording mode. Currents across non-junctional membranes were investigated using single cells and a conventional voltage-clamp method. Currents across gap junctional membranes were assessed using cell pairs in conjunction with a double voltage-clamp approach. All measurements of membrane potentials were corrected for the liquid junction potentials between pipette solution and bath solution (-12 mV). Details about the experimental approach, data acquisition, and data analysis have been described before (Weingart, 1986; Niggli, Rildisilli, Maurer & Weingart, 1989; Rudsiili & Weingart, 1989). The results are presented as means+ standard error of the mean (S.E.M.). RESULTS

Non-junctional membrane Measurements of membrane potentials revealed a value of - 70O2 + 2 1 mV (n= 31). Figure 1 illustrates an example of a steady-state current-voltage relationship obtained from a single cell. The amplitude of the current flowing at the end of a 3 s voltage-clamp pulse (Im) was measured and plotted vs. test potential (Vm). The resulting plot was linear at negative voltages and grew progressively steeper at positive voltages. The slope of the current-voltage relationship, determined at the zero current intercept, revealed an input conductance of 0-2 + 003 nS (n = 14).

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F. BUKAUSKAS, C. KEMPF AND R. WEINGART

Gap junctional communication Cell pairs were visually selected under the microscope. Functional tests allowed us to distinguish between the following configurations: preparations whose intercellular contacts contain (i) gap junctions (41 pairs), (ii) cytoplasmic bridges (13 pairs), (iii) o

60 -/m (pA) 40

-150

-100

/ /

~~ .-~~~~-20

~

100 ~~~~50

vm (MV

.--40 -60

Fig. 1. Steady-state current-voltage relationship of non-junctional membrane, studied in a single cell. Currents flowing at the end of a 3 s voltage-clamp pulse (I.) were plotted vs. voltage across the non-junctional membrane (VT). V,h= -42 mV. Input conductance= 027 nS.

gap junctions and cytoplasmic bridges (8 pairs), or (iv) no conducting path (19 pairs). The identification occurred by means of electrical and pharmacological criteria, as will be shown.

Dependence of gj on junctional membrane potential To perform electrical measurements, both cells of a cell pair were clamped to the same holding potential (Vh), i.e. around -50 mV. Subsequently, the voltage of cell 1 was stepped to a test potential (V,), while the voltage of cell 2 was maintained (V2 = Vh). The associated currents were determined with pipettes 1 (I,) and 2 (I2), respectively. The difference V1,- V2 corresponds to the voltage gradient across the gap junction (Ij), and I2 to the current flow through the gap junction (Ij). Figure 2 illustrates a standard voltage-clamp experiment examining the effects of Jj on Ij. Voltage pulses were administered to cell 1 every 30 s. The pulses lasted 15 s and were made to vary in amplitude and polarity. On the one hand, Ij pulses of small amplitude (25 mV) were associated with Ii signals with little time dependence. Depolarization gave rise to a small decrease in Ij (Fig. 2A), hyperpolarization to a marginal increase (Fig. 2B). On the other hand, fl pulses of large amplitude (100 mV) were accompanied by Ij signals with pronounced time dependence, irrespective of the polarity. The initial peak, Ij inst' was followed by a monotonic decay to a new steady level, I, s. The amplitudes of the former revealed comparable values of gj inst' i.e.

GAP JN(&1-TIOXS IN IN'S'ECT CELLS

325

5 0 nkS (Fig. 2C) and 4 85 nS (Fig. 2D). The amplitudes of the latter yielded different values of gj ssX i.e. 0 85 and 1-4 nS. Hence, gj decreased by factors of 5 9 and 3 45, respectively, during the 15 s pulses. The analysis of these records and others from the same experiment is illustrated in Fig. 3. The amplitudes of lI w,ere determined at the beginning (1j,inst) and end A

v,

B

C

Lr

V1

D

-

'1 rL

V2 /2

150 mV 1250 pA

10 s

Fig. 2. Response of gap junctioni current to transjunctional voltage gradient, determined in a cell pair. Each panel shows four traces representing the voltages and currents of cell 1 (V11 I,) and cell 2 (V2' IO2), respectively. Voltage deflections in V1 correspond to transjunctional voltage gradients (Vj), current deflections in I2 to transjunctional currents (I). Small voltage pulses (A and B, 25 mV) were associated with Ij signals exhibiting a weak time-dependent decay. Large voltage pulses (C and D, 100 mV) were accompanied by Ij signals with a pronounced time-dependent decay. I' = -40 mV .

(ViJs) of a voltage-clamp pulse and plotted vs. [. Figure 3A depicts the resulting current-voltage relationships. I'lotting 1J inst revealed the instantaneous currentvoltage relationship (0). Over the voltage range examined, i.e. from -100 to + 100 mV, it was linear. Plotting Ij ss yielded the steady-state current-voltage relationship (A). This graph showed a clear voltage dependency. At small voltages, it followed closely the instantaneous plot. At larger voltages, however, it deviated substantially irrespective of the polarity. The deviation from linearity was usually more pronounced for depolarizing voltages. Figure 3B illustrates the dependency of gj on voltage across the gap junction. The ratios Ij inst/ Vj (0) and j ss/ Vj (A) were calculated and plotted vs. Vj. The graph of gj, inst approximated a horizontal line (O; gj = 5-3 +0 09 nS; n = 10). This indicates that immediately after establishment of a voltage gradient, gj is not affected by Vj. The graph of gj ss yielded a bell-shaped curve (A) which was asymmetrical. On the one hand, depolarization led to a decrease in gj. It declined rapidly for Vj values ranging from 0 to + 50 mV. Above +50 mV, gj decreased slowly, however, without reaching a steady level, even at Vj = + 100 mV. On the other hand, hyperpolarization gave rise to a biphasic modulation of gj. Small changes in Vj provoked an increase in gj, establishing a maximum at V = -25 mV. Between Vj values of -25 and -75 mV,

326

F. BUKAUSKAS, C. KEMPF AND R. WEINGART

gj decreased steeply, levelling off near - 100 mV. Similar results were obtained in five additional preparations. Two features of gj s are of particular interest: (i) it is not maximal at a holding potential of -40 mV; (ii) it settles to a steady level at negative voltages, but not at positive ones. This suggests that gj must be controlled

-100

100

Vj (mV) Fig. 3. Electrical properties of gap junction. A, current-voltage relationships. Junctional currents (Ij), determined early (AJ1inst) and late (I; S) during voltage-clamp pulses, were plotted vs. junctional voltage (j). The Ij inst graph (0) revealed a linear relationship with a slope of 0 19 mV/pA. The Ij ss graph (A) yielded a relationship which follows closely the instantaneous plot for small voltages, but deviates substantially for large voltages. B, voltage dependence of gap junction conductance (gj). Values of g; inst and gj,8 were calculated and plotted vs. Tj. The instantaneous plot (0) described a horizontal line owing to a constant g, (mean gj = 5-29 nS). The steady-state plot (A) revealed a strong voltage dependence of gj. Filled symbols correspond to data points gained from records illustrated in Fig. 2. Vh=-40 mV.

not only by f, but also by the non-junctional membrane potential, Vm (see Dependence of gj on non-junctional membrane potential below). The transition from Ij, inst to Ij s occurred with a time course which depended on Vj. For small changes in Ij, i.e. + 12-5 mV, Ij remained constant, at least during the 15 s pulses (not shown). For larger changes in ., however, the decay of Ij became

G-AP JUNCTIONS IN INSECT CELLS

327

progressively faster. In addition, Ij signals associated with depolarizing voltage steps relaxed more quickly and more completely than those accompanied by hyperpolarizing pulses (compare C and D of Fig. 2). Examination of individual Ij records revealed that the decline of Ij does not follow a single exponential. A

B

innijprnv wFfnnnmnw

V,-in vw

m

UTUT

V2

12

uu

50 mV 100 pA

los Fig. 4. Effect of non-junctional membrane potential (Vm) on current flow across gap junction (Ij). A conditioning pulse of 75 mV was administered simultaneously to cell 1 (V1) and cell 2 (V2), first in the hyperpolarizing (A) and then in the depolarizing direction (B), while test pulses (- 12-5 mV; 1 s; 0-2 Hz) were applied repetitively to cell 1 to assay g1. Hyperpolarization produced constant Ij deflections (I2 signal in A); depolarization gave rise to a time-dependent decrease in Ij deflections which was reversible (I2 signal in B). Vh = -42 mV.

Dependence of gj on non-junctional membrane potential As outlined above (see Fig. 3B), the decay of gj ss was not symmetrical with respect to Vj, implying that gj depends on additional factors. Hence, we examined the effects of the non-junctional membrane potential (Vm) using the following protocol. Starting from a common Vh, in this case -40 mV, identical conditioning pulses were delivered concomitantly to both cells. Small test pulses were then superimposed on cell 1 (- 12-5 mV; 1 s; 0-2 Hz) to determine gj. The conditioning pulses lasted for 10-30 s, sufficient for gj to reach a steady-state. Figure 4 shows representative signals, indicating that the junctional membrane responded differently to directional changes in Vm. Hyperpolarization by 75 mV (Fig. 4A) evoked a small increase in Ij test, Before, during and after application of the conditioning pulse, the mean values of gj were 5-9, 641 and 5-9 nS, respectively. Depolarization by 75 mV (Fig. 4B) produced complex current changes. The holding current grew more outward with time, reflecting a time-dependent change in non-junctional membrane conductance, while the amplitude of IJ test declined, thus leading to a decay of gj from 5-9 to 0-4 nS within 25 s. Upon return to Vh, gj recovered to 5-5 nS. The restoration of gj occurred more slowly than the decay. Both decay and recovery of gj were faster at larger depolarizing conditioning pulses (not shown). The records depicted in Fig. 4 and others from the same experiment were assayed

328

F. BVIKA U7SKAS, C.. KEMIPF ANI) R. I'l EING6ART

for g, prevailing at the end( of the conditioning pulses. Values of j, obtained in this way were plotted vs. Vm. As shown in Fig. 5. y ss remaiie(d constant when I'm was changed from - 165 to -40 mV (mean value 6 0 nS). Howvever, it decreased progressively when 1, was depolarized towards positive voltages (Im = + 60 mY'; j 7.5

25

-150

-100

-50

gj (nS)

-

0

50

Vm (mV) Fig. 5. Dependence of gap junction conductance (j) on non-junictional membrane potential (Vm). Vm of both cells was varied simultaneouslyr (increment 25 mV duration 10-30 s; frequency 0 02 Hz) and yj assaved by consecutive application of test pulses to onie of the cells. Values of gjy,, were determined at the en(d of a step change in fI'. The plot shows a decrease in yj for v-alues of lnmore positive than -40 mYV. The filled symbols correspond to values extracted from the records shown in Fig. 4. E} = -42 mV.

= ± 5 mV. This suggests = 02 nS). The half-maximnal decay of yj sswas reached at +m that gj depends strongly on ll during steady-state conditions. Similar results were obtained in ten additional cell pairs. On average, half-maximnal decrease in yj was established at Vm = + 21 + 5 mXY (n = I 1).

Modulation of gj by heptanol Cell pairs were further characterized using heptanol, an alkanol which impairs gap junctions dose dependently and reversibly (e.g. Riidisiili & Weingart, 1989). Figure 6A illustrates the result of such an experiment. Starting from a Vh of -40 mV, a standard voltage-clamp pulse (amplitude - 125 mV; duration 1 s) was administered to cell 1 every 6 s. Vralues of gj were calculated and plotted vs. time. Exposure to 3 mM-heptanol (horizontal bar) gave rise to a dramatic decrease in gj. The first sign of a change was visible within 1 min; the maximal effect occurred 5 min later. The response was fully reversible after wash-out of the alkanol. Immediately before complete uncoupling and early during recovery from uncoupling, the Ij signals showed discrete current steps, suggesting the operation of single gap junction channels. In this study, forty-one cell pairs were found to obey the criteria of gap junctions, i.e. dependency of gj on Vj, Vm, and uncoupling by heptanol. In these cases the mean

GAP JL ENCTIONS IN LMSCEGT CELLS

329

out to be 5 0 + 1 2 nS. To avoid interference from Vj and Vm, gj was determined using a negative Vh (-40 to -70 mV) and small transjunctional voltage gradients (12 5 to 25 mV).

gj turned

Single gap junction channels In a related study, cell pairs were exposed to 3 mM-heptanol to reduce the amplitude of Ij and thereby visualize putative single-channel activity. Examination A

8

-

3 mM-heptanol I --I

I

6 _

2

0 0

2

4

6 8 Time (min)

10

12

14

3 mM-heptanol

B 4

2

0 0

4

2

Time

6

8

10

(min)

Fig. 6. Effects of heptanol on intercellular junctions. Transjunctional voltage steps (Vj) were administered repetitively (- 12-5 mV; 1 s; 0-17 Hz) to one cell of a cell pair and the associated junctional currents (Ij) recorded. The junctional conductance (gj) was calculated and plotted vs. time. A, response of a cell pair with putative gap junctions. Exposure to 3 mM-heptanol (horizontal bar) provoked a reversible decrease in gj. Vh =-40 mV. B, response of a cell pair with putative cytoplasmic bridges. Exposure to 3 mM-heptanol had no effect on gj. VT -40 mV. =

of IJ signals at large magnification revealed discrete current steps attributable to random opening and closing of gap junction channels. In order to analyse these events, the following procedure was adopted. Voltage steps of 1 s duration were applied repetitively (0 3 Hz) to one cell of a pair. In most cases, the associated Ij

330

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BUKAUSKAS, C. KEMPF AND R. WEINGART

signals exhibited repetitive channel activity. A representative record is shown in Fig. 7A. The upper trace shows the transjunctional voltage gradient (Vj), the lower trace the accompanying gap junction current (Ij). In this record, three discrete current levels could be identified (dashed lines), the lowest level reflecting leak current. A

I50 mV

I

-,-L UP1|I

/i

B

_

10 pA J

- s

0.06

0.04

0.02

0

10 20 Amplitude (pA) Fig. 7. Electrical activity of gap junction channels visualized by exposure to 3 mMheptanol. A, transjunctional voltage gradient (Vj) and associated gap junctional current (I). Horizontal lines refer to discrete current levels extracted from the analysis outlined in B. B, amplitude histogram of current signal, plotting the normalized frequency of occurrences vs. amplitude of I,. Bin width = 01 pA. The interpeak intervals revealed a single-channel conductance, yj, of 160 pS. Vh = -46 mV.

Figure 7B shows the amplitude histogram of the current record (sampling interval 0 7 pA). The amplitude of the peaks corresponds to the dwell time at particular current levels, the distance between the peaks to the size of current levels. The first corresponds to the background fluctuations when Vlj was zero (peakbackground), the second peak to the leak fluctuations when Vj was -50 mV (peakleak), respectively, the subsequent peaks to the open channel fluctuations (peakehannel). The distances between (peakchannel) and (peakleak) are multiples of the unitary current amplitude. In this case, the analysis yielded a unitary current of 8 pA (horizontal lines in Fig. 7A), corresponding to a single-channel conductance, yj, of 160 pS. Such single-

G AP JUNCTIONS IN INSECT CELLS 331 channel events were investigated in five cell pairs clamped at a Vj of -50 to -70 mV. To determine yj, current transitions were analysed by means of a cursor method. Events which produced current deflections of opposite polarity in I1 and I2 (see Veenstra, 1990) were accepted for the analysis. Examining eighty records, we obtained a mean yj of 132-7 + 141 pS (n = 334). -8

n~

ginS

-6

A

.4 -2

-50

-100

50

0

100

Vj (mV) 7.5

B

gj (nS)

.5

.2.5

-150

-100

-50

0 50 Vm (mV) Fig. 8. Electrical properties of cytoplasmic bridges. A, dependence of cytoplasmic bridge conductance (gj) on junctional membrane potential (T'). Values of gj,inst and gj,8S were calculated and plotted V8. Vj. The instantaneous (0) and steady-state plot (A) revealed horizontal lines (mean gj, 7-2 and 71 nS, respectively). Vh = -42 mV. B, dependence of cytoplasmic bridge conductance (gj) on non-junctional membrane potential (Vm). Vm of both cells was varied concommitantly (duration 15 s; frequency 003 Hz) and gj was assayed by repetitive application of test pulses (Vj -25 mV; 1 s; 03 Hz) to one of the cells. Values of gj were determined at the end (gj .8) of each Vm step. The plot revealed a constant gj (mean values 5-7 nS). Vh = -42 mV.

Cytoplasmic communication

Current-voltage relationships In some cell pairs, the Ij signals exerted properties different from those described so far. Such preparations were examined for a dependency of gj on Y. Figure 8A

illustrates the result of a typical experiment. V. pulses of constant duration (15 s), variable amplitude and either polarity, were applied to one of the cells and the associated Ij signals recorded. Values of gj were calculated and plotted vs. Vj. The

332

F. BUKA USKAS, C. KEMPF AND R. WEINVGART

graphs for g9 inst (0) and gj,ss (A) were virtually superimposable. Over the voltage range examined (- 100 to + 100 mV), the relationships were linear and without slope. Statistical analysis revealed mean values of 7 2 + 0-1 and 741 + 0-2 nS, respectively. This behaviour is compatible with the presence of cytoplasmic bridges between the cells. Similar results were obtained in twelve other preparations. Dependence of gj on non-junctional membrane potential Cell pairs with putative cytoplasmic bridges were used to investigate the effects of Vm on gj. Figure 8B shows a representative result. Vm of both cells was stepped to different levels with Vj maintained at 0 mV. At each level, gi was determined by applying small test pulses to cell 1 (Vj; amplitude -25 mV; duration 1 s; frequency 0 3 Hz). Values of gj were extracted and plotted vs. Vm. Over the voltage range investigated (-142 to +58 mV), gj ss remained constant (mean value 5-7 +0-1 nS), suggesting that gj is not controlled by Vm. This result was confirmed in all cell pairs whose gj was insensitive to Vj. Again, this finding cannot be reconciled with the properties of gap junctions (see Fig. 5).

7Modulation of gj Cell pairs with a gj insensitive to t' or Vm were further characterized pharmacologically. For this purpose, the response of gj to heptanol was examined. Figure 6B documents the result of such an experiment. A standard Vj pulse (- 125 mV; 1 s) was administered every 6 s to assess gj. Values of gj obtained in this way were plotted vs. time. It turned out that 3 mM-heptanol (horizontal bar), a supramaximal dose for impairment of gap junctions (Riudisiili & Weingart, 1989), had no influence on gj even after 5 min of exposure. This finding has been confirmed in four other preparations using exposure times up to 10 min. This behaviour is not compatible with gap junctions. It suggests the presence of cytoplasmic connections between the cells. During the course of this study, thirteen cell pairs were identified as possessing cytoplasmic bridges. The mean gj turned out to be 19-6+331 nS. Co-existence of gap junctions and cytoplasmic bridges Eight cell pairs showed signs of a co-existence of gap junctions and cytoplasmic bridges. Figure 9A illustrates the effect of Vm on Ij in such a cell pair. V1 refers to the voltage signal of cell 1, 12 to the associated current signal of cell 2. In order to determine g,, small test pulses (- 125 mV; 1 s; 0 5 Hz) were applied alternately to cell 1 and cell 2. In cell 2 this gave rise to the currents Ij (upward deflections) and membrane current of cell 2). Ij +Ilm, 2 (downward deflections; m, 2' non-junctional = were held at With this procedure, the cells Vh -62 mV initially. Thereafter, Vm of both cells was stepped to +38 mV. This produced a 31% decrease in Ij which developed with time. According to Fig. 5. gap junctions would have responded with a complete block of lI. Return of Vm to -62 mV led to a complete recovery of I. Subsequently, this cell pair was exposed to 3 mM-heptanol, a dose which usually provokes complete uncoupling of gap junctions (Fig. 6A; see also Riidisiili & Weingart, 1989). As shown in Fig. 9B, in this case it exerte(d a moderate effect only: gj decreased from 10 to 6 nS, i.e. by 40%. Assessment of the influence of Vm on I

(AP JUNCTIONS IN ILNSECT CELLS

333

revealed no additional change in Ij (not shown). The decrease in gj was fully reversible after wash-out of heptanol. These observations are compatible with the concomitant existence of gap junctions and cytoplasmic bridges. In this group of preparations, determined under control conditions. gj averaged 17-7+46 nS. A

B

10

V1

'2

3 mM-heptanol

12 111 1M1 Jil

5-5

___ITIW

$1

0

150 mV 10 s

0-1 nA

°

22

4

4

Time (min) Fig. 9. Co-existenice of gap junctions and cytoplasmic bridges. A, voltage signal of cell 1 (I'l) and current signal of cell 2 (2) . Ij was assessed by small test pulses applied alterniately, to cell 1 and cell 2 (- 125 mV; 1 s; 05 Hz). Upward deflections in '2 correspond to IP downward deflections to Ij +Im 2 (non-junctional membrane current of (ell 2). Changing Vm in both cells simultaneously from -62 to +38 mV led to a 32% dec rease in I gj, control = 8 6 nS. B, exposure to a supramaximal dose of heptanol (3 mM) pro(luced a 40% decrease in gj' gj,co.ntrol = 10 nS. h =62 mV. DISCUSSION

Insect cells (Aedes albopictus, clone C6/36; Igarashi, 1978), grown at low density (- 104 cells/Cm2), form cell pairs suitable to study the biophysics of intereellular communication. Interestingly, not all cell pairs exhibited the same physiological and pharmacological properties. Electrical measurements suggested the existence of two different types of intercellular contacts. gap junctions and cytoplasmic bridges. The following criteria were used for identification: dependence of intercellular current (Ij) on (i) transjunctional potential (Vj), or (ii) non-junctional membrane potential (JV); (iii) response of Ij to heptanol. By light microscopy, it was impossible to distinguish between the different types of cell pairs.

Gap junctions

Dependence of gj on V Transjunctional currents exhibiting a Vj-dependent gating were associated with gap junctions. The instantaneous current-voltage relationship was linear and hence 9i ss constant. However, the steady-state current-voltage relationship deviated from linearity, i.e. gj,ss was voltage-dependent (Fig. 3). The gj ss vs. Vj relation was bell shaped and asymmetrical. On the one hand, gj decreased to low levels for large Vj

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F. BUKA ULWKAS, C. KEMPF AND R. WEIANGART

gradients, leaving a Vj-insensitive gj. This residual gj was smaller for depolarizing voltages, indicating that Vm may affect gj as well. On the other hand, small positive Vj steps produced a decrease in gj, small negative steps an increase. Again, this implies that gj is not controlled by Vj alone. Indeed, the asymmetry of the gj plot disappears when gj ss is determined at more negative values of Vh (not shown). The time course of the transitions from gj inst to gj ss was dependent on Vj. The larger Vj, the faster was the decay in gj. irrespective of polarity. However, the decline in gj was complex, suggesting that gj may be regulated by several mechanisms. Kinetic analyses are needed for a better understanding. A similar Vj dependence of gj has been observed in other tissues including salivary glands of insect larvae (Chironomus, Obaid et al. 1983; Drosophila, Verselis et al. 1991), amphibian blastulae (Spray, Harris & Bennett, 1981), hearts of chicken embryos (Veenstra, 1990), and liver of adult rats (Moreno, Campos de Carvalho, Verselis, Eghbali & Spray, 1991).

Dependence of gj on Vm Figures 4 and 5 document that yj is also controlled by V1§. These graphs show that gj ss remained constant when Vm was negative to -40 mV, but declined progressively at more positive voltages. In contrast to the relationship gj = f(Vj), gj = f(Vm) decreased to zero. Complete uncoupling by V7m excludes the possibility that the Vj-insensitive residual gj is caused by cytoplasmic bridges (see below). The time course of changes in gj was sensitive to Vm. The larger the depolarizing step, the faster was the decay and recovery of gj. Again, the time-dependent changes in gj were complex. A full analysis requires more experiments. While dependency of gj on Vj is a widespread phenomenon, dependency of gj on Vm has been observed but in few tissues, i.e. squid blastomeres (Spray et al. 1984), and salivary glands of Chironomus (Rose, 1970; Socolar & Politoff, 1971; Obaid et al. 1983) and Drosophila larvae (Verselis et al. 1991). Conceivably, these cells may use Vm to modify the degree of intercellular coupling and thereby modulate cellular functions. Hence, the insect cells studied possess gap junctions whose gj is regulated by Vj and Vm. Differences in voltage range and degree of uncoupling suggest that the two parameters act via separate mechanisms.

Effects of heptanol To further distinguish between gap junctions and cytoplasmic bridges, cell pairs were exposed to heptanol. This alkanol is known to interfere reversibly with gap junctions (e.g. Niggli et al. 1989). Hence, in case of gap junctions, a supramaximal dose of heptanol (3 mM) is expected to inhibit Ij completely. This is exactly what was observed experimentally (Fig. 6A). In cell pairs which showed heptanol-dependent uncoupling, the existence of gap junctions was routinely confirmed by determining the relationships gj = f(Vj) and/or gj = f(Vm). All preparations examined yielded electrical and pharmacological data which were consistent. Gap junction channels Heptanol reversibly uncoupled cell pairs with gap junctions (Fig. 6A). This approach was used to visualize elementary currents carried by gap junction channels. Signals of Ij, recorded immediately before complete uncoupling and early during

GAP JUNCTIONS IN INSECT CELLS 335 recovery from uncoupling, showed discrete current steps attributable to the random opening and closing of single gap junction channels (Fig. 7). Signal analysis yielded a single-channel conductance (yj) of 133 pS. As shown before, effects of heptanol on yj per se can presumably be excluded (see Burt & Spray, 1988). The value of yj is comparable with an estimate from another arthropod tissue (lateral giant axon of earthworm, 100 pS; Brink & Fan, 1989) and various vertebrate tissues (e.g. Brink, 1991). The latter is surprising since diffusion studies established a cut-off limit for channel permeation of 2-45 kDa for insect salivary glands and 0-86 kDa for mammalian heart muscle (for references, see Imanaga, 1987; Weingart, 1987). An independent estimate of yj may be obtained from the size of the permeating molecules. Assuming a uniform cylindrical pore, the resistance of a channel can be expressed as Rchannel = Rpore +Raccess (Rpore = p(l/7(d/2)2); Raccess = 2p/ir d, where p = resistivity of solution inside the pore; d = diameter of pore; I = length of pore; Hille, 1984). Hence, Rchannel turns out to be 2-5 GQ for Chironomus salivary glands (d = 2-9 nm, see Zimmerman & Rose, 1985; 1 = 15 nm, i.e. two unit cell membranes; p = 100 Qcm, i.e. resistivity of a 130 mm salt solution), and 10 GQ for adult mammalian heart muscle (d = 1-4 nm; see Riidisiili & Weingart, 1991), corresponding to conductances (Ychannel) of 400 and 100 pS, respectively. The computed values Of Ychannel may be regarded as upper limits. Usually, membrane channels have conductances which are smaller than those predicted by this formalism (Hille, 1984). In the case of insect salivary glands, the ratio Ychannel/Yj is 3; in the case of mammalian heart muscle it is 2-9.

Cytoplasmic bridges The cytoplasm is an electrically passive element which obeys Ohm's law. Hence, cytoplasmic bridges are expected to show no voltage-dependent gating and to be inert pharmacologically. We have found cell pairs whose Ij signals were consistent with these predictions. Varying Vj or Vm (voltage range + 100 mV and - 150 to + 50 mV, respectively), we found that gj was voltage and time independent (Fig. 8A and B). Furthermore, when exposed to 3 mM-heptanol, these cell pairs showed no decrease in gj (Fig. 6B). These findings are compatible with the existence of cytoplasmic bridges, an alternative type of intercellular contact. Hence, functional differences provide a means to distinguish between these structures. Physiologists have observed cytoplasmic bridges in cell lines of vertebrates (Flagg-Newton, Dahl & Loewenstein, 1981) and insects (Spray, Cherbas, Cherbas, Morales & Carrow, 1989), but not in differentiated cells. Spray et al. (1989) established functional properties of cytoplasmic bridges similar to ours. Anatomists have previously detected such structures in dividing cells in culture (Beams & Kessel, 1976; Mullins & Biesele, 1977). During cell division, nuclear division (i.e. mitosis) is followed by cytoplasmic division (i.e. cytokinesis). During the latter, the membrane around the middle of a cell is drawn inward to form a cleavage furrow, which gradually deepens until it consists of a narrow bridge, the midbody. The midbody may persist for some time before it narrows and finally breaks leaving two separate daughter cells (see Karasiewicz, 1981). Some cell pairs could not be classified in terms of having gap junctions or cytoplasmic bridges. In these preparations, 3 mM-heptanol gave rise to a marginal

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uncoupling and V,-dependent gating of gj was rather weak (Fig. 9). These moderate effects could be explained by the co-existence of gap junctions and cytoplasmic bridges, the latter being more conductive. This view is supported by the finding that the mean 9j control in this group of cell pairs was 17-7 nS, a value close to that for pure cytoplasmic bridges (19-6 nS), but considerably larger than that for pure gap junctions (5 0 nS). Co-existence of gap junctions and cytoplasmic bridges implies that de novo formation of gap junction channels occurs during cytokinesis when DNA synthesis is at rest. Thus, mother cells dispose of sufficient channel proteins to supply the daughter cells. In addition, it suggests that formation of operational gap junction channels is not controlled by the nucleus. The observation that Aedes albopictus cells possess gap junctions and/or cytoplasmic bridges is relevant to the interpretation of cell-to-cell coupling studies performed on cultured cells. Criteria commonly used to identify gap junctions, e.g. the presence of intercellular current flow or tracer diffusion, are not sufficient. The existence of more than one type of intercellular junction demands more sophisticated experimental tests. The authors acknowledge the technical assistance of Marlis Herrenschwand and Pia Jentsch. They also thank Silvio Weidmann for valuable comments on the manuscript. This work was supported by the Swiss National Science Foundation (grants 3.253-0.85, 31.25333.88, and 31.25732.88). This paper is dedicated to Silvio Weidmann on the occasion of his 70th birthday. REFERENCES

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