JPhysiology (1992) 86, 109-115 © Elsevier, Paris

109

Regulation of potassium conductance in the cellular membrane at early embryogenesis P Bregestovski, I Medina*, E

Goyda*

INSERM U29, Hdpital de Port-Royal, 123 Bd de Port-Royal, 75014 Paris, France

Summary - At the early stages of development of the fresh water fish loach (Misgurnus fossilis) the resting membrane potential (Er) of cleaving cells oscillates periodically with an amplitude of 8-12 inV. Er oscillation correlates with the cell cycle and is accompanied by changes of K+ conductivity. Two types of K÷-selective ionic channels with conductance of approximately 70 and 25 pS in symmetrical (150 mM KC1) solution were observed in the membrane of cleaving loach embryos. 'High' conductance and 'low' conductance channels were recorded in approximately 90% and 10% of patches investigated (n = 275), respectively? The activity of 'high' conductance channels was regulated by the application of pressure to the membrane, ie these channels were stretch-activated (SA). The activity of SA channels changes dramatically during the cell-cleavage cycle. At the beginning of interphase the probability of SA channels being in the open state (P0) was minimal, while at prometaphase the probability was increased 10-100-fold. Application of ATP to the cytoplasmic inside-out patches induced a reversible elevation of stretch sensitivity of the SA channels in 50% of the patches, while the nonhydrolyzable analogue of ATP was not effective. Combined application of ATP, cAMP and cAMP-dependent protein kinase (PK) induced a reversible elevation in the SA channel activity while inhibitors of PK prevented its activating effects. Phosphatase inhibitors prolonged the activating effect of PK on SA channels. We propose that oscillations of the resting potential during the cell-cleavage cycle arise due to modulation of SA channel sensitivity to stretch through cAMP-dependent phosphorylation. potassium 1 embryogenesis I Misgurnus fossilis

Introduction Nerve cells and many other excitable cells are sensitive to mechanical stimuli or to changes in hydrostatic pressure (Wann and McDonald, 1980). Apparently, mechanical responses are transduced into electrical signals through stretch-activated ionic (SA) channels (Sachs, 1986). This type o f channel has been described first on cultured embryonic skeletal muscles (Guharay and Sachs, 1984) and since that mechanosensitive singlechannel ionic currents were observed in more than 30 cell types (for review see Morris, 1990). The physiological role o f SA channels was not clearly demonstrated although involvement in membrane depolarization (Katz, 1950), mechanoreception (Guharay and Sachs, 1984) initiation of

stretch-induced contraction (Kirber et al, 1988) and permeability o f vascular endothelium (Lansman e t al, 1987; Otesen et al, 1988) have been suggested. Here we present a review o f recent studies of SA ionic channels in the m e m b r a n e of fresh water fish (loach, M i s g u r n u s f o s s i l i s ) . The data reveal the existence of at least two physiological ways o f remarkable modulation o f this channel activity. Firstly, sensitivity o f SA channels to applied pressure changes dramatically during the cell-cleavage cycle (Medina and Bregestovski, 1988; Bregestovski and Medina, 1988; Medina et al, 1989). Secondly, the activity o f SA channels m a y be regulated through c A M P - d e p e n d e n t phosphorylation (Medina and Bregestovski, 1991). We suggest that the second way is the key factor to SA channel modulation during the cell cycle.

* Permanent address: AV Palladin Institute of Biochemistry, Ukrainian Academy of Sciences, Lvov Branch, Dragomanov Street 14/16, Lvov 290005, Ukraine.

110

Resting potential of embryos' oscillatory changes during the cell-cleavage cycle The period of fertilization and, consequently, cleavage phase of embryo development are accompanied by changes in a number of physiological processes. Intracellular free calcium concentration (Poenie et al, 1985), intracellular pH (Lee and Steinhardt, 1981), metabolism of phosphoinositide lipids, activity of protein kinases and protein phosphatases (Whitaker and Patet, 1990) cyclically increase during this period. There are also oscillatory changes in the membrane properties of cleaving embryos. Cell cycledependent modulation of the resting membrane potential and membrane currents has been observed in amphibian, fish, ascidian and other cleaving embryos (Woodward, 1968; Slack et al, 1973; Bozhkova et al, 1974; Lee and Steinhardt, 1981; Block and Moody, 1990). Figure 1A shows a typical intracellular recording of resting potential amplitude from the fish (loach) embryo during the first hours after fertilization. It can be seen that the resting potential increases from the value of approximately-40 mV at the first division (arrow 2) to approximately - 7 0 mV after the fifth division (arrow 32). However, resting potential rises non-monotonously: it oscillates periodically with an amplitude of 8 12 inV. The period of oscillations is approximately 30 min, which strongly correlates with the cycle of the cell division. Combined voltage-clamp and current-clamp experiments show that these oscillations are accompanied by changes in the membrane conductance. Maximal conductance corresponds to the m a x i m u m amplitudes of the resting potential and maximal values of outward currents (fig 1B) (Medina et al, t988). There is evidence indicating that these changes in membrane permeability are apparently associated with modulation of conductance for potassium ions (Woodward, 1968; Morimoto and Sato, 1984; Webb and Nuccitelli, 1985; De Laat et al, 1974; Slack and Warner t973).

The membrane of loach embryos contains channels sensitive to pressure To understand the mechanism underlying the modulation of cell resting potentials and input conductance we studied the behaviour of single

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Fig 1. Resting potential, ionic current and membrane conductance of the cleaving loach embryos oscillate during the cell cycle. Arrows correspond to prometaphase of the cell cycles and show number of forming cells. A. Microelectrode recording of the resting membrane potential. B. Double microelectrode voltage-clamp recording of the membrane conductivity and transmembrane ionic currents. Holding potential: --40 mV. Eggs were fertilized and embryos prepared as described early (Medina et al, 1988; Medina and Bregestovski, 1988, 1991). Resting potential, membrane conductivity and ionic current recording were made using conventional voltage-clamp and current-clamp microelectrode techniques. Microelectrodes were filled with 3 M KCl and had a resistance of 10-20 mOhms.

K + ionic channels in the membrane of loach embryos (Medina and Bregestovski, 1988; Bregestovski and Medina, 1988; Medina et al, 1989). Two types of K+-selective ionic channels with conductance of approximately 70 and 25 pS in symmetrical (150 mM KC1) solution were observed in the membrane of these cells. 'High' conductance and ' l o w ' conductance channels were recorded, respectively, in 91% and 13% of patches investigated (n = 275). We found out that the activity of 'high' conductance channels is regulated by the application of pressure to the membrane, ie these channels

111 are stretch-activated (SA). Its main properties are illustrated in figure 2. In the cell-attached configuration spontaneous appearance of currents was observed. Application of suction to the pipette increased the frequency of activation and duration of bursts (fig 2B, C). Probability of the channel being open (P0) increased with the degree of suction. A suction of 1.5 cm Hg produced > 100-fold increase in P0 (Medina and Bregestovski, 1988). The distribution of open time was approximated by the sum of two exponentials and the slow component, which describes the distribution of burst durations, increased with the negative pressure (fig 2C). The distribution of closed times was approximated by the sum of three exponential functions. The fastest component was not stretch-dependent while the sum of the other two components:Was the most sensitive to suction (fig 2C). Appiying positive pressure to the pipette diminished.the probability of channel activation compared witti the control.

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It was shown that these SA channels have preferentially K+-selectivity (Medina and Bregestovski, 1988; Bregestovski and Medina, 1988). When bathed on both membrane surfaces by 140 mM KC1 the channel conductance was about 70 pS. The kinetic behaviour did not depend markedly on either membrane potential (in the range from -70 to +70 mV) or calcium concentration (up to 1 mM) on the cytoplasmic side of the membrane. The presence of K÷-selective SA channels in about 90% of recorded patches allowed us to suggest that these channels represent the main means for modulation of K + permeabiltiy in the membrane of loach embryos (Medina et al, 1988).

Spontaneous activity and stretch-sensitivity of SA channel changes during the cell cycle lit has been shown that at continuous recording the P0 of SA channels changes periodically 5 to

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Fig 2. Stretch-activated channel currents in the membrane of cleaving loach embryo. Cell-attached patch clamp recording. Pipette potential: +20 inV. Inward currents are shown as downward. 256-cell phase. Filter t kHz. A. Patch current recorded at slow time sweep. Infrequent spontaneous channel activity was observed in control. Applying of suction to the pipette (-1.5 cm Hg) produced almost continuous activation of channel. The release of negative pressure decreased channel activity. B. Examples of stretch-activated ion channel currents obtained at different negative pressures in the pipette. Note the increased frequency and burst duration with increased suction. C, The distribution of burst open (left) and closed times (right) of the stretch-activated channel in control (top histograms) and during application of -1.5 cm Hg suction to the pipette (bottom histograms). Continuous lines are calculated by a sum of two or three exponentials. The distribution of burst times had two time constants in control: ~cl = 3.2, "~2 = 8.4 ms and after suction: "Cl = 4.4, "c~_= 27.2 ms. The distribution of closed times had three time constants in controll: "c[ = 0.9, "c2 = 3.7. "c3 = 58.3 ms and after suction: "tl = 0.8, "~2= 1.4, x3 = 32.5 ms (from Medina and Bregestovski, 1988, with permission.)

112 20 times (fig 3A). During the increase in P0 the kinetics o f the channel changed in a similar manner as observed upon suction. It was shown that these variations in SA channel activity strongly correlated with changes in the resting potential and membrane conductance during cell cycle (Medina et al, 1988; Medina and Bregestovski, 1988). Moreover, it was discovered that during the cell-cleavage cycle sensitivity of SA channels to stretch changes dramatically. At the beginning of interphase P0 and the stretch sensitivity of SA channels were minimal, while at prometaphase they were increased 10-100-fold (Medina and Bregestovski, 1991). Figure 3B illustrates records of SA channel activity at different times during the cell-cleavage cycle and the responses of these channels to pressure application. During the period o f a high spontaneous activity (prometaphase, point m in fig 3A), the channel displayed marked activation at relatively low suction. In contrast, during a phase of low spontaneous activity (early stage of interphase, point k), the channel showed an absence o f response even at much stronger suction. As a result, the slope o f the pressure dependence of P0 in semi-logarithmic coordinates changed markedly at different points in the cell cycle (fig 3C). At m, P0 increased monotonically as the pressure in the pipette was made more negative. In contrast, at point k P0 did not change over a wide range o f applied pressures. At this point pressure dependence o f P0 was nearly parallel to abscises axis, ie the channel b e c a m e insensitive to stretch. This type of SA channel activity might be explained either by periodic changes in the internal cell pressure and in the tension o f the membrane

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Fig 3. Open probability and stretch-sensitivity of SA channels in the membrane of loach embryos oscillate during the cleavage cell cycle. Cell-attached recording, pipette potential: +20 mV. A. Time-dependent changes of Po of SA channels at continuous recording. Arrows designate moments of embryo cleavage observed under the light microscope. B. Single channel ion currents recorded at different stages of the cell cycle corresponding to points k (early stage of interphase), and m (prometaphase) in A. Note that application of suction to the pipette caused an increase in activation at m but not a k. Expanded traces show that suction resulted in longer burst durations of SA channels. C. Pressure dependence of the SA channel activity at different stages of the cell-cleavage cycle, corresponding to points k, and m in A. Note changes in the slope of the plots which indicates an alteration in the stretch sensitivity during the cell cycle (modified from Medina and Bregestovski, 1991).

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113 surface of the intact cell (Hiramoto, 1974; Yoneda

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et al, 1978; Schoeder and Otto, 1984; Aszalos et al, 1986) or by periodic changes in intracellular second messengers activity (Lasareva et al, 1984; Poenie et aI, 1985; Goyda et al, 1989; for review

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see Whitaker and Patel, 1990) and consequently protein phosphorylation (Peaucellier et al, 1984). Our analysis shows that the first explanation does not appear to account for the observed variations in SA channel activity (Medina and Bregestovski, 1991).

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Evidence for regulation of SA channel activity by phosphorylation Several lines of evidence show that SA channels may be modulated due to phosphorylation. Firstly, in about 50% of inside-out patches stretch sensitivity of SA channels increased on the application to the cytoplasmic side of the membrane of ATP but not its non-hydrolyzable analogue (Medina and Bregestovski, 1991). Indeed, as illustrated in figure 4A, the addition of 0.3 mM ATP to the cytoplasmic part of the membrane resulted in an approximately 10-fold elevation of the channel activity and maintenance of P0 at this level throughout time ATP was present in the solution. After substitution of the non-hydrolyzable analogue of ATP 5'-adenylylimidodiphosphate (AMP-PNP, 0.3 mM) for the ATP, activity returned to control level. Thus, AMP-PNP did not alter channel activity and ATP action on SA channels might involve either an energy-dependent process or protein phosphorylation. Secondly, on patches not sensitive to ATE cAMP-dependent protein kinase (PK) increased stretch sensitivity only in the presence of cAMP. Figure 4B shows that combined application of ATP + cAMP had no effect on the channel activity. However, addition to this mixture of PK (10 units/ml) induced an approximate 300-fold increase in P0. This effect was reversible. Subsequent application of PK and ATP for 6 rain to this same patch was not effective. When cAMP was subsequently added to the PK and ATP, there was an immediate elevation in channel activity. Thirdly, inhibitors of cAMP-dependent protein kinase reversibly prevented the activating effect of PK and dibutyryl-cAMP transiently elevated activity of SA channels in intact cells. Moreover, application of phosphatase inhibitors (orthovanadate and para-nitrophenyl) to excised patches

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TIME (rain) Fig 4. SA channels can be modulated by phosphorylation. Inside-out patch clamp recording. Membrane potential: -40 mV. A. Changes of SA channel open probability on addition of 0.3 mM ATP (ATP-Na2, Sigma) or 0.3 mM AMP-PNP (5'adenylylimidodiphosphate, Sigma) to the cytoplasmic side of the excised patch. Each point represents the result of analysis of 50 s duration. B. Changes in P0 resulting from combined application of 0.3 mM ATP, 0,1 mM cAMP and 10 units/ml PK (cAMP-dependent protein kinase from rabbit muscle, Sigma). Originally this patch was not sensitive to application of only ATP. Each point represents the result of analyzing records of 60 s duration. Bars indicate duration of application of designated agents. PK was applied 6 min after addition of ATP and cAMP. Note that the probability increased only in the presence of all three agents (modified from Medina and Bregestovski, 1991).

markedly prolonged the activity of SA channels (Medina and Bregestovski, 1991).

Conclusions Regulation of the ionic channel activity by cAMP-dependent phosphorylation was shown on several preparations (Doroshenko et al, 1982; Siegelbaum et al, 1982; Cachelin et al, 1983;

114 Ewald et al, 1985; Nakamura and Gold, 1987; Sadoshima et al, 1988; Kume et al, 1989). It has also been shown that cAMP levels, cAMP-dependent protein kinase activity and protein phosphorylation change periodically during the cell cycle (Lasareva et al, 1984; Peaucellier et al, 1984). Results obtained on cells of loach embryos suggest that the activity of K+-selective SA ionic channels is modulated by phosphorylation. SA channels were also observed on oocytes or cleaving embryos of amphibian and ascidian (for review see Moody et al, 1991). However, ionic selectivity of these channels is different from that demonstrated on the loach. In oocytes of X e n o p u s (Metthfessel et al, 1986; Yang and Sachs, 1990) and ascidian Boltenia villosa these channels are cation-selective and pass Na ÷, Ca 2+, and K + but not C1- ions. On the other hand, it was shown that processes of the early embryogenesis in the X e n o p u s laevis are determined by mechanical interaction between cells (Beloussov et al, 1988). Thus, it is possible that SA channels are involved in the regulation of embryonic development.

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Doroshenko PA, Kostyuk PG, Martynyuk AE (1982) Intracellular metabolism of adenosine 3,5-cyclic monophosphate and calcium inward current in perfused neurones of Helix pomatia. Neuroscience 7, 21252134 Ewald, DA, Williams A, Levitan IB (1985) Modulation of single Ca2+ dependent K+ channel activity by phosphorylation. Nature 315, 503-506 Goyda EA, Medina IR, Rott NN (1989) Periodic changes of intracellular pH in developing loach embryos do not correlate with activity of Na/H metabolism. Ontogenes 20, 443-446 (in Russian) Guharay F, Sachs F (I984) Stretch-activated single ion channel in tissue cultured embryonic chick skeletal muscle. J Physiol (Lond) 352, 685-701 Hiramoto Y (1974) Mechanical properties of the surface of the sea urchin egg at fertilization and during cleavage. Exp Cell Res 89, 320-326 Katz B (1950) Depolarization of sensory terminals and the initiation of impulses in the muscle spindle. J Physiol (Lond) 1tl, 261-282 Kirber MT, Walsh JV Jr, Singer JJ (1988) Stretch-activated ion channels in smooth muscle: a mechanism for the initiation of stretch-induced contraction. Pfliigers Arch 412, 339-345 Kume H, Takai A, Tokuno H, Tomita T (1989) Regulation of Ca2+-dependent K+-channel activity in tracheal myocytes by phosphorylation. Nature 34I, t52-154 Lansman JB, Hallam LA, Rink TJ (1987) Single stretchactivated ion channels in vascular endothelial cells as mechanotransducers? Nature 325, 811-813 Lasareva AV, Rott NN, Goyda EA, Shiyan RB, Mikhailova GC (1984) Changes in cAMP content during the cell cycle in the cleaving loach embryos. Ontogenes 15, 171-176 (in Russian) Lee SC, Steinhardt RA (1981) Observation on intracellular pH during cleavage of eggs of Xenopus laevis. J Cell Biol 91, 414-419 Medina IR, Bregestovski PD (1988) Stretch-activated ion channels modulate the resting membrane potential during early embryogenesis. Proc R Soc Lond B Biol Sci 235, 95-102 Medina IR, Bregestovski PD (1991) Sensitivity of stretch-activated K+ channels changes during cellcleavage cycle and may be regulated by cAMP-dependent protein kinase. Proc R Soc Lond B Biol Sci 245, 159-164 Medina IR, Goyda EA, Bregestovski PD (1988) Stretchactivated potassium channels are one of the factors of resting potential oscillation at early embryogenesis of loach. Biologicheskie Membrany (Biological Membranes, Russian) 5, 960-969 Medina IR, Goyda EA, Bregestovski PD (1989) Stretchsensitivity of potassium channels changes during mythotic cycle of embryonic cells. In: Proc Symposium: "Single ionic channels in biological membranes', Moscow, p 67

115 Methfessel C, Witzemann V, Takahashi T, Mishina M, Numa S, Sakmann B (1986) Patch clamp measurement on Xenopus laevis oocytes: current through endogenous channels and implanted acetylcholine receptor and sodium channels. Pfiigers Arch 407, 577588 Moody WJ, Simoncini L, Coombs JL, Spruce AE, Villas M (199l) Development of ion channels in early embryos. J Neurobiot 22, 674-684 Morimoto K, Sato E (1984) Cell cycle-dependent changes of membrane permeability in white micromere. Dev Growth Diff 26, 372-384 Morris CE (1990) Mechanosensitive ion channels J Membr Biol 113, 93-107 Nakamura T, Gold G (1987) A cyclic nucleotide-gated conductance in olfactory receptor cilia. Nature 325, 443-444 Otesen SP, Clapham D, Davies PF (1988) Haemodynamic shear activates a K ÷ current in vascular endothelial cells. Nature 331, 168-170 Peaucellier G, Doree M, Picard A (1984) Rise and fall of protein phosphorylation during meiotic maturation in oocytes of Sabellaria alveotata (Polychaeta Annelid). Dev Biol 106, 267-274 Poenie M, Alderton J, Tsien RY, Steinhardt RA (1985) Changes of free calcium levels with stages of the cell division cycle. Nature 315, 147-149 Sachs F (1986) Biophysics of mechanoreception. Membr Biochem 6, 173-192 Sadoshima J, Akaike N, Kanaide H, Nakamura M (1988) Cyclic AMP modulates Ca-activated K chart-

nel in cultured smooth muscle cells of rat aortas. Am J Physiol 255, H754-H759 Schoeder TE, Otto JJ (1984) Cyclic assembly-disassembly of cortical microtubules during maturation and early development of starfish oocytes. Dev Biol 103, 493-503 Siegelbaum SA, Camardo JS, Kandel ER (1982) Serotonin and cyclic AMP close single K ÷ channels in Aplysia sensory neurones. Nature 299, 413-417 Slack C, Warner A (1973) Intracellular and intracellular potentials in the early amphibian embryo. J Physiol (Lond) 232, 313-330 Wann KT, MacDonald HG (1980) The effects of pressure on excitable cells. Comp Biochem Physiol 64A, 1-12 Whitaker M, Patel R (1990) Calcium and cell cycle control. Development 108, 525-542 Webb PJ, Nuccitelli R (1985) Fertilization potential and electrical properties of the Xenopus Iaevis egg. Dev Biol 107, 395-406 Woodward PJ (1968) Electrical signals of new membrane production during cleavage of Rana pipiens eggs. J Gen Physiol 52, 509-531 Yang XC, Sachs F (1990) Characterization of stretchactivated channels in Xenopus oocytes. J Physiol 431, 103-122 Yoneda M, Ikeda M, Wasitani S (1978) Periodic change in tension at the surface of activated nonnucleated fragments of sea-urchin eggs. Dev Growth Differ 20, 329-336

Regulation of potassium conductance in the cellular membrane at early embryogenesis.

At the early stages of development of the fresh water fish loach (Misgurnus fossilis) the resting membrane potential (Er) of cleaving cells oscillates...
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