Abnormal chloride and potassium conductances in cultured embryonic tongue muscle from trisomy 16 mouse

Developmental Brain Research 122 (2000) 193–197 www.elsevier.com / locate / bres

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Abnormal chloride and potassium conductances in cultured embryonic tongue muscle from trisomy 16 mouse S. Peng, S.I. Rapoport, R.J. Pearce, Z. Galdzicki* Laboratory of Neurosciences, National Institute on Aging, National Institutes of Health, Room 6 C-103, Building 10, 9000 Rockville Pike, Bethesda, Maryland, MD 20892, USA Accepted 9 May 2000

Abstract Trisomy 16 (Ts16) mouse is considered an animal model of Down syndrome (human trisomy 21). Whole-cell patch-clamp was used to evaluate potassium and chloride currents of cultured tongue muscle cells from fetal Ts16 and diploid mice. No difference was found in membrane capacitance between the two groups. K 1 and Cl 2 currents were pharmacologically isolated. K 1 conductance was reduced by 31% in Ts16 cells (373 pS / pF) compared with diploid cells (539 pS / pF). Cl 2 conductance was 51% larger in Ts16 cells (103 pS / pF) compared with diploid cells (68 pS / pF). However kinetics for K 1 and Cl 2 currents did not differ between the cell types. An increase in Cl 2 conductance and a decrease in K 1 conductance in Ts16 muscle cells, if present in muscle of Down syndrome subjects, might account for the observed hypotonia in these subjects.  2000 Elsevier Science B.V. All rights reserved. Keywords: Muscle; Tongue; Hypotonia; Down syndrome; Ts16 mouse; Potassium conductance, Chloride conductance

Down syndrome (trisomy 21, Ts21), a human genetic disorder caused by an additional copy of chromosome 21 [20], is responsible for multiple neurological and physiological abnormalities including mental retardation [11], early onset of Alzheimer disease [30] and hypotonia [13]. The hypotonia is characterized by muscle weakness and decreased tendon reflexes, and is prominent throughout life [22,26]. Partial glossectomy has been advocated to ameliorate the lingual effects of hypotonia [29].The trisomy 16 (Ts16) mouse is considered an animal model of Down syndrome because of genetic homology between human chromosome 21 and the distal portion of the long arm of mouse chromosome 16 [12]. In addition to this genetic homology, multiple phenotypic abnormalities have been observed in this model, including brain hypocellularity [28], transient edema, anasarca, and cardiac, craniofacial, audio vestibular, skeletal, endocrinological and neuropathological defects [16,21]. Because the Ts16 fetus does not survive to birth [21], electrophysiological responses from these animals can only be studied by employing cell culture techniques. In cell culture both mouse Ts16 and *Corresponding author. Tel.: 11-301-594-3990; fax: 11-301-4020074. E-mail address: [email protected] (Z. Galdzicki).

human Ts21 dorsal root ganglia (DRG) neurons showed an increase in Na 1 and K 1 conductances [4,23]. Abnormal electrical properties of excitable Ts16 cells led us to postulate: that abnormal electrical properties might exist in Ts16 striated muscle, and might contribute to hypotonia, if these abnormalities occur in DS patients [22,26]. Therefore, in the present study, we investigated electrophysiological parameters of cultured tongue muscle cells from Ts16 and diploid mouse embryos. We found significant abnormalities in both K 1 and Cl 2 conductances of the Ts16 cells. Some of these results have been presented in abstract form [25]. Ts16 mouse fetuses were obtained by breeding males, doubly heterozygous for Robertsonian translocation of chromosome 16 (Rb(6.16)24Lub / Rb(16.17)7BnrF1), with normal C57BL / 6 females. Embryos were recognized as diploid or Ts16 as previously described [15]. Tongue muscle from Ts16 and diploid control fetuses was removed and dissected into small pieces, which then were digested at 378C for 30 min in Puck’s solution containing 0.125% trypsin (without Ca 21 or Mg 21 ). After digestion, cells and tissue clumps were pelleted and re-suspended in Dulbecco’s modified Eagle medium (DMEM) with L-glutamine (Gibco RBL Life Tech., Grand Island, NY) supplemented with 10% (in vol) horse serum, 10% fetal bovine serum,

0165-3806 / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved. PII: S0165-3806( 00 )00058-4

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penicillin (100 units / ml) and streptomycin (100 mg / ml). The tissue was dissociated into a cell suspension by trituration through a fire-polished Pasteur pipette. Single cells were plated at a density of 5310 5 cells per 35 mm Nunc culture dish that had been coated with 20 mg Vitrogen 100 (Celtrix, Santa Clara, CA). Cultures were maintained in an incubator at 378C with 5% CO 2 for at least 7 days before recording. At this stage cells revealed spontaneous contractions. Colcemid (Gibco) was used in a final concentration of 10 27 M to induce formation of myoballs a few hours before recording [31]. Patch pipettes were pulled to a resistance between 2 MV and 3 MV. Whole-cell patch-clamp recordings were performed using an Axopatch 200A amplifier (Axon Instruments, Foster City, CA) and data were stored on-line. Only data from cells with a seal resistance in excess of 5 GV were analyzed. Passive cell membrane parameters were measured from the current response to a 5 mV, 12 ms hyperpolarization voltage step from a holding potential of 260 mV, cell membrane resistance (R m ) and capacitance (Cm ) were calculated as previously described [15]. Capacitance was calculated as the integral of the current response between the onset of the pulse and 5-fold of its initial decay time. A steady-state current was used to calculate membrane resistance. Total membrane currents were measured by applying 10 mV depolarization voltage steps from a holding potential of 260 mV. Except when measuring isolated Cl 2 current, the linear leakage component was subtracted on-line using a -P/ 8 protocol [6]. All the current values were estimated using a third order polynomial fit to the late component of the outward current (I stst ). Membrane conductance (Gmax.out ) was defined as the maximum slope of the (I stst 2Vm ) relations [15]. Curve fits were done with pClampfit software (Axon Instruments). Data are presented as mean6S.E.M., with number of cells in parenthesis. Statistical analysis were performed using Student’s t-test. The significance level was set at P,0.05. The solutions used were based on those applied in studies of currents in human skeletal muscle cells [32] and they are listed below with salt concentrations expressed in mM; (1) chloride current solution: (pipette) CsCl, 133; CaCl 2 , 1; MgCl 2 , 2; HEPES / OH, 10; EGTA, 10; pH titrated to 7.4 with CsOH; (bath solution) CsCl, 145; CaCl 2 , 1; MgCl 2 , 2; HEPES / OH, 10; dextrose, 10; CdCl 2 , 1; tetrodotoxin (TTX), 10 23 ; pH titrated to 7.4 with CsOH; (2) potassium current solution: (pipette) Na-aspartate, 5; K-aspartate, 120; CaCl 2 , 1; MgCl 2 , 2; HEPES / OH, 10; EGTA, 10; dextrose, 20; pH titrated to 7.4 with KOH; (bath) Na-aspartate, 150; K-aspartate, 5; CaCl 2 , 1; MgCl 2 , 2; HEPES / OH, 10; TTX, 10 23 ; CoCl 2 , 4; pH titrated to 7.4 with NaOH. Our measurements showed that mean Cm and R m did not differ significantly between Ts16 and diploid cells, indicating comparable cell sizes and membrane resistances (at 260 mV) in the two cell groups (data not shown). To

examine the contribution of K 1 to the outward current, a ‘potassium current solution’, as defined above, was used. Current traces shown in Fig. 1 are from a diploid (1A) and a Ts16 myocyte (1B). I-V curves were constructed and Gmax.K /Cm were obtained (Fig. 1C). The mean Gmax.K /Cm was significantly reduced by 31% in Ts16 cells (from 539 pS / pF in diploid to 373 pS / pF in Ts16 cells). These results along with kinetic data are summarized in Table 1. Tail current measurements confirmed that the outward current was indeed predominantly carried by K 1 , as the reversal potential was 283 mV (data not shown), close to the K 1 equilibrium potential of 284 mV under these conditions. We also compared K 1 current activation and inactivation time constants between Ts16 and diploid cells. At two potentials (150 and 170 mV), significant differences were found in tK, inact between the two cell groups. At the other four potentials (130, 140, 160 and 180 mV) no significant difference was found, indicating that channels in the two cell types likely function similarly (see Table 1 for summary). To see if the normalized maximum outward Cl 2 conductance (Gmax.Cl /Cm ) was abnormal in the Ts16 muscle cells, we isolated the Cl 2 current by excluding K 1 salt ‘chloride current solution’. Fig. 2 shows sample current traces measured from a diploid (Fig. 2A) and a Ts16 cell (Fig. 2B). Because the Cl 2 component contributed substantially to the outward leak current, we did not perform leak subtraction. From recordings such as those shown in Fig. 2A and 2B, I–V curves were constructed to calculate a slope conductances for each cell (Fig. 2C). The data are summarized in Table 2. Mean Gmax.Cl /Cm in Ts16 cells was increased by 51% to 103 pS / pF, compared with 68 pS / pF in diploid cells. In some recordings, rectification in the outward Cl 2 current was observed when membrane potential exceeded 160 mV (2 out of 26 diploid cells and 6 out of 25 Ts16 cells), possibly due to the presence of more than one type of Cl 2 channels. This was not investigated further in the present study. Channel activation (tCl, act ) and inactivation (tCl, inact ) time constants are summarized in Table 2. No significant difference in these parameters was found between the two cell types. Sample curves are shown in Fig. 2. Puff application of 1 mM of 9-AC (anthracene-9-carboxylic acid), a chloride channel blocker [8], inhibited outward current (n55) at all tested potentials this block begins almost immediately and reaches steady almost complete inhibition after 100–400 s. During a normal muscle contraction the action potential causes a depolarization of membrane potential that spreads over the cell surface membrane. Subsequently, through the network of transverse tubules, this change in membrane potential is carried into the depth of the muscle fiber. This constitutes an electrical trigger for contraction and causes a charged sensor of T-tubule membrane to open the

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Fig. 1. Sample potassium current traces (recorded in ‘potassium current solutions’). Holding potential was 260 mV, and 14 voltage steps between 260 mV and 170 mV, in 10 mV increment, of depolarization were applied to evoke membrane current. In (A) (diploid): R m , 200 MV; Cm , 95 pF; Gmax.K /Cm , 579 pS / pF. In (B) (Ts16): R m , 254 MV; Cm , 117 pF; Gmax.K /Cm , 337 pS / pF. In (C), I-V curves of outward K 1 currents were constructed from A and B.

ryanodine receptor and release Ca 21 to initiate contraction. Thus, any alteration in membrane properties which affects the propagation of this depolarization or modulates action potential firing frequency could modify the ability of muscle to contract or maintain tension. Initiation of an action potential depends on resting membrane potential, which in muscle is determined predominately by membrane permeabilities to K 1 and Cl 2 7 . The Cl 2 conductance acts to minimize the depolarizing effect of K 1 accumulation in the T-tubular system during activity, as has been reported in striated muscle of amphibians, fish, and mammals [1,7,17,24]. Because of the absence of neurons under these culture conditions, tongue muscle in culture may undergo changes characteristic of denervated muscle in vivo. Denervation in vivo results in a reduction in Cl 2 conductance and an increase in K 1 conductance [9]. As a consequence, the contribution of Cl 2 conductance to resting membrane conductance is lost. We did not observe a significant difference in inward Na 1 current between Ts16 and diploid muscle cells (data not shown here). The outward current in these cells

consisted of K 1 and Cl 2 components, and we pharmacologically isolated both of them. K 1 conductance was found to be decreased by 31% in the Ts16 muscle cells. Cl 2 conductance was increased by 51% in Ts16 cells in comparison with the diploid cells, although under our experimental conditions this difference was evident only at positive membrane potentials. We surmise that if a similar relation exists between K 1 and Cl 2 conductance in Ts21 muscle, these changes would increase the threshold for excitability. Although Cl 2 is a dominant component in innervated muscle, the membrane resting potential would shift if intracellular Cl 2 or K 1 concentrations are changed. Repolarization of the action potential would be altered as well. Interestingly, the differences in K 1 and Cl 2 conductances between Ts16 and diploid cells were in opposite directions to those reported for denervation of normal muscle [9]. This suggests that a common regulatory mechanism for K 1 and / or Cl 2 conductances following denervation is disrupted in fetal Ts16 muscle. One such possible mechanism involves phosphorylation of channel

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Table 1 Mean membrane conductance and kinetic parameters: activation time constant tK,act and inactivation time constant tK,inact of potassium in Ts16 and diploid cultured muscle cells a

Table 2 Mean whole-cell membrane conductance and kinetic parameters: activation time constant tCl,act and inactivation time constant tCl,inact of chloride current in diploid and Ts16 cultured tongue muscle cells a

Parameter

Diploid

Ts16

Parameter

Gmax K /Cm (S / F)

539.0635.8 (54)

373.1633.0 (42)**

Gmax

Membrane potential (mV)

tK,act (ms)

80 70 60 50 40 30

3.760.3 4.160.3 4.860.3 6.060.5 7.260.5 11.061.1

(45) (45) (45) (44) (38) (27)

3.360.3 3.760.3 4.460.4 5.760.5 7.260.8 8.960.8

(28) (28) (28) (28) (25) (20)

Cl

/Cm (S / F)

Diploid

Ts16

6866.6 (26)

Membrane potential (mV)

tCl,act (ms)

80 70 60 50 40

89611.8 (6) 101613.2 (11) 96613.6 (11) 98611.2 (10) 171685.4 (5)

103610.1 (25)**

91616.1 (11) 82613.3 (11) 82614.9 (13) 81612.6 (10) 8268.8 (7)

tCl,inact (ms) tK,inact (ms) 80 70 60 50 40 30 a

75.064.5 (34) 80.165.4 (34) 83.466.1 (31) 97.066.5 (26) 100.2614.4 (12) 102.4624.9 (6)

68.864.4 (22) 64.862.9 (22)* 68.063.8 (21) 72.964.4 (19)** 107.0613.5 (18) 159.0631.3 (9)

Data were obtained in ‘potassium current solutions’. * Significantly different from diploid values at P,0.05, and ** at P,0.01.

70 60 50 40

4.260.2 3.661.2 2.161.0 2.560.6

(2) (2) (2) (2)

2.460.6 2.060.4 2.661.1 5.163.7

(5) (5) (4) (4)

a

Data were obtained in ‘chloride current solutions’. ** Significantly different from diploid at P,0.01. No statistical comparison was performed on channel inactivation time constants, tCl,inact , because of the small number of cells.

Fig. 2. Chloride current in Ts16 and diploid cultured muscle cells. Current was evoked by 22 consecutive 400-ms voltage steps between 2100 mV and 1110 mV, at 10 mV increments from a holding potential of 260 mV (recorded in ‘chloride current solutions’). The interval between two adjacent episodes is 5 s. (A), from a diploid cell, (B), from a Ts16 cell. In the diploid cell, membrane parameters are: R m , 527.9 MV; Cm , 56.9 pF; Gmax.Cl /Cm , 74.9 pS / pF; in Ts16 cell, R m , 280 MV; Cm , 89.4 pF; Gmax.Cl /Cm , 137 pS / pF. In (C), I-V curves were constructed from A and B.

S. Peng et al. / Developmental Brain Research 122 (2000) 193 – 197

proteins by protein kinase A (PKA) activated by cAMP, a common second messenger in cellular signal transduction pathways. Levels of cAMP and PKA activity were shown to increase in parallel after denervation of rat muscle [10]. In rabbit vascular smooth muscle, elevated PKA activity has been reported to enhance delayed-rectifier K 1 channel activity leading to increased current [3]. Thus, it is possible that some step in the sequence of events initiated by muscle denervation, resulting in K 1 phosphorylation via increase in cAMP levels and an elevation of PKA activity, is aberrant in murine Ts16 muscle. Cl 2 channels play an important role in muscle and they have been shown to affect muscle function in several genetic disorders [5,14,18,19]. An example is myotonia, where skeletal muscle relaxation is impaired due to enhanced membrane excitability [18]. In certain myotonias, Cl 2 conductance is decreased [2,5,14]. Additionally, a 2-fold increase in K 1 conductance in myotonic goat muscle fibers has been reported [8]. As may be expected for hypotonia, our findings in Ts16 tongue muscle are opposite to those reported for myotonia [8]. If our present findings in murine fetal Ts16 muscle are confirmed for Down syndrome muscle, they could add a new light to understanding hypotonia in DS. Our results show that the Ts16 conditions affects excitable fetal muscle cells as well as central and peripheral neurons, although in different cell-specific manner [27]. Acknowledgements We thank to Dr. Gerald Ehrenstein (NINDS, NIH) and Dr. Fabio Ruzzier (Physiol. Depart., Univ. Trieste) for their critical comments on this paper.

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