 |
INTRODUCTION |
Delayed rectifier potassium channels are responsible for the repolarizing current that brings membrane potential back to its resting value after the depolarizing phase of action potential. Modifications of the electrophysiological parameters of this class of channels, because of gene mutations for example, might be implicated in some human diseases like ataxia and myokymia (Browne et al. 1994
) and cardiacarrhythmia (Long QT Syndrome) (Curran et al. 1995
;Sanguinetti et al. 1995
).
The gating properties of delayed rectifier potassium channels have been extensively investigated in in vitro preparations from mouse, rat, and fly nervous system. Various voltage-gated potassium channels were cloned and characterized from different tissues of human origin. An insulinoma potassium channel (hPCN1) was expressed and studied in Xenopus oocytes (Philipson et al. 1991
). A Shaker potassium channel, classified as Kv1.5, was cloned from human heart and transfected in a mouse cell line (Snyders et al. 1993
; Tamkun et al. 1991
). The gene for h-DRK1, the human homologous of rat DRK1 (Frech et al. 1989
) and mouse mShab (Pak et al. 1991
), was recently cloned from a human genomic DNA library (Albrecht et al. 1993
) and its electrophysiological properties were investigated after cDNA injection in Xenopus oocytes (Benndorf et al. 1994
). Because it is possible that host cells used for transfection confer to expressed exogenous channels properties that are not present in native channels (see also Shi et al. 1994
), it is of interest to examine the properties of channels in their tissue of origin. A quantitative study of potassium channels in native tissues however, is sometimes complicated by the presence of multiple overlapping ionic currents.
The human neuroblastoma cell line SH-SY5Y, established by repetitive subcloning of the SK-N-SH cell line (Biedler et al. 1978
), exhibits the morphological and biochemical features of cells derived from the neural crest, like sympathetic neurons (Barnes et al. 1981
; Ross et al. 1981
). These cells can be induced to acquire a neuronal phenotype by prolonged treatment, with the differentiating agent retinoic acid (Påhlman et al. 1984). Because of their capacity to differentiate in mature ganglion-like cells and to their cellular homogeneity, SH-SY5Y cells are a highly suitable model for studying the role ion channels play in the excitability of cells of human origin. The use of this cell line would also permit to combine molecular biological and electrophysiological approaches.
It was previously shown that in SH-SY5Y cells depolarizing voltage steps initially evoke a fast activating and full inactivating inward current, followed by an outward current component (Brown et al. 1994
; Johansson 1994
; Toselli et al. 1996
). The inward current, identified as a tetrodoxin (TTX)-sensitive Na+ conductance, was studied in detail, and changes in its gating properties during differentiation were related to the ability of SH-SY5Y differentiated cells to generate overshooting action potentials (Toselli et al. 1996
).
The aim of the present study was to characterize and compare the nature, gating properties, and electrophysiological role of the native voltage-dependent outward current in both undifferentiated and differentiated SH-SY5Y cells. Channels expressed in the two types of cells were found to differ in their electrophysiological parameters, which might be related to the observed distinct ability of the two cell types to generate regular action potentials.
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METHODS |
Cell culture
SH-SY5Y cells were grown as monolayer in RPMI 1640 medium (GIBCO) supplemented with 10% heat inactivated fetal calf serum, 100 µg/ml streptomycin and 100 IU/ml penicillin. The medium was replaced three times a week. Cells were cultured in 75 ml plastic flasks in a 5% CO2 atmosphere at 37°C; when confluent they were split in 35-mm plastic Petri dishes to be used for electrophysiological experiments. Differentiation of SH-SY5Y cells was achieved by treatment with 10 µM retinoic acid (RA) (Påhlman et al. 1984). Differentiated cells were used after 14-18 days of RA treatment to obtain a high percentage of cells that showed clear morphological differentiation (Påhlman et al. 1984). Undifferentiated cells were used 2-6 days after plating and no RA was added.
Solutions
During voltage- and current-clamp recording the cells were bathed in 125 mM NaCl, 4 mM KCl, 2 mM CaCl2, 1.2 mM MgSO4, 10 mM glucose, 0.3 µM TTX, 10 mM N-2-hydroxyethylpiperazine-N
-2-ethanesulfonic acid (HEPES)/NaOH (pH 7.4). The patch pipette contained 140 mM KCl, 4 mM NaCl, 0.02 mMCaCl2, 0.8 mM ethylene glycol-bis(
-aminoethyl ether)N,N,N
,N
-tetraacetic acid (EGTA), 2 mM MgCl2, 4 mM Mg-ATP, 10 mM HEPES/KOH (pH 7.4). Contamination by voltage-gated calcium currents was negligible in most undifferentiated cells but sizeable in most differentiated cells; 200 µM CdCl2 was therefore added to the bath to suppress calcium currents when present. Chemicals were purchased from Sigma Chemical (St. Louis, MO). Changes of extracellular solutions were obtained by a fast multibarrel delivery system positioned close to the cell tested.
Electrophysiology
Electrophysiological recordings were carried out by using the whole cell patch clamp configuration (Hamill et al. 1981
). Experiments were performed at room temperature (21 ± 2°C).
Stimulation, acquisition, data analysis, and curve fitting were performed with pCLAMP software (Axon Instruments, Burlingame, CA) and the packages ASYSTANT (Macmillan Software, New York) and ORIGIN (Microcal Software, Northampton, MA). Linear components of leak and capacitive currents were first reduced by analog circuitry and then almost completely cancelled with the P/N method. Patch pipettes had a resistance of 3-6 M
and occasionally, especially in current clamp experiments, their tips were coated with Sylgard to reduce capacitance.
Experiments were performed on small cells with short processes to guarantee sufficient voltage and space clamp control. Cells were discarded if current tracings showed signs of notchlike discontinuities. A sampling interval of 25 µs/point and series resistance compensation of 60-70% were applied when tail currents were studied. Currents were filtered at 3 kHz.
 |
RESULTS |
Under voltage-clamp conditions and using the solutions described in METHODS, a series of depolarizing voltage steps from
90 mV to potentials between
30 and +80 mV elicited an outward current in both undifferentiated and differentiated SH-SY5Y cells, as shown, respectively, in Fig. 1, A and B. This current was observed in ~93% of the clamped cells (n = 293). In undifferentiated cells, currents activated at about
30 mV, activated with sigmoidal time course and showed a slow but sizeable inactivation, particularly evident at potentials positive to +30 mV. In differentiated cells, the outward conductance activated at voltages positive to
20 mV and its time course of activation was similar to that observed in undifferentiated cells but its inactivation was less evident.

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| FIG. 1.
Voltage-dependent activation of outward currents in undifferentiated and differentiated SH-SY5Y cells. A and B: superimposed current traces were elicited at potentials indicated, from a holding potential of 90 mV, in an undifferentiated SH-SY5Y cell (A) and in a cell differentiated with retinoic acid (B), using extra- and intracellular saline appropriate to isolate K current (see METHODS). C: average of peak amplitude of outward current densities against voltage obtained from undifferentiated cells ( ,n = 11) and from differentiated cells ( , n = 13). Bars denote standard deviations; holding potential, 90 mV.
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In Figure 1C the current-voltage relations are shown. They were obtained by averaging current density, obtained dividing peak current amplitude by cell area, measured at each test potential in 11 undifferentiated (
) and 13 differentiated (
) cells. In both types of cells the outward current displayed inward rectification
more pronounced in undifferentiated cells
at voltages positive to +60 mV.
Currents elicited from both undifferentiated and differentiated cells were suppressed by replacing K+ with 140 mM Cs+ in the patch pipette (n > 60) (see also Toselli et al. 1996
), or by adding 20 mM tetraethylammonium (TEA) to the extracellular solution (Fig. 2A). On the other hand, focal perfusion with 5 mM 4-aminopyridine (4-AP) or lowering the holding potential from
90 to
40 mV had little or no effect on these currents, as shown in the sample tracings of Fig. 2, B and C, respectively. The peak current measured at +50 mV was 98 ± 4% of control after application of 4-AP (n = 6) and it was 91 ± 7% of control after shifting from a Vh of
90 mV to a Vh of
40 mV (n = 6). Furthermore, addition to the extracellular solution of 200 µM Cd2+, which blocks completely voltage-dependent calcium channels in SH-SY5Y cells (Toselli et al. 1991
), affected neither the shape nor the amplitude of the outward current, as shown in Fig. 2D (100 ± 3% of control at +50 mV, n = 4).

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| FIG. 2.
Pharmacology of outward currents in SH-SY5Y cells. A: block of outward current by extracellular tetraethylammonium (TEA, 20 mM) in an undifferentiated cell. B: effect of extracellular 4-aminopyridine (4-AP, 1 mM) on outward current of an undifferentiated cell. C: effect of change of holding potential from 90 to 40 mV on outward current of an undifferentiated cell. D: action of cadmium (200 µM) on outward current of an undifferentiated cell. Test potential was +50 mV for all cells. A, B, and D: outward currents recorded in control saline before and after drug application are labeled Contr and Wash, respectively.
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The time course and voltage-dependence of activation, the absence of a sizeable decrease in current amplitude at a given test potential after changing the holding potential from
90 to
40 mV, and the pharmacological evidences previously described strongly suggest that the outward current elicitable in both undifferentiated and differentiated SH-SY5Y cells is classifiable as a delayed rectifier potassium current (IK) and also exclude contamination from fast inactivating (IA) or calcium-activated potassium conductances.
Conductance and steady-state activation
The voltage dependence of IK steady-state activation was studied using the following protocol: a test potential of amplitude variable from
30 to +80 mV was applied and lasted the appropriate time for the current to reach its maximum; then the test pulse was followed by a repolarizing pulse to
50 mV. An example of currents elicited using this protocol is shown in Fig. 3A.

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| FIG. 3.
Steady-state activation parameters of potassium current. A: tail currents associated with step repolarization to 50 mV from depolarizing pulses of various amplitudes. Duration of depolarizing pulses was that at which current reached a peak at different potential levels, as indicated in protocol. B: normalized steady-state conductance (gnorm) against membrane potential as derived from tail current measurements like those shown in (A). Data points were interpolated by an equation of form: gnorm = 1/{1 + exp[(Vm + V1/2)/k]}, where V1/2 = 19.5, k = 13.8 for undifferentiated cells and V1/2 = 37.0 and k = 14.8 for differentiated cells. C: steady-state activation parameter (ninf) vs. membrane potential. Data points were interpolated by an equation of form: ninf = 1/{1 + exp[(Vm + V1/2)/k]}, where V1/2= 5.5, k = 19.0 for undifferentiated cells and V1/2 = 8.2 and k = 21.1 for differentiated cells. Data in B and C are represented as means ± SD forn = 7 undifferentiated cells ( ) and n = 13 differentiated cells ( ).
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Tail currents, generated by relaxation of IK on return from different test potentials to
50 mV, could be fitted by a single exponential function. The amplitude of IK at the beginning of the repolarizing step was determined by extrapolating the current tail at time 0 of repolarization and correcting for current inactivation at the time of test pulse offset, following the equation h(t) = hinf
(hinf
ho) exp(
t/
h) (see Steady-state inactivation and Kinetics of inactivation). IK was converted to the conductance value (gK) with the following equation
|
(1)
|
where Vm is the repolarization potential and EK is the potassium equilibrium potential (
89.6 mV in the ionic conditions used for this study).
The instantaneous current-voltage (I-V) relation was determined by using the protocol illustrated in Fig. 4A and measuring the tail current amplitudes at 0.3 ms after the repolarization onset. The instantaneous I-V relation was linear between
60 and 0 mV in both differentiated and undifferentiated cells (not shown) and the use of the above equation was therefore justified. The reversal potential evaluated from the instantaneous I-V relation was
77 ± 2 mV for differentiated cells (n = 7) and
76 ± 3 mV for undifferentiated cells (n = 12); both potentials are ~10 mV more depolarized to the theoretical value given by Nernst equation; this discrepancy could be because of an intracellular K+ concentration lower than expected.

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| FIG. 4.
Activation kinetics of K currents. A: time course of deactivation of K currents at different repolarizationpotentials in an undifferentiated cell. Membrane repolarization at potentialsindicated followed a step depolarization to +50 mV of 3.5 ms duration from a holding potential of 90 mV; pulse protocol is indicated over current traces. A period of 300 µs was blanked after termination of test pulses. Tail currents were fitted by nonlinear least-squares curve fit with Hodgkin-Huxley type equation:Itail = A[ninf (ninf n0) exp( t/ n)]3[hinf (hinf h0) exp( t/ h)]. n values measured at 60, 40, 20, 0, and +20 mV were respectively 3.52, 4.71, 6.43, 3.50, and 2.18 ms. B: onset of potassium currents in an undifferentiated cell on depolarization at different test potentials, fitted with a power function of third order; pulse protocol is indicated over current traces. n values measured at 10, 0, +10, +20, +30, +40, and +50 mV were respectively 4.83, 4.73, 2.75, 1.81, 1.28, 1.22, and 1.09 ms. C: voltage dependence of time constants of activation ( ) and deactivation ( ) represented as means ± SD of 8 undifferentiated cells. ( ): least-squares best fit withequation n = 0.75 + 1/[0.16·exp(V·0.06) + 0.05·exp( 0.03·V)]. D: voltage dependence of time constants of activation ( ) and deactivation ( ) represented as means ± SD of 6 differentiated cells.( ): least-squares best fit with equation: n = 0.95 + 1/[0.16·exp(V·0.05) + 0.09·exp( 0.02·V)].
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For each cell tested, the maximal value of conductance (gmax) was evaluated by interpolating the sets of conductance values with the Boltzmann type equation gK = gmax/{1 + exp[(Vm
V1/2)/k]}. The mean gmax values obtained were4.3 ± 3.2 nS (0.34 ± 0.21 mS/cm2, obtained by averaging single densities) for undifferentiated cells (n = 7) and15.2 ± 8.1 nS (0.78 ± 0.50 mS/cm2) for differentiated cells (n = 13), the differences being statistically significant (Student's t-test, unpaired, confidence level 0.05). The gmax value for each cell was used to calculate the normalized conductance (gnorm), and the mean gnorm values from differentiated and undifferentiated cells are plotted versus membrane potential in Fig. 3B. When sets of gnorm values measured for each cell were fitted individually to a Boltzmann function, the mean parameters of the function were as follows: for undifferentiated cells, V1/2= 22.1 ± 1.2 and k =
14.1 ± 1.0; for differentiated cells V1/2= 37.0 ± 4.4 and k =
14.4 ± 1.5. The 15-mV difference in V1/2 between undifferentiated and differentiated cells was statistically significant (t-test, confidence level 0.05).
According to the Hodgkin and Huxley model (Hodgkin and Huxley 1952
) the conductance gK could be described by the equation
|
(2)
|
where ninf is the steady-state value of the activation parameter n. In both undifferentiated and differentiated cells the activating phase of the potassium current was best fitted by a single exponential raised to the third power (x = 3) and therefore the steady state activation parameter resulted equal to ninf = (gK/gmax)1/3.
Mean ninf values for undifferentiated and differentiated cells are drawn versus membrane potential in Fig. 3C. For each cell the ninf curve was best fitted by a Boltzmann equation and the average half activation potentials resulted to be
4.6 ± 7.2 mV (undifferentiated cells, n = 7) and 11.3 ± 6.5 mV (differentiated cells, n = 13), while the average slope factors were
17.3 ± 2.3 mV and
20.6 ± 2.4 mV, respectively.
Kinetics of activation and deactivation
To investigate the activation kinetics of potassium currents in the voltage range between
90 and +80 mV, the values of the time constant of activation (
n) were estimated by using two separate methods.
The first method measured
n values in the voltage range between
70 and +20 mV from relaxation of IK during repolarization to variable potentials after a 6 ms depolarizing step to +50 mV. The pulse protocol and tail currents obtained at five distinct repolarizing potentials are illustrated in Fig. 4A.
n values were then obtained by fitting potassium tail currents (nonlinear least squares curve fit) with a Hodgkin and Huxley type equation (Hodgkin and Huxley 1952
)
|
(3)
|
where A = Gmax(V
VK), n0, and h0 are the n and h values determined at the end of the depolarizing pulse and ninf, hinf, and
h are the activation and inactivation parameters at the repolarization potential. n, h, and
h parameters were fixed when experimentally available, otherwise they were free to vary between reasonable upper and lower bounds while fitting tail currents.
In the voltage range examined, the time constant of deactivation was voltage dependent for both undifferentiated and differentiated cells:
n increased with voltage and peaked at
20 mV for both cell types [
n = 8.2 ± 3.5 ms (n = 8) and 6.2 ± 2.6 ms (n = 6), respectively].
In the voltage range between
30 and +50 mV for undifferentiated cells and between
20 and +50 mV for differentiated cells
n was calculated directly from the activation phase of IK. To this end, current tracings were fitted by a function of the type used for calculation of
n from tail currents. Similar values of
n were also obtained by fitting tracings with an exponential function raised to the third power
(see tracings in Fig. 4B), suggesting that, in the voltage and time range examined, the contribution of inactivation parameters to calculation of
n was negligible. Between
20 and +50 mV,
n decreased monotonically in both undifferentiated and differentiated cells:
n = 8.6 ± 3.2 ms at
20 mV and 1.2 ± 0.3 ms at +50 mV for undifferentiated cells (n = 8), while for differentiated cells
n = 5.7 ± 0.6 ms at
20 mV and 1.4 ± 0.5 ms at +50 mV (n = 6).

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| FIG. 5.
Voltage dependence of potassium current steady-state inactivation. A: superimposed current traces, elicited at +50 mV, were obtained from an undifferentiated cell (top) and from a differentiated cell (bottom) after conditioning potentials of variable amplitude and 660 ms duration; pulse protocol is indicated over current traces. B: average steady-state inactivation (hinf) vs. membrane potential obtained by applying protocol shown in (A) to 12 undifferentiated cells ( ) and to 16 differentiated cells ( ). To obtain values of hinf, peak currents at +50 mV were normalized to their largest measured values. Continuous lines through data points are least squares best fit of Boltzmann type equation: hinf = S + (1 S)/{1 + exp[(V + V1/2)/k]} where S = 0.09, V1/2 = 14.0 mV, andk = 10.8 mV for undifferentiated cells and S = 0.36, V1/2 = 9.5 mV, and k = 14.0 mV for differentiated cells. Bars indicatemeans ± SD.
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Mean
n values for undifferentiated and differentiated cells, obtained from both activation and deactivation and plotted versus membrane potential are shown in Fig. 4, C and D, respectively.
Steady-state inactivation
The voltage dependence of IK steady-state inactivation(hinf) was studied by measuring the peak potassium current elicited at +50 mV after a conditioning potential of varying amplitude and 660 ms duration; this protocol allowed the inactivation variable to reach its steady-state value at any investigated potential. hinf was determined by normalizing the peak current to the maximum current evoked with a conditioning hyperpolarizing step to
90 mV. The voltage protocol and two families of current tracings obtained from an undifferentiated and a differentiated cell are shown in Fig. 5A.

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| FIG. 6.
Time course of potassium current inactivation at potentials indicated in an undifferentiated cell (A) and in a differentiated cell (B). Continuous lines superimposed to current tracings represent best fit obtained from least-squares nonlinear regression analysis to a single exponential function. h is 106, 72, and 50 ms, respectively, at 0, +20, and +40 mV for undifferentiated cell and 415, 200, and 168 ms in differentiated cell.
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In Fig. 5B, average steady-state inactivation values for both undifferentiated and differentiated SH-SY5Y cells are plotted versus conditioning potential. Inactivation had a threshold of about
30 mV in both undifferentiated and differentiated cells; in neither of the two types of cells IK however reached full inactivation, even at very positive potentials: at +60 mV, IK was 11 ± 4% of control in undifferentiated cells (n = 12) and 40 ± 15% of control in differentiated cells (n = 16), the difference being statistically significant (t-test, confidence level 0.05). Both steady-state inactivation curves showed a sigmoidal dependence on voltage that fitted well the Boltzmann type function
|
(4)
|
where V1/2 is the voltage at which the steady-state variable hinf is half-maximal, k is a slope factor and S is a dimensionless variable that represents the noninactivating portion of the current. Averaging the values obtained from single cell fits, the mean parameters resulted to be as follows: for undifferentiated cells V1/2 =
13.8 ± 6.7, k = 9.6 ± 3.5 and S = 0.086 ± 0.042 (n = 12); for differentiated cells, V1/2 =
9.7 ± 5.8,k = 13.2 ± 5.2 and S = 0.365 ± 0.151 (n = 16).

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| FIG. 7.
Voltage dependence of potassium current inactivation kinetics. A and B: time course of recovery from inactivation of K channels at repolarization potentials of 100 and 40 mV for an undifferentiated cell (A) and at repolarization potential of 60 mV for a differentiated cell (B). Currents were measured on depolarization to +50 mV after a prepulse of variable amplitude (Vc) and duration ( t), as shown by pulse protocol over current tracings. C: onset of recovery from inactivation obtained from current tracings shown in A and B as indicated: peak potassium currents at +50 mV were plotted vs. prepulse duration. Continuous lines represent best fit obtained from least-squares nonlinear regression analysis to a single exponential function for recovery at 100 mV ( = 44 ms), 40 mV ( = 230 ms), and 60 mV ( = 30 ms). D: time constants of inactivation ( h) vs. membrane potential. h values for potentials negative to 40 mV were measured as indicated in C (n = 6 for undifferentiated cells and n = 7 for differentiated cells); h values positive to 20 mV were derived from decay of K current (n = 11 for undifferentiated cells and n = 12 for differentiated cells). Bars indicate means ± SD. ( ): least-squares best fit with equations: h = 45.5 + 10100/[61.1·exp(V·0.033) + 0.063·exp( 0.146·V)] for undifferentiated cells and h = 20.0 + 8943/[30.5·exp(V·0.029) + 0.091·exp( 0.177·V)] for differentiated cells.
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Kinetics of inactivation
At voltages positive to
20 mV the time constant of inactivation (
h) was evaluated by fitting a single exponential to the falling phase of the current trace elicited at each test potential with a 660 ms step, as shown in the sample tracings of Fig. 6. The values of
h obtained with this method decreased monotonically with increasing voltages for both undifferentiated and differentiated cells. In a few differentiated cells the time course of inactivation displayed a biexponential decay, and a
h at least 10 times slower was also detectable (not shown). Because this slow
h was observed only occasionally, it was not taken into further consideration.
The time course of the recovery from inactivation of IK was evaluated in the voltage range between
100 and
40 mV. Sample tracings of IK recorded on step depolarizations to +50 mV after conditioning pulses of variable duration to hyperpolarizing potentials are shown in Fig. 7, A and B. Peak potassium currents obtained from these sample tracings were plotted as a function of the duration of the conditioning pulse (Fig. 7C). In both undifferentiated and differentiated cells, recovery from inactivation had a single exponential time course at all voltages tested. The averaged values of the time constants of inactivation and recovery from inactivation expressed as a function of membrane potential are shown in Fig. 7D.
Electrophysiological role of the outward conductance
It is known that, in SH-SY5Y cells, voltage-gated Na+ conductances are present and are involved in the generation of overshooting action potentials in differentiated cells (Johansson 1994
; Toselli et al. 1996
) or abortive action potentials in undifferentiated cells (Toselli et al. 1996
). Hereby we investigated whether or not the potassium conductance previously described is also involved in the excitability of these cells. Indeed, in the few cells expressing a neglegible K current, the shape of the spikes or spikelets elicited under current clamp conditions was dramatically altered, because of the lack of the repolarizing phase. In cells where both the Na and the K conductance were available, the same effect could be reversibly obtained by adding 20 mM TEA to the extracellular solution (Fig. 8A). This clearly demonstrates that the outward current is the one responsible for the repolarizing phase of spikes and abortive action potentials in differentiated and undifferentiated cells respectively.

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| FIG. 8.
Action potentials generated by SH-SY5Y cells under current-clamp conditions. A: superimposed voltage responses were obtained using intra- and extracellular saline for current clamp experiments as described in METHODS, before, during, and after focal application of 20 mM TEA. Cell was stimulated with rectangular current pulses of 200 pA amplitude. B: voltage responses to a current pulse stimulus, as indicated on top, for an undifferentiated cell (top) and for a differentiated cell (bottom). Note different repolarizing phases in two cases. C: voltage response to prolonged current injection, as indicated on top, in a differentiated cell. Holding potential in A-C was about 80 mV.
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In a previous work we have shown that the rate of rise of action potentials increases during cell differentiation, as a consequence of changes in sodium channel gating (Toselli et al. 1996
). Here the decaying phase of action potentials was investigated. In undifferentiated cells the average slope of spikelet decay (dV/dt) was
4.3 ± 1.7 mV/ms (n = 24), while in differentiated cells the slope of action potential decay was
11.5 ± 4.7 mV/ms (n = 28) (Fig. 8B). Cell capacity (Cm), measured in the same two groups of cells, was 12.5 ± 4.3 pF and 21.3 ± 7.3 pF in undifferentiated and differentiated cells, respectively. Because the rate of repolarization was about three times greater and Cm nearly doubled in differentiated cells, we can conclude that the repolarizing capacitive current (Ic= -Cm · dV/dt) was more than threefold faster in cells treated with retinoic acid. Finally, whereas multiple firing or even multiple spikelet generation was never observed in any undifferentiated cell in response to prolonged stimulations, trains of action potentials could be elicited in a fraction (5/7) of differentiated cells (Fig. 8C).
 |
DISCUSSION |
The present results show that the outward current measurable from the human neuroblastoma cell line SH-SY5Y is carried by potassium ions, on account of its reversal potential and of its block by external TEA and intracellular cesium ions.
In both differentiated and undifferentiated SH-SY5Y cells, this conductance had features apparently similar to the features of a "delayed rectifier" potassium conductance, according to its threshold and time course of activation and to its pharmacological properties. The potassium current was almost insensitive to 4-AP and cadmium and its amplitude did not change significantly on shifting the holding potential shift from
90 to
40 mV, indicating no sizeable contamination from A-type or calcium-dependent potassium conductances. A detailed analysis of kinetic properties and voltage dependence of activation and inactivation showed however differences between the currents measured in undifferentiated and differentiated cells.
Comparison of potassium-current properties in undifferentiated and differentiated cells
In undifferentiated cells, the potassium current was relatively small in amplitude; the current amplitude increases significantly in differentiated cells: maximum conductance changes from 4.3 to 15.2 nS. Because of a gradual increase in cell surface during differentiation, current density increases only about a factor of two (from 0.34 to 0.78 mS/cm2) but the difference is still statistically significant. These values are similar to those measured in guinea pig hippocampal neurons (Sah et al. 1988
), undifferentiated IMR-32 neuroblastoma cells (Ginsborg et al. 1991
), and rat suprachiasmatic nucleus neurons (Bouskila and Dudek 1995
) but are more than 10-fold smaller than those measured in N1E-115 differentiated neuroblastoma cells (Moolenaar and Spector 1978
) and rat sympathetic neurons (Belluzzi and Sacchi 1988
). This fact could partly reflect a relatively low channel density even in differentiated cells. An increased expression of the related gene might be responsible of the higher K+ conductance after differentiation.
At both stages of differentiation the time course of activation was sigmoidal and best described by a power function of the third order. In differentiated cells however, we measured a shift of the potential for half-maximal activation of ~15 mV toward more positive potentials, compared with that of undifferentiated cells (see Fig. 3, B and C).
Kinetics of activation and deactivation looked similar in both differentiated and undifferentiated cells. In both cases the relation between time constant of activation and voltage was bell shaped with a maximum at about
20 mV. No significant differences between the two sets of values were measured in the two types of cells.
Potassium currents displayed a relatively slow but sizeable voltage- and time-dependent inactivation in both undifferentiated and differentiated cells. This has been considered to be a true gating property of the channels and not an artifact caused by K+ accumulation; although a small reduction in EK cannot be excluded on prolonged depolarization, such effect cannot account for the magnitude of the outward current decline and for its voltage and time dependence.
Our results show that both steady-state properties and kinetics of potassium current inactivation changed during differentiation. For voltages positive to
30 mV the amount of steady-state current inactivation was progressively greater in undifferentiated cells than in differentiated cells; for instance at +60 mV steady-state inactivation was almost complete in undifferentiated cells, while it reached <70% in differentiated cells (see Fig. 5B).
In both types of cells, current inactivation was a relatively slow process when compared with activation: the smallest values of
h were measured between
100 and
60 mV (~20 ms in undifferentiated cells and 50 ms in differentiated cells) and the highest values at about
20 mV (333 and 517 ms in undifferentiated and differentiated cells, respectively). The time constants of inactivation (
h) displayed a bell shaped voltage dependence in both differentiated and undifferentiated cells;
h curve for undifferentiated cells, however, reached a maximum at about
30 mV, whereas for differentiated cells the curve was shifted by ~10 mV toward more positive potentials, with a maximum at about
20 mV.
In a recent characterization of potassium currents in differentiated SH-SY5Y cells, Johansson et al. (1996)
reported values for steady-state activation and inactivation much closer to those that we measured in undifferentiated cells. Furthermore, they reported a great variability from cell to cell concerning the time course of inactivation. These discrepancies are probably the result of the much longer period of differentiation of the cells tested in this study. With respect to this, it is noteworthy to observe that expression of functional voltage-gated Ca channels becomes also maximal and some gating properties of TTX-sensitive sodium channels change significantly after treatment of SH-SY5Y cells with RA for 15 days or longer (Toselli et al. 1991
, 1996
).
In conclusion, the outward conductances expressed in differentiated and undifferentiated cells display several distinct biophysical properties. Most of the features that characterize the K+ conductance of differentiated cells, rather than those observed in undifferentiated cells, are similar, at least qualitatively, to those displayed by several native potassium channels in mature mammalian neurons (Belluzzi and Sacchi 1988
; Bouskila and Dudek 1995
; Sah et al. 1988
).
Channel properties alterations during differentiation could be the result of expression of different channel subunits (Swanson et al. 1990
). Alternatively, different posttranslational processes could also contribute to generate potassium channels with distinct properties. For instance, Covarrubias et al. (1994)
found that protein kinase C (PKC) specifically eliminates rapid inactivation of the cloned hKv3.4 K+ channel and that mutating one serine to aspartic acid in the N-terminal domain mimics the action of PKC. Concerning activation, it was shown that deletions at the C-terminus resulted in a shift of the voltage dependence of activation of drk1 channels (Van Dongen et al. 1990
).
Electrophysiological implications
The change of the electrophysiological properties of the potassium conductance during in vitro differentiation of SH-SY5Y cells is not a surprising result, because a similar event also occurs to the TTX sensitive Na+ conductance present in the same cell line. Furthermore it was shown that alterations in sodium channel gating properties during differentiation are paralleled by the ability of these cells to generate overshooting action potentials (Toselli et al. 1996
). Concerning the potassium conductance studied here, the marked prolongation of the action potential during focal application of TEA (see Fig. 8A) indicates that this current is responsible for the falling phase of action potential. Furthermore the different features acquired by the potassium conductance during prolonged treatment with retinoic acid might contribute, together with the changes in sodium channel gating, to modify the excitability properties of these cells. This hypothesis is corroborated by the following observations: 1) during differentiation, potassium channel activation is shifted by ~20 mV toward positive potentials; this could contribute to speed up the depolarizing phase of action potential and delay repolarization; 2) in differentiated cells, the extent of steady-state inactivation at positive potentials is much lower, current inactivation is slower and recovery from inactivation is faster than in undifferentiated cells; 3) a sizeable increase in the outward current occurs during differentiation. These observations suggest that in differentiated cells repolarization could be faster and more complete after an action potential. Indeed, in current-clamp experiments, we have measured in differentiated cells an action potential repolarization rate about threefold faster than that measured in the abortive action potentials observed in undifferentiated cells. A faster and more pronounced repolarization might also contribute to generate trains of spikes during prolonged stimulation. Indeed this firing pattern was observed in some differentiated cells but never in undifferentiated cells. These simple considerations can give a qualitative explanation of the contribution of the studied potassium conductances to changes in excitability during differentiation in SH-SY5Y cells.