Voltage-Dependent Gating of ATP-Activated Channels in PC12 Cells
Ken Nakazawa,
Min Liu,
Kazuhide Inoue, and
Yasuo Ohno
Division of Pharmacology, National Institute of Health Sciences, Setagaya, Tokyo 158, Japan
 |
ABSTRACT |
Nakazawa, Ken, Min Liu, Kazuhide Inoue, and Yasuo Ohno. Voltage-dependent gating of ATP-activated channels in PC12 cells. J. Neurophysiol. 78: 884-890, 1997. The possibility that P2X receptors exhibit voltage-dependent gating in a similar manner to nicotinic receptors was investigated in rat pheochromocytoma cells with the use of whole cell voltage-clamp techniques. In the presence of extracellular ATP, slowly activating inward currents were elicited by stepping from
50 mV to potentials more negative than
80 mV; these currents had a time constant of ~60 ms at
120 mV. This slowly activating component (as a fraction of the total ATP-induced current) increased with membrane hyperpolarization from
80 to
100 mV and was much increased when depolarizing prepulses were applied, although the time constant of activation was not altered by depolarizations. The fraction of the slowly activating current and its time constant were decreased as the ATP concentration was increased from 10 to 300 µM. Thus it has been demonstrated that voltage-dependent gating of ATP-activated channels does occur in PC12 cells, and that this gating is modified by agonist concentration. It is possible that such gating may serve as a postsynaptic mechanism to facilitate excitatory neurotransmission by contributing to the inward rectification of the ATP-activated currents.
 |
INTRODUCTION |
Extracellular ATP has been regarded as one of the neurotransmitters in neuronal, muscular, and nonexcitable tissues (for reviews see Abbracchio and Burnstock 1994
; Dubyak and El-Moatassim 1993
; Edwards and Gibb 1993
; Evans and Surprenant 1996
). Fast neurotransmission by extracellular ATP is achieved by the activation of nonselective cation channels in postsynaptic membrane. These ATP-activated channels have now been termed "P2X receptors," and cDNAs encoding the members of this receptor/channel family have recently been successfully cloned (for reviews see North 1996
; Surprenant et al. 1995
). Physiological significance of the ATP-activated channels in autonomic neurons, sensory systems, and the CNS has also been suggested (e.g., Dubyak and El-Moatassim 1993
; Edwards and Gibb 1993
; Thorne and Housley 1996
).
Among ligand-gated channels, nicotinic acetylcholine receptor channels have been shown to exhibit a voltage-dependent gating. This phenomenon was first demonstrated by Magleby and Stevens (1972)
from an observation of slowing of the decay time course of end-plate currents at negative potentials, and has been well characterized by various investigators (e.g., Ascher et al. 1978
; Auerbach et al. 1996
; Colquhoun and Sakmann 1985
; Marchais and Marty 1979
). As for the ATP-activated channels, the existence of such voltage-dependent gating was speculated from the observation of time-dependent increase in the current on hyperpolarizing steps under whole cell voltage clamp (Nakazawa 1994
), but quantitative analysis has not been attempted. The comparison of the voltage-dependent gating of the ATP-activated channels with that of nicotinic acetylcholine receptors may also be of interest because the molecular structure of the ATP-activated channels deduced from the cloned cDNAs was completely different from that of the so-called "ligand-gated channel superfamily" including nicotinic receptors (North 1996
; Surprenant et al. 1995
). For the present study, we therefore aimed at clarifying basic properties of the voltage-dependent gating of ATP-activated channels in rat pheochromocytoma cells, where the properties of the ATP-activated channels (Nakazawa et al. 1990
, 1991
) are similar to those in peripheral neurons (e.g., Evans et al. 1992
; Nakazawa 1994
) and the expression of neuronal type P2X receptors has been demonstrated (Brake et al. 1994
; Wang et al. 1996
).
 |
METHODS |
PC12 cells (passage 55-70) were cultured according to Inoue and Kenimer (1988)
. Cells were plated on collagen-coated coverslips placed on the bottom of 35-mm polystyrene dishes. Current recordings were made with conventional whole cell voltage-clamp methods (Hamill et al. 1981
) under the conditions described elsewhere (Nakazawa et al. 1990
). The cells were placed in an experimental chamber (volume ~1 ml) and bathed in an extracellular solution containing (in mM) 140 NaCl, 5.4 KCl, 1.8 CaCl2, 1.0 MgCl2, 10 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid (HEPES), and 11.1 D-glucose, pH adjusted to 7.4 with NaOH. Tip resistances of heat-polished patch pipettes ranged between 3 and 5 M
when the pipettes were filled with an intracellular solution containing (in mM) 150 CsCl, 10 HEPES, and 5 ethyleneglycol-bis(2-aminoethylether)-N,N,N
,N
-tetraacetic acid, pH adjusted to 7.3 with CsOH. Application of ATP was made from an emitting tube (2 mm ID). Cells located near the mouth of the emitting tube (~1 mm distance) were selected for recordings, and the ATP-containing extracellular solution was applied rapidly (~0.4 ml/s). To avoid desensitization of ATP-activated channels, the period of each ATP application was brief (~10 s), and each application was separated by 1 min. Experiments were performed at room temperature (~25°C). Electrical signals were recorded with a patch-clamp amplifier (Nihon Kohden CEZ-2400, Tokyo, Japan), filtered at 5 kHz, and stored on magnetic tape for later analysis.
Off-line analysis of membrane current was made with the use of software for patch/whole cell clamp data (Nihon Kohden, QP-120J) on a personal computer (NEC PC9801RA2). Data were sampled at 1 kHz. Activation kinetics of the current was determined by plotting sampled data in a logarithmic manner, and time constants were calculated with the use of the least-square method programmed in the software. When determining time constant of the currents with hyperpolarizing steps, the current amplitude was measured as differences from that at the end of 200-ms steps. Because the activation time constants were normally <70 ms in the present study, it is assumed that >94% of channels should have been activated by the end of a 200-ms voltage step. For current traces with relatively rapid activation kinetics, the amplitude of voltage-dependent component was routinely measured from 2 ms after the beginning of voltage steps.
All the data are given as means ± SE.
Drugs
ATP (ATP disodium salt) was purchased from Sigma (St. Louis, MO). All the other compounds were of a reagent grade.
 |
RESULTS |
Voltage-dependent slow component of ATP-activated current
Figure 1A compares current traces in response to a ramp pulse changing from
80 to +40 mV before (control) and during the application of 30 µM ATP (+ATP). The current-voltage relationship was obtained for the ATP-activated current by subtracting the current without ATP from that with ATP (Fig. 1B). The ATP-activated current exhibited a strong inward rectification, as previously reported (Nakazawa et al. 1990
, 1991
), and the outward current component at positive potentials was not clearly resolved.

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| FIG. 1.
Current-voltage relationship for ATP-activated current. Currenttraces obtained with ramp pulse changing from 80 to +40 mV (0.5 V/s)before (control) and during application of 30 µM ATP (+ATP) in PC12 cell are shown. B: difference of these 2 current traces.
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Figure 2A illustrates current traces obtained with a hyperpolarizing step to
120 mV from a holding potential of
50 mV. The current trace during the application of 10 µM ATP (+ATP) is superimposed with the trace just before the ATP application (control). The current at
120 mV did not instantaneously reach its steady-state level, but gradually increased with time. This slowly activating component (Fig. 2A, Islow) was observed in all the cells tested when they were stepped to more negative than
80 mV. In Fig. 2B, the slowly activating current is normalized to the instantaneous current at the beginning of the hyperpolarizing steps and plotted against membrane potential. The relative amplitude of the slow current was increased at more negative potentials in a range between
80 and
100 mV, but at potentials more negative than
100 mV it was not further facilitated. The results suggest that 1) voltage-dependent gating occurs to the ATP-activated channels, 2) the gate is more permissive at more negative potentials, and 3) the gating is saturated around
100 mV.

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| FIG. 2.
Slowly activating component of ATP-activated current observed when hyperpolarizing steps were applied from holding potential of 50 mV. ATP (10 µM) was administered for 10 s while a hyperpolarizing step was applied to cells every 2 s. A: superimposition of current traces before (control) and during application of ATP (+ATP). Cell was hyperpolarized to 120 mV in this case. Note current component slowly appearing during hyperpolarizing step (Islow). Dashed line: 0 current level. Vm, membrane potential; Im, membrane current. B: slow current component at various hyperpolarizing test potentials. Slow component was normalized to remaining instantaneous component at each potential and plotted against test potentials. Each symbol and bar represent mean and SE from 5 cells tested.
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Figure 3 illustrates current traces obtained with a depolarizing prepulse to +100 mV. A current trace obtained with a prepulse to
100 mV is superimposed for comparison. At
120 mV, the amplitude of the slowly activating current was remarkably larger with the prepulse to +100 mV than with the prepulse to
100 mV (compare double-headed arrows), although the absolute current level at the end of the hyperpolarization was still more inward with the prepulse with
100 mV. Similar results were obtained from four cells tested. The results suggest that the increased slow component with depolarization is not due to activation of some additional inward current, but that the voltage-dependent gate of the ATP-activated channels has closed during preceding depolarization.

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| FIG. 3.
Slowly activating component at 120 mV with prepulse to +100 or 100 mV. Cell was held at 50 mV and alternately stepped to +100 and 100 mV for 200 ms every 2 s before application of 200-ms hyperpolarizing step to 120 mV. During this repeated voltage protocol, ATP (10 µM) was applied for 10 s. Membrane currents with these 2 prepulses at peak response to ATP are superimposed. Double-headed arrows: slow component. Dashed line: 0 current level.
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The idea of the voltage-dependent gating of the ATP-activated channels was also supported by a "tail current" observed when membrane potential was depolarized from
120 to
50 mV (Fig. 4A,
). When the duration of the hyperpolarizing step to
120 mV was gradually increased from 50 to 200 ms, the tail current developed along with the development of the slowly activating current at
120 mV in all of six cells tested. The result suggests that the slow current component unequivocally reflects hyperpolarization-dependent induction of an inward current but not inhibition of an outward current. Figure 4B compares the time course of the slowly activating current and that of the tail current. The time courses could be fitted by a single exponential, and the time constants (
) were almost identical in this case (67 vs. 63 ms). These two time constants were similar in all of four cells tested (mean: 59.9 ± 4.8 ms, mean ± SE, vs. 62.9 ± 5.7 ms).

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| FIG. 4.
Tail current observed when membrane potential was changed from 120 to 50 mV in presence of 10 µM ATP. A: development of tail current along with that of slowly activating current. Cell was held at 50 mV, and 200-ms depolarizing step to +100 mV, followed by hyperpolarizing pulse of various durations (50-200 ms) to 120 mV, was applied every 2 s. During application of this voltage protocol, ATP (10 µM) was administered for 10 s to elicit inward current. With development of slow current component at 120 mV, tail current at 50 mV ( ) also became larger. B: time course of activation of slow current and decay of tail current. Amplitude of slow current at 120 mV and that of tail current at 50 mV obtained with 200-ms hyperpolarizing step shown in A are semilogarithmically plotted against time. Straight lines are fitted to data points with least-square method, and calculated time constants ( ) are shown. One-sixteenth (slow current) or 1/8 of data points (tail current) used for calculation are shown.
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Voltage dependence of the slow current
In the following sections, current traces just before the application of ATP are subtracted from the traces during the ATP application, and the current components induced by ATP alone are illustrated to facilitate the perception of the fraction of the slowly activating current (see Fig. 5A, for example). Voltage dependence of the slow component of the ATP-activated current was examined with the use of various prepulses (Fig. 5) and test pulses for the activation of the slow component (Fig. 6). In these experiments, a single step protocol (e.g.,
50 to +100 to
80 to
50 mV) was repetitively applied every 2 s, and the current response around the peak ATP-activated current was analyzed. Figure 5 shows changes in the slowly activating current when the prepulse was varied between
80 and +140 mV (ATP was separately applied for each prepulse). The slow component was measured at
120 mV to induce the maximal opening of the channels (see Fig. 2). The fraction of the slowly activating component was increased with a prepulse of a more positive potential (Fig. 5B). This result, combined with the result shown in Fig. 3, suggests that the gate progressively closes with depolarization. On the other hand, the time course of the slowly activating current was not affected by the change of the prepulse (Fig. 5C).

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| FIG. 5.
Slow current component obtained with various prepulses. Cells were held at 50 mV and stepped to various potentials for 200 ms before application of 200-ms hyperpolarizing step to 120 mV every 2 s. During this repeated voltage protocol, ATP (10 µM) was applied for 10 s. A: slow current at 120 mV obtained with prepulse to 20 mV. Current before ATP application was subtracted. Dashed line: 0 current level. B: fraction of slow current at 120 mV obtained with various prepulses. Amplitude of slow current (Islow in A) was divided by that of total current (Itotal in A), and values are plotted against voltage of prepulses. Each symbol and bar represent mean and SE obtained from 6-10 cells tested. C: activation time constant for slow current at 120 mV. Time constant was determined as in Fig. 4B and plotted against voltage of prepulses. Data are mean and SE from 6-10 cells tested.
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| FIG. 6.
Slow current component at various test potentials. Cells were held at 50 mV and stepped with 200-ms prepulse to +100 mV, followed by various test potentials for 200 ms every 2 s. During this repeated voltage protocol, ATP (10 µM) was applied for 10 s. A: slow current at 80, 100, and 120 mV. Current before ATP application was subtracted. Dashed line: 0 current level. B: fraction of slow current at various test prepulses ( ). Slow current was normalized to total current as in Fig. 5 and plotted against test potentials. From results shown in Fig. 5B, instantaneous current component was calculated by subtracting slow current from total current, and is also plotted (see text; ). Each symbol and bar represent mean and SE obtained from 4-10 cells tested. Curve was fitted to data assuming parameter for voltage-dependent gating (h ) as described in text. C: activation time constant for slow current at various test potentials. Time constant was determined as in Fig. 4B and plotted against test potentials. Data are mean and SE from 4-10 cells tested.
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Figure 6 shows changes in the slow current when the test pulse for the activation was changed. A prepulse of +100 mV was applied to induce the maximal closing of the channels in this case (see Fig. 5B). Test pulses more positive than
60 mV did not produce a current large enough to analyze. For test pulses of
60 mV or more negative, the fraction of the slowly activating current was smaller at
60 mV compared with other test potentials tested, but it was almost identical between
80 and
140 mV (Fig. 6B). The time course of the activation tended slightly to be accelerated at more negative potentials (Fig. 6C).
The slow current component shown in Fig. 5B may reflect the fraction (f) that has been closed during the prepulses. Thus the remaining fraction (1
f) can be regarded as the fraction that remains open during the prepulses. On the basis of this assumption, the remaining component obtained from the experiments shown in Fig. 5 is also plotted in Fig. 6B. Assuming a single voltage-dependent gate (h), the data were fitted by a curve predicted from the scheme of Hodgkin and Huxley (1952)
, namely
|
(1)
|
where h
is the parameter for the gating at steady state, E1/2 is the voltage for the half-maximal opening, Em is membrane potential, and k is a slope factor reflecting an energy barrier. The data were well fitted when E1/2 =
30 mV and k =
35 mV were assumed (Fig. 5B).
Dependence of the slow current on the concentration
of ATP
Figure 7A compares the voltage-dependent slow components of the current activated by 10 and 30 µM ATP. The component induced by 30 µM ATP more readily approached its maximal level than that by 10 µM ATP. Figure 7, B and C, illustrates the fraction of the slow component and the time course of the component when the concentration of ATP was varied between 10 µM and 1 mM. The fraction of the slowly activating component was reduced with the increased concentrations of ATP (Fig. 7B). The time constant was also decreased, or, in other words, the activation was accelerated depending on the concentration of ATP up to 300 µM (Fig. 7C). The rate constant (k+1) was calculated from
(k+1 = 1/
) and is logarithmically plotted against the concentration of ATP in Fig. 7D. The relation was linear, and could be fitted by a straight line with a slope of 0.35 ms
1/M
1.

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| FIG. 7.
Dependence of slow current on concentration of ATP. Slow current was activated at 120 mV with 200-ms prepulse of +100 mV every 2 s. Holding potential: 50 mV. During repeated voltage protocol, various concentrations of ATP were applied for 10 s. A: slow current induced by 10 and 30 µM ATP in PC12 cell. Dashed line: 0 current level. B: fraction of slow current obtained with various concentrations of ATP. Fraction was obtained as in Fig. 5. Each symbol and bar represent mean and SE obtained from 7-10 cells tested. C: dependence of activation time constant for slow current on concentration of ATP. Time constant was determined as in Fig. 4B. Data are mean and SE from 7-10 cells tested. D: double logarithmic plot of rate constant for activation of slow current vs. concentration of ATP. Rate constant (k+1) was calculated from mean values of time constant ( ). Straight line was drawn by eye.
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|
 |
DISCUSSION |
We have characterized the voltage-dependent slow component of the ATP-activated current in PC12 cells and demonstrated that a voltage-dependent gate opens at negative potentials and that the gating is influenced by the concentration of ATP.
When estimated from the fraction of the slowly activating current component, the half-maximal opening of the voltage-dependent gate may occur around
30 mV, and the maximal opening was achieved around
100 mV. The fraction could be fitted by a curve predicted from a single voltage-dependent gate of the Hodgkin-Huxley type (Fig. 6B). The gating may be due to movement of a single charged group involved in the channel protein. This view may be supported by the finding that the time course of the activation of the slow component and that of the tail current were comparable (Fig. 4B): the translocation of the charge group between two positions in a to-and-fro manner may account for the voltage-dependent opening and closing of the channels. Unlike the fraction of the slow current component, the time constant of the current activation did not exhibit dependence on the voltage (Figs. 5 and 6). This does not accord with the Hodgkin-Huxley type model, where the gating kinetics are affected by membrane potential (Hille 1992
; Hodgkin and Huxley 1952
). It is possible that the movement of the charge group is fast, and that the transition between the open and the closed state is governed by another slow ("rate-limiting") voltage-independent process. If this scheme is expressed as a sequential model

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where C1 and C2 are closed states, O is an open state, and
,
, k+1, and k
1 are rate constants.
When the concentration of ATP was increased, the fraction of the slow current component was decreased, and the activation of this component was accelerated (Fig. 7). If the acceleration of the activation is attributed to the change in the second slow process in Scheme 1, the voltage-independent process (C2
O) may be divided into two steps, namely the binding of ATP to the closed state (C2) and the transition from the closed state to the open state (O)

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where A is ATP. In this scheme, the rate constant k+1 can be expressed as a direct function of the concentration of ATP ([ATP]): k+1 = k
+1 [ATP]. A linear relation between k+1 and [ATP] is supported by the result shown in Fig. 7D. The reduction of the fraction of the slow current with the increased concentrations of ATP may be explained in the following manner: a large fraction of the ATP-activated channels has already been opened by high concentrations of ATP, and thus the ratio of channels available for the voltage-dependent opening is decreased. In other words, "voltage-independent" opening may preferentially occur with high concentrations of ATP. This process may also be included in the scheme for the channel behavior. For example, if it is assumed that this voltage-independent opening is attributed to the binding of ATP to C1 in Scheme 1, an overall scheme for the channel behavior can be expressed as follows

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This may be the simplest scheme to explain the results obtained in the present study. It is, however, also noted that precise determination of the beginning of the slow component is difficult when the activation is too rapid. In the present study, we routinely adopted data points 2 ms after the beginning of test pulses when the current exhibited fast activation kinetics (see METHODS), and determination in this way may have underestimated the fraction of the slow component in the presence of higher concentrations of ATP.
Judging from the data shown in Fig. 6B, the full opening or full closing of the channels was not achieved even at very negative or positive potentials. The results suggest that a small part of the ATP-activated channels is independent of the voltage-dependent gating. We cannot conclude at present whether this voltage-independent fraction is attributed to a qualitatively different population of the ATP-activated channels or not. Because the gating was also affected by the concentration of ATP (Fig. 7), it is assumed that all the channels are controlled by the voltage-dependent gate if the concentration of ATP is sufficiently low. In this regard, we have already performed a test in which hyperpolarizing steps were applied in the presence of a lower concentration of ATP (3 µM), but the current under this condition was not large enough for quantitative analysis (unpublished observations).
Nicotinic acetylcholine receptor channels also exhibit voltage-dependent gating. For muscle-type nicotinic receptors, the decay time course of the end-plate currents is slowed (Magleby and Stevens 1972
; Sheridan and Lester 1977
), and the opening of single channels is increased at more negative potentials (Auerbach et al. 1996
; Colquhoun and Sakmann 1985
; Neher and Sakmann 1976
). Interestingly, like the voltage-dependent gating of the ATP-activated channels in this report, the activation kinetics of the voltage-dependent component mediated through nicotinic receptors is also accelerated when the concentration of agonists is increased, whereas the kinetics is not affected by membrane potentials (Sheridan and Lester 1977
). Auerbach et al. (1996)
recently showed, with the use of mutants of nicotinic receptors lacking the binding site for agonists, that the voltage-dependent gating requires the binding of agonists. Unlike muscle-type nicotinic receptors, the voltage-dependent activation of nicotinic receptors is accelerated at more positive potentials in Aplysia neurons (Ascher et al. 1978
).
The voltage-dependent gating reported here may be responsible for the inward rectification of macroscopic currents permeating through ATP-activated channels in PC12 cells (Nakazawa et al. 1990
, 1991
) or other neuronal cells (Bean et al. 1990
; Evans et al. 1992
; Nakazawa 1994
). In addition to this gating, the inward rectification may also be attributed to single-channel conductance because unitary currents through the ATP-activated channels have been shown to exhibit inward rectification (Bean et al. 1990
; Krishtal et al. 1988
; Nakazawa and Hess 1994
). Although the physiological significance of the voltage-dependent gating is unclear at present, it may serve as a postsynaptic mechanism for efficient excitatory neurotransmission. On neurotransmission by ATP, postsynaptic neurons or nonneuronal cells may be more easily depolarized when they are largely hyperpolarized, whereas they are less easily depolarized when they have already been partially depolarized.
 |
ACKNOWLEDGEMENTS |
We thank T. Obama for culturing cells with skilled techniques, Dr. S. Ueno for stimulating discussion, and Dr. K. Fujimori for continuous encouragement. M. Liu is a visiting researcher supported by Japan China Medical Association.
 |
FOOTNOTES |
Address for reprint requests: K. Nakazawa, Division of Pharmacology, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya, Tokyo 158, Japan.
Received 12 March 1997; accepted in final form 21 April 1997.
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