Retardation of cation channel deactivation by mitochondrial
dysfunction in adrenal medullary cells
M.
Inoue,
N.
Fujishiro, and
I.
Imanaga
Department of Physiology, School of Medicine, Fukuoka University,
Fukuoka 814-0180, Japan
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ABSTRACT |
The
mechanism for cyanide (CN) activation of a nonselective cation (NS)
channel coupled with a muscarinic receptor in a guinea pig chromaffin
cell was studied with the perforated-patch method. Bath application of
a protein kinase inhibitor resulted in a dose-dependent inhibition of
muscarine-induced current (IM) but had no apparent effect on the CN-induced current (ICN). On the
other hand, production of ICN occluded muscarine
activation of NS channels in an amplitude-dependent manner.
Deactivation of IM after washout was retarded while
ICN was also active, and the extent of the
retardation increased with an increase in the relative production of
ICN on muscarinic stimulation. Restoration of
Na+ pump activity from CN suppression was conspicuously
retarded below 19-20°C, and the apparent diminution of
IM and ICN after washout was
retarded in parallel with a decrease in temperature. The results
suggest that CN activation of NS channels is due to suppression of
deactivation of the channel.
muscarinic receptor; mitochondria; temperature; cyanide
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INTRODUCTION |
SECRETION OF CATECHOLAMINES in response to hypoxia is
vital for the body to deal with the life-threatening event. Secreted catecholamines, especially adrenaline, increase cardiac output and
enhance gluconeogenesis and glycogenolysis in the liver with the
consequent increase in blood glucose (6). Although carotid body type I
cells are a sensing site for O2 tension, recent studies demonstrated that adrenal medullary cells are also capable of promptly
detecting a decrease in O2 tension with a subsequent secretion of catecholamines (14, 24). Accumulating evidence indicates
that hypoxia produces a depolarization with the consequent activation
of voltage-dependent Ca2+ channels and that the subsequent
increase in intracellular Ca2+ concentration
([Ca2+]i) is responsible for
catecholamine secretion in type I and chromaffin cells (2, 3, 14, 26).
How hypoxia is detected is controversial, and two main hypotheses have
been proposed. One is the membrane ion channel hypothesis that a
K+ channel itself or its closely associated regulator in
the membrane senses O2 levels (11, 22). Although
K+ channel activity in isolated patch membranes was
documented to be suppressed by hypoxia (11), it remains to be
determined whether the hypoxia-sensitive K+ channel is
active at resting membrane potentials and whether suppression of its
activity is responsible for depolarization in response to hypoxia (2).
The other is that mitochondria play a primary role for the detection
(8, 9). This proposal is principally based on findings that the effects
of hypoxia can be mimicked by various types of mitochondrial inhibitors.
Thompson and Nurse (25) reported that anoxia suppressed two distinct
voltage-evoked K+ currents, Ca2+-dependent and
delayed rectifier type, in adrenal medullary cells obtained from
newborn rats. However, a depolarizing response or receptor potential to
anoxia was not suppressed by bath addition of 10 mM tetraethylammonium,
which completely suppressed the anoxia-sensitive K+
currents. On the other hand, Mojet et al. (24) reported that the
depolarization of mitochondrial membrane potential preceded an increase
in [Ca2+]i in response to hypoxia
in the rat chromaffin cell, and they proposed that mitochondria can
serve as a site for detection of a decrease in O2.
Consistent with this hypothesis, cyanide (CN) and anoxia induced
activation of a nonselective cation (NS) channel and inhibition of the
Na+ pump in guinea pig adrenal chromaffin cells (15). This
cation channel may be the same as that activated by muscarinic receptor stimulation (19), because muscarine failed to induce a further inward
current during the full production of a current in response to CN (14).
If this notion is tenable, then anoxia and CN might activate the
channel through phosphorylation because the muscarinic activation of
channels and deactivation may be mediated by a protein kinase and a
Mg2+-dependent phosphatase, respectively (16, 20). In the
present experiment, we examined the mechanism for CN activation of NS channels. Our findings are consistent with the notion that exposure to
CN diminishes the deactivation process with the consequent dominance of
the activation.
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METHODS |
Whole cell recordings.
Experiments on dissociated adrenal medullary cells were done, as
described elsewhere (17). Briefly, female guinea pigs weighing 250-300 g were killed by a blow to the neck, and the adrenal
glands were eliminated and immediately put into ice-cold
Ca2+-free solution in which 1.8 mM Ca2+ was
simply removed from a standard saline containing (mM): 137 NaCl, 5.4 KCl, 1.8 CaCl2, 0.5 MgCl2, 0.53 NaH2PO4, 5 D-glucose, 5 HEPES, and
4 NaOH. Adrenal medullas were cut into three to six pieces and
incubated for 30 min with 0.25% collagenase dissolved in the
Ca2+-free solution. After the incubation, the tissues were
washed three times in the Ca2+-free solution and then kept
in it at room temperature (23-25°C). A few pieces of the
tissues were put in the bath apparatus, which was placed on an inverted
microscope, and adrenal chromaffin cells were dissociated mechanically
with fine needles. Then, dissociated cells were allowed to adhere to
the bottom for a few minutes before the bath apparatus was perfused
with saline at a rate of 1 ml/min. The whole cell current was recorded
with the perforated-patch method (13). The current was recorded with an
Axopatch 200A amplifier (Axon Instruments) and then fed into a brush
recorder after low-pass filtering at 3 or 5 Hz and into a video tape
after being digitized with an analog-to-digital converter. The pipette solution contained (mM): 120 potassium isethionate, 20 KCl, 10 NaCl, 10 HEPES, and 2.6 KOH. On the day of the experiment, nystatin dissolved in
dimethyl sulfoxide (10 mg in 50 µl) was added to the pipette solution
at a final concentration of 100 µg/ml. Glucose and NaCl in the
standard saline were equimolarly replaced with sucrose and NaCN in a CN
solution. The pH of the pipette solution and external solutions was
adjusted to 7.2 and 7.4 with KOH and NaOH, respectively. All chemicals
were bath applied, and a CN-induced current (ICN)
or muscarine-induced current (IM) was evoked by perfusion with the CN solution or 3 µM muscarine-containing solution, unless otherwise noted. The membrane potential was corrected for a
liquid junction potential of
12 mV between the pipette solution and the standard solution. Experiments except for those at low temperatures were carried out at 23-25°C. When bath
temperature was lowered from room temperature, the perfusate was cooled
with a Peltier device. Thus the temperature around the
cell examined was expected to be lower than that at the outflow, where
temperature was measured. In a separate experiment, the temperature at
the center of bath was lower by ~3°C than the 19.7°C at the
outflow, by 1.6°C than the 20.8°C, and by 1.4°C than the
21.8°C. Data are expressed as means ± SD, and statistical
significance was determined with Student's t-test.
Fluorescence recordings.
To label the cell surface with a fluorescence dye, we incubated
dissociated cells for 30 min in a standard solution to which 5 µM
di-8-ANEPPS and 0.05% Pluronic F-127 were added. The dish in which the cells settled was placed on a Zeiss Axiovert microscope attached to a Zeiss LSM 410 laser confocal scanning unit (Carl Zeiss).
The objective lens was an oil-immersion lens with a magnification of
×63 and a numerical aperture of 1.25. Illumination with 488 nm
was provided by an argon laser, and emission was monitored above 570 nm
because the peak emission wavelength was reported to be 570-580 nm
(4). The theoretical spatial resolution given by the equation d =
/(2 × NA), where d represents the smallest resolvable
distance,
is the wavelength of emission light (~580 nm), and NA
is the numerical aperture (1.25), is ~0.2 µm. Thus images and line-scan images were obtained in all experiments with a
pixel size of <0.2 µm and with a full width at half-maximal intensity of ~0.7 µm. The actual value of d may be twice or three times larger than the theoretical one because of diffraction (23). To
study effects of CN, one-half of the 2-ml solution in the dish was
replaced with the CN solution and the administration was completed within 8 s.
Chemicals.
Nystatin, (±)-muscarine chloride, and Pluronic F-127 were obtained
from Sigma; di-8-ANEPPS was from Molecular Probes;
N-(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride
(HA-1004) was from Seikagaku (Tokyo, Japan); collagenase was from
Yakult (Tokyo, Japan); NaCN was from Hayashi Pure Chemical (Tokyo, Japan).
 |
RESULTS |
Retardation of IM deactivation by CN.
One of the findings for involvement of phosphorylation in the
activation of NS channels is a reversible suppression of the muscarinic
production of a nonselective cation current (INS)
by protein kinase inhibitors (16, 18). Consistent with the previous result, addition of 300 µM HA-1004, a general protein kinase
inhibitor (12), to a 3-µM muscarine-containing perfusate resulted in
a gradual decline of IM in a reversible manner,
whereas the inhibitor failed to suppress ICN (Fig.
1A). In the previous experiment
(15), 57 and 43% of ICN were estimated to be due
to activation of NS channels and inhibition of the Na+ pump
current (Ipump), respectively.
ICN in this particular cell, however, might have
comprised only inhibition of the Ipump, but this
possibility may not be feasible because the subsequent application of 3 and 10 µM muscarine did not induce a further inward current, as was
noted previously. In a total of eight cells, HA-1004 suppressed IM in a concentration-dependent manner but did not
affect ICN (Fig. 1B). This finding raises
the possibility that exposure to CN activates NS channels independent
of phosphorylation. One of such possibilities is that a change in
membrane tension is responsible for the NS channel activation, because
hypoxia and metabolic suppression were reported to increase volume in
various types of cells (10, 21). Thus we investigated whether or not a
short exposure to CN would induce any alteration in cell size. To this
end, the cell surface was labeled with di-8-ANEPPS and fluorescence was observed in line-scan or z-axis images. Figure
2 shows results of line-scan analysis. The
line marked in the image (Fig. 2A) was scanned every 0.1 s, and
these scans were used to construct the line-scan image (Fig.
2B). Addition of 1 ml CN solution to 1 ml of dish solution
(final CN concentration, 2.5 mM) did not induce any change in cell
diameter for 200 s in nine of ten cells tested, as noted in one
cell (Fig. 2, A and B, left). However, in
another cell (Fig. 2, A and B, right), the
diameter began to increase 26 s after the onset of CN exposure and the
increase continued. Figure 2C summarizes relative diameters of
10 cells during 2-min exposure to CN. It is evident that short exposure to CN did not alter cell size. Similarly, z-axis images were
not altered during the CN exposure (not shown). The results indicate that short application of CN does not induce an increase in diameter at
least in the order of ~0.5 µm.

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Fig. 1.
Failure of protein kinase inhibitor to suppress cyanide (CN)-induced
current. Values are means ± SD of 2-5 observations. A:
chart record of a 3 µM muscarine (M)-induced current and a 5 mM
CN-induced current (IM and ICN,
respectively). Whole cell current was recorded at a holding potential
of 67 mV with perforated-patch method. Here and in subsequent
figures, horizontal arrows are zero-current level. M, CN, and HA-1004
(HA: protein kinase inhibitor) were bath applied during the period
indicated by the bar. Nos. are µM concentrations used. B:
summary of relative amplitudes of IM and
ICN in presence of 100, 150, and 300 µM HA.
Amplitudes are expressed as fractions of currents in absence of HA in
same cell.
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Fig. 2.
No change in cell volume during short exposure to CN. Values are
means ± SD of 10 cells. A: fluorescence image of
chromaffin cells obtained in confocal mode. Image was obtained
approximately in middle of cells. Straight line is a site for scanning.
B: line-scan image. Line scan was performed every 100 ms.
Intensity of fluorescence was indicated by heights. Arrows, onset of
addition of CN solution. C: summary of relative diameters of
cells during CN exposure. Relative diameters are expressed as fractions
of those before exposure.
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If phosphorylation is indeed involved, then the activation of channels
might be attributed to a decrease in phosphatase activity with the
consequent dominance of kinase activity. If this is the case, then
IM should diminish slowly after washout of
muscarine while ICN is also active. This inference
was examined by applying muscarine at a maximum of the current evoked
by 0.5 mM CN; thus IM was expected to be reversible
under such conditions because the amplitude of the fully developed
ICN was about one-half of IM
and activation of the channel may not have been saturated (14). Figure
3A shows that deactivation of
IM after washout was considerably retarded in
ICN production, an effect that rapidly disappeared after washout of CN. To elucidate the relation between retardation of
IM deactivation and ICN
production, half-decay times of IM in the
production of ICN were expressed as a fraction of
those of control IM in the same cells and the ratio
was plotted against the relative amplitude of ICN,
which had developed on muscarinic stimulation. The relative amplitude
of ICN was expressed as a fraction of
IM evoked in the absence of CN. Figure 3B
shows that as the relative amplitude of ICN
increased, deactivation of IM slowed. Furthermore,
the amplitude of IM evoked in the presence of CN
diminished with an increase in the relative amplitude of ICN (not shown).

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Fig. 3.
Retardation of IM deactivation in
ICN production. A: chart records of whole
cell currents. Current was recorded at 62 mV with
perforated-patch method. CN and muscarine were bath applied during
indicated period (double line for CN and bars for M). B:
relative amplitudes of ICN are plotted against
ratios of half-decay times for IM in presence of CN
to those for IM in absence of CN. Amplitudes of
ICN, which were evoked in 2 cells by 0.1 mM CN, in
6 by 0.25, in 2 by 0.5, and in 2 by 5, were measured on application of
muscarine, and values were expressed as fractions of
IM elicited in absence of CN.
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Effects of low temperature.
The foregoing observations suggest that suppression of mitochondrial
function retards the deactivation of IM. We then
asked if low temperatures have a similar retarding action on
IM, because mitochondrial F1-ATPase
activity diminished markedly below ~20°C (1, 7). First, to
determine how low temperature affects mitochondrial function,
restoration of the Na+ pump activity from CN inhibition was
examined. The ICN elicited in the presence of 6 mM
Ba2+ was in a major part attributed to inhibition of
Ipump, and suppression of the pump activity by CN
was thought to be due to a decrease in intracellular ATP contents (15).
Thus, if ATP production in mitochondria is indeed impaired by a
decrease in temperature, then restoration of the pump activity should
be slow. Figure 4 shows that this is the
case. As the temperature in the outflowing perfusate increased from
19.6°C (actual temperature around the cell, ~16.6°C),
restoration of the pump activity became rapid (Fig. 4A). This
rapid event could not be ascribed to a general effect of temperature on
kinetics. The time required for half-suppression of
Ipump after CN administration increased only
slightly with decreasing temperature, whereas the time for
half-restoration after washout increased steeply (Fig.
4B). Furthermore, the temperature dependence of
half-times for production and diminution of ICN in
the absence of Ba2+ did not apparently differ from those
for ICN in Ba2+ (Fig. 4B). In
Fig. 4C, amplitudes of ICN in the presence
and the absence of 6 mM Ba2+ were plotted against
temperature. The apparent x-intercepts for ICN with and without Ba2+ were 18.7 and
17.8°C, respectively. Because restoration of the pump activity is
likely to reflect that of mitochondrial functions, a decrease in
temperature below 21°C (actual temperature, ~19.5°C) may
impair the restoration.

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Fig. 4.
Retardation of restoration of Na+ pump activity from CN
inhibition at low temperatures. A: chart records of
ICN in presence (Ba) and absence of 6 mM
Ba2+. Whole cell current was recorded at 65 mV with
perforated-patch method. Nos. beside traces are temperature in
outflowing perfusate. CN was applied during indicated period (bar).
Sequence of application was from top to bottom.
B: half-times for production ( and ) and diminution ( and ) of ICN are plotted against temperatures.
Half-times are times required for half-production or-diminution of
ICN. Triangles and squares mean
ICN in presence and absence of 6 mM
Ba2+, respectively. ICN in presence of
Ba2+ was mainly due to suppression of Na+ pump
current. C: amplitudes of ICN in presence
( ) and absence ( ) of Ba2+ are plotted against
temperatures.
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Whether or not deactivation of IM is retarded at
low temperatures was then examined. IM was
successively evoked as the temperature in the outflow decreased from
28.5 to 15.7°C and then increased up to
29°C (Fig. 5, B-D). It is evident that the
half-decay time markedly increased with decreasing temperature below
21°C (Fig. 5C), whereas the half-rise time only slightly
increased. A similar difference in temperature dependence of the
half-rise time and the half-decay time (Fig 5, B and C)
of ICN was noted below 21°C. The half-rise
times of the first two ICNs elicited at 28.5 and 22.7°C were apparently smaller; these results, however, may not indicate the marked temperature dependence of the half-rise time, because results of ICN elicited at 22.2 and
26.4°C in the increasing phase of temperature did not differ from
those of ICN below 21°C. The time course of the
developing process of ICN sometimes slowed with
time of the recording and then became stable. A comparison of
temperature dependence of the decay time for ICN
with that for IM reveals that the decay process of
IM and ICN had the same temperature dependence. In Fig. 5D, amplitudes of
IM and ICN are plotted against
temperature. The regression line for ICN had a steeper slope than that for IM, and the apparent
x-intercepts for IM and
ICN were 9 and 13°C, respectively.

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Fig. 5.
Parallel retardation of diminution of IM and
ICN at low temperatures. A: chart records
of IM and ICN at various
temperatures. Whole cell currents were recorded at 62 mV with
perforated-patch method. M and CN were bath applied during indicated
period (double line for CN and bar for M). No. beside each trace is
temperature in outflow. B and C: half-rise times and
half-decay times of IM ( ) and
ICN ( ) after washout are plotted against
temperatures, respectively. D: amplitudes of
IM ( ) and ICN ( ) are
plotted against temperatures. Open and closed symbols correspond to
decreasing and increasing phases of temperatures, respectively. Note
that a temperature decrease from 22.7 to 15.7°C reversibly induced
an inward shift of holding current by 0.9 pA.
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To facilitate analysis of temperature effects in different cells,
slopes and x-intercepts for ICN in the
presence of 6 mM Ba2+ and for IM were
expressed as fractions of those for ICN in its absence in the same cells. The relative x-intercept of
ICN in the presence of Ba2+ and that of
IM thus obtained significantly differed from one, and similarly, the relative slope of IM was
noticeably different from one (Fig. 6,
A and B). These results indicate that
Ipump inhibition and INS, which
constitute ICN, have a different temperature dependence. On the other hand, ratios of half-decay times at 20°C (actual temperature, ~17°C) to those at 24°C for
ICN in the presence of Ba2+ and for
IM did not differ appreciably, compared with those
for ICN in its absence, thereby suggesting the
involvement of a common mechanism for the retardation of both
restoration of pump activity and diminution of INS.

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Fig. 6.
Temperature dependence of ICN in absence and
presence of Ba2+ and IM. Slopes of
temperature-amplitude relations were calculated with a least-squares
method. Relative slopes and x-intercepts of
ICN in presence of Ba2+ (Ba-CN) and of
IM (M) are expressed as fractions of
ICN in its absence in same cells. Half-decay times
of ICN in presence and absence of Ba2+
and IM after washout were measured at 24 and
20°C (actual temperature, ~18°C). Values at 20°C are
expressed as fractions of those at 24°C, and then ratios for
ICN in presence of Ba2+ and for
IM are calculated as fractions of those for
ICN in its absence. Asterisks are statistical
significance (P < 0.05). Values are means ± SD of 3 and 5 cells for Ba-CN and M, respectively.
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DISCUSSION |
Our previous study (15) suggested that 57 and 43% of 5 mM CN-induced
inward currents can be attributed to activation of NS channels and
inhibition of the Ipump, respectively. Because amplitude of INS diminished progressively in the
absence of Na+ pump activity and this decrease was enhanced
by replacement of sucrose with glucose, i.e., by glycolytic production
of ATP, activation of the channel was presumed to be due to an ATP
decrease that resulted from consumption by energy-dependent processes,
such as Na+-K+-ATPase. Thus, to obtain a
maximum stimulation of NS channels, CN activation of the channel was
observed as ICN without isolation of the
INS. In the present experiment, the notion that
ICN comprises production of INS
and inhibition of the Na+ pump was further supported by a
difference in temperature dependence of IM and
Ipump inhibition. The relative values of
x-intercept for IM and
Ipump inhibition significantly differ from one.
Similarly, IM production and
Ipump inhibition had relative slopes of 0.82 and
1.05, respectively, and the former appreciably differed from one. Thus
temperature dependence of ICN is thought to reflect a combination of those related to IM production and
Ipump inhibition.
In carotid body type I cells, a decrease in O2 tension was
proposed to be sensed by an O2-sensitive K+
channel or its closely associated regulator (11, 22) or by mitochondria
(8, 9). Similarly, these two mechanisms were suggested to be involved
in O2 sensing in adrenal medullary cells (14, 24, 25). The
findings that CN-induced activation of NS channels and inhibition of
the Na+ pump were reproduced by exposure to anoxia are
consistent with the mitochondria hypothesis. The rapid secretion of
catecholamines by hypoxia would be accounted for readily by the
membrane ion channel hypothesis, whereas how the dysfunction of
mitochondria promptly induces a change in membrane excitability would
be a challenging issue for the mitochondrial hypothesis. The sequence of our experiments revealed that dysfunction of mitochondria indeed induces a rapid depolarization through inhibition of the
Na+ pump and activation of NS channels. In particular,
anoxia or CN activation of NS channels is estimated to correspond to
~60% of anoxia- or CN-induced currents and exposure to anoxia or CN even in the absence of Na+ pump activity results in
activation of the channel. Thus mechanisms for stimulation of NS
channels by anoxia or chemical hypoxia seem to develop specially for
transduction of O2 signal to catecholamine secretion. The
present results suggest that CN activation of NS channels is due to
suppression of the deactivation process for the channel. First, the
diminution of IM after washout was retarded in
ICN generation, and the degree of retardation
depended on the relative production of ICN on
muscarinic stimulation. As the relative amplitude of
ICN compared with that of IM
increased, IM diminution was even more retarded.
This close correlation between relative production of
ICN and retardation of IM decay
indicates that the deactivation process for the NS channel diminishes
in generation of ICN. Because the biophysical
properties of CN or anoxia-sensitive channels resemble those of the
muscarinic one and the muscarinic stimulation of the channel was
occluded in an amplitude-dependent manner by generation of
ICN (Figs. 1 and 3; Ref. 13), there would be no
doubt that exposure to CN or hypoxia activates the same NS channel as
that regulated by the muscarinic receptor. Thus it is likely that
diminished deactivation process for NS channels is responsible for CN
activation of the channel. The decrease in rate constant for
deactivation is expected to shift the equilibrium between activation
and deactivation toward the former with the consequent production of
INS. Secondly, the failure of HA-1004 to suppress
ICN is consistent with our hypothesis. The
muscarinic activation of NS channels was reversibly suppressed by
various isoquinoline sulfonamide derivatives with different potencies
(16, 18), and this inhibition was assumed to be due to inhibition of
the protein kinase involved and the consequent shift of equilibrium
between phosphorylation and dephosphorylation toward the latter (20).
The fact that HA-1004 failed to suppress ICN
suggests that the rate constant for deactivation decreased almost to
null during exposure to 5 mM CN. Thirdly, deactivation of
IM was retarded at low temperatures. This
low-temperature effect is probably due to the dysfunction of
mitochondria, because restoration of the Na+ pump activity
from CN inhibition was retarded below 21°C (actual temperature,
~19.5°C). Because the CN inhibition of the pump activity is
probably attributed to a decrease in ATP contents, mitochondrial production of ATP in chromaffin cells may have a steep temperature dependence below 19-20°C. This temperature dependence of ATP
production might be ascribed to that of ATP synthase in the terminal
step of oxidative phosphorylation, because temperature dependence of F1-ATPase activity obtained from heart mitochondria was
biphasic with a steep decrease below 20°C (1, 7). Because it took a
few minutes to change the bath temperature in 1°C and thus studying quantitative effects of low temperatures on the whole cell current was
difficult, we did not consistently investigate whether or not a
decrease in temperature induces an inward current. In one cell shown in
Fig. 4, decreasing the bath temperature to 15.7°C reversibly
induced an inward current of 0.9 pA. The fact that metabolic inhibition
with CN and low temperatures induce a similar retardation of
IM deactivation indicates that the deactivation process of NS channels is closely associated with the mitochondrial function. Based on three lines of evidence, it would be rational to
assume that exposure to CN or anoxia activates NS channels through
suppression of the deactivation process. Our previous studies (16, 20)
suggest that a Mg2+-dependent phosphatase is responsible
for deactivation of the NS channel. Thus our notion means that exposure
to CN results in a decrease in an apparent activity of the
Mg2+-dependent phosphatase. This thesis was not examined
directly in the present experiment, because the
Mg2+-dependent phosphatase has not been identified
molecularly and there is no specific inhibitor for protein phosphatase
IIC (5), a candidate for the phosphatase involved. The best means to
inhibit the phosphatase activity is removal of Mg2+. In
preliminary experiments, we found that the CN potency to induce an
inward current was almost abolished by decreasing concentrations of
free Mg2+ inside the cell.
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FOOTNOTES |
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: M. Inoue,
Department of Physiology, School of Medicine, Fukuoka University,
Fukuoka 814-0180, Japan
(E-mail:minoue{at}fukuoka-u.ac.jp).
Received 10 May 1999; accepted in final form 26 August 1999.
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