(Received for publication, September 22, 1995; and in revised form, January 15, 1996)
From the
The biochemical properties of the mitochondrial K channel are very similar to those of plasma membrane K
channels, including inhibition by low concentrations of ATP and
glyburide (Paucek, P., Mironova, G., Mahdi, F., Beavis, A. D.,
Woldegiorgis, G., and Garlid, K. D.(1992) J. Biol. Chem. 267,
26062-26069). Plasma membrane K
channels are highly
sensitive to the family of drugs known as K
channel
openers, raising the question whether mitochondrial K
channels are similarly sensitive to these agents. We addressed
this question by measuring K
flux in intact rat liver
mitochondria and in liposomes containing K
channels
purified from rat liver and beef heart mitochondria. K
channel openers completely reversed ATP inhibition of
K
flux in both systems. In liposomes, ATP-inhibited
K
flux was restored by diazoxide (K
= 0.4 µM), cromakalim (K
= 1 µM), and two developmental cromakalim
analogues, EMD60480 and EMD57970 (K
= 6
nM). Similar K
values were observed in
intact mitochondria. These potencies are well within the range observed
with plasma membrane K
channels. We also compared the
potencies of these K
channel openers on the plasma
membrane K
channel purified from beef heart myocytes. The
K
channel from cardiac mitochondria is 2000-fold more
sensitive to diazoxide than the channel from cardiac sarcolemma,
indicating that two distinct receptor subtypes coexist within the
myocyte. We suggest that the mitochondrial K
channel is
an important intracellular receptor that should be taken into account
in considering the pharmacology of K
channel openers.
K channel openers (KCOs) (
)activate
ATP-inhibited K
channels. As described in several
excellent reviews(1, 2, 3) , members of this
drug family exhibit a rich and clinically important pharmacology. Thus,
cell membrane K
channels (cellK
) in
different tissues are considered to mediate the hypotensive and
diabetogenic effects of diazoxide (4) and the cardioprotective
effects of cromakalim and its derivatives(5) . It is important
to determine whether these drugs also act on mitochondrial K
channels (mitoK
) in their therapeutic range.
In
the first reports of KCO actions in mitochondria, Belyaeva et al.(6) and Szewczyk et al.(7) observed
stimulation of K uptake by KCOs in respiring
mitochondria. RP66471 was the most potent KCO studied (K
= 50 µM), whereas P1060
and diazoxide were only weakly active at 700 µM. Because
these concentrations are much higher than K
values observed with cellK
(1) , these
results appear to imply that mitochondrial actions of KCOs are not
pharmacologically important.
We now report that diazoxide,
cromakalim, and two experimental benzopyran derivatives are very potent
activators of K flux through ATP-inhibited
mitoK
, with K
values similar to
those observed with cellK
. KCO activation of K
flux was observed in both intact mitochondria and proteoliposomes
containing reconstituted mitoK
. No effect was observed on
uninhibited K
flux, which likely explains the low
potencies observed by previous workers (6, 7) in
assays that did not include Mg
and ATP. We also found
that mitoK
and cellK
from beef heart
differed strongly in their sensitivity to diazoxide, indicating
distinct receptor subtypes among K
channels from the same
cell. Our results indicate that mitoK
may be an important
intracellular receptor for K
channel openers, and they
raise the possibility that mitoK
is the site of action of
cardioprotective KCOs. (
)
Figure 1:
Activation of K flux
by K
channel openers in liposomes reconstituted with
mitoK
. Dose-response curves and Hill plots (inset) for activation of K
flux through
mitoK
by four KCOs: EMD60480 (
), EMD57970 (
),
diazoxide (
), and cromakalim (
).
``
J
'' is the maximum
ATP-sensitive K
flux, i.e. the difference
between control fluxes in the absence and presence of saturating ATP
(0.5 mM). ``
J'' is the difference
between fluxes in the presence or absence of the drug, both measured in
the presence of 0.5 mM ATP. In nine separate preparations,
J
ranged between 400 and 600
µM/s, similar to values previously reported using these
protocols(8) . Observed K
values and
Hill slopes are given in the text.
Observed K values (mean and S.D.) were 1.05 ± 0.06
µM for cromakalim (n = 5), 0.37 ±
0.03 µM for diazoxide (n = 4), 6.1
± 1.3 nM for EMD60480 (n = 2), and 6.20
± 0.02 nM for EMD57970 (n = 2). As
shown in the inset to Fig. 1, cromakalim and diazoxide
exhibited indistinguishable Hill slopes of 2.0 ± 0.5, and the
benzopyranyl derivatives yielded Hill slopes of 3.5 ± 0.3. Hill
slopes greater than 1.0 may reflect a tetrameric structure of the
channel, as observed with other K
channels(16) , or the existence of multiple binding sites
on a regulatory ATP binding cassette, as has been proposed for the
sulfonylurea receptor of the pancreatic
cell (17) .
We
also measured KCO activation of K flux in
proteoliposomes reconstituted with mitoK
purified from
beef heart mitochondria. Observed K
values from
two experiments were 1 µM for cromakalim and 0.4
µM for diazoxide. These results extend a previous
observation that cardiac and hepatic mitoK
behave very
similarly.
We stress that these drugs stimulated K flux only when K
flux was inhibited by
Mg
and ATP. Control (uninhibited) K
flux is observed in media containing Mg
alone,
ATP alone, and lacking both Mg
and ATP(8) .
In each of these conditions, KCOs had no effect on control K
flux at doses up to 30-fold higher than their respective K
values.
Figure 2:
Activation of K flux by
K
channel openers in liposomes reconstituted with
cardiac sarcolemmal K
channels (cellK
).
Dose-response curves and Hill plots (inset) for activation of
K
flux through cellK
by four KCOs:
EMD57970 (
), EMD60480 (
), cromakalim (
), and diazoxide
(
). ``
J
'' and
``
J'' are defined as in Fig. 1, but with
reference to rates in 2 mM ATP. Observed K
values and Hill slopes are given in the text. Duplicate
experiments on an independent preparation yielded similar
results.
Fig. 3contains representative light-scattering traces from
rat liver mitochondria respiring on ascorbate-TMPD. Swelling in
K salts (trace a) was sharply inhibited by
addition of 100 µM ATP (down arrow to trace
b) to levels close to those observed in TEA
salts (trace c). In agreement with previous results(10) ,
higher [ATP] had no further effect in K
medium, and ATP had no effect on TEA
flux (not
shown). When 20 µM cromakalim was included in the assay
medium containing 100 µM ATP, ATP inhibition was prevented (up arrow to trace d, Fig. 3). In the absence of ATP, cromakalim had no effect on the control rate
up to 100 µM, the highest dose tested. When cromakalim was
added during the inhibited state, ATP inhibition was reversed (not
shown).
Figure 3:
Activation of K flux by
cromakalim in intact mitochondria. Light-scattering kinetics for
mitochondria suspended in K
media (see
``Experimental Procedures''). Trace a, K
medium without ATP or cromakalim. Trace b, K
medium containing 0.1 mM ATP. Trace c,
TEA
medium without ATP. Trace d, K
medium with 0.1 mM ATP and 20 µM cromakalim. The down arrow from trace a to trace b shows the inhibitory effect of ATP on swelling
kinetics, and the up arrow from trace b to trace
d shows the opening effect of cromakalim. Ascorbate-TMPD was used
as the respiratory substrate; entirely similar results were obtained
with succinate.
Fig. 4contains dose-response curves for activation
of K flux by diazoxide, cromakalim, and EMD60480 in
mitochondria. Activation was measured relative to rates in the presence
of 100 µM ATP and 1 mM Mg
,
conditions in which the K
for ATP inhibition is
2-3 µM(13) . The estimated K
values were 2.3 µM for diazoxide,
6.3 µM for cromakalim, and 5.4 nM for EMD60480.
Figure 4:
Dose-response curves for activation of
K flux in intact mitochondria. Relative ATP-sensitive
K
uptake into respiring rat liver mitochondria;
J/
J
, is plotted versus drug concentration. ``
J
''
and ``
J'' are defined as in Fig. 1. The
dose-response curves reflect activation by EMD60480 (
), diazoxide
(
), and cromakalim (
). Assay medium for
J contained 1 mM Mg
and 0.1 mM ATP, which maximally inhibited K
uniport in
mitochondria(10) , and ascorbate-TMPD as respiratory
substrates. The K
values reported in the text
are means of two independent experiments. For each drug, duplicate K
values were within 5% of each
other.
As in proteoliposomes, these drugs stimulated K flux only when K
flux was inhibited by
Mg
and ATP. In doses 20-fold higher than their
respective K
values, these KCOs had no effect on
flux through the uninhibited channel. This effect was verified in media
containing Mg
alone, ATP alone, and lacking both
Mg
and ATP.
This is the first report showing that KCOs activate
mitoK over the same dose range as they activate
cellK
. This finding was observed in mitochondria and in
proteoliposomes reconstituted with mitoK
and raises the
possibility that mitoK
may be activated by KCOs in
vivo. Kinetic parameters differed between intact mitochondria and
the reconstituted preparations. As previously reported (13) ,
the K
for ATP inhibition is lower in
mitochondria (2-3 µM) than in proteoliposomes
(20-25 µM). We now show that the K
values for diazoxide and cromakalim are about 6-fold higher in
mitochondria than in liposomes. On the other hand, the K
for EMD60480 is about the same in the two
preparations. These differences may reflect regulatory complexity in
intact mitochondria, which is lost upon extraction and reconstitution.
In the dose ranges studied, KCOs had no effect on K flux when Mg
and/or ATP were omitted from the
assay medium. The lack of effect of KCOs on the open channel is also
characteristic of cellK
(20) . The finding that
KCOs in low doses have no effect on the uninhibited channel is also
consistent with the results of Belyaeva et al.(6) and
Szewczyk et al.(7) , who did not include
Mg
and ATP in the assay medium used for their
studies.
A particularly exciting development
in heart is the finding by Grover and colleagues (5, 25) and others (26, 27) that KCOs
are cardioprotective during experimental ischemia. KCO-treated hearts
maintained higher ATP levels and exhibited reduced infarct size and
enhanced post-ischemic recovery upon reperfusion. All of these effects
were blocked by glyburide, which is contraindicated in patients
susceptible to cardiac ischemia. Preconditioning, in which a period of
brief ischemia reperfusion protects the heart against subsequent
ischemic damage(28) , was also blocked by
glyburide(29) . These pharmacological effects point to a role
of K channels in myocardial protection; but, again, the
receptor for these effects has not been identified, and a mitochondrial
site of action cannot be excluded(25) .
Exploration of this
possibility is aided by the existence of receptor subtypes among
K channels(1) . For example, cromakalim is a
potent activator of cellK
from heart and vascular smooth
muscle (29) but has a minimal effect on insulin
secretion(4, 30) . Diazoxide is a potent vasodilator (4) and also reduces insulin secretion (31) but has
little effect on cardiac cellK
(18) . This raises
the question whether mitoK
and cellK
from
the same cell differ pharmacologically. Accordingly, we have
compared drug sensitivities of cardiac mitoK
and
cellK
reconstituted from beef heart. These experiments
yielded the following preliminary results: (i) mitoK
from
heart and liver do not differ significantly in their drug sensitivities (K
values); (ii) cardiac mitoK
and
cardiac cellK
exhibit similar sensitivities to benzopyran
derivatives; however, (iii) cardiac mitoK
is about 2000
times more sensitive to diazoxide than cardiac cellK
. The
low sensitivity of reconstituted cardiac cellK
to
diazoxide is entirely consistent with previous reports(18) .
The inferred existence of receptor subtypes within the cardiac myocyte
may provide a means to determine the site of cardioprotective action of
K
channel openers.