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Vol. 54, Issue 1, 197-206, July 1998
Unit of Pharmacology,
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Summary |
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Summary
Introduction
Materials & Methods
Results
Discussion
References
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We evaluated the involvement of the sarcolemmal ATP-sensitive K+ channel in the depolarization of skeletal muscle fibers occurring in an animal model of human hypokalemic periodic paralysis, the K+-depleted rat. After 23-36 days of treatment with a K+-free diet, an hypokalemia was observed in the rats. No difference in the fasting insulinemia and glycemia was found between normokalemic and hypokalemic rats. The fibers of the hypokalemic rats were depolarized. In these fibers, the current of sarcolemmal ATP-sensitive K+ channels measured by the patch-clamp technique was abnormally reduced. Cromakalim, a K+ channel opener, enhanced the current and repolarized the fibers. At channel level, two open conductance states blocked by ATP and stimulated by cromakalim were found in the hypokalemic rats. The two states could be distinguished on the basis of their slope conductance and open probability and were never detected on muscle fibers of normokalemic rats. It is known that insulin in humans affected by hypokalemic periodic paralysis leads to fiber depolarization and provokes paralysis. We therefore examined the effects of insulin at macroscopic and single-channel level on hypokalemic rats. In normokalemic animals, insulin applied in vitro to the muscles induced a glybenclamide-sensitive hyperpolarization of the fibers and also stimulated the sarcolemmal ATP-sensitive K+ channels. In contrast, in hypokalemic rats, insulin caused a pronounced fiber depolarization and reduced the residual currents. Our data indicated that in hypokalemic rats, an abnormally low activity of ATP-sensitive K+ channel is responsible for the fiber depolarization that is aggravated by insulin.
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Introduction |
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Top
Summary
Introduction
Materials & Methods
Results
Discussion
References
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The
familial HOPP is an inherited muscle disease characterized by episodes
of flaccid paralysis and muscle weakness accompanied by lowering of
serum K+ concentration (Lehmann-Horn et
al., 1994
). The HOPP is transmitted as an autosomal dominant
inheritance with a reduced penetrance in the woman (Fouad et
al., 1997). For many years, HOPP has been classified as a
metabolic disease. However, recently, it has been included in the
muscle diseases caused by an abnormal functionality of ion channels and
therefore defined as a channelopathy (Lehmann-Horn and Rüdel,
1996). Linkage studies have recently shown that the HOPP gene is
colocalized with the gene encoding the 
1 subunits of the skeletal
muscle L-type Ca2+ channel (Fontaine et
al., 1994; Jurkat-Rott et al., 1994; Ptacek et
al., 1994). Three points mutations were found within the coding sequence of the 1 subunit of muscular L-type
Ca2+ channel of patients with HOPP (Lehmann-Horn
and Rüdel, 1996). However, these mutations do not affects the
macroscopic Ca2+ current of myotubes cultured
from muscle of patients with HOPP or Ca2+ current
of channels expressed in cell line (Lapie et al., 1997). Furthermore, the Ca2+ channel mutations found in
HOPP patients do not correlate with the depolarization of the fibers,
the characteristic muscle paralysis induced by insulin (Tricarico
et al. 1997b; Lehmann-Horn et al., 1994), and the
lowering of serum K+ concentration occurring
during the attacks (Cannon, 1996; Fouad et al., 1997). These
observations suggest that the phatophysiological mechanisms responsible
for HOPP are complex, possibly involving different factors other than
the Ca2+ channel.
Preliminary data showed that in an animal model of HOPP, the
K+-depleted rats (Dengler et al.,
1979; Bond and Gordon, 1993), the activity of the sarcolemmal
KATP channels is abnormally reduced (Tricarico
et al., 1997b). In these animals as in the humans affected by HOPP, insulin depolarizes the fibers and provokes muscle paralysis (Dengler et al., 1979; Bond and Gordon, 1993; Lehmann-Horn
et al., 1994; Tricarico et al., 1997b). In
contrast, in normokalemic rats, the hormone stimulates the sarcolemmal
KATP channels and leads to a
glybenclamide-sensitive hyperpolarization of the fibers (Iannaccone
et al., 1989; Lehmann-Horn et al., 1994;
Tricarico et al., 1997a). These findings suggest the
possible involvement of this type of channel in the human HOPP. This is
supported by the observations that the K+ channel
openers, cromakalim, and pinacidil are, respectively, capable of
repolarizing the skeletal muscle fibers from humans affected by HOPP
and restoring the muscle strength in the same patients (Spuler et
al., 1989; Grafe et al., 1990; Links et al., 1993; Ligtenberg et al., 1996).
In the current work, we investigated the properties of the KATP channel of skeletal muscle fibers of K+-depleted rats. In particular, we measured the macropatch current, the single-channel conductance, and its sensitivity to the specific blockers ATP and glybenclamide and to the agonist cromakalim. Experiments were devoted to evaluation of the effects of insulin on resting potentials and on sarcolemmal KATP channels of hypokalemic and normokalemic rats.
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Materials and Methods |
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Top
Summary
Introduction
Materials & Methods
Results
Discussion
References
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Rat housing and diet. Male Wistar rats (280 ± 20 g of body weight, 3 months old) were divided into two groups and housed in three rats per cage. The rats were fed 30 g of pellets/day based on different recipes for 23-36 days of treatment. The first group of rats was made hypokalemic by feeding them with a special food free of K+ (Mucedola, Settimo Milanese, Milan, Italy) composed of 21.3% casein, 15% sucrose, 3% grain, 2% multivitamin mixture, 3% mineral water free of K+, 15% DL-methionine, 0.25% choline, 5% corn oil, 5% lard, and 43.35% dextrin. The normokalemic rats were fed with food containing a normal concentration of K+ (0.8%).
For the evaluation of the serum concentration of K+ and Na+, blood samples were collected from the tail vein of the animals on randomly selected rats from each group at the beginning and throughout the period of the treatment. At the time of death, intracardiac blood samples were collected from the rats after an overnight fast for the evaluation of the levels of the two ions and for serum glucose and insulin determinations. The rats were considered hypokalemic when the serum K+ level was
3.2 mEq/liter. In our
condition, 
18 days of treatment were needed to induce measurable
hypokalemia in the rats.
Spectrophotometry and radioimmunoassay.
The blood samples
were centrifuged at 2500 rpm at 7° for 15 min. After this time, a few
microliters of clear serum samples were collected and used for the
analysis. Only serum without evident emolysis was used for the
analysis. Standard flame spectrophotometry (EEL 450 flame photometer;
Corning Glassworks, Corning, NY) was used for detection of the serum
K+ and Na+ levels. The
values were expressed as mEq/liter concentration of ions. The glucose
levels were measured on serum samples using a spectrophotometric method
(Shimadzu 7000 Poli CL, Tokyo, Japan) based on enzymatic reaction
(Tricarico and Conte Camerino, 1994b). The activity of the antibodies
directed against insulin was evaluated by radioimmunoassay using a
standard kit (Ct-Cis Bio International; CIS Diagnostici, Padova,
Italy).
Muscle preparations and single fiber isolation.
The EDL and
FDB muscles were dissected from the bones with the animals under
urethane anesthesia (1.2 g/kg). Resting potential measurements were
performed mainly on EDL muscles, whereas patch-clamp data were obtained
from single fibers of FDB and EDL muscles. After dissection, the
muscles were immersed in Ringer's solution. Single fibers were
obtained by enzymatic treatment of the muscles (Tricarico and Conte
Camerino, 1994a).
Solutions. The pipette solution contained 150 mM KCl, 2 mM CaCl2, 10 mM MOPS, pH 7.2. The bath solution contained normal Ringer's, 145 mM NaCl, 5.5 mM KCl, 1 mM MgCl2, 0.5 mM CaCl2, 5 mM glucose, and 10 mM MOPS, pH 7.2. The low K+ solution contained 145 mM NaCl, 0.5 mM KCl, 1 mM MgCl2, 0.5 mM CaCl2, 5 mM glucose, and 10 mM MOPS, pH 7.2. The symmetrical K+ solution contained 150 mM KCl, 0.5 mM EGTA, and 10 mM MOPS, pH 7.2.
Other solutions were prepared by lowering the concentration of the KCl in the bath from 150 to 30 mM with the addition of a small amount of sucrose as needed to obtain a final osmolarity of 298 mOsM on both sides of the membrane. Stock solutions (5 mM) of the nucleotide tested, Na2ATP, AMP-PNP, MgATP, and MgADP, were prepared by dissolving the chemicals in the bath solution (symmetrical K+). Cromakalim (Sigma Chemical, St. Louis, MO) and glybenclamide (Sigma) were first dissolved in dimethylsulfoxide at concentrations of 0.28 M and 4.05 mM. In the range of the cromakalim and glybenclamide concentrations tested, the corresponding dimethylsulfoxide concentrations did not mimic the effects of the drugs on KATP channels and on resting potentials (solvent control). Microliter amounts of the stock solutions of the nucleotides and drugs tested then were diluted in the bath solutions as needed. Insulin (bovine pancreas; Sigma) was dissolved at 4 units/liter concentration in the low K+ solution and the normal Ringer's solution.Microelectrode technique.
The resting potentials of EDL
muscle fibers were measured by using one intracellular microelectrode
in current clamp mode (Tricarico et al., 1994b). The fibers
were impaled by an intracellular voltage electrode filled with KCl (3 mM) of resistance of 10-15 M
and connected to an
holder/amplifier (WPI Instruments, New Haven, CT). To evaluate the
effects of insulin and of cromakalim on resting potentials, the muscles
were incubated for 30 min at 30° with low K+
solution or normal Ringer's solution enriched with the compounds under
study.
Patch pipettes.
Pipettes were prepared as described
previously (Tricarico and Conte Camerino, 1994a). The tip opening area
of the pipettes were measured by scanning electron microscopy
(Cambridge Instruments). Measurements of micro- and macropatch
conductance and tip opening area were performed on the same pipettes
according to the method of Sakmann and Neher (1983). A linear
correlation between the pipette conductance and the tip opening area
has been found in the range of conductance of 50-1600 nS. The slope of
the straight line was 0.00698, the intercept was 0.302, and the
coefficient of correlation was 0.778. Macropipettes with an average tip
opening area of 5.2 ± 1 µm2 (number of
macropatches, 340) were used to measure the current sustained by
multiple channels (25-35 channel/patch area) and the pharmacological
properties of KATP channels. However, the single-channel conductance and the channel open probability
(Popen) were measured using micropipettes
having a tip opening area of 0.9 ± 0.1 µm2 (number of micropatches, 64). Using this
type of pipette, no more than two or three open channels were observed
in the patches. A few micropatches (3 of 41 excised from normokalemic
rat fibers and 3 of 23 patches excised from hypokalemic rats) contained
only single units. This was tested by observing the single-channel transitions for long periods of time (123 sec-257 sec) in the presence
of internal 50 µM Mg ADP, a physiological stimulator of
the KATP channels, in the bath condition that ensures the
maximum stimulation of the channel open probability (Allard and
Lazdunski, 1992).
Recordings of macropatch currents and single-channel
currents.
Experiments were performed in cell-attached and
inside-out configurations using standard patch-clamp techniques.
Recordings of KATP current were performed during
voltage step of 53 sec from 0 mV of holding potential to different
voltages (from 
60 to +40 mV) after 20 sec from patch excision in the
presence of 150 mM KCl on both sides of the membrane
patches at 20°.
70 to +70 mV). The macropatch current and
single-channel current were recorded at 20 kHz of sampling rate and
filtered at 2 kHz using Axon Instruments (Burlingame, CA) hardware and
the pClamp software package (Tricarico and Conte Camerino, 1994a).
The effects of the cromakalim on the current were evaluated at 60 mV
(Vm), at 20°, in the presence of 150 mM KCl on both sides
of the membrane. The K+ channel opener was tested
in the presence or in the absence of ATP in the bath. Before
recordings, the macropatches were exposed to the agonists for ~20
sec.
To evaluate the effect of insulin on KATP
currents, the FDB and EDL muscles were incubated at 30° with a normal
Ringer's solution enriched with the hormone (4 units/liter) for 45 min. After this time, the muscles were exposed to the enzyme solution
containing insulin for single fiber dissociation.
Analysis of the macropatch current and single-channel
current.
The currents flowing through the macropatches were
calculated subtracting the base-line level of the current defined as
the closed state of the channels, measured in the presence of ATP, from
the open-channel level. Two methods were used to evaluate the rundown
of the current. First, after patch excision, the time-dependent decay
of the current was followed during voltage steps from 0 to 60 mV (Vm)
of 16 min at 20°. We found that no time-dependent decay of the
current of the normokalemic (number of observations, 12) rat fibers
(number of observations, 11) was observed during the first 3 min after
excision. Therefore, the patch-clamp data of hypokalemic and
normokalemic rat channels were collected during this period of time.
Second, we evaluated whether in normokalemic and hypokalemic rats the
maximum amplitude of the current recorded in the presence of 50 µM MgADP matched those recorded in the absence of the
nucleotide.
70 to +70 mV), using the cursor method provided by the Fetchan
program (pClamp software package). The single-channel conductance was
calculated as the slope of the voltage-current relationship of the
channel in the range of potentials from 70 to 10 mV and in the
range of potentials from +10 to +70 mV. No correction of liquid
junction potential was made, estimated to be <+1.8 mV in our
experimental conditions.
The Popen was measured as the ratio between
the time spent by the channel in an open state over the total time of
recording.
Statistics.
The data are expressed as mean ± standard
error unless otherwise specified. The frequency of finding
KATP channels in the macropatches was calculated
as reported previously (Tricarico et al., 1997c).
1)exp(t/1) + P2(1/2)exp(t/2),
where P1 and P2
are the fractional contributions for the respective components to the
area under the curves, 1 and
2 are the time constants of each component,
and t is the time. This type of analysis was performed
within the bursts of openings. The close time interval between bursts
was calculated testing different time intervals of variable durations
to find the minimum one at which the number of closing events were
relatively insensitive to further increase of this parameter. In the
patches from normokalemic rats containing only one open conductance
level, we calculated a mean time interval between bursts of 5.3 ± 0.6 msec (three patches). This parameter was assumed to be the same in
the hypokalemic rats.
The current distributions of normokalemic rats could be fitted with the
Gaussian function f(A) = P·exp{[(A µ)2][2(
2)]}/[
(2
)], or with the sum of two terms, where P is the
fractional contribution of the component(s) to the area under the
curves, A is the bin center amplitudes of the component or
components, µ is the mean of the component, and is the standard
deviation of the component or components.
The agonist effect of cromakalim on the channel current was evaluated
as the ratio between the current in the presence of drug + ATP and the
current in the presence of ATP alone (Icontrol). The concentration-response relationship could be fitted with the equation Idrug/Icontrol = (1 k)/[1 + (ED50/[drug])n], where
Idrug/Icontrol is the ratio
between the current measured in the presence of the drug and that
measured in the absence of drug, ED50 is the
concentrations of the drug needed to enhance the current by 50%,
[drug] is the concentration of the drug tested, n is the
slope of the curves, and k is the maximum change observed in
the current. However, the antagonist effect of ATP and glybenclamide was described by an equation in which the factor
(ED50/[drug])n was
replaced by
([drug]/IC50)n, where
IC50 is the concentration of the compound needed
to reduce the current by 50%.
The algorithms of the fitting procedures used were based on Marquardt
least-squares fitting routine. Significant differences between
individual pairs of mean values were determined by Student's t test.
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Results |
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Top
Summary
Introduction
Materials & Methods
Results
Discussion
References
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In vivo observations. After 23-36 days of treatment with the K+-free diet, the serum K+ concentration significantly decreased from 5.1 ± 0.3 mEq/liter (nine rats) in the normokalemic rats to 2.5 ± 0.2 mEq/liter (nine rats) (p < 0.001) in the hypokalemic rats. The serum Na+ level was unaltered by hypokalemia with 142 ± 3 mEq/liter (nine rats) and 144 ± 4 mEq/liter (nine rats) in the normokalemic and hypokalemic rats, respectively. No significant differences between hypokalemic and normokalemic rats were observed in the serum fasting insulinemia and glycemia states. The insulin concentration was 166 ± 12 pM (five rats) in normokalemic animals and 170 ± 17 pM (five rats) in hypokalemic rats. The glucose levels were 168 ± 11 mg/ml (five rats) in normokalemic animals and 164 ± 12 mg/ml (five rats) in hypokalemic rats.
Resting potentials of skeletal muscle fibers of normokalemic and
hypokalemic rats and effects of insulin.
In normal Ringer's
solution (5.5 mEq/liter K+ ion), the EDL muscle
fibers of hypokalemic rats were depolarized compared with the
normokalemic rat fibers (Table 1). The
perfusion of the muscles with low K+ solution
(0.5 mEq/liter K+ ion) caused a more pronounced
depolarization that was enhanced further after the addition of insulin.
The exposure of the muscle to normokalemic K+
solution containing insulin did not reverse the depolarization (Table
1), indicating that this effect was due to insulin action rather than
to changes in K+ concentrations. In contrast, the
in vitro administration of the hormone to normokalemic rats
(Table 1) caused a marked hyperpolarization of the muscle fibers (Table
1). In agreement with in vivo study (Tricarico et
al., 1997a), this effect was antagonized dose-dependently by
in vitro application of glybenclamide (100 nM to
1 µM).
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, at 60 mV (Vm), calculated by the fitting
routine was 4 ± 2.1 pA/µm2 (54/9
macropatches/rats) and 11.1 ± 2.5 pA/µm2 (goodness of fit, 1.9) (98/9
macropatches/rats) for the first and second populations, respectively
(Fig. 2, B, D, and E). In contrast, only one type of current was found
in the normokalemic rat fibers showing µ ± of 21.5 ± 3 pA/µm2 (goodness of fit, 2) (170/9
macropatches/rats) (Fig. 2, A and C).
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Single KATP channel currents of skeletal muscle fibers of normokalemic and hypokalemic rats. At the channel level, at least two open conductance states (O1 and O2) that were blocked by ATP and opened by cromakalim were recorded routinely by micropatches in the hypokalemic rat fibers (Fig. 4B), but only one conductance level blocked by ATP and opened by cromakalim was detected in the normokalemic rat fibers (Fig. 4A).
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60 mV (Vm), was 0.18 ± 0.08 (3 patches) and 0.13 ± 0.07 (3 patches) for O1 and O2, respectively, and 0.437 ± 0.06 (3 patches) in normokalemic rats.
The duration of the bursts of openings of the two conductance states
present in the hypokalemic rats was significantly reduced compared with
that of the normokalemic rats. The mean ± standard deviation of
the burst duration was 9.5 ± 9 msec, 8.3 ± 8 msec for O1
and O2 levels, and 246.5 ± 21 msec (p < 0.001) for the normokalemic rat channel, respectively. Kinetic analysis
performed within the bursts of openings revealed that the mean open
time did not differ significantly being 2.31 ± 1.5 msec (three
patches), 2.22 ± 1.9 msec (three patches), and 2.36 ± 2.1 msec (three patches) for O1 and O2 levels and for that of the
normokalemic rat channel, respectively. The distribution of the open
dwell-time of hypokalemic and normokalemic rat channels could be fitted
by the sum of two exponential functions (Fig.
6, A, C, and E) (goodness of fit, 1.8-2.05). The 1 was 0.311 ± 0.08 and 0.321 ± 0.08 msec
for O1 and O2, respectively, and 0.351 ± 0.09 msec for the
channel of normokalemic rats, where 2 was 2.5 ± 0.8 and
2.3 ± 0.6 msec for O1 and O2, respectively, and 2.1 ± 0.9 msec for the channel of the normokalemic rats. No significant changes
were observed in the mean close time of 2.01 ± 1.7 msec (three
patches), 1.99 ± 1.8 msec (three patches), and 2.26 ± 2.0 msec (three patches) for O1 and O2 levels and for that of the
normokalemic rat channel, respectively. Also, the distribution of the
close dwell-time of hypokalemic and of the normokalemic rat channels
could be fitted by the sum of two exponential functions (Fig. 6, B, D,
and F) (goodness of fit, 1.9-2.1). The 1 was 0.211 ± 0.06 and
0.198 ± 0.08 msec for O1 and O2 levels, respectively, and
0.195 ± 0.09 msec for that of the normokalemic rats channel,
whereas 2 was 2.4 ± 0.8 and 2.3 ± 0.6 msec for O1 and O2
levels, respectively, and 2.1 ± 0.9 msec for that of the
normokalemic rat channels. In our experiments, it was not possible to
accurately evaluate the long close times separating the bursts because
longer recording periods were needed to collect sufficient data and the
channel activity often decreased with time, leading to a considerable patch-to-patch variability (Ashcroft and Ashcroft, 1990).
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Effects of insulin on KATP currents of the
skeletal muscle fibers of normokalemic and hypokalemic rats.
In
hypokalemic rats, the incubation of FDB and EDL muscles with insulin
reduced the residual KATP current. In these
fibers, only one population of KATP current was
detected with µ ± calculated by fitting routine of 3.6 ± 2 pA/µm2 at 60 mV (Vm) (101/17
macropatches/rats) (goodness of fit, 1.89) (Fig.
8B). In contrast, in normokalemic rats,
insulin enhanced the currents from 21.5 ± 3 pA/µm2 (170/9 macropatches/rats) to 29 ± 5 pA/µm2 (139/7 macropatches/rats) (goodness
of fit, 2.1) (Fig. 8A).
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), ATP and glybenclamide inhibited the
KATP current of muscle fibers preincubated with insulin but with less efficacy; the corresponding
IC50 values were 380 ± 10 µM
and 110 ± 11 nM.
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Discussion |
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Top
Summary
Introduction
Materials & Methods
Results
Discussion
References
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KATP channel in hypokalemic rats. We report the presence of an abnormal KATP channel in the skeletal muscle fibers of the hypokalemic rats. This channel fluctuates between at least two open conductance states that could be distinguished on the basis of their slope conductance (at negative membrane potential) and open probability. None of these states were detected on fibers from EDL and FDB muscles of normokalemic rats. Low conductance channel states may have originated either from substates of a single population of channel or from separate channel populations. The frequent fluctuations observed between the fully open (O1 level) and the low conductance state (O2 level) and the occurrence of direct transitions from a fully open to a closed state crossing the intermediate O2 level (Fig. 5, arrows) suggest that the two levels arise from the same pore. The fact that the kinetic parameters of the two levels found in the hypokalemic rats measured within the bursts of openings were not different in respect to that of the normokalemic rat channel supports this view.
Subconductance states in KATP channels are observed rarely, and the molecular mechanisms that explain the appearance of these states in KATP channel are not known. Fan et al. (1993) and Vivaudou and Forestier
(1995) have shown that the lowering of internal pH below 6.5 leads to
the appearance of several subconductance states in cardiac and skeletal
muscle KATP channels, respectively. This
phenomenon is reverted by rising the internal pH to physiological values (Fan et al., 1993). Weik et al. (1989)
have shown that the internal block by high concentration of adenine
produces subconductance states in the skeletal muscle
KATP channel (Weik et al., 1989). Both
situations imply reversible modifications of the channel pore, such as
changes in the surface charge distribution or internal block of the
pore. Although it is unlikely that a low internal pH or adenine is
responsible for the appearance of subconductance states in our
experiments (subconductance states were observed in excised patches on
which the effects of endogenous factors can be excluded), it is still
possible that a small molecule or ion directly plugs the conduction
pathway and, by shuttling in and out rapidly, reduces the unitary
current. This is supported by the fact that the subconductance state
(O2) disappeared when the patches were depolarized.
From our experience, an homogeneous distribution of
KATP current can be detected in the normokalemic
rats of 3-4 months of age. No silent macropatches were detected in
these animals. In contrast, in hypokalemic rats of the same age, two
populations of currents of lower amplitude were detected. The reduced
KATP currents that we recorded in the fibers of
the hypokalemic rats seem to be the result of the lower
Popen rather than of the two open
conductance states, although an additional mechanism is required to
explain the presence of the two current populations. Recently, Inagaki
et al. (1997) and Bryan et al. (1997) showed that
an optimal molar ratio of the and 
monomers that compose the
KATP channel complex of 1:1 is required to have
functional KATP channel in cell line (Clement
et al., 1997; Inagaki et al., 1997). These authors also showed that the coexpression of ( ) + results in
a sort of negative dominant effects of the monomer on the complex
so that a very low KATP current can be recorded
(Inagaki et al., 1997). The coexpression of the alone
elicits a high KATP current of well known
properties. On the basis of these findings, we can hypothesize that in
hypokalemic rats an erroneous assembly occurring in same fractions of
the membrane of the and monomers results in a complex
responsible for the first current population of lower amplitude.
There was a good correlation between the effects of cromakalim at
single-channel and macroscopic levels. This is supported by two
findings. First, the concentrations of cromakalim effective on
KATP channels also were effective on the resting
potentials of the hypokalemic rat fibers. Second, the repolarization of
the hypokalemic rat fibers induced by the K+
channel opener was abolished by nanomolar concentrations of
glybenclamide. However, the agonist effects of the compound seemed to
be more pronounced on the hypokalemic rat channels than on the
normokalemic one, suggesting a state-dependent effect of the drug. The
lack of effects of cromakalim on the resting potential of fibers of normokalemic rats was due to the fact that this parameter was close to
the equilibrium potential for K+, so there was no
favorable electrical gradient allowing an efflux of
K+ from the fibers.
Effects of insulin on resting potentials and on KATP
channels of the skeletal muscle fibers of normokalemic and hypokalemic
rats.
The relevance of our findings resides in the fact that
insulin exerted opposite electrical effects in the hypokalemic and normokalemic rat fibers at macroscopic and single-channel levels. In
normokalemic rats, insulin enhanced the current and induced a
glybenclamide-sensitive hyperpolarization of the fibers. In hypokalemic
rats, the hormone almost completely abolished the current and strongly
depolarized the fibers. The relationship among the
KATP channel, resting potential, and the effects
of insulin is consistent with the existence of a functional coupling between KATP channels and
Na+/K+-ATPase. There is
evidence that in epithelial cells (Hurst et al., 1993) and
pancreatic cells (Ding et al., 1996), the
KATP channels are functionally coupled to
Na+/K+-ATPase so a
stimulation of the pump promotes different effects; for example, it
leads to an activation of the outward KATP
current that sustains the hyperpolarization of the cell membrane and
controls the duration of the spike burst intervals (Hurst et
al., 1993; Ding et al., 1996). An inhibition of the
pump down-regulates the KATP channels (Hurst
et al., 1993). In skeletal muscles, the existence of a
functional coupling between these two macrocomplexes is supported by
the fact that the in vivo or in vitro
administration of insulin to the normokalemic rats caused an
hyperpolarization of the fibers that is antagonized by glybenclamide
and ouabain (Iannaccone et al., 1989; Bond and Gordon, 1993;
Tricarico et al., 1997a). We believe that the functional
coupling between
Na+/K+-ATPase and
KATP channels helps to explain the mechanism by
which insulin produces depolarization and possibly the paralysis in hypokalemic rats. It is known that insulin hyperpolarizes the skeletal
muscle fibers of normokalemic rats by a direct action on the
Na+/K+-ATPase, producing an
influx of K+ into the muscle and transient
hypokalemia (Iannaccone et al., 1989; Lehmann-Horn et
al., 1994). We propose here that in normokalemic rats, the effects
of insulin are buffered by an activation of the sarcolemmal
KATP channel that sustains the hyperpolarization of the fibers, whereas in hypokalemic rats, insulin stimulates the
influx of K+ into the muscles but no efflux of
K+ occurs due to the abnormally low basal
activity of the KATP channels. This precipitates
the hypokalemia leading to depolarization of the fibers and possibly to
the paralysis. An additional contribution to the depolarization of the
fibers came from the inhibitory effects exerted by insulin on the
residual KATP current and from the fact no
significant change in the sensitivity of the channel to ATP was found
after insulin treatment.
Relationship between the K+-depleted rats and human
HOPP.
A point of interest is the relationship between the effects
of the hypokalemia tested in the rats and patients with HOPP. In
normokalemic solution, the fibers of K+-depleted
rats and of humans affected by HOPP are already depolarized, although
in patients, a less pronounced depolarization occurs (Rudel et
al., 1984; Lehmann-Horn et al., 1994). In both
K+-depleted rats and patients, further
depolarization occurs after in vitro exposure of the muscles
to low K+ solution, insulin, or both (Dengler
et al., 1979; Lehmann-Horn et al., 1994). Muscle
fiber depolarization and flaccid paralysis occur in
K+-depleted rats and in other secondary forms of
the disorder after in vivo administration of insulin and
glucose (Lehmann-Horn et al., 1994; Tricarico et
al., 1997b). This also represents a useful diagnostic test in
humans with HOPP (Lehmann-Horn et al., 1994). The fact that
insulin causes depolarization and paralysis in the primary and
secondary forms of the disorder is consistent with the hypothesis that
this phenomenon is not directly related to the mutations of the
Ca2+ channel detected in humans with HOPP.
). This often is observed in young
male patients (9-18 years of age) in whom the attacks of weakness and
paralysis last 6-7 days.
We found in the hypokalemic rats that the abnormalities are limited to
muscular defects (abnormal KATP channel complex,
altered insulin pathway involved in the regulation of the channel, or both) rather than changes in the serum insulin and glucose levels. This
is supported by the finding that no significant differences in the
fasting insulinemia and glycemia were found between normokalemic and
hypokalemic rats. Similar conclusions have been drawn from studies in
humans (Ligtenberg et al., 1996). In addition to these observations, we showed that cromakalim, through openings of
KATP channels, repolarized the skeletal muscle
fibers of the K+-depleted rats. Similarly, the
K+ channel opener is effective in producing a
significant repolarization and in increasing in vitro the
force of contraction of skeletal muscle from humans with HOPP (Spuler
et al., 1989; Grafe et al., 1990).
In conclusion, even with the knowledge that HOPP is linked to mutations
of the 1 subunit of skeletal muscle Ca2+
channel, the lack of sarcolemmal KATP channel
activity helps to explain most of the symptoms of the primary and
secondary forms of these disorders.
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Acknowledgments |
|---|
We are grateful to Dr. Mariagrazia Barbieri and Dr. Roberto Poli for their helpful assistance.
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Footnotes |
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Received July 22, 1997; Accepted March 16, 1998
This work was supported by Telethon-Italy (Grant 579).
Send reprint requests to: Prof. D. Conte Camerino, Dipartimento Farmacobiologico, Facoltà di Farmacia, via Orabona n°4, 70126, Università di Bari, Italy. E-mail: conte{at}farmbiol.uniba.it
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Abbreviations |
|---|
KATP, ATP-sensitive
K+ channel;
EGTA, ethylene glycol bis(-aminoethyl
ether)-N,N,N',N'-tetraacetic
acid;
AMP-PNP, adenylylimidodiphosphate;
EDL, extensor digitorum
longus;
FDB, flexor digitorum brevis;
MOPS, 3-(N-morpholino)propanesulfonic acid;
HOPP, hypokalemic
periodic paralysis.
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References |
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Top
Summary
Introduction
Materials & Methods
Results
Discussion
References
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-cells.
Biochem Biophys Acta
1279:
219-226[Medline].1-subunit gene mutations causing hypokalemic periodic paralysis.
Neurol Disord
7:
33-38.-cell ATP-sensitive K+ channel.
FEBS Lett
409:
232-236[Medline].This article has been cited by other articles:
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 D. Tricarico, A. Mele, A. L. Lundquist, R. R. Desai, A. L. George Jr., and D. C. Camerino Hybrid assemblies of ATP-sensitive K+ channels determine their muscle-type-dependent biophysical and pharmacological properties PNAS, January 24, 2006; 103(4): 1118 - 1123. [Abstract] [Full Text] [PDF]
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),
responses of the normokalemic rat currents to the blockers.
Continuous lines, responses of the two components, of
lower (
) and higher amplitude (
), of the hypokalemic rat currents
to the blockers. The macropatch currents were recorded after 20 sec
from macropatch excision during voltage steps from 0 to 
