Effects of temperature on slow and fast inactivation of rat
skeletal muscle Na+
channels
Robert L.
Ruff
Departments of Neurology and Neuroscience, Case Western Reserve
University School of Medicine, Louis Stokes Cleveland Veterans
Affairs Medical Center, University Hospitals of Cleveland,
Cleveland, Ohio 44106
 |
ABSTRACT |
Patch-clamp
studies of mammalian skeletal muscle
Na+ channels are commonly done at
subphysiological temperatures, usually room temperature. However, at
subphysiological temperatures, most
Na+ channels are inactivated at
the cell resting potential. This study examined the effects of
temperature on fast and slow inactivation of
Na+ channels to determine if
temperature changed the fraction of Na+ channels that were excitable
at resting potential. The loose patch voltage clamp recorded
Na+ currents
(INa) in vitro
at 19, 25, 31, and 37°C from the sarcolemma of rat type IIb
fast-twitch omohyoid skeletal muscle fibers. Temperature affected the
fraction of Na+ channels that were
excitable at the resting potential. At 19°C, only 30% of channels
were excitable at the resting potential. In contrast, at 37°C, 93%
of Na+ channels were excitable at
the resting potential. Temperature did not alter the resting potential
or the voltage dependencies of activation or fast inactivation.
INa available at
the resting potential increased with temperature because the
steady-state voltage dependence of slow inactivation shifted in a
depolarizing direction with increasing temperature. The membrane
potential at which half of the Na+
channels were in the slow inactivated state was shifted by +16 mV at
37°C compared with 19°C. Consequently, the low availability of
excitable Na+ channels at
subphysiological temperatures resulted from channels being in the slow,
inactivated state at the resting potential.
mammalian skeletal muscle; sodium channel; sodium current; fast
inactivation; slow inactivation; paramyotonia congenita; hyperkalemic
periodic paralysis
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INTRODUCTION |
MAMMALIAN SKELETAL MUSCLE and other excitable tissues
have two inactivation processes with different kinetics and voltage dependencies (1, 2, 14, 21, 34-36, 38, 39, 41). Fast inactivation
closes channels on a millisecond time scale, whereas slow inactivation
takes seconds to minutes. In rat and human skeletal muscle, fast
inactivation helps to terminate the action potential (1, 34, 36). Slow
inactivation is too slow to affect action potential termination.
However, in mammalian skeletal muscle, slow inactivation operates at
more negative potentials than fast inactivation, so that the
distribution of channels between the closed and slow inactivated state
regulates the number of excitable
Na+ channels as a function of the
membrane potential (32, 35, 36, 38, 39, 41). Slow inactivation changes
the number of excitable channels but does not change the single-channel
conductance or single-channel open time (32). Physiological studies
suggest that fast and slow inactivated states are distinct (5, 30, 32,
42, 45). Na+ channel mutations can
independently change fast or slow inactivation (10, 16, 17, 46).
Therefore, the slow and fast inactivated states probably represent
distinct Na+ channel
conformations. Although Na+
channels may undergo slow inactivation from either open, closed, or
fast-inactivated states (32), the rate of development of slow
inactivation or recovery from slow inactivation and the completeness of
slow inactivation may be influenced by the conformations of either the
activation or fast inactivation gates (13, 43).
Voltage-gated Na+ channels are
responsible for the rising phase and subsequent propagation of action
potentials in muscle, nerve, and secretory tissues. One of the
important determinants of membrane excitability is the availability of
Na+ channels. The density of
excitable Na+ channels depends on
the density of channels in the membrane and the fraction of channels
that are excitable (34). Most patch-clamp studies of
Na+ channels are performed at room
temperature. A consistent and puzzling finding in room temperature
studies of cloned Na+ channels
expressed in a variety of cell systems is that a low fraction, 50% or
less, of Na+ channels are
excitable at membrane potentials of
80 mV to
90 mV (20,
26, 43, 47). Similarly, in vitro studies performed at room temperature
on fast-twitch skeletal muscle fibers from rats (32, 34, 35, 41),
rabbits (21), or humans (34, 38, 39) indicate that more than 50% of
Na+ channels are inexcitable at
the resting potential due to Na+
channel slow inactivation. In vivo patch-clamp data on rat fast-twitch skeletal muscle fibers indicate that at physiological temperature most
Na+ channels are excitable at the
cell resting potential. The amplitudes of
Na+ current density
(INa) at the
resting potential of rat fast-twitch fibers studied in vivo (37) are
similar to the maximum values of
INa obtained from
in vitro recordings at 19°C (34). However, to obtain the maximum
value of INa for
in vitro studies, the membrane must be sufficiently hyperpolarized for
a prolonged time to recover Na+
channels from slow inactivation (34). Consequently, a physiologically unreasonably large fraction of Na+
channels are inexcitable at room temperature because they are in the
slow inactivated state.
Temperature could influence the slow inactivation process in several
ways. In some studies of cloned
Na+ channels studied in different
expression systems, only 80-85% of
Na+ channels were subject to slow
inactivation (13, 16, 17, 29, 43). Elevated temperature could increase
the fraction of excitable Na+
channels by reducing the fraction of
Na+ channels that can be slow
inactivated. Alternatively, temperature could change the operative
voltage range for slow inactivation. The high proportion of slow
inactivated Na+ channels at the
resting potential found at subphysiological temperatures could result
if at low temperatures the slow inactivation-membrane potential
relationship was shifted in a hyperpolarized direction.
This study used a loose patch voltage clamp to examine fast and slow
inactivation of
INa in
fast-twitch, type IIb, rat skeletal muscle fibers at temperatures from
19°C to 37°C. The fraction of
Na+ channels that were excitable
at the resting potential increased with temperature. This study
examined the hypothesis of whether temperature altered the fraction of
Na+ channels that were excitable
at the resting potential by changing the steady-state voltage
dependence of slow inactivation.
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METHODS |
Male Wistar rats (290-320 g) were anesthetized with
intraperitoneally injected pentobarbital, omohyoid muscles were
removed, and the rats were killed by pentobarbital overdose.
Tissue handling.
Muscle fibers were dissected and studied in a solution composed of 19 parts Tyrode solution (95% by volume) and 1 part rat serum (5% by
volume). The presence of 5% rat serum in the bathing solution greatly
increased the survival time for muscle fibers at 31 and 37°C. In
bathing solution with 5% rat serum, omohyoid muscle fibers maintained
resting potentials of less than
90 mV for more than 1 h. The
rat serum was obtained by exsanguinating rats, allowing the removed
blood to clot and collecting the clear serum. Serum was discarded if
any visual evidence of hemolysis was present. Serum was centrifuged at
5,000 rpm to remove any cellular particulate material. The supernatant
serum was filtered through a 0.01-µm Millipore filter and stored at
70°C. Tyrode solution contained (in mM) 135 NaCl, 3.5 KCl, 1 MgCl2, 6 CaCl2, 10 HEPES and 10 glucose.
Solutions were vigorously gassed with O2, and the pH was 7.4.
Dissections were performed at 19°C. Surface connective tissue was
removed as completely as possible by a combination of dissection and
collagenase treatment (32, 38, 39). The dissection began after the
muscle had been in oxygenated, circulated Tyrode solution containing 2 mg/ml of collagenase (Sigma type I) and 1 mg/ml of bovine albumin
(Sigma fraction V) for 45 min. In the later stages of dissection, the
collagenase-containing solution was perfused only over the middle of
the fibers so that the tendon attachments remained intact. The
enzymatic treatment enabled dissection of the muscle to a preparation
that was only a few fibers thick and greatly improved the quality of
the seals between the voltage-clamp pipettes and the sarcolemma. Before
current recordings were made, the collagenase was diligently washed
from the bathing chamber. INa recordings
were made >200 µm from the endplate region to avoid the region of
high Na+ channel density near the
endplate (38, 39).
Temperature regulation.
Experiments were performed at 19, 25, 31, and 37°C. The recording
chamber had three thermistors that monitored bath temperature. The
three temperature values had to all be within 0.2°C of the target
temperature for an experiment to proceed. The temperature of the
recording chamber was controlled with a Peltier device (Cambion,
Cambridge, MA) with a direct current feedback controller. The chamber
used for recording
INa was modified
to reduce the exposed solution-air interface to 5 mm × 7 mm. In
addition, the chamber was perfused by one of four reservoirs of bathing
solution. Each reservoir was maintained at one of the target
temperatures by a surrounding temperature-controlled water bath. The
temperature of the recording chamber could be changed within 10 s. A
muscle preparation was kept at a recording temperature for at least 5 min before recording
INa or membrane potentials.
Loose patch voltage clamp.
Technical details of the loose patch voltage clamp technique used to
measure INa were
previously described (32, 38, 39). With the bathing solution described
in Tissue handling, the
resistive seals between the cleaned muscle membranes and pipettes were
>20 M
at all temperatures studied. The high seal resistance
reduced the fraction of membrane current that passed across the seal
resistance rather than through the pipette and improved the frequency
resolution and response time of the patch clamp. The fraction of
membrane current lost across the seal was corrected for by analog and
digital compensation. Micropipettes had tip diameters after fire
polishing of ~10 µm and resistances of 200-300 k
. These
sizes of pipettes were chosen to permit sampling from a sufficiently
large patch of membrane to reduce local variations in
INa density and
yet not to stimulate too large a current so that voltage control of the
cell was maintained. The pipettes were coated with a double layer of
Sylgard (Dow Corning 184, Midland, MI) to within 100 µm of the tip to
reduce capacitive coupling between the bath and pipette. Minimal
suction was applied to the loose patch micropipettes to avoid the
formation of membrane blebs (24, 38).
Measurement of membrane current with the loose patch.
A potential applied to the micropipette changed the transmembrane
potential of the small patch of membrane under the pipette. Analog and
digital corrections compensated for the current that flowed across the
seal between the pipette and the sarcolemma so that the transmembrane
potential was controlled and the transmembrane current could be
measured (35, 38, 39, 41). The maximum inward
INa from a patch
of membrane at a given holding potential, INa max,
was determined by a group of six depolarizing test pulses. Test pulses
were 4 ms long at 19, 25, and 31°C and 2 ms long at 37°C. Each
test pulse was preceded by a 20-ms, 50-mV hyperpolarizing prepulse,
relative to the holding potential, to remove fast inactivation of
INa. In some
experiments, the 20-ms hyperpolarizing prepulses to remove fast
inactivation were scaled so that all prepulses were to
120 mV.
The depolarization of the test pulses were incremented in 6-mV steps.
Voltages of the test pulses were chosen to bracket the membrane
potential that elicited
INa max,
which is called VINa max.
Details of the pulse protocols used to measure
INa and the
calculations of
INa max and
VINa max from the six pulse protocols were previously described (38, 39).
INa max was
measured every 15 s or every 30 s to assay the state of slow
inactivation of macroscopic
INa.
Measurement of the voltage dependence of fast inactivation.
The steady-state voltage dependence of fast inactivation was studied by
applying 20-ms conditioning prepulses that were immediately followed by
4-ms depolarizing test pulses to about
VINa max at 19, 25, and 31°C (35, 36, 38, 39, 41). At 37°C, the test
pulses were 2-ms long. A prepulse duration of 20 ms was sufficiently long to study fast inactivation in rat skeletal muscle fibers at the
membrane potentials examined in this study (35, 36, 38, 39, 41). The
steady-state voltage dependence of fast inactivation, the
"h
"
curve, was described by a Boltzmann distribution
|
(1)
|
where
INa/INa max
is the relative amplitude of
INa during a test
pulse, Vm is the
membrane potential during a prepulse,
Vh1/2 is the
potential of the prepulse that inactivates half the
Na+ channels by the fast
inactivation process, and
Ah is a parameter determining the steepness of the
h
-membrane
potential relationship.
Slow inactivation.
Slow inactivation was studied by measuring the size and time course of
the change in
INa max
after the holding potential changed. The steady-state values of
INa max at
a given membrane potential and the time constant of the change in
current density following a change in potential were obtained by a
least squares fit of the following function to
INa max
|
(2)
|
where
INa initial
is the initial
INa max,
INa final
is the steady-state
INa max
after changing the potential,
INa(t)
is the current density at each time, and
is the time constant.
Equation 2 was also used to determine
the time constant for development of fast inactivation,
h, from records of
INa.
Due to the time it took for slow inactivation to reach steady state
after a change in membrane potential, the voltage sensitivity of slow
inactivation could not be completely described for a single fiber.
Therefore, the holding potential at which the maximal
INa max was
obtained and the relative value of
INa max at
other holding potentials for fibers within a given group of fibers were
plotted together. The smooth curves were the least squares fits of a
Boltzmann distribution to the data
|
(3)
|
where
s
is the
steady-state slow inactivation of
INa,
INa max/maximal
INa max is
the steady-state
INa max
obtained at a given holding potential relative to the maximal
INa max
that could be obtained when slow inactivation was completely removed, Vs1/2 is the
potential at which 50% of Na+
channels were closed due to slow inactivation, and
As describes the
steepness of the voltage dependence of slow inactivation.
Membrane Na+
conductance.
Membrane Na+ conductance was
calculated from a least squares of the following equation to the data
|
(4)
|
where
G(Vm)
is the Na+ conductance as a
function of membrane potential,
Ipk(Vm)
is the peak inward
INa produced by a
depolarizing test potential to
Vm, and
Erev is the
reversal potential for
INa. The membrane
potential at which
G(Vm)
was half-maximal was called VG1/2.
Erev was
calculated as the zero current intercept for current-voltage relationships obtained from current traces such as those shown in Fig.
1. For the current traces in Fig. 1,
Erev was +49 mV
at 19°C and +47 mV at 37°C.
Resting potentials.
Resting potentials were measured with an intracellular microelectrode
filled with 3 M KCl at a distance of 0.1-0.15 mm from the patch
electrodes. Resting potential was measured for each cell after
completing INa
recordings. Vh1/2
was measured before and after impalement with the voltage electrode to
determine the depolarization produced by the impalement. The actual
resting potential was the potential measured by the voltage electrode minus the depolarization associated with the impalement (35, 38, 39,
41). The membrane potential throughout an experiment was determined
from three factors: 1) the directly
measured membrane potential at the end of an experiment,
2) the effect of temperature on the
resting potential (see Table 1), and
3) changes in
Vh1/2, which were
used to determine shifts in the membrane potential over time for
experiments at a single temperature. Current recordings were stopped if
the compensation of the resting membrane potential for different
temperatures combined with changes in
Vh1/2 showed that
the membrane potential had changed by more than 5 mV from the beginning
of an experiment.
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Table 1.
Effects of temperature on the resting membrane potential and the
voltage dependencies of conductance and fast inactivation
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Histochemical fiber type.
Histochemical fiber type was determined at the completion of current
recordings, as previously described, from analysis of a segment of the
fiber that INa
was recorded from (38, 39).
Statistical analysis.
Data were analyzed with ANOVA using two-tailed tests with
set at
0.05 (38, 39). Curve fitting was performed using a commercial product,
SigmaPlot (Jandel Scientific, San Rafael, CA). Values are shown as
means ± SE.
 |
RESULTS |
This study examined
INa from rat
omohyoid type IIb skeletal muscle fibers at 19, 25, 31, and 37°C.
The effects of temperature on the voltage dependencies of activation
and fast inactivation of
INa were studied
in 12 cells at each of the four study temperatures. Seven cells were
studied at three temperatures, and 2 cells were studied at only two
temperatures. INa
measurements were made on 18 cells at 19°C, 19 cells at 25°C,
19 cells at 31°C, and 18 cells at 37°C. The voltage dependence
and kinetics of slow inactivation were determined from 15 fibers at
19°C, 13 fibers at 25°C, 15 fibers at 31°C, and 14 fibers
at 37°C.
Effects of temperature on resting membrane potential.
The resting potential of a fiber, determined by impaling the cell with
an intracellular microelectrode, could be measured only at the end of a
current recording session because cells studied with the loose patch
clamp would depolarize after the membrane was impaled (35, 41). For
studies at a single temperature, changes in
Vh1/2 were used
to measure alterations of the cell resting potential during the course
of an experiment (32). Because it was not known if
Vh1/2 varied with
temperature, the strategy used to determine the membrane potential of a
cell during the course of an experiment was modified when the cell was
studied at several temperatures. First, this study determined the
effect of temperature on the resting potential of the omohyoid muscle fibers. Second, to ensure that changes in the voltage dependence of
INa at different
temperatures were reversible and reflected only the effects of
temperature, INa
data were accepted only from fibers that were studied at two or more
temperatures when at least one temperature was studied twice and the
voltage dependencies of
INa values for
recordings at the same temperature were comparable.
This study determined the effect of temperature on the resting membrane
potential of cells by measuring the resting potential of 40 or more
fibers at each study temperature. Table 1 shows a tendency for fibers
to hyperpolarize with increased temperature, but the resting potentials
were not significantly altered by temperature. This study determined
the resting potential of a fiber at each temperature during the course
of an experiment using the following steps:
1) the membrane potential was
determined at the end of an experiment by direct measurement,
2) changes in the value of Vh1/2 were used
to determine changes in the resting potential during portions of an
experiment performed at a specific temperature, and
3) the differences among the resting
potentials shown in Table 1 were used to determine changes in the
membrane potential associated with changing the experiment's temperature.
Temperature effects on activation and fast inactivation of
INa.
Figure 1 shows inward
INa traces
generated at 19 and 37°C from the same type IIb fiber. On the basis
of the resting potential values shown in Table 1, the membrane
hyperpolarized by 2 mV when the temperature was increased from 19 to
37°C. At 19°C, the inward currents were stimulated by
depolarizing pulses from
55 mV to +35 mV in 10-mV increments. At
37°C, the inward
INa traces were
stimulated by depolarizing pulses from
57 mV to +33 mV.

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Fig. 1.
Na+ current density
(INa) recorded
with the loose patch voltage clamp from a single patch on the
extrajunctional membrane rat type IIb omohyoid skeletal muscle fiber at
19 and 37°C. Although the pipette was not moved during the
experiment, slight change in the fiber geometry associated with change
in temperature may have slightly altered the amount of membrane that
was being sampled by the patch pipette. Currents were elicited at the
resting potential of the fiber, which was 103 mV at 19°C and
105 mV at 37°C. Currents were elicited by 4-ms depolarizing
pulses in increments of 10 mV from 55 mV to +35 mV at 19°C
and by 2-ms duration pulses from 57 mV to +33 mV at 37°C.
Note that top time calibration applies
to currents at 19°C and bottom
time calibration applies to currents at 37°C. At 19°C, maximum
inward INa from a
patch of membrane at a given holding potential
(INa max)
was 9.4 mA/cm2 at 103 mV
and was 17.3 mA/cm2 with slow
inactivation removed. At 37°C,
INa max was
19.2 mA/cm2 at 105 mV and
was 20.3 mA/cm2 with slow
inactivation removed.
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Currents activated faster, and fast inactivation developed more rapidly
at 37°C compared with at 19°C. Figure
2 shows the voltage dependence for the
apparent rate constant for development of fast inactivation,
1/
h, for the current traces
shown in Fig. 1. Over the voltage ranges shown in Fig. 2,
1/
h varied linearly with
voltage. The slope of the
1/
h-voltage relationship was
more than 10-fold steeper at 37°C. At 19°C, the slope of the
1/
h-voltage relationship was
0.0267 ms
1 · mV
1
and at 37°C the slope was 0.300 ms
1 · mV
1.
The apparent rate of development of fast inactivation at 0 mV (1/
h 0mV) was 15-fold
faster at 37°C compared with that at 19°C.

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Fig. 2.
Voltage dependence of 1/time constant for development of fast
inactivation ( h) for the
declining phases of the current traces from the rat type IIb omohyoid
muscle fiber shown in Fig. 1. Equation 2 (see text) was fit to the declining phase of each
current trace to calculate h.
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Figure 3 shows the temperature dependence
of
h, as an Arrhenius plot, for
12 cells studied at 19, 25, 31, and 37°C. In Fig. 3, the logarithm
of the mean value of
h 0mV, was plotted
against temperature. The linear decline of the logarithm of
h 0mV with temperature
corresponded to a
Q10 value of 4.13 for 1/
h over the temperature
range of 19 to 37°C.

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Fig. 3.
Temperature dependence of h at
0 mV ( h 0mV), as an
Arrhenius plot, for the 12 cells studied at each of the study
temperatures (19, 25, 31, and 37°C).
Q10 for the
decline of h 0mV with
temperature over the range from 19 to 37°C was 4.13.
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Temperature did not appreciably alter the voltage dependencies of
activation or fast inactivation. Figure 4
shows the voltage dependencies of activation and fast inactivation at
19 and 37°C for the 13 cells studied at both temperatures.
VG1/2
occurred at
36.0 ± 0.7 mV at 19°C and at
35.2 ± 0.8 mV at 37°C. The voltage dependencies of fast inactivation
were similar at 19 and 37°C. Vh1/2 was
75.1 ± 1.8 mV at 19°C and
78.5 ± 2.1 mV
at 37°C. Ah values were also similar (5.1 ± 0.8 mV at 19°C and 5.2 ± 0.7 mV at 37°C). Table 1 shows the values for
Vh1/2,
Ah, and
VG1/2 for the 21 cells that were studied at two or more temperatures. The values of
Vh1/2,
Ah, and
VG1/2 in Table 1
were not significantly different at 19°C compared with those at
37°C and were not significantly different when values at 19°C
were compared with those at any other temperature.

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Fig. 4.
Steady-state voltage dependence for fast inactivation and voltage
dependence of peak Na+ conductance
for 13 cells studied at 19°C and 37°C. Smooth curves are fits
of Eq. 1 (see text) to the data for
fast inactivation and Eq. 4 (see text)
to the conductance data. For the conductance data, reversal potential
for INa was +50
mV at 19°C and +48 mV at 37°C. Membrane potential at which the
Na+ conductance as a function of
membrane potential was half maximal was 36.0 ± 0.7 mV at
19°C and 35.2 ± 0.8 mV at 37°C. For fast
inactivation, potential that inactivates half the
Na+ channels by fast inactivation
of INa was
75.1 ± 1.8 mV at 19°C and 78.5 ± 2.1 mV at
37°C. Parameter determining steepness of the steady-state fast
inactivation of
INa
(h )-membrane
potential relationship was 5.1 ± 0.8 mV at 19°C and 5.2 ± 0.7 mV at 37°C.
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Temperature increased the amount of
INa available at the
resting potential.
In contrast to temperature not affecting the voltage dependencies of
activation and fast inactivation, temperature appreciably altered the
amount of INa
elicited by depolarizations from the resting potential. The maximum
inward current at the resting potential (INa RP)
increased with temperature. In Fig. 1, at 19°C,
INa RP was
9.4 mA/cm2 at
103 mV and,
at 37°C,
INa RP was
19.2 mA/cm2 at
105 mV. The
increase in
INa RP
could have resulted from a shift in the voltage dependence of slow
inactivation or a reduction in the fraction of channels affected by
slow inactivation. Either alteration in slow inactivation would have
reduced the number of channels at the resting potential that were slow
inactivated at higher temperature. Alternatively, increased temperature
could have unmasked hidden channels or increased the maximum
probability that a channel would open with depolarization
(Po). If
temperature acted by shifting the voltage dependence of slow
inactivation or by reducing the fraction of channels susceptible to
slow inactivation, then the density of excitable
Na+ channels measured after
prolonged hyperpolarizations that removed slow inactivation should be
comparable at different temperatures. If increased temperature unmasked
Na+ channels or increased
Po, then the
maximum density of open channels in response to a depolarizing pulse
would be larger at 37°C compared with at 19°C.
INa max
provided an experimental assay of the density of open channels.
The single-channel conductance should increase with a
Q10 value of
~1.3 (18, 19, 23). Consequently, if temperature did not change the
density of excitable channels, then
INa max
should have increased ~1.6-fold from 19 to 37°C, corresponding to
the Q10 value of
1.3. For the cell shown in Fig. 1,
INa max
after slow inactivation was removed was 17.3 mA/cm2 at 19°C and 20.3 mA/cm2 at 37°C. The maximal
INa max
increased only 1.2-fold in response to a 18°C increase in
temperature for the cell shown in Fig. 1. Table
2 shows that maximal
INa max
increased slightly with temperature. The temperature-dependent increase
in maximal
INa max
shown in Table 2 corresponded to a
Q10 of 1.21. Therefore, temperature did not increase the maximum density of open
channels produced by a depolarizing pulse. Consequently, the effect of
temperature on
INa max was
probably mediated by an effect of temperature on slow inactivation and
not the result of unmasking Na+
channels or increasing
Po.
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Table 2.
Effects of temperature on the peak INa available at RP and
the steady-state voltage dependence of slow inactivation
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Table 2 shows that the fraction of
INa available at
the resting potential compared with the maximal value of
INa with slow inactivation removed
(INa RP/maximal
INa max)
increased with temperature. At 37°C, 93% of
Na+ channels were excitable at the
resting potential compared with only 30% of channels being excitable
at 19°C.
Temperature changed the voltage dependence of slow inactivation of
INa.
To determine if temperature changed the voltage dependence of slow
inactivation or altered the fraction of
Na+ channels that were susceptible
to slow inactivation, slow inactivation was studied at each of the
study temperatures. Because of the time required to study slow
inactivation, it was not practical to study slow inactivation in the
same cell at different temperatures. Table 2 shows the number of cells
evaluated at each temperature at which slow inactivation was studied.
Figure 5 shows the development of and
recovery from slow inactivation of
INa for a fiber
at 19°C. Note that
INa max at
100 mV was only 60% of the maximal value of
INa max at
150 mV when slow inactivation was removed. Figure 6 shows the development of and recovery
from slow inactivation of
INa for a fiber
at 37°C. For this fiber,
INa max at
the resting potential of
105 mV was 95% of the maximal value of
INa max and INa max at
90 mV was 55% of the maximal value of
INa max.
Note that, in Fig. 5, slow inactivation reduced
INa max to
<1% of the maximal value of
INa max and
that, in Fig. 6, slow inactivation reduced
INa max to
<5% of the maximal value of
INa max.
The data in Figs. 5 and 6 show that all or almost all
Na+ channels were susceptible to
slow inactivation.

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Fig. 5.
Development of and recovery from slow inactivation of
INa for a fiber
at 19°C. Slow inactivation reversibly changed
INa max in
response to alterations in the holding potential.
INa max,
measured with the loose patch clamp, changed as the holding potential
was successively changed from 100 mV to 75 mV to
100 mV to 150 mV. Arrows
(top) indicate the times when
holding potentials were changed. Values for the time constant
associated with slow inactivation
( s) were obtained by a least
squares fit of Eq. 2 (see text) to the
data. Values of s were 122 s
for 100 mV to 75 mV, 298 s for 75 mV to 100
mV, and 54 s for 100 mV to 150 mV.
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Fig. 6.
Development of and recovery from slow inactivation of
INa for a fiber
at 37°C. Slow inactivation reversibly changed
INa max in
response to alterations in the holding potential.
INa max,
measured with the loose patch clamp, changed as the holding potential
was successively changed from 105 mV to 90 mV to
70 mV to 105 mV and finally to 135 mV. Arrows
(top) of indicate the times when
holding potentials were changed. Values of
s were obtained by a least
squares fit of Eq. 2 (see text) to the
data. Values of s were 22 s for
the transition from 105 mV to 90 mV, 18 s for 90
mV to 70 mV, 16 s for 70 mV to 105 mV, and 41 s
for 105 mV to 135 mV.
s ,
Steady-state slow inactivation of
INa.
|
|
For the experiments shown in Figs. 5 and 6,
INa max was
measured by removing fast inactivation using 20-ms-long hyperpolarizing prepulses. The prepulses amplitudes were 50 mV hyperpolarized relative
to the holding potential. Four different cells were studied at 19 and
37°C using a different protocol in which the hyperpolarizing prepulses were set to
120 mV. The amplitudes of the prepulses needed to bring the membrane potential to
120 mV during these four experiments were determined from the estimated potential of the
membrane patch. The potential of the membrane patch was estimated by
comparing the change in potential of the tip of the patch pipette that
was needed to fast inactivate 50% of the channels with the values of
Vh1/2 shown in
Table 1. The data on the development of and recovery from slow
inactivation obtained from the four cells studied at 19 and 37°C
using prepulses to
120 mV to remove fast inactivation were
similar to the data shown in Figs. 5 and 6.
The voltage dependence of slow inactivation was determined by plotting
the steady-state values for
INa max/maximal
INa max vs.
membrane potential for all of the fibers studied at a given temperature. Figure 7 shows the voltage
dependence of
s
for 15 fibers at 19°C and 14 fibers at 37°C. At both temperatures, slow inactivation could eliminate
INa max,
which indicates that all of the
Na+ channels were susceptible to
slow inactivation.
Vs1/2 occurred at
104.0 ± 2.1 mV at 19°C and at
87.9 ± 2.7 mV
at 37°C (P < 0.001). The slopes
of the steady-state slow inactivation curves were similar with
As = 5.7 ± 0.8 mV at 19°C and 5.4 ± 0.9 mV at 37°C. Table 2 shows the
values of Vs1/2
and As at 19, 25, 31, and 37°C. Figure 8 shows that
Vs1/2 shifted
linearly toward more positive potentials with increasing temperature.
Vs1/2 increased by 16.1 mV from 19°C to 37°C. The slope of the temperature
dependence of
Vs1/2 was 0.894 mV · °C
1.

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Fig. 7.
Voltage dependence of steady-state slow inactivation for 15 fibers at
19°C and 14 fibers at 37°C. Smooth curves are least squares
fits of Eq. 3 (see text) to the data
points for 19°C (dashed line) and 37°C (solid line). Potential
at which 50% of Na+ channels were
closed due to slow inactivation
(Vs1/2) was
104.0 ± 2.1 mV at 19°C and 87.9 ± 2.7 mV at
37°C (P < 0.001). Parameter
determining the steepness of the
s -membrane
potential relationship was 5.7 ± 0.8 mV at 19°C and 5.4 ± 0.9 mV at 37°C.
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Fig. 8.
Temperature dependence of
Vs1/2. Mean
values of Vs1/2
at each of the study temperatures are plotted vs. temperature.
|
|
Figures 4 and 7 show that, despite the positive shift in
Vs1/2 with
increasing temperature,
Vs1/2 was more
negative than
Vh1/2 from
19°C to 37°C. At 19°C,
Vs1/2
Vh1/2 was
29.2 mV, and, at 37°C,
Vs1/2
Vh1/2 was
9.6 mV.
Temperature accelerated the kinetics of slow inactivation of
INa.
Figures 5 and 6 show that slow inactivation developed and recovered
more rapidly at 37°C compared with at 19°C. Figure
9 plots
s vs. membrane potential for
slow inactivation at 19°C and 37°C. The configuration of the
s-voltage relationship changed
in two ways with temperature. First,
s decreased with increasing
temperature. Second, the maximal value of
s
(
s max) shifted in a
depolarizing direction with increasing temperature. To determine
s max at each of the
study temperatures, a fourth-degree polynomial was fitted to the
s-membrane potential
relationships, such as are shown in Fig. 9. The
s max occurred at about
the same voltage as
Vs1/2. At
19°C,
s max was
10-fold greater than that at 37°C. Figure
10, an Arrhenius plot of
s max, shows the linear
decline in the logarithm of
s max with increasing
temperature. The
Q10 for the
decline of
s max with
temperature over the range from 19°C to 37°C was 3.60.

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Fig. 9.
Values for s, the time
constants for the development of or recovery from slow inactivation,
are plotted vs. membrane potential for slow inactivation at 19 and
37°C.
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Fig. 10.
Temperature dependence of
s max, which is the
maximum value of s at a
specific temperature, as an Arrhenius plot, for the 12 cells studied at
each of the study temperatures (19, 25, 31, and 37°C).
Q10 for the
decline of s max with
temperature over the range from 19 to 37°C was 3.60.
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 |
DISCUSSION |
This study demonstrated that temperature profoundly affected the
fraction of Na+ channels that were
excitable at the resting potential in fast-twitch mammalian skeletal
muscle. As shown in Table 2, only 30% of
Na+ channels were excitable at the
resting potential at 19°C compared with 93% of channels being
excitable at 37°C. The reason more Na+ channels were excitable at the
resting potential was that the midpoint of the
s
-membrane
potential relationship shifted by 16.1 mV from
Vs1/2 =
104 mV at 19°C to
Vs1/2 =
87.9 mV at 37°C.
The kinetics of fast inactivation (Figs. 2 and 3) and slow inactivation
(Figs. 9 and 10) became faster with increased temperature. The absolute
values and temperature dependence of
h 0mV found in this study
were similar to the values found for cloned wild-type rat skeletal
muscle Na+ channels (rSkM1, µ1)
expressed in human embryonic kidney (HEK) cells (15), wild-type human
skeletal muscle Na+channels
(hSkM1) expressed in the tsA201 line of transformed human kidney cells
(8), and hSkM1 Na+ channels in
human myoballs (28). The
Q10 for
h 0mV was 4.13 in this
study compared with 3.34 for rSkM1
Na+ channels studied between 12 and 37°C (15), 3.2 for the hSkM1 Na+ channels expressed in tsA201
cells studied between 14 and 30°C (8), and 3.6 for hSkM1
Na+ channels in myoballs studied
between 10 and 37°C (28).
This study found only slight effects of temperature on the voltage
dependencies of activation of conductance and
h
(Fig. 4,
Table 1). Prior studies on mammalian skeletal muscle, cardiac muscle,
and peripheral nerve Na+ channels
found variable effects of temperature on the voltage dependencies of
activation and fast inactivation. Human myoballs express both hSkM1 and
hSkM2 Na+ channels (28). The hSkM2
channel is relatively resistant to TTX, expressed on immature and
denervated skeletal muscle cells, and is the same at the most commonly
found cardiac Na+ channel (20).
The voltage dependencies of activation and fast inactivation did not
vary over the temperature range from 10 to 37°C for the hSkM1
Na+ channels in myoballs. In
contrast, for the hSkM2 Na+
channels in myoballs, fast inactivation shifted in a hyperpolarizing direction with reduced temperature, but the voltage dependence of
activation did not vary with temperature (28). In guinea pig
ventricular myocytes, fast inactivation of
INa shifted in a
hyperpolaring direction, as temperature was reduced from 36 to 15°C
(25). In sheep cardiac Purkinje fibers, temperature did not appreciably
alter the voltage dependence of peak
Na+ permeability over a range from
10 to 24°C or
h
over a range
from 16 to 26°C (9). In rat peripheral nerve nodes of Ranvier, the
voltage dependencies of activation and fast inactivation did not change
with temperature between 22 and 37°C (40). In summary, the lack of
effect of temperature on the voltage dependencies of activation and
fast inactivation found in this study is consistent with prior findings
for hSkM1 Na+ channels in human
myoballs and Na+ channels in rat
peripheral myelinated nerve fibers.
Prior studies on mammalian skeletal muscle and cardiac
Na+ channels suggested that
reducing temperature might decrease the fraction of excitable
Na+ channels at the cell resting
potential. Resealed human skeletal muscle fiber segments studied at
21°C had to be maintained at holding potential of
110 mV for
2-5 min to recover Na+
channels from slow inactivation (14). In guinea pig cardiac ventricular
myocytes, the maximum Na+
conductance elicited by depolarizing steps from the resting potential increased by 69% at 25°C compared with 15°C and peak
Na+ conductance was 41% larger at
35°C compared with that at 25°C (25). In sheep cardiac Purkinje
fibers, the amplitude of peak inward
INa elicited by
depolarizations from the resting potential increased by 50% at
26°C compared with at 16°C (9). Dudel and Rüdel (11)
found that very long duration hyperpolarizing prepulses were required
to recover INa
for recordings at 10°C and below. The findings described in the
above experiments on mammalian cardiac and skeletal muscle cells are
consistent with increased inactivation of
INa at lower temperatures.
In this study, the
s
-membrane
potential relationship was shifted in hyperpolarized direction relative
to the
h
-membrane potential relationship. Prior in vitro loose patch voltage-clamp studies on rat and human fast-twitch skeletal muscle fibers from this
laboratory (32, 34, 38, 39), from Dr. Walter Stühmer's laboratory (41), and collaborative studies between this laboratory and
Dr. Stühmer's laboratory (35, 36) found similar separations in
the voltage dependencies of fast and slow inactivation for fast-twitch
muscle fibers. In vitro studies of
Na+ channel inactivation in frog
twitch skeletal muscle fibers (2) and crayfish giant axons
(42) also showed that slow inactivation developed at hyperpolarized
potentials compared with fast inactivation. In contrast, in studies of
cloned skeletal muscle Na+
channels, expressed in a variety of nonmuscle cell systems, the operating voltage ranges of fast and slow inactivation overlapped (10,
13, 16, 17, 29, 43, 46). Consequently, when skeletal muscle
Na+ channels were expressed in
nonmuscle cells, the operating voltage ranges of fast and slow
inactivation were not distinct, whereas, when skeletal muscle or nerve
Na+ channels were studied in the
native tissue, the voltage dependence of slow inactivation was shifted
in a hyperpolarizing direction compared with fast inactivation.
The causes for the differences in the relative voltage dependencies of
fast and slow inactivation of Na+
channels studied in expression systems compared with native tissues are
not known. However, precedents exist for variations in the voltage-dependent behavior of skeletal muscle
Na+ channels based on the cell in
which the Na+ channel was
expressed. Na+ channels are formed
from the same glycoprotein in fast- and slow-twitch fibers (20).
Several laboratories reported differences in the voltage dependencies
of Na+ channel activation and
inactivation between mammalian fast- and slow-twitch skeletal muscle
fibers (12, 21, 34-36, 41). In mammalian slow-twitch fibers, slow
inactivation develops at hyperpolarized potentials compared with fast
inactivation; however, when studied at room temperature, the separation
between the fast and slow inactivation curves is smaller for
slow-twitch fibers compared with fast-twitch fibers (34-36, 38,
39). It is not known why the voltage dependencies of activation and
fast and slow inactivation differ between mammalian fast- and
slow-twitch muscle fibers (34).
This study showed that, at a physiological temperature of 37°C,
most skeletal muscle Na+ channels
in type IIb fibers were excitable. Comparing type IIa fibers with type
IIb mammalian skeletal muscle fibers,
Vs1/2 was similar
and Vh1/2 was
positively shifted for type IIa fibers (34, 39). Both fast and slow
inactivation developed at more positive potentials for type I fibers
compared with type IIb fibers (34, 38). Consequently, most
Na+ channels on type I and IIa
skeletal muscle fibers should be excitable at the resting potential at
37°C.
This study and prior studies of slow inactivation of
INa in mammalian
skeletal muscle fibers from rabbits (21), rats (32, 34), and humans
(34, 38, 39) found that slow inactivation could eliminate
INa. In addition,
Na+ channels were completely
susceptible to slow inactivation in frog twitch muscle
fibers (2) and crayfish axons (42). Cummins and Sigworth (10) reported
that slow inactivation could reversibly eliminate
INa for rat
skeletal muscle Na+ channels
(rSkM1, µ1) expressed in HEK cells. Conversely, in some studies of
Na+ channels expressed in
nonmuscle cell lines, slow inactivation was incomplete, with
~15-20% of
INa not affected
by slow inactivation (13, 16, 29, 43). In some studies, slow
inactivation would eliminate
INa only if the
expressed Na+ channels had
impaired fast inactivation (13, 43). The findings in this study are
compatible with those of Cummins and Sigworth (10) for rSkM1
Na+ channels expressed in HEK
cells and with the findings from crayfish axons and muscle fibers from
frogs, rabbits, rats, and humans.
Skeletal muscle membrane excitability is a complex interplay of
Na+,
K+, and
Cl
conductances. In cold
environments, extremity skeletal muscle cools to conserve core body
temperature. Reduction of the population of excitable
Na+ channels at lower temperature
may help to prevent skeletal muscle membrane hyperexcitability. At
physiological temperatures, Na+
channels open slightly faster than delayed rectifier
K+ channels. However, at 37°C,
mammalian skeletal muscle delayed rectifier
K+ channels activate sufficiently
rapidly to assist in terminating the action potential (44). When
activation is modeled according to Hodgkin-Huxley kinetics (18, 19),
the time constants for the activation parameter for
Na+ channels,
m, and for delayed rectifier
K+ channels,
n, both decrease with
increasing temperature. The Q10 values for
the decline of
m and
n with temperature have been
reported as similar in rat skeletal muscle (4). The activation rate for
Na+ channels varied as

3m and the activation rate of
delayed rectifier K+ channels
varied as 
4n. Therefore,
reduced temperature produced a greater slowing of activation for
delayed rectifier K+ channels
compared with Na+ channels.
Consequently, as the temperature of skeletal muscle declined, the
difference in the rate of activation of
Na+ channels and delayed rectifier
K+ channels increased. At
23°C, mammalian skeletal muscle delayed rectifier
K+ channels opened too slowly
compared with Na+ channels to
affect action potential termination (1). Consequently, reducing the
number of excitable Na+ channels
at lower temperatures may compensate for the slower opening of delayed
rectifier K+ channels and allow
the muscle membrane to maintain an appropriate level of excitability.
Possible roles of slow inactivation in inherited clinical disorders
of skeletal muscle membrane excitability.
Clinical disorders of skeletal muscle membrane excitability are caused
by mutations of SCN4A, the gene for
the adult form of the human skeletal muscle
Na+ channel (3, 6, 31).
Hyperkalemic periodic paralysis (HPP) is an autosomal dominant disorder
characterized by attacks of weakness that are commonly associated with
elevated serum K+ levels. Myotonia
was also present in some families with HPP. Paralytic attacks in HPP
may be induced by K+ loading, cold
environment, or rest after exercise. Several point mutations in
SCN4A are associated with HPP. Two
mutations, Thr704Met and Met1592Val, account for ~90% of the
genotyped kindreds. Studies on muscle biopsies showed that paralysis
resulted from membrane depolarization that rendered the membrane
inexcitable due to inactivation of
Na+ channels. A persistent
INa caused the
depolarization (31). Single-channel studies of mutant
Na+ channels demonstrated
persistent INa
resulting from 1) disrupted fast
inactivation with an excessive amount of slow mode
Na+ channel gating (7, 15, 17) or
2) window currents created by a
membrane potential range over which a small fraction of
Na+ channels could open and not
fact inactivate (10, 47). Window currents were created by shifts in the
steady-state voltage dependence of activation (10) or both activation
and fast inactivation (47). For the pathological persistent
INa to last >10
s, the mutations producing HPP could disrupt slow inactivation (10, 33). Mutations associated with weakness in addition to myotonia may
have altered slow inactivation, enabling a paralyzing persistent INa to exist (17,
29). Alternatively, the mutations would not have to involve slow
inactivation if slow inactivation was always incomplete so that a
fraction of INa
was not subject to slow inactivation (16, 17).
Slow inactivation was disrupted in two of four studied
Na+ channel mutations associated
with HPP, including the most common mutations Thr704Met and Met1592Val
(10, 16, 17). Two mutations, Met1360Val and Ala1156Thr, displayed the
same pattern of slow inactivation of
INa as wild-type
channels when studied at room temperature (16, 17). The slow
inactivation results for the Met1360Val and Ala1156Thr mutations can be
reconciled in two ways. First, as discussed above, slow inactivation
may be incomplete in vivo so that slow activation inactivation need not
be disrupted for mutant Na+
channels to produce persistent depolarizing
INa (16, 17). The
second way of reconciling the Met1360Val and Ala1156Thr slow inactivation data is that these mutations may have abnormal temperature dependencies for slow inactivation that would reduce the impact of slow
inactivation at physiological temperatures but not at room temperature.
For example, the voltage dependence of slow inactivation of the mutant
channels at 37°C could have a positive voltage shift and the
mutations could have a larger temperature dependence compared with
normal channels. Consequently, at physiological temperatures, larger
than usual depolarizations would be required to slow inactive mutant
Na+ channels. Hence, slow
inactivation would not terminate the pathologically persistent
INa. A larger
temperature dependence could result in slow inactivation for the mutant
Na+ channels being similar to
normal channels when studied at room temperature.
Paramyotonia congenita (PC) is an autosomal dominant clinical disorder
of skeletal muscle membrane excitability (3, 6, 31). PC overlaps
clinically with HPP, and it is associated with mutations of
SCN4A. Patients with PC may have
myotonia at normal temperature, but the prominent features are
1) myotonia that is aggravated or
elicited by skeletal muscle cooling,
2) cold-induced weakness that
follows myotonia, and 3) worsening
of myotonia by exercise (paradoxical myotonia). Muscle biopsies from PC
patients demonstrated normal membrane properties at 37°C but
cooling to 27°C triggered depolarization to about
40 mV. A
pathologically persistent
INa caused the
depolarization (3, 6, 22, 31). Single-channel studies of mutant
Na+ channels usually demonstrated
alterations in fast inactivation, but these studies did not explain why
pathologically persistent INa and paralysis
were elicited by cooling. One suggestion for the PC mutations was that
mutant channels could directly transit from the fast inactivated state
to the open state, thereby enabling a fraction of channels to be open
at depolarized potentials (22). However, the hypothesis that
depolarized mutant channels would transit between fast inactivated and
open channel states does not consider that, unless slow inactivation
was perturbed, depolarized mutant channels would accumulate in the slow
inactivated state, which would terminate the pathological
INa.
Plassart-Schiess et al. (27) found that fast inactivation was not
appreciably altered by the Ile693Thr mutation, which was associated
with the clinical manifestations of cold-induced weakness without
stiffness. They suggested that the cold-induced weakness might result
from altered slow inactivation, and Hayward et al. (17) found impaired
slow inactivation in the Ile693Thr mutation. A reduction in the
temperature dependence or a reversal in the effect of temperature on
the voltage dependence of slow inactivation, so that
Vs1/2 shifted in
a positive voltage direction with cooling, would enhance a cold-induced
persistent INa by
preventing PC mutant channels from entering the slow, inactivated state
at reduced temperatures. Normal
Na+ channels are more susceptible
to slow inactivation with cooling (Fig. 8), which would facilitate the
ability of a pathologically persistent
INa to produce
inactivation-induced paralysis.
 |
ACKNOWLEDGEMENTS |
This work was supported by the Office of Research and Development,
Medical Research Service of the Department of Veterans Affairs.
 |
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: R. L. Ruff,
Neurology Service 127(W), Cleveland VAMC, 10701 East Blvd., Cleveland,
OH 44106 (E-mail: ruff.robert{at}cleveland.va.gov).
Received 24 March 1999; accepted in final form 18 June 1999.
 |
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