Temperature-sensitive gating of cation current in guinea pig
ileal muscle activated by
hyperpolarization
Hiroe
Yanagida1,
Ryuji
Inoue1,
Masao
Tanaka2, and
Yushi
Ito1
Departments of 1 Pharmacology and
2 Surgery and Oncology, Graduate School of
Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan
 |
ABSTRACT |
The temperature dependence of hyperpolarization-activated
current (Ih)
was investigated in freshly isolated guinea pig ileal smooth muscle
cells, using the nystatin-perforated whole cell recording technique.
Hyperpolarizing pulses (
50 to
120 mV) from
40 mV
evoked time-dependent inward rectifying currents with a reversal
potential of
33 mV and a slow activation time course well
approximated by a single exponential. The properties of these currents,
such as steady-state variables, dependence on external K, modification
by norepinephrine, and blockade by Cs or ZD-7288, coincide well with
those of the "classical"
Ih discovered in
the sinoatrial node. Raising the temperature (range: 22-33°C)
accelerated the activation time course of this
Ih and shifted
its 50% activation potential positively (12 mV/10 degree) with much
less change in the maximum conductance. Based on a simple closed-open
model, this can be explained by a high temperature dependence of the opening rate constant (temperature coefficient: 3.4). The activation profile of reconstructed
Ih at 36°C
suggests that a considerable overlap could occur between the ranges of
Ih activation and
physiological membrane potential.
norepinephrine; ZD-7288; smooth muscle; hyperpolarization-activated
current
 |
INTRODUCTION |
HYPERPOLARIZATION-ACTIVATED cation current
(Ih) was first
clearly described in sinoatrial nodal cells (4) and has been identified
in a variety of excitable cells that generate spontaneous action
potentials, including cardiac muscle, smooth muscle, and some neurons
(3-5, 9, 17, 22, 24, 25, 28). Originally, this current was noted
in the cardiac Purkinje fiber and sinoatrial node and was thought to
reflect a K conductance that deactivates during hyperpolarization (11,
21, 26) but later was shown to be a nonspecific cation conductance that
is induced upon membrane hyperpolarization (9, 11, 34).
Ih flows through
a class of nonselective cation channels, since its reversal potential ranges between
40 and
13 mV, and its amplitude is greatly
affected by changing the external Na or K concentrations and is
suppressed by millimolar concentrations of external Cs (e.g., see
Refs. 3, 9, 11, 15, 16, 24, 28, 30). The physiological
significance of
Ih as a pacemaker
current has been most firmly established in the Purkinje fiber and
sinoatrial nodal cells, where selective blockade of this current
decreases the rate of slow diastolic depolarization. An elaborate
computer simulation model has been constructed to confirm this role (8,
12, 14, 30). Ih has also been reported to be regulated by several physiologically important neurotransmitters such as norepinephrine, serotonin, adenosine, and neurotensin (4, 5, 25, 28) and even a synthetic agent
E-4080 (22), thus suggesting a pivotal role of this current in finely
tuning the membrane excitability in a manner dependent on the state of
the body.
The first report of
Ih in smooth
muscle came from the work of Benham et al. (3), who identified in
rabbit jejunum a cationic conductance that is evoked in response to
hyperpolarization with slow activation kinetics and is effectively
blocked by Cs but much less by Ba. This work and others showed,
however, that the activation range of
Ih was relatively
negative to the resting membrane potential, thus making it unlikely to
account for the pacemaking in smooth muscle. More recently, the
pacemaker role of an
Ih-like current
has been evaluated by patch-clamp and contractile experiments in the
rat urinary bladder smooth muscle, using ZD-7288, which has been found
to potently block
Ih in some
tissues (17, 24). However, the results obtained were not conclusive,
showing that, despite the selective inhibition of
Ih-like currents
by ZD-7288, the compound augmented the amplitude of spontaneous contractions.
In the preliminary stage of our experiments, we noticed that the
activation range of
Ih shifts
positively on raising the temperature, and a high temperature
coefficient (Q10) value
(2-4) for the Ih activation
time constant has already been reported in the sinoatrial node (15).
This implies that there may be considerable overlap between the
activation range of
Ih and the
dynamic range of the membrane potential if a physiological temperature
is employed and that the data obtained at room temperature (which
varies considerably) may be inappropriate and may result in
underestimation. The aim of the present study is therefore to delineate
the temperature dependence of the gating kinetics of
Ih in gut smooth
muscle under experimental conditions in which the temperature is well
controlled. To this end, we employed the nystatin-perforated recording
technique, which minimally disturbs the intracellular milieu (19), and performed experiments at temperatures from 22 to 33°C, the upper maximum at which the experimental data of
Ih could be
reproducibly acquired (see METHODS). Based on these data,
we further estimated the kinetic profile of
Ih at the
physiological temperature of 36°C. Some of the results of the
present study have been communicated to the 71th annual meeting of the
Japanese Pharmacological Society (33).
 |
METHODS |
Cell dispersion. Guinea pigs of either
sex (500-1,000 g) were stunned by striking the back of the head
and then were decapitated with a guillotine. After opening the
abdominal cavity, a cylindrical segment of ileum (5-10 cm long)
5-10 cm proximal from the ileocecal valve was excised and quickly
transferred to nominally Ca-free Krebs solution prewarmed to 35°C.
This was subdivided into two or three short segments (2-3 cm long)
in which glass rods (ca. 10 cm in length, 2 mm in diameter) were
inserted and fixed with thin silk thread. These were consecutively
incubated in Ca-free Krebs solution and a solution containing 1 mg/ml
collagenase (type I; Sigma) at 35°C for 5 and 20 min, respectively,
and then were stored in 0.75 mM Ca-containing Krebs solution
supplemented with 1 mg/ml albumin and kept in a refrigerator at
4-10°C until use. Just before an experiment, a digested
segment was cut open, and the thin longitudinal layer was carefully
peeled off with two fine forceps and minced into small pieces. These
were triturated with a blunt-tipped Pasteur pipette until a sufficient
number of cells were released. Dispersed cells were used within 6 h
from enzymatic digestion. All experiments were carried out according to
the Guidelines for Animal Experiments at Kyushu University School of Medicine.
Electrophysiology. The patch-clamp
system employed in this study was essentially the same as that
described elsewhere (31). In brief, a high-impedance, low-noise
patch-clamp amplifier (AxoPatch 1-D; Axon Instruments) was used to
apply voltages to and sample voltage or current signals from the
clamped cell through an analog (A)-to-digital (D), DA converter (TL-1;
Axon Instruments) that was driven by a 32-bit computer (Aptiva; IBM)
using a commercial software package (pClamp version 5.0). Data analysis
was performed, and Figs. 1-7 were made using the software Clampfit
version 6.03 (Axon Instruments), Kaleida Graph version 3.04, and Origin
version 4.1 (Microbal). The temperature of superfusing solution was
controlled at the desired value, using a temperature-controlling unit
(accuracy: ±0.5°C; MT-1). The details of the
nystatin-perforated recording have been described elsewhere (6). The
series resistance
(Rs) after full
membrane permeabilization by nystatin ranged between 15 and 30 M
(usually 15-30 min required for this), and cells having larger
Rs values were
not taken into evaluation. Raising the temperature sometimes caused a
slight reduction in
Rs (by <10
M
), but this was >500-fold smaller than the whole cell input resistance (ca. 5 G
) and thus was not corrected.
Data fitting. To facilitate analyzing
genuine Ih
currents, leak currents were digitally corrected on off-line analysis.
Thus the actual traces shown in Figs. 1-7 have already been leak
subtracted, except for Figs. 1A,
2B, and
3A,
inset. Two types of fitting were performed after leak subtraction using a nonlinear least squares routine. For exponential fitting of the
Ih activation
time course
|
(1)
|
where
I(t),
I0, and
I1 denote the
amplitudes of Ih
at time t, time
0, and at steady state, respectively, and
is the
time constant of
Ih activation. In
some cases in which fitting was not satisfactory due to small
Ih amplitudes,
the deactivation time courses of the
Ih tail currents
were fitted by the following equation
|
(2)
|
For
Boltzmann fitting of the steady-state activation curve of
Ih (for details,
see the legend to Fig. 1)
|
(3)
|
where
P
(Vm)
denotes steady-state open probability
(P
) of
Ih at membrane
potential (Vm),
Vh is the
half-Ih
activation potential, and k is the
slope factor.
Model. Experiments at temperatures
higher than ca. 30°C were often unstable due to development of a
nonspecific leak and the instability of the clamped membranes, i.e., no
complete reverse of the effects of temperature increase was obtained.
We made numerous trials to overcome this problem but could get at most
only a limited number of available data up to 33°C. Thus, to
estimate the kinetic parameters of
Ih at the body
temperature of 36°C, an adequate model quantitatively describing
the gating characteristics of Ih was required
(for results see Fig. 7). We employed the minimal two-state
[closed (C)-open (O)] model that has frequently been used
to describe the voltage-dependent gating of ionic currents that exhibit
virtually no inactivation [e.g., nicotinic nonselective cation
channels (1),
Ih (34),
muscarinic K (27), and muscarinic nonselective cation channels
(20)], although
Ih in the
Purkinje fiber has been shown to exhibit more complicated kinetics (11)
where C and
O denote the closed and open states of
Ih and
and
represent the rate constants of opening and closing transitions, respectively. The relevance of this model for the theoretical approximation of
Ih amplitude can
be justified by the following considerations:
1) the time course of
Ih shows no
obvious lag at the beginning of hyperpolarization within our tested
range (compare with Ref. 30) and 2)
the time course of
Ih could be fitted by a single exponential with high correlation coefficients (>0.95 at
120 to
90 mV; fitting with multiple
exponentials or multiple power functions of several exponentials
produced poor or sometimes meaningless results). It has also been
suggested that there is little significant difference between the
two-state and more complex multiple-state models for simulating the
time-dependent change of
Ih current (14).
Furthermore, because our aim was not to deduce a precise model of
Ih gating from
the experimental data but to calculate the time-dependent change in
Ih amplitude with
satisfactory precision, we employed the above approach throughout the study.
Defining the probability of being open
(Po) at time
t and
Vm as
Po(t,Vm)
[and that of being closed as 1
Po(t,Vm)]
and that at steady state as
P
(Vm),
the value of
Po(t,Vm)
can be expressed using the Hodgkin-Huxley formalism as follows
|
(4)
|
|
(5)
|
The
results of these expressions were compared with the values obtained
from exponential fitting of the
Ih time course
(Eq. 1 vs.
4) or Boltzmann fitting of
normalized steady-state activation curve (Eq. 3 vs. 5), by which
the rate constants
and
at each membrane potential were calculated.
Solutions. The following solutions
were used (in mM): modified Krebs solution (137 Na+, 5.9 K+, 1.2 Mg2+, 1.5 Ca2+, 15.5 HCO
3, 2.5 H2PO3
4,
130.3 Cl
, and 12 glucose,
continuously aerated with 97% O2
and 3% CO2) and
K+ internal solution for
nystatin-perforated recording [140
K+, 1.2 Mg2+, 142.4 Cl
, 10 glucose, and 10 HEPES (adjusted at pH 7.2 with Tris base)].
Chemicals. ZD-7288 was purchased from
Tocris (Bristol, UK), EGTA was from Dojin (Kumamoto, Japan), and
nystatin, norepinephrine (HCl salt), 3-isobutyl-1-methylxanthine
(IBMX), and
N-methyl-D-glucamine were
from Sigma (St. Louis, MO).
Statistics. All statistical data are
expressed as means ± SE. Statistical significance of difference
between given sets of data was evaluated by Student's
t-test.
 |
RESULTS |
General properties of Ih in
ileal smooth muscle cells at room temperature.
We first investigated the biophysical and pharmacological properties of
hyperpolarization-evoked inward currents in guinea pig ileal smooth
muscle at an ambient temperature of 25°C. As demonstrated in Fig.
1A,
voltage step pulses (7.5 s in duration) to various hyperpolarizing
levels (from
50 to
120 mV) from the preceding
depolarization (
40 mV) resulted in slow development of inward
currents, which then deactivated upon repolarization to
60 mV.
The amplitude of these currents was increased, and the activation time
course accelerated by stronger hyperpolarizations. Consistent with
this, fitting of current traces with a monoexponential function
revealed that the time constant of activation decreases at more
negative potentials, e.g., being 3,786 and 1,812 ms at
90 and
120 mV, respectively (also see Fig. 4). These values are 10- to
100-fold larger than those of any voltage-dependent Na, Ca, and K
currents so far investigated in smooth muscle, thus suggesting the
involvement of channels with extremely slow kinetics.

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Fig. 1.
Hyperpolarization-activated current
(Ih) recorded
from guinea pig ileal smooth muscle at 25°C. Nystatin-perforated
recording. Bath contained physiological saline solution (PSS).
A: actual traces of
Ih
(right) during hyperpolarization to
50 to 120 mV (10-mV step;
left). Solid curves are exponential
fits of actual traces (see Eq. 1).
B: instantaneous and steady-state
current-voltage
(I-V)
relationships averaged from 8 cells. Amplitudes of
Ih at the
beginning ( ) and end ( ) of hyperpolarizing pulse are plotted
against the membrane potential. Difference between steady-state and
instantaneous
I-V
curves is indicated by . C:
steady-state activation curve of
Ih at 25°C,
obtained by tail current analysis. Time course of tail current (upon
repolarization to 60 mV; see
inset for example of 120 mV)
was fitted by a single exponential and was extrapolated to the
beginning of the repolarization pulse to estimate the initial amplitude
(indicated by arrow in inset). In
each experiment, the tail current amplitude evaluated in this way was
plotted against the membrane potential to determine the maximally
activated level
(Imax) by
Boltzmann fitting. Amplitude of tail current
(I) at a given conditioning
hyperpolarizing pulse
(Vc) was then
normalized to
Imax (i.e.,
I/Imax)
and averaged for 8 different cells, and the mean ( ) was plotted
against Vc,
together with SE (bars). Solid sigmoid curve is the best fit of data
points to the Boltzmann equation (Eq. 3), where the 50% activation potential
(Vh) and slope
factor (k) are 91 and 9 mV,
respectively.
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|
Figure 1B shows the instantaneous and
steady-state current-voltage relationships
(I-V
curve) of inward currents evoked by hyperpolarizing pulses (averaged
from 8 cells). The apparent activation threshold of the time-dependent
component was found to be between
60 and
70 mV, and its
I-V
curve shows an inward-going rectification. The degree of activation
appears to saturate at very negative potentials, as indicated by the
nearly ohmic portion of the steady-state I-V
curve below
100 mV. The maximum conductance
estimated between
100 and
120 mV is ~1.2 nS, which is
about five times larger than the resting leak conductance of 0.22 nS.
The degree of steady-state activation of the hyperpolarization-evoked
inward current can be described well by a Boltzmann-type equation
(Eq. 3; see METHODS). As
shown in Fig. 1C, the values of
Vh and
k evaluated from eight pooled data
sets were
91 and 9 mV, respectively. Calculation using these
parameters suggests that the availability of channels responsible for
these inward currents would be extremely low near the resting membrane
potential of this muscle at 25°C (~1% at steady state at
50 mV).
The reversal potential of the hyperpolarization-evoked current was
estimated by tail current analysis in the presence of 10 µM
nifedipine, 10 mM 4-aminopyridine (4-AP), and 10 mM tetraethylammonium (TEA; Fig.
2B; see
also the legend to Fig. 2). As shown in Fig. 2C, linear regression analysis of the
normalized current amplitude gives a reversal potential of about
35 mV (
33 ± 2 mV, n = 5; averaged from individual measurements) under normal ionic
conditions. Similar values were also obtained in the absence of 10 mM
4-AP and TEA (
30 ± 1 mV, n = 5) or in the presence of 1 mM TEA (
30 ± 2 mV,
n = 6). The reversal potential became
more negative (
41 ± 1 mV, n = 9; equivalent to
26.6-mV shift per 10-fold decrease; P < 0.05 with unpaired
t-test) when the external Na
concentration was cut in half, whereas it became more positive
(
22 ± 2 mV, n = 4; Fig. 2B;
P < 0.05 with unpaired
t-test) when the external K
concentration was raised from 5.9 to 40 mM (equimolar replacement of
Na; this corresponds to an Na ion-to-K ion permeability ratio of
~0.29; compare with Ref. 16). In addition, the slope conductance of
the fully activated inward tail current was increased more than twofold
with elevated external K solution (17.5 pS/pF with 40 mM K vs. 8.1 pS/pF with physiological saline solution). On the other hand, total
substitution of external monovalent cations with
N-methyl-D-glucamine
resulted in almost complete abolition of the inward currents (data not
shown). These observations strongly suggest that the conductance
underlying the hyperpolarization-evoked currents in this muscle is a
nonselective cation conductance and may have a higher permeability to K
than Na.

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Fig. 2.
Reversal potential of
Ih in guinea pig
ileum. A: protocol for evaluating the
reversal potential and corresponding actual traces of
Ih. Membrane was
first hyperpolarized to 120 mV
(Ba; almost full activation of
Ih occurred) or
90 mV (Bb; activation of
Ih was much less
than at 120 mV) and then was repolarized to various levels
( 110 to 20 mV). To evaluate the genuine
Ih current, the
amplitude of the tail currents upon repolarization from a preceding
hyperpolarization to 90 mV was subtracted from that from
120 mV (Bc), by which
contamination from the currents other than
Ih was minimized.
Nifedipine (10 µM) was applied to eliminate voltage-dependent Ca
currents, and 10 mM 4-aminopyridine (4-AP) and 10 mM tetraethylammonium
(TEA) were also added in the bath to minimize the activation of
transient K current (see, e.g., Ref. 2). However, even in the presence
of these K channel blockers, residual K currents were activated (ca.
greater than 30 mV), and thus the subtraction method mentioned
above was needed. C:
I-V
relationships of tail currents normalized by cell capacitance. , PSS
(n = 5); , 40 mM K external
solution (n = 4).
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|
The hyperpolarization-evoked inward current in guinea pig ileal smooth
muscle was almost completely suppressed by the monovalent cation Cs (1 mM; data not shown) or by the recently synthesized blocker of
Ih ZD-7288 (10 µM; Fig.
3A; 2 ± 2% of control, n = 5). As shown
in Fig. 3A, the
I-V
relationship in the presence of 10 µM ZD-7288 can be nearly
superimposed on that of the instantaneous leak current. The inhibitory
effects of ZD-7288 seem to be specific to these inward currents, since
voltage-dependent inward Ca (91 ± 4% of control at 0 mV,
n = 3) and outward-rectifying currents (96 ± 4% of control at 40 mV, n = 5) were only slightly or not significantly affected by the presence of
this compound (10 µM).

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Fig. 3.
Effects of ZD-7288 and norepinephrine on
Ih in guinea pig
ileum. A: instantaneous ( ) and
steady-state ( , control; , in the presence of 10 µM ZD-7288)
I-V
relationships obtained from the same cell.
Inset: actual traces at 120 mV.
B: steady-state activation curve
evaluated by slow rising ramp voltages ( 120 to 20 mV, 10 s) in
the absence ( ) and presence (x) of 10 µM norepinephrine. Curves
are drawn according to the results of Boltzmann fitting:
G(Vm) = Gmax/{1 + exp[(Vm Vh)/k]},
where
G(Vm)
denotes chord conductance of
Ih at membrane
potential (Vm)
and where maximal conductance
(Gmax),
Vh, and
k are 0.81 nS, 82 mV, and 6 mV
for control and 0.83 nS, 75 mV, and 6 mV for 10 µM
norepinephrine, respectively.
|
|
Finally, we tested a possible modulatory effect of norepinephrine on
the hyperpolarization-evoked inward currents in guinea pig ileal smooth
muscle. As shown in Fig. 3B, the
steady-state activation curve was shifted more positively in the
presence of 10 µM norepinephrine (shift of
Vh: 6.8 ± 0.5 mV, n = 4;
P < 0.05 with paired
t-test), whereas
k (8.8 ± 1.0 vs. 8.0 ± 0.7 mV
for control and 10 µM norepinephrine, respectively,
n = 4) and maximal conductance
(Gmax; 1.04 ± 0.03-fold of control, n = 4) remained almost constant. A similar extent of
Vh shift, without
noticeable changes in
Gmax and
k values, was also obtained by
application of 100 µM IBMX (7.1 ± 1.3 mV,
n = 8).
As has been described above, the hyperpolarization-evoked inward
cationic currents observed in guinea pig ileal smooth muscle exhibit a
high degree of similarity to
Ih so far
identified in other types of smooth muscle [rabbit jejunum (3)
and rabbit portal vein (22)] and other tissues, including
sinoatrial nodal cells and some neurons (Ref. 11; see also the
Introduction). This fact strongly indicates that the
hyperpolarization-evoked current in guinea pig ileum belongs to the
Ih class of
cation channels and can be designated as
Ih.
Temperature dependence of
Ih in guinea pig ileal
smooth muscle.
It has been reported that the time constant of
Ih activation is
highly sensitive to temperature (15). We therefore investigated changes
in the gating parameters of
Ih in guinea pig
ileum at temperatures between 22 and 33°C. At temperatures lower
than this, the amplitude of
Ih was too small
to evaluate, whereas at higher temperatures, the recordings were
unstable, hampering reproducible evaluation of the data (see
METHODS).
Figure 4A
demonstrates an example of
Ih currents (leak
corrected) at two different temperatures (25 and 30°C) recorded
from the same cell. Raising the temperature slightly increased the amplitude and clearly accelerated the time course of
Ih at very negative potentials such as
120 mV. As summarized in Fig.
4B, the steady-state activation curve
evaluated from pooled data was clearly shifted toward more positive
potentials by an increase in the temperature (see also Fig.
5B). The
time constants of
Ih activation
showed more complicated dependence on the temperature. At very negative
potentials, they became shorter at higher temperatures, whereas at less
negative potentials, they were prolonged (Fig. 4C; compare with the reconstructed
time constant curve for 36°C in Fig.
7C). In contrast,
Gmax was less sensitive to temperature. The mean
Gmax averaged from pooled data was 15.5 ± 1.2 (n = 9), 13.3 ± 1.1 (n = 8), and 12.8 ± 2.9 (n = 3) pS/pF, for 30, 25, and 22°C,
respectively. In addition, the reversal potential of Ih seemed to be
unaffected by changing the temperature (
30 ± 2 mV,
n = 5 at 30°C and
32 ± 2 mV, n = 4 at 22°C vs. 25°C;
P > 0.05 with unpaired
t-test).

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Fig. 4.
Ih currents
recorded at different temperatures in guinea pig ileal smooth muscle
cells. Nystatin-perforated recording. Bath contained saline.
A: superimposed actual traces of
Ih in response to
hyperpolarizing pulses ( 50 to 120 mV) from 40 mV
at 25 and 30°C (for voltage protocol, see Fig.
1A).
B: steady-state activation curves
evaluated from pooled data at 22 ( ;
n = 3), 25 ( ;
n = 8), and 30°C ( ,
n = 9; for details, see the legend to
Fig. 1C). Data for 25°C are from
Fig. 1C. Curves are drawn according to
the results of Boltzmann fitting (Eq. 3), where
Vh and
k are 96.6, 90.9, and
85.5 mV and 8.5, 9.0, and 8.9 mV for 22, 25, and 30°C,
respectively. C: time constants of
Ih activation at
22, 25, and 30°C ( , , and , respectively) are plotted
against the membrane potential. For 60 mV, the value was
obtained from the deactivation time course of tail current (see
METHODS and Eq. 2).
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Fig. 5.
Relationships of and vs. membrane potential at two different
temperatures (A) and relationship
between the observed and predicted
Vh values
(B).
A: ( ) and ( ) at various
membrane potentials at 30°C (n = 9); ( ) and ( ) at various membrane potentials at 25°C
(n = 8). Solid and dashed curves are
drawn according to the results of best fits to a single exponential of
the data points for and , respectively (see text for results).
B: observed
Vh values ( )
with SE (bars) are plotted against temperature, together with values
predicted from the points where and become equal ( ). Solid
and dashed lines are results of linear regression of data points.
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To delineate temperature dependence of
Ih gating in our
preparation more unequivocally, we calculated the rate constants of opening (
) and closing (
) by comparing the results of
exponential and Boltzmann fitting of observed data
(Eqs. 1 or 2 and 3) with theoretical expressions
derived from the two-state (C-O) model (Eqs.
4 and 5; for further
detail, see METHODS), as performed elsewhere (e.g., see
Ref. 20). As graphically summarized in Fig.
5A (at 25 and 30°C), the values
of
and
can be expressed as decaying and growing
exponential functions of membrane potential,respectively
where
the crossing point of the curves for
and
(i.e.,
=
; equivalent to
Vh of Boltzmann
curve) gives values of
91 and
86 mV for 25 and 30°C,
respectively. Good accordance was found over all of the tested
temperature ranges (22-33°C) between values obtained from
Boltzmann fitting
(Vh) and those
calculated from the point
=
(Fig.
5B). The degree of positive shift in Vh was 12 mV/10° increase in temperature.
To determine the relationship between membrane potential and rate
constants (
and
) at 36°C, we next constructed Arrhenius plots for averaged
and
values at given membrane potentials and
then fitted them to the following
equation
|
(6)
|
where kb, T,
h, R,
S, and
H denote the Boltzmann constant, absolute temperature,
Planck constant, gas constant, and entropic and enthalpic changes,
respectively. An example at
120 mV is shown in Fig.
6A. Quite
large
H values were obtained for
(22.4 ± 3.5 kcal/mol,
n = 6), whereas those for
were
relatively small (8.0 ± 1.2 kcal/mol,
n = 5). The
H value of
is
equivalent to a Q10 of 3.4 between
25 and 35°C (that for
: 1.6), thus suggesting that the rate of
opening may be much more susceptible to temperature change than that of
closing. Extrapolation of the Arrhenius plot to the point corresponding
to 36°C allows the estimation of
and
at this temperature.
For example, at
120 mV, 2.26 and 0.01 s
1 are obtained for
and
, respectively (indicated by arrows in Fig.
6A). As shown in Fig.
6B, the rate constants at 36°C
estimated in this way can be described by the following exponential
functions of membrane potential
|
(7)
|
|
(8)
|
With
the use of these relationships, the
Ih current traces
and steady-state activation and time constant curves at 36°C were reconstructed (Fig. 7). The reconstructed
steady-state activation curve
(Vh:
81.1
mV) overlaps considerably the physiological range of membrane
potential. For example, the availability of
Ih at physiologically attainable potentials such as
70,
60, and
50 mV is 24, 10, and 4% in the steady state, respectively.
These values correspond to the conductance of 0.28, 0.12, and 0.04 nS, respectively (taking 1.2 nS as the maximum), thus being comparable to
the resting conductance of this cell (ca. 0.22 nS; see
above). In addition, the reconstructed time constant curve
shows a biphasic dependence on the membrane potential with a peak
around
70 mV, the pattern being also evident at lower
temperatures (e.g., Fig. 4C).

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Fig. 6.
Arrhenius plots of rate constants and (A) and reconstructed relationships
of and vs. membrane potential at 36°C
(B).
A: mean values of ( ) and ( ) averaged from 3-9 measurements at 120 mV are plotted
on logarithmic scale against the inverse of the absolute temperature
(T). Solid and dashed lines are the best fits of data points to
Eq. 6.
B: values of ( ) and ( )
at 36°C estimated by extrapolating Arrhenius plots (see arrows in
A) are plotted against the membrane
potential. Solid and dashed curves are drawn according to the results
of best fits to a single exponential of data points for and ,
respectively (see Eqs. 7 and 8).
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Fig. 7.
Reconstructed activation time course and steady-state activation and
time constant curves for
Ih in guinea pig
ileum. A: reconstructed
Ih activation
time course at 120 to 50 mV (thick solid curves) at
36°C. Dotted line indicates 0-current level.
B: reconstructed steady-state
activation curves at 25, 30, and 36°C. Dotted line indicates the
level of 50% activation. Arrows are drawn from intersecting points of
this line with activation curves, thus corresponding to
Vh.
C: reconstructed time constant curve
at 36°C. Reconstruction was performed using the experimental
expressions for and (see, e.g., Eqs.
7 and 8) and
Eqs. 4 [multiplied by the
driving force
(Vm + 30 mV) and mean Gmax
of 1.2 nS] and 5. Time constant
was calculated as the inverse of the sum of and at each
membrane potential.
|
|
 |
DISCUSSION |
The present results have clearly shown that a
hyperpolarization-activated cationic current closely resembling
Ih is present in
guinea pig ileal smooth muscle (thus we have referred to it as
Ih), and its
gating kinetics are greatly dependent on the temperature. The first
conclusion has been supported by several lines of evidence. First, the
ionic properties of
Ih in guinea pig
ileum are highly consistent with those of
Ih identified in
the sinoatrial node, Purkinje fibers, and other tissues, including
rabbit jejunum smooth muscle; the reversal potential measured under
normal ionic conditions is intermediate between the K and Na
equilibrium potentials [
33 mV (current study);
25
mV (34), between
20 and
30 mV (11),
24 mV (30),
24.5 mV (3), and
29 mV (17)] and was shifted negatively and positively by decreased Na and increased K
concentrations in the bath, respectively (3, 10, 11). The degree of
observed negative shift caused by decreased Na, which is equivalent to 27 mV per 10-fold change, is also similar to that obtained in the calf
Purkinje fiber (29-35 mV per 10-fold change in external Na
concentration; see Ref. 10). Furthermore, the fully activated conductance of Ih
in our preparation
(Gmax) was
increased more than twofold when the external K concentration was
raised from 5 to 40 mM (Fig. 2C),
whereas it was almost unaffected by a decrease in external Na
concentration (unpublished data). These results agree well with those
observed, e.g., in the Purkinje fiber (11) and jejunum smooth muscle
(3). Second, the activation characteristics of
Ih in guinea pig
ileum, such as an apparent activation threshold of
60 to
70 mV, activation time constants of the order of seconds in the
range from
50 to
120 mV, and Boltzmann parameters
(Vh =
91
mV, k = 9 mV at room temperature), are
comparable to those so far found for
Ih (3, 11, 24,
28, 34). Finally, effective inhibition by 1 mM Cs and 10 µM ZD-7288
(Fig. 3A) and modulation by
norepinephrine of
Ih in our
preparation (Fig. 3B) are also commonly observed features of
Ih in other
tissues (e.g., see Refs. 4 and 25). Although other types of
hyperpolarization-activated inward rectifying currents with similar
kinetic properties to Ih have very
recently been discovered [slowly activating, Cs-inhibitable K-selective currents in a cultured murine hippocampal cell line, (32);
DIDS-inhibitable Cl-selective currents with very similar kinetics to
Ih in rat
sympathetic neurons (7)], the evidence discussed above strongly
supports the view that
Ih in guinea pig ileum pertains to the same class of
Ih that has been
evaluated by DiFrancesco and co-workers (4, 11) and also by Yanagihara and Irisawa (34) and Noma and co-workers (16).
High temperature dependence was most evident in the activation kinetics
of Ih, such as
the activation time constants (Fig. 4C
or the corresponding rate constant
;
Q10: 3-4; Fig.
6A) and Vh (+12 mV
shift/10° increase). In contrast, the fully activated conductance
(Gmax) showed
much less dependence on the temperature (~1.2-fold change in the
range of 25-30°C). The latter finding could be interpreted to
suggest that the conductive properties of
Ih channels do
not change much with temperature, with a degree comparable to that
expected for passive aqueous diffusion (18). No significant change in
the reversal potential of
Ih at two
different temperatures also supports this view. In contrast, the strong temperature dependence of activation time constants (or of the rate
constant
; Figs. 4C and
6A) suggests that the opening
kinetics of Ih
may be highly susceptible to temperature change, paralleled with a
marked positive shift in
Vh. Assuming that
the C-O model is quantitatively sufficient to approximate our
experimental data, this implies that the rate of opening of
Ih channels may
be the major determinant of the high temperature sensitivity of gating of Ih in our preparation.
The mechanism underlying the temperature dependence of
Ih remains to be
elucidated. It is however interesting to note that changes in the
kinetic parameters, such as the positive shift of
Vh and minimal
modification of
Gmax and slope
factor k by norepinephrine, resemble
those of raising the temperature (although
Gmax increased slightly, this effect could be attributed to thermodynamic effects on
ionic conductivity of
Ih). Recent
single channel recordings of
Ih in the rabbit
sinoatrial node have revealed that cAMP applied at the cytosolic side
of the patch membrane significantly shortens the latency of the first
Ih channel
opening in response to a hyperpolarizing pulse that would be governed
mainly by closed-to-open state transitions, without noticeable
modification of the unitary conductance (13). These findings are
apparently consistent with the speculated effects of temperature on the
Ih gating
mentioned above. Furthermore, because it appears that there is
substantial basal production of cAMP in our preparation (a
phosphodiesterase inhibitor, IBMX, produces similar positive shifts in
Ih activation
curves), it may be possible that increased temperature enhances the
cAMP production, thereby modifying the
Ih activation
kinetics. It is thus worthy to examine whether some common mechanisms
are involved in the effects of temperature and norepinephrine on
Ih.
In summary, the gating properties of
Ih in guinea pig
ileal smooth muscle are greatly dependent on temperature. Particularly, the rate of activation is enhanced and the activation curve shifted positively to a level that the physiological membrane potential can
actually reach as the temperature goes up. This has not unequivocally been shown in previous studies carried out at room temperatures [but see a brief suggestion by Benham et al. (3)].
Considering that in physiological situations gut smooth muscle cells
are under tonic influence of various inhibitory neurotransmitters and
hormones that could hyperpolarize the membrane toward the K ion
equilibrium potential [e.g., inhibitory junction potentials
(23)], the role of
Ih in modulating
the electrical activities of this muscle might be more significant than
has previously been envisaged. Obviously, further systematic studies
taking into account all possible elements participating in the genesis,
propagation, and modulation of gut electrical activities [e.g.,
interstitial cells of Cajal (29)] will be required to elucidate
this intriguing issue.
 |
ACKNOWLEDGEMENTS |
We are grateful to A. F. Brading, Department of Pharmacology,
Oxford University, for critical reading of our manuscript.
 |
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. Inoue, Dept.
of Pharmacology, Graduate School of Medical Sciences, Kyushu Univ.,
Fukuoka 812-8582, Japan (E-mail:
inouery{at}pharmaco.med.kyushu-u.ac.jp).
Received 30 December 1998; accepted in final form 24 August 1999.
 |
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