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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
REFERENCES

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).


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INTRODUCTION
METHODS
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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 MOmega (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 MOmega ), but this was >500-fold smaller than the whole cell input resistance (ca. 5 GOmega ) 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
<IT>I</IT>(<IT>t</IT>) = <IT>I</IT><SUB>0</SUB> + <IT>I</IT><SUB>1</SUB> × [1 − exp (−<IT>t</IT>/&tgr;)] (1)
where I(t), I0, and I1 denote the amplitudes of Ih at time t, time 0, and at steady state, respectively, and tau  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
<IT>I</IT>(<IT>t</IT>) = <IT>I</IT><SUB>0</SUB> + <IT>I</IT><SUB>1</SUB> × exp (−<IT>t</IT>/&tgr;) (2)
For Boltzmann fitting of the steady-state activation curve of Ih (for details, see the legend to Fig. 1)
<IT>P</IT><SUB>∞</SUB>(<IT>V</IT><SUB>m</SUB>) = 1/{1 + exp[(<IT>V</IT><SUB>m</SUB> − <IT>V</IT><SUB>h</SUB>)/<IT>k</IT>]} (3)
where Pinfinity (Vm) denotes steady-state open probability (Pinfinity ) 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)
<AR><R><C>&agr;</C></R><R><C>C ⇄ O</C></R><R><C>&bgr;</C></R></AR>
where C and O denote the closed and open states of Ih and alpha  and beta  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 Pinfinity (Vm), the value of Po(t,Vm) can be expressed using the Hodgkin-Huxley formalism as follows
<IT>P</IT><SUB>o</SUB>(<IT>t</IT>,<IT>V</IT><SUB>m</SUB>) = <IT>P</IT><SUB>∞</SUB>(<IT>V</IT><SUB>m</SUB>) × {1 − exp[−(&agr; + &bgr;) × <IT>t</IT>]} (4)

<IT>P</IT><SUB>∞</SUB>(<IT>V</IT><SUB>m</SUB>) = &agr;/(&agr; + &bgr;) (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 alpha  and beta  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.


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ABSTRACT
INTRODUCTION
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DISCUSSION
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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 (open circle ) and end () of hyperpolarizing pulse are plotted against the membrane potential. Difference between steady-state and instantaneous I-V curves is indicated by triangle . 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 (open circle ) 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.

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. open circle , PSS (n = 5); , 40 mM K external solution (n = 4).

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 (open circle , control; triangle , 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 (open circle ) 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 (open circle ; n = 3), 25 (; n = 8), and 30°C (black-triangle, 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 (open circle , , and black-triangle, 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 alpha  and beta  vs. membrane potential at two different temperatures (A) and relationship between the observed and predicted Vh values (B). A: alpha  () and beta  (open circle ) at various membrane potentials at 30°C (n = 9); alpha  (black-triangle) and beta  (triangle ) 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 alpha  and beta , respectively (see text for results). B: observed Vh values () with SE (bars) are plotted against temperature, together with values predicted from the points where alpha  and beta  become equal (black-triangle). Solid and dashed lines are results of linear regression of data points.

To delineate temperature dependence of Ih gating in our preparation more unequivocally, we calculated the rate constants of opening (alpha ) and closing (beta ) 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 alpha  and beta  can be expressed as decaying and growing exponential functions of membrane potential,respectively
&agr;(<IT>V</IT><SUB>m</SUB>) = exp[−0.0706 × (<IT>V</IT><SUB>m</SUB> + 117.4)] s<SUP>−1</SUP> for 30°C and

&agr; (<IT>V</IT><SUB>m</SUB>) = exp[−0.0677 × (<IT>V</IT><SUB>m</SUB> + 124.5)] s<SUP>−1</SUP>

for 25°C

&bgr;(<IT>V</IT><SUB>m</SUB>) = exp[0.0394 × (<IT>V</IT><SUB>m</SUB> + 29.5)] s<SUP>−1</SUP> for 30°C and

&bgr;(<IT>V</IT><SUB>m</SUB>) = exp[0.0395 × (<IT>V</IT><SUB>m</SUB> + 32.5)] s<SUP>−1</SUP>

for 25°C
where the crossing point of the curves for alpha  and beta  (i.e., alpha  = beta ; 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 alpha  = beta  (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 (alpha  and beta ) at 36°C, we next constructed Arrhenius plots for averaged alpha  and beta  values at given membrane potentials and then fitted them to the following equation
&agr; (or &bgr;) = (<IT>k</IT><SUB>b</SUB> × T/<IT>h</IT>) × exp(&Dgr;S/<IT>R</IT>)

× exp[−&Dgr;H/(<IT>R</IT> × T)] (6)
where kb, T, h, R, Delta S, and Delta 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 Delta H values were obtained for alpha  (22.4 ± 3.5 kcal/mol, n = 6), whereas those for beta  were relatively small (8.0 ± 1.2 kcal/mol, n = 5). The Delta H value of alpha  is equivalent to a Q10 of 3.4 between 25 and 35°C (that for beta : 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 alpha  and beta  at this temperature. For example, at -120 mV, 2.26 and 0.01 s-1 are obtained for alpha  and beta , 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
&agr;(<IT>V</IT><SUB>m</SUB>) = exp[−0.0668 × (<IT>V</IT><SUB>m</SUB> + 107.9)] s<SUP>−1</SUP> (7)

&bgr;(<IT>V</IT><SUB>m</SUB>) = exp[0.0393 × (<IT>V</IT><SUB>m</SUB> + 35.6)] s<SUP>−1</SUP> (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 alpha  and beta  (A) and reconstructed relationships of alpha  and beta  vs. membrane potential at 36°C (B). A: mean values of alpha  (open circle ) and beta  () 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 alpha  () and beta  (black-triangle) 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 alpha  and beta , 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 alpha  and beta  (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 alpha  and beta  at each membrane potential.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha ; 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 alpha ; 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.


    REFERENCES
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RESULTS
DISCUSSION
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