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Laboratory of Physiology, Department of Biomedical Sciences, the Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan
Submitted 8 May 2003; accepted in final form 1 July 2003
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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-S, also produced a positive shift in the voltage-dependence of Ih activation, suggesting that the effect of ATP results from its direct action on the channel protein. These results indicate that cytosolic ATP directly regulates the voltage dependence of Ih activation as an intracellular modulating factor. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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; Lamas 1998; Tokimasa and Akasu 1990) and in cardiac tissues (where it was named If, Brown et al. 1979; DiFrancesco 1981a,b, 1993). The channel underlying Ih is activated by membrane hyperpolarization and slowly depolarizes the membrane potential. Therefore this current has been proposed to control neuronal excitability and its functional role and modulating mechanism have been studied by many investigators (Pape 1996).
It has been shown that intracellular cAMP caused the depolarizing shift in the voltage dependence of Ih (DiFrancesco and Tromba 1988; Ingram and Williams 1996; Pedarzani and Storm 1995; Raes et al. 1997). In recent years, five different genes encoding Ih channels have been isolated from mouse brain (mHCN1–3, Ludwig et al. 1998), rabbit heart sino-atrial node (HAC4, Ishii et al. 1999), and a sea urchin testis (spHCN) cDNA library (Gauss et al. 1998). The HCN family contains a cyclic nucleotide binding domain (CNBD) in its cytoplasmic C-terminus that is highly homologous to that of cAMP- and cGMP-dependent protein kinases (Biel et al. 1999). The heterologously expressed HCN channels were shown to be regulated by the direct effect of cytosolic cAMP on its CNBD (Ludwig et al. 1998; Santoro et al. 1998). The regulation of Ih or If by the direct action of cAMP was also reported in guinea-pig primary afferent neurons (Ingram and Williams 1996), rat hippocampal pyramidal cells (Pedarzani and Storm 1995), and rabbit sino-atrial node cells (DiFrancesco and Tortora 1991). In contrast, cAMP was reported to indirectly control the Ih through the phosphorylation by cAMP-dependent protein kinase (PKA) in bull-frog sympathetic neurons and canine cardiac Purkinje fibers (Chang et al. 1991; Tokimasa and Akasu 1990). The presence of Ih and its modulation by cAMP have been described in the growth cone and soma of neonatal rat dorsal root ganglion (DRG) neurons (Wang et al. 1997). In addition, it has been reported that rat DRG neurons express high levels of HCN1 and HCN2 and relatively low levels of HCN3 and HCN4 (Chaplan et al. 2003). However, in rat DRG neurons, the modulating pathway for Ih and the possibility that other intracellular modulating factors affect Ih channel activity have not been studied yet. It was reported that ATP applied intracellularly in the presence of a saturating concentration of cAMP, which could fully activate PKA and Ih, acted as a substrate for the phosphorylation by PKA and caused a further positive shift of the Ih activation curve in addition to a direct effect by cAMP in mouse DRG neurons. However, no paper has investigated whether cytosolic ATP itself can affect Ih activation. In this study, therefore, we investigated the effect of intracellular dialysis of cAMP and ATP on the voltage dependence of Ih in rat DRG neurons to confirm whether intracellular ATP itself acts as a modulating factor for Ih.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Rat DRG neurons were isolated from adult male Sprague-Dawley rats (7–12 wk old), using procedures that have been reported previously (Kim et al. 1980; Yong et al. 1988).
Briefly, rats were killed by cervical dislocation and ganglia were dissected from the full length of the vertebral column. The ganglia were incubated at 37°C first in calcium-magnesium free phosphate-buffered saline (CMF-PBS) containing collagenase type IV (500 U/ml, Worthington Biochemicals) and DNAse I (0.12 µg/ml, Sigma) for 2 h and then in CMF-PBS containing trypsin (0.25% wt/vol) for 15 min. After the trypsin digestion, cells were gently agitated with a silicon-coated Pasteur pipette and centrifuged to remove trypsin.
The isolated cells were suspended in Dulbecco's modified Eagle's medium (DMEM, Gibco BRL) and cultured on coverslips coated with poly-D-lysine.
The cells were kept at 37°C in a humidified atmosphere of 95% air-5% CO2 and cultured for 1 wk until use in each experiment. The DMEM was supplemented with 10% fetal bovine serum (ICN Biochemicals), 100 U/ml penicillin, 100 µg/ml streptomycin, 2.5 µg/ml fungizone, and 10 µM cytosine arabinoside. The culture medium was changed every 2 days.
Whole cell recording
Whole cell recordings were made at room temperature. Heat-polished glass electrodes with 2- to 5-M
tip resistance were used. The cultured DRG neurons have neurites. We used the round shape neurons to minimize a space-clamp problem in the whole cell recordings.
The bath solution contained (mM): 140 NaCl; 6 KCl; 1.2 MgCl2; 2.5 CaCl2; 10 D-glucose; 10 HEPES (pH =7.2 adjusted with NaOH).
The pipette solution contained (in mM) 140 K-methansulfonate (KMeSO4), 2 MgCl2, 1.1 EGTA, and 10 HEPES (pH = 7.2 adjusted with KOH).
In a number of experiments, ATP, cAMP, ATP--S, and AMP-PNP were added to the pipette solution, and BaCl2, 4-aminopyridine (4-AP), tetraethylammonium (TEA), tetrodotoxin (TTX), and SQ-22536 (adenylate cyclase inhibitor, Sigma) were applied to the bath solution. The liquid junction potential (approximately 3.3 mV) between the pipette solution and the bath solution was not corrected. Neurons were continuously superfused with the bath solution at a flow rate of 1 ml/min throughout the experiments.
Whole cell currents were measured with a patch-clamp amplifier (CEZ-2400, Nihon Koden) and sampled using an analog/digital converter (MacLab, AD Instruments). When Ih was recorded, a sampling frequency of 0.4 kHz was used, and data were stored in a personal computer (Macintosh, Apple). The built-in four pole bessel filter was used at the frequency higher than 0.2 kHz if needed. Activation curves were fitted with a Boltzman function using the Igor Pro program (Wave Metrics). Cell capacitance was determined by integrating the area under a capacity transient current elicited by a –10-mV voltage step from the holding potential, and the amplitude of a current was normalized to the cell membrane capacitance. A series resistance (Rs) was also calculated from the capacity transient and the cell capacitance (9.9 ± 0.6 M, n = 121). A potential error caused by Rs was corrected for each recording when the activation curves of Ih were determined.
Data are presented as mean ± SE. The significance of differences between the means of two experiments was assessed with Student's t-test. The level of significance chosen was 0.05.
Drugs
The following drugs were dissolved in the pipette solution to the desired concentration: ATP (Sigma), cAMP (Sigma), AMP-PNP (Sigma), and ATP--S (Sigma).
Forskolin (Wako Pure Chemicals) was dissolved in a dimethyl sulfoxide stock solution (10 mM). SQ-22536 was dissolved in a distilled water stock solution (100 mM). The stock solutions were stored at –20°C and thawed immediately prior to use and diluted with the external solutions to desired final concentrations.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Rat DRG neurons with diameters ranging from 20 to 50 µm (28.3 ± 1.0 µm, n = 53) were used for the experiments. The mean cell capacitance was 110.2 ± 5.7 pF (n = 110). Under conventional whole cell voltage-clamp conditions, rat DRG neurons were held at –60 mV, and the potentials were raised step-wise to between –70 and –130 mV (Fig. 1A). Hyperpolarizing voltage pulses elicited slowly activating inward currents without inactivation (Ih).
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Figure 1B shows the current voltage relationships of Ih. The open circles indicate the amplitudes of the initial currents measured at the beginning of hyperpolarizing pulses (instantaneous current). The closed circles indicate the steady-state I-V relationship measured at the end of 2-s pulses (steady-state current). The net amplitudes of Ih activated by hyperpolarizing voltage pulses were determined by measuring the difference in the instantaneous and steady-state currents achieved at the beginning and the end of the pulse, respectively. This current began to be activated at –70 mV and almost fully activated at –130 mV. The reversal potential was evaluated by determining the intersection of the instantaneous I-V relationships stepping up from –60 and –130 mV (Mayer and Westbrook 1983). The reversal potential was –20.3 ± 5.0 mV (n = 10). This value was consistent with the report that Ih channel in various neuronal cells passes both Na+ and K+ (Pape 1996). The permeability ratio of Ih channel in rat DRG neurons for Na+ and K+ (PNa/PK) determined with the Goldman constant-field equation was 0.40.
Ih and the current through HCN clone channels have been reported to be almost abolished by extracellular Cs+ and only partially inhibited by extracellular Ba2+ (Ludwig et al. 1998; Pape 1996). In mHCN2 channels expressed in human embryonic kidney (HEK) 293 cells, the effects of TEA and 4-AP on Ih were also examined (Ludwig et al. 1998). The pharmacological profile of Ih in cultured rat DRG neurons was investigated to compare with that of Ih in other tissues. The magnitude of the inhibition was estimated by measuring the amplitudes of Ih in the presence and absence of the blocking agents. Extracellular applications of 2 mM Cs+, 2 mM Ba2+, and 20 mM TEA reduced the amplitude of Ih by 79.2 ± 6.4% (n = 6), 34.5 ± 7.1% (n = 8), and 19.2 ± 8.6% (n = 7), respectively (Fig. 1C). However, this current was insensitive to 1 mM 4-AP.
Effects of cyclic AMP and ATP on the voltage dependence of Ih
It is well known that intracellular cAMP produces a positive shift in the activation curve of Ih in DRG neurons and other neuronal cells (Cardenas et al. 1999; Pape 1996; Raes et al. 1997). Furthermore, it was reported that cAMP induced a positive shift in HEK293 cells with mHCN2 channels (Ludwig et al. 1998).
The effects of intracellular dialysis of nucleotides on Ih in rat DRG neurons were examined. BaCl2 (0.5 mM), 4-AP (2 mM), TEA (3 mM), and TTX (0.5 µM) were added to the extracellular solution to minimize contamination from voltage-gated Na+ and K+ currents. We used cAMP at a concentration of 1 mM, which produced the maximal effect in heterologously expressed HCN channels (Ishii et al. 1999; Ludwig et al. 1998; Santoro et al. 1998).
Voltage pulses to various potentials, followed by the pulse to –130 mV, where Ih appeared to be fully activated, were applied to cells. To examine the voltage dependency of the Ih activation more closely, hyperpolarizing voltage steps were applied to rat DRG neurons from a holding potential of –30 mV. This voltage protocol elicited Ih followed by tail currents (Itail) as shown in Fig. 2A. The peak amplitude of the tail current from –30 mV was defined as Itailmax (Fig. 2A, bottom). The degree of the activation of Ih at the various voltages was determined from normalizing 
Itail with Itailmax. Itail was defined as the difference between Itail from various voltage steps and Itailmax (Fig. 2A, bottom). The data points were fitted with the Boltzmann function: Itail/Itailmax = (Amax – Amin)/{1 + exp [(Vm – Vhalf)/k]} + Amin, where Amax is the maximal activation value, Amin is the minimal activation value, Vm is the test potential, Vhalf is the membrane potential for half-maximal activation, and k is the slope factor. Under this condition, Vhalf and k with the control pipette solution were –97.4 ± 1.9 (n = 22) and 7.4 ± 0.5 mV (n = 22), respectively. With the pipette solution containing 1 mM cAMP, the amplitude of Ih increased between –80 and –90 mV (Fig. 2B). Vhalf obtained from cAMP-dialyzed cells was –82.3 ± 2.6 mV (n = 11, Fig. 2A). This value of Vhalf was significantly different from that obtained with the control pipette solution (P < 0.001). The mean current density measured at –130 mV was –2.3 ± 0.4 pA/pF (n = 21) under the control condition and –3.1 ± 0.6 pA/pF (n = 11) in the presence of cAMP. The peak amplitude of Ih was not influenced by cAMP (P = 0.68).
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In mouse DRG neurons, it was reported that intracellular perfusion with solution containing ATP (5 mM) also induced a depolarizing shift of Vhalf of Ih (Raes et al. 1997). In rat DRG neurons, when 1 mM ATP was added to the pipette solution, Vhalf was –97.6 ± 2.3 mV (n = 10), which was not significantly different from that obtained in the absence of ATP (Fig. 2C). When the concentration of ATP was raised to 2 mM, Vhalf was a more positive value (–86.8 ± 1.6 mV, n = 19, P < 0.001) than that measured with the pipette solution containing 1 mM ATP or no nucleotides (Fig. 2C). As shown in Fig. 2B, in neurons dialyzed with 2 mM ATP, the current amplitude elicited by voltage steps to –90 mV increased. The amplitudes of maximum current density (–3.0 ± 0.4 pA/pF, n = 19) did not significantly change (P = 0.89). In rat DRG neurons, 2 mM ATP added to the intracellular solution produced a depolarizing shift of Vhalf of Ih, almost equal to that produced by 1 mM cAMP. The Vhalf of Ih using the pipette solution containing both ATP (2 mM) and cAMP (1 mM) together was –86.0 ± 2.0 mV (n = 6), a value that was not significantly different from that obtained in the presence of ATP (2 mM, P = 0.79) or cAMP (1 mM, P = 0.36) alone. In addition in the presence of 5 mM ATP in the pipette solution, the Vhalf (–88.2 ± 1.5 mV, n = 4) was not significantly different from that obtained in the presence 2 mM ATP (P = 0.69). Although the membrane permeable analog of cAMP (db-cAMP, 8-bromo cAMP, 8-cpt cAMP) was also tested at the concentration range of 1–5 mM, no analog added to the bath solutions had an effect on Ih in rat DRG neurons.
Effect of adenylate cyclase inhibitor on the ATP-induced positive shift in the voltage dependency of Ih
The effect of intracellular cAMP on the voltage dependency of Ih was explained to be caused by its direct action on the channel protein in some neuronal cells (Ingram and Williams 1996; Pedarzani and Storm 1995) and Xenopus oocytes and HEK293 cells expressing the mHCN1 and mHCN2 channel, respectively (Ishii et al. 1999; Ludwig et al. 1998). To investigate the modulating pathway of ATP in rat DRG neurons, we examined whether the activation of adenylate cyclase contributes to the ATP-induced positive shift of Vhalf of Ih in rat DRG neurons. An adenylate cyclase activator, forskolin (10 µM), significantly increased the amplitude of Ih induced by hyperpolarizing voltage steps between –70 and –90 mV by 29.1 ± 9.0% (n = 6), suggesting that Ih in rat DRG neurons could be regulated through the modulating pathway including adenylate cyclase. However, this effect of forskolin was not observed in the presence of a membrane permeable adenylate cyclase inhibitor, SQ-22536 at 100 µM (Goldsmith and Abrams 1992; Grupp et al. 1980; Madison and Nicoll 1986). This result indicated that SQ-22536 certainly inhibited the adenylate cyclase activity in rat DRG neurons under our experimental conditions. The effect of SQ-22536 on Ih activation was tested with the pipette solution containing 2 mM ATP. After the activation curve was determined using the normal external solution, SQ-22536 (100 µM) was applied to the extracellular solution. Vhalf was –90.4 ± 1.7 (n = 8) and –91.5 ± 1.2 mV (n = 8) before and after application of SQ-22536 (100 µM), respectively (Fig. 3). These values were not significantly different from each other (P = 0.58). The effect of ATP on the Ih activation was not influenced by the adenylate cyclase inhibitor. Another adenylate cyclase inhibitor, MDL 12,330A, was also tested. MDL 12,330A almost abolished the Ih amplitude at –130 mV, with the pipette solution containing 2 mM ATP. However, a similar effect was observed regardless of the presence of ATP or cAMP. Therefore this result was probably caused not by an inhibitory effect on adenylate cyclase but by an unknown effect of this compound.
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Effects of nonhydrolysable ATP analogs on the voltage dependency of Ih
In several neuronal tissues, it was reported that the phosphorylation process was involved in the modulation pathway of Ih by intracellular cAMP (Chang et al. 1991; Tokimasa and Akasu 1990). If cytosolic ATP shifted the Vhalf of Ih in rat DRG neurons without being converted to cAMP by adenylate cyclase and phosphorylation by PKA, a nonhydrolysable ATP analog would be expected to produce a similar effect on Ih activation. To test this hypothesis, we observed Ih with a pipette solution containing 2 mM AMP-PNP (Leventhal and Bertics 1991; Tokimasa 1995) or ATP--S (Chrysogelos et al. 1983; Goody et al. 1972) and compared its voltage dependency in the presence and absence of these analogs. LiCl (6.4 mM) was added to the control pipette solution when the effect of AMP-PNP was tested because AMP-PNP was supplied in the form of a lithium salt (AMP-PNP: Li+ = 1:3.2). The current density and voltage dependency of Ih in rat DRG neurons were not influenced by LiCl. When DRG neurons were dialyzed with AMP-PNP (2 mM), the amplitude of the slow activating current at –90 mV was greater than that with the control pipette solution (Fig. 4A). The peak current density was –2.1 ± 0.5 (n = 5) and –2.6 ± 0.5 pA/pF (n = 5) in the absence and presence of AMP-PNP (2 mM), respectively, and they were not significantly different (P = 0.45). Vhalf was –99.5 ± 0.5 mV (n = 10) with the control pipette solution, whereas the Vhalf in AMP-PNP-dialyzed cells was shifted to a more positive potential (–89.6 ± 1.0, n = 5, P < 0.01; Fig. 4B).
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Another nonhydrolysable ATP analog, ATP--S (2 mM), also mimicked the effect of ATP. In ATP--S-dialyzed cells, the Vhalf of Ih shifted toward positive potential (–91.3 ± 3.9 mV, n = 5, P < 0.01) without changing the fully activated current density (–3.4 ± 1.7 pA/pF in control vs. –3.5 ± 0.6 pA/pF in the presence of ATP--S, P = 0.98).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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; Santoro et al. 1998). In addition, in many types of neuronal cells, it has been shown that cAMP causes a positive shift in the voltage dependence of Ih (McCormick and Pape 1990; Pape 1996; Raes et al. 1997), and its effect was attributed to the direct action of cAMP against the channel protein (Pedarzani and Storm 1995). The direct effect of cAMP on If in cardiac tissue was also demonstrated in inside-out patches (DiFrancesco and Tortora 1991). On the other hand, it was reported that similar effects of cAMP on Ih in bull-frog sympathetic neurons (Tokimasa and Akasu 1990) and canine Purkinje fibers were explained by the phosphorylation through PKA activity (Chang et al. 1991). In rat DRG neurons, serotonin (5-HT) shifted the activation curve of Ih toward the positive potential by increasing the cAMP concentration and this effect was mimicked by forskolin (Cardenas et al. 1999). In this study, we found that cytosolic ATP regulates the voltage dependency of Ih in rat dorsal root ganglion neurons. The application of ATP (2 mM) to the pipette solution caused the positive shift of the activation curve of Ih.
The electrophysiological properties of Ih in rat DRG neurons almost agree with those of Ih in other neuronal cells and HCN channels (Biel et al. 1999; Pape 1996). On the other hand, the pharmacological properties of Ih in rat DRG neurons differ from those previously reported. The magnitude of the inhibition by extracellular Ba2+ was rather greater than that reported in acutely isolated rat DRG neurons (Scroggs et al. 1994) and mHCN2 channels (Ludwig et al. 1998). It was shown that in mouse DRG neurons, TEA (25 mM) applied extracellularly failed to block Ih (Mayer and Westbrook 1983). However, in cultured rat DRG neurons, Ih was slightly but not significantly reduced by TEA. In this study, although intracellular application of cAMP shifted the Ih activation curve toward a more depolarized voltage compared with that in the absence of cAMP in rat DRG neurons, membrane permeable analogs of cAMP (db-cAMP, 8-bromo cAMP and 8-CPT cAMP) applied extracellularly were ineffective against Ih activation in contrast with the report in neonatal rat DRG neurons (Wang et al. 1997) and in guinea-pig primary afferent neurons (Ingram and Williams 1996). The reason for this is unclear at present. In mouse DRG neurons, Raes et al. (1997) reported that 5 mM ATP added to a pipette solution containing 100 µM cAMP induced a 7-mV additional shift of Vhalf. This "additional" shift was explained to be caused not by the direct action of ATP on the channel but by the action of PKA activated by cAMP. In rat DRG neurons, we could not demonstrate an additional positive shift of Vhalf with the pipette solution containing 1 mM cAMP and 2 mM ATP. We do not have an explanation for the difference between our result and that in mouse DRG neurons. It might be related to the difference in the nucleotides concentrations.
In rat DRG neurons and other neuronal cells, an adenylate cyclase activator, forskolin, activates Ih by shifting its activation curve to a more positive voltage (Cardenas et al. 1999; Ingram and Williams 1996; Tokimasa and Akasu 1990). In this study, extracellularly applied forskolin (10 µM) significantly increased the Ih amplitude at –90 mV by about 30%. Therefore adenylate cyclase-dependent steps seem to be included in the modulation cascade of Ih in rat DRG neurons. If the effect of ATP observed in rat DRG neurons resulted from the phosphorylation by PKA, cAMP would be produced from ATP by adenylate cyclase (Richards et al. 1981). However, this is not consistent with this study. The effect of ATP on Ih in rat DRG neurons was not influenced by the adenylate cyclase inhibitor, SQ-22536, indicating that adenylate cyclase activity does not contribute to the ATP-induced shift in the activation curve of Ih. Application of a nonhydrolysable ATP analog, ATP--S (2 mM), to the pipette solution also shifted the Vhalf in the positive direction, suggesting that ATP modulates the voltage dependency of Ih in rat DRG neurons without conversion to cAMP. However, it is well known that ATP--S can act as a substrate for some kinds of protein kinases and cause an irreversible protein thiophosphorylation (Eckstein 1985; Liou et al. 1999). Therefore the possibility that the change in the phosphorylation balance caused by ATP--S leads to a modulation of Ih could not be eliminated. Moreover, the effect of ATP was also mimicked by another nonhydrolysable ATP analog, AMP-PNP. Thus ATP, AMP-PNP, and ATP--S may act directly on the channel protein. If the direct action of ATP against the channel protein facilitated Ih in rat DRG neurons, the free ATP concentration in the intracellular solution is important. Then, we calculated free ATP concentration with Maxchelator software (Stanford University), which was discussed in detail by Bers et al. (1994). The parameters of temperature, pH, and ionic strength that we used in this software were 25°C, 7.2, and 0.16 N, respectively. The free ATP concentrations in the pipette solutions containing totally 1 and 2 mM ATP were 91.3 and 417 µM, respectively. The free ATP concentration is about four times higher in the pipette solution containing 2 mM ATP than that containing 1 mM ATP. This may cause the positive shift in the voltage dependence of Ih when the concentration of ATP added to the pipette solution is increased. In adult guinea pig and chick embryo dorsal root ganglion neurons, the total intracellular ATP concentration was calculated as 1.7 and 1.5–2 mM, respectively (Fukuda et al. 1983; Schousboe et al. 1970). In rat DRG neurons, the total ATP concentration is presumably similar to that in guinea pig or chick DRG neurons. Because ATP at the concentration beyond 2 mM modulated the activation of Ih in this study, it is highly probable that ATP contribute to regulating the voltage dependency of Ih in intact DRG neurons. In many neurons, it has been reported that Ih contributes to setting the resting membrane potential (Pape 1996). In rat DRG neurons, it was shown that the application of CsCl (2 mM), which could completely abolish Ih, hyperpolarized the membrane potential reversibly by approximately 5 mV, suggesting that Ih played an indispensable role in setting membrane potential (Wang et al. 1997). Thus it is possible that change in the voltage dependency of Ih by ATP contributes to regulating the membrane potential. However, it is still unknown what extracellular signals can regulate Ih activation via change in the intracellular ATP concentration. For example, under the condition in which supply of ATP is stopped, such as hypoxia, the neuronal excitability might be influenced by the inhibition of Ih. Further experiments are necessary to clarify whether the regulation of Ih by cytosolic ATP has physiological or pathophysiological role in the neuronal excitability.
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DISCLOSURES |
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FOOTNOTES |
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Address for reprint requests and other correspondence: N. Kitamura, Laboratory of Physiology, Dept. of Biomedical Sciences, The Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan (E-mail: nakit{at}vetmed.hokudai.ac.jp).
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, instantaneous current, n = 10) and the end (
, steady-state current, n = 10) were plotted against the membrane potentials. Symbols and vertical bars indicate mean values ± SE. C: pharmacological property of Ih in rat DRG neurons. CsCl (2 mM), BaCl2 (2 mM), TEA (20 mM), and 4-aminopyridine (4-AP, 1 mM) were applied extracellularly to voltage-clamped neurons. Left: typical records of Ih evoked by hyperpolarizing pulse to –130 mV from holding potential of –60 mV before (
) application of Cs+, Ba2+, 4-AP, and TEA. Right: columns and vertical bars indicate the mean and SE of Ih inhibition rates at –130 mV by Cs+ (n = 6), Ba2+ (n = 8), 4-AP (n = 7), and TEA (n = 6).
, n = 5). Symbols and vertical bars indicate the mean values ± SE of activation rates at each potential.