Introduction

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K_ATP channels, which are present in various tissues (Noma, 1983; Cook and Hales, 1984; Spruce et al., 1987; Standen et al., 1989), play fundamental roles in various physiological and pathophysiological conditions (Ashcroft and Ashcroft, 1990; Edwards and Weston, 1993). In the cardiovascular system, activation of K_ATP channels causes vascular relaxation (Samaha et al., 1992), shortening of cardiac action potential duration and a decrease of contractility (Fosset et al., 1988; Nichols et al., 1991). These effects would be cardioprotective by prohibiting excessive ATP consumption and Ca⁺⁺-overload (Escande and Cavero, 1992; Lawson and Downey, 1993; Grover, 1994).

Several pharmacological agents, known as K⁺ channel openers, can activate K_ATP channels and therapeutic usage of these drugs has been suggested for cardiovascular diseases such as hypertension, arrhythmia, angina pectoris, ischemic heart disease and congestive heart failure (Anderson, 1992). The K⁺ channel openers consist of compounds with diverse chemical structures. These include pyridine derivatives such as pinacidil and nicorandil, benzopyran derivative (e.g., cromakalim), butenoic acid (e.g., ER001533) and so on (Edwards and Weston, 1993). KRN4884 is a novel pyridine derivative related to nicorandil, and it was recently reported that KRN4884 produced a long-lasting hypotensive effect compared with levcromakalim in anesthetized dogs (Izumi et al., 1995). KRN4884 had greater specificity for the coronary vasculature than Ca⁺⁺ channel blockers such as nifedipine and nilvadipine (Izumi et al., 1995). We have found potentiation by nitric oxide of K_ATP channel current induced by KRN4884 or cromakalim (Shinbo and Iijima, 1997). In rat isolated aorta, the effects of KRN4884 were inhibited by glibenclamide (1 µM) (Izumi et al, 1995; Shinbo and Iijima, 1997). These findings suggest that activation of K_ATP channels might be responsible for the pharmacological effects of KRN4884. However, no systematic study has been performed regarding the cellular mechanism of its action.

In the present study, single channel currents were recorded in guinea pig ventricular cells to determine the mechanism of actions of KRN4884. We show that KRN4884 activates cardiac K_ATP channels both by decreasing the affinity of the channels to ATP and by a mechanism independent of intracellular ATP.

	Methods

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Cell preparation. Single ventricular cells of guinea pig hearts were obtained by an enzymatic dissociation procedure (Isenberg and Klöckner, 1982). Guinea pigs weighing 250 to 590 g were anesthetized with pentobarbital (50-60 mg/kg i.p.). Under artificial respiration, the heart was quickly removed from the thorax and hung on a Langendorff type apparatus to start the coronary perfusion with a Ca⁺⁺-free Tyrode's solution. The heart was then perfused with Ca⁺⁺-free Tyrode's solution containing 0.04% (w/v) collagenase (200-230 units/mg; Wako Pure Chemical Industry, Osaka, Japan) for 25 to 35 min at 35°C. After the enzyme treatment, the heart was rinsed with a high K⁺, low Cl storage solution. Then the left ventricle was dissected from the digested heart, and stored in the storage solution at 4°C for later use.

Solutions and drugs. The normal Tyrode's solution contained (in mM): NaCl, 136.9; KCl, 5.4; CaCl₂, 1.8; MgCl₂, 0.53; NaH₂PO₄, 0.33; HEPES, 5.0; and glucose, 5.5; pH = 7.4, adjusted with NaOH. The high K⁺, low Cl storage solution contained (in mM): taurine, 10; oxalic acid, 10; L-glutamic acid, 70; KCl, 25; KH₂PO₄, 10; EGTA, 0.5; glucose, 11; and HEPES, 10; pH = 7.4, adjusted with KOH. The pipette solution contained (in mM): KCl, 150; CaCl₂, 1; HEPES, 5; pH = 7.4, adjusted with KOH. The bath solution contained (in mM): KCl, 150; MgCl₂, 0.5; HEPES, 5; EGTA, 1; pH = 7.2, adjusted with KOH. ATP disodium salt (0-10 mM) was added into the bath solution to give the concentration described in the text. The concentration of free Mg⁺⁺ in the bath solution was kept constant at 0.5 mM.

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Activation of KATP channels by KRN4884 in guinea pig ventricular cells. (A) Original current trace. Immediately after formation of inside-out patch in the ATP-free bath solution, activation of outward KATP channel current was observed at 50 mV. The pipette and bath solutions contained 150 mM KCl. The protocols are shown as bars at the top of the trace, and the arrow head indicates the zero current level. The low-pass filter was set at 300 Hz for the trace reproduction. Note that KRN4884 (0.3 and 3 µM) increased the outward current in a concentration-dependent manner and that this current was inhibited by glibenclamide (3 µM). (B) The concentration-response relationship for KATP channel activation by KRN4884. All data were obtained in the presence of ATP (1 mM). The smooth curve was obtained by fitting the data with the Hill equation 1 (see "Methods"). Data points indicate mean ± S.E. Chemical structure of KRN4884 is shown in the inset.

Electrophysiological recordings. The patch-clamp technique (Hamill et al, 1981) was used to record currents from inside-out membrane patch using a patch-clamp amplifier (Nihon Kohden CEZ-2300, Tokyo, Japan). Patch pipettes were pulled with a micropipette puller (model P-97, Sutter Instrument Co., Novato, CA) from glass capillary tubing (Corning 7052; Warner Instrument Co., CT), and the electrode resistance was 4 to 10 megaohm. The data were stored on digital audio tape with a DAT recorder (TEAC, Tokyo, Japan) for later computer analysis.

Data analysis. The data were low-pass filtered at 1 to 3 kHz, and digitized at 2 to10 kHz into a computer (IBM-compatible 386, Proside, Tokyo, Japan or PC-98, NEC, Tokyo, Japan) with either pClamp software (Axon Instruments, Foster City, CA) or in-house programs made by us. For analyzing the open- and closed-time distributions, the threshold for event detection was set at half of the unitary single channel amplitude. Usually the patch membrane contained more than two active channels, and it was quite difficult to obtain patches with only one active channel. Therefore, the analysis was confined within bursting activity where no overlap of the channel opening was detected, and closed-time more than 30 ms was discarded.

	Results

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Activation by KRN4884 of K_ATP channels in guinea pig ventricular cells. After the gigaohm-seal was established, the bath solution was switched from the normal Tyrode's solution to the high-K⁺, ATP-free bath solution, and the membrane potential was held at 50 mV. Then the patch membrane was excised in the ATP-free bath solution, resulting in rapid activation of outward channel currents (fig. 1A). The current was inhibited by addition of 1 mM ATP into the bath solution, confirming that the current was caused by the K⁺ current flowing through K_ATP channels. KRN4884 (0.3 and 3 µM) increased the outward current in a concentration-dependent manner, and the unitary current amplitudes were similar to that of K_ATP channels in the ATP-free solution. Furthermore, channel activity was abolished by 3 µM glibenclamide (fig. 1A).

Effects of KRN4884 on the K_ATP channel kinetics. Single-channel properties were compared between the K_ATP channel current in the ATP-free bath solution and the channel current induced by KRN4884 (0.3 µM) in 1 mM ATP-containing bath solution. As shown in figure 2A, both channel currents were outward at positive potentials and inward at negative potentials, and reversed at approximately 0 mV. At negative potentials, inwardly rectifying K⁺ channels with relatively small unitary amplitude and long opening time overlapped with either K_ATP channels (left panels) or KRN4884-induced channels (right panels).

View larger version (24K): [in this window] [in a new window]   Fig. 2.   (A) Single-channel current carried through KATP channels (left panels) and KRN4884-induced channels (right panels). The excised patch membrane was exposed to ATP-free high-K+ solution (left panels) or high-K+ solution containing KRN4884 (0.3 µM) plus ATP (1 mM) (right panels). The pipette solution contained 150 mM KCl. The membrane potential is indicated at the left of each row. The downward deflection indicates inward current. The dotted line indicates the current level where all channels were closed. The inward currents with small amplitudes and long open time, which were caused by the inwardly rectifying K+ channel, were evident at negative potentials. Data were low-pass filtered at 2 kHz and sampled every 0.2 ms. (B) The amplitude histograms of KATP channel current in ATP-free condition (left panels) and the KATP channel current induced by KRN4884 (0.3 µM) plus ATP (1 mM) (right panels). These histograms correspond to the actual traces shown in panel A. Histograms were constructed from a 2-min continuous recording at a given membrane potential indicated in each panel, and fitted with a sum of two (upper two panels; 60 mV) and three (lower two panels; 60 mV) Gaussian distributions. Each peak represents the time spent in the closed level (dotted line), in the inward rectifier K+ channels (asterisks) and KATP channels (arrows). (C) Average current-voltage relationship for the KATP channel current (open circles) and the KRN4884-induced KATP channel current (0.3 µM, closed circles). Data points indicates mean ± S.E. (Error bars are shown only when they were larger than symbols.)

View larger version (31K): [in this window] [in a new window]   Fig. 3.   Distribution of the open time (upper rows) and closed time (lower rows) for the KATP channel current (left panels) and the channel current induced by KRN4884 (0.1 µM) in the presence of 1 mM ATP. The membrane potential was held at 60 mV. The currents were sampled at 10 kHz through a low-pass filter of 3 kHz. The histograms were first constructed with a bin of 2 ms for open-time distributions and with a bin of 1 ms for closed-time distributions, and the slow components were fitted with single exponentials. The excess number of events above the theoretical curve were replotted with a 0.1-ms bin for both the open- and closed-time histograms (fast components). o,s and o,f indicate the time constant of slow and fast components of the open time, respectively; and c,s and c,f are those of the closed time, respectively.

Effect of KRN4884 on ATP inhibition of K_ATP channels. After confirming the activation of the K_ATP channels at 50 mV in the ATP-free bath solution, ATP (0.01-1 mM) was cumulatively applied. ATP inhibited the channel current in a concentration-dependent manner (fig. 4A). In the presence of KRN4884 (0.3 µM), the concentration of ATP required to inhibit the channel current increased (fig. 4B). As shown in figure 4B (expanded time scale), it is obvious that KRN4884 (0.3 µM) enhanced the K_ATP channel current activated in the ATP-free bath solution. Similar results were obtained in five of six patches.

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Effect of KRN4884 on ATP inhibition of KATP channels. (A) Inhibition by ATP of KATP channels in the absence of KRN4884. After confirming the activation of the KATP channel current at 50 mV, ATP was cumulatively applied to the patch membrane as indicated by the protocol shown on the top. The arrow head indicates the zero current level. The low-pass filter was set at 300 Hz for the trace reproduction. (B) Inhibition by ATP of KATP channels in the presence of KRN4884 (0.3 µM). The explanation is the same as in panel A, except that KRN4884 (0.3 µM) was present during this experiment. In the inset, the initial part of the KRN4884 application was shown in an expanded time scale, to show that the drug increased KATP channels even in the absence of ATP. (C) The ATP concentration-KATP channel activity relationships in the absence (open circles) and presence (closed circles) of KRN4884 (0.3 µM). The smooth curves were fitted by the least squares to a Hill equation 2 (see "Methods").

Effects of KRN4884 on K_ATP channels after rundown. After activation of K_ATP channels in the ATP-free bath solution, the channel activity spontaneously declined with time, which is known as rundown (Trube and Hescheler, 1984). This phenomenon was thought to be caused by dephosphorylation of the channel (Misler et al., 1986; Findlay, 1987) and/or by depolymerization of actin cytoskeleton (Furukawa et al., 1996). It appeared that KRN4884 partially restored the channel activity after rundown and that this effect largely depended on the extent of rundown. As demonstrated in figure 5, KRN4884 was applied to the patch membrane during various degrees of rundown. When rundown was incomplete (fig. 5A), i.e., NP_oi decreased to 20% at 1.5 min after the exposure to ATP-free solution, KRN4884 (3 µM) remarkably increased the K_ATP channel activity. On the other hand, KRN4884 reactivated the K_ATP channels only slightly when it was applied after long exposure to ATP-free solution (fig. 5B). No reactivation was produced by KRN4884 (~10 µM) after much more prolonged exposure to ATP-free solution (fig. 5C). This was not caused by loss of the K_ATP channels during the course of experiments, because the subsequent application of 5 mM ATP for 3 min could restore the channel activity (fig. 5C). To quantitatively analyze this phenomenon, the mean patch current (NP_oi) was measured at the peak response to ATP-free solution (NP_oi_Peak), and just before (NP_oi_Rundown) and during application of 3 µM KRN4884 (NP_oi_KRN4884). The extent of rundown and the reactivation by KRN4884 were quantified as the ratios NP_oi_Rundown/NP_oi_Peak and NP_oi_KRN4884/NP_oi_Peak, respectively. The data obtained from 19 patches are shown in figure 5D. A positive correlation is evident ( = 0.907, P < .0001).

View larger version (22K): [in this window] [in a new window]   Fig. 5.   Effects of KRN4884 on KATP channels after spontaneous rundown. After various degrees of rundown, KRN4884 partially reactivated KATP channels in panels A and B, whereas there was little or no reactivation in panel C. The protocols are shown at the top of each panel. The membrane potential was held at 50 mV. The arrow head indicates the zero current level. The data were low-pass filtered at 300 Hz. (D). The NPoiKRN4884/NPoipeak ratio was plotted against the NPoiRundown/NPoipeak ratio. The line was determined by linear regression analysis; P < .05 was considered significant.

View larger version (13K): [in this window] [in a new window]   Fig. 6.   Effects of KRN4884 on KATP channels after Ca++-induced rundown. KRN4884 (1 and 3 µM) partially reactivated KATP channels in a concentration-dependent manner. The protocol is shown at the top of the panel. The membrane potential was held at 50 mV. The arrow head indicates the zero current level. The data were low-pass filtered at 300 Hz for the trace reproduction.

Synergistic effect of intracellular NDP and KRN4884 on K_ATP channels after rundown. The effects of KRN4884 on K_ATP channels, described so far, are similar to those of intracellular NDP. That is, binding of NDPs to their own receptor on K_ATP channels not only stimulates opening of the channels that had been closed by ATP but also directly activates the channel after rundown (Lederer and Nichols, 1989; Tung and Kurachi, 1991). It is thus important to know whether the effects of NDP and KRN4884 are additive or synergistic. In this experiment we used UDP because UDP was thought to bind selectively to the NDP site without affecting the ATP binding site of the channel (Tung and Kurachi, 1991). Prolonged exposure to ATP-free solution led the K_ATP channel to almost complete rundown. This was confirmed by applying KRN4884 (0.3 µM) without any change of the channel current (fig. 7). Under this condition, UDP (3 mM) alone reactivated K_ATP channels. Subsequently, the same concentration of KRN4884 (0.3 µM) clearly facilitated the channels. In figure 7B, the data obtained from seven cells are summarized. In five patches examined, KRN4884 (0.3 µM) did not increase NP_oi in the absence of UDP but consistently facilitated the openings of the K_ATP channels in the presence of UDP. On the average the relative NP_Oi was 0.437 ± 0.100 for UDP and 0.909 ± 0.184 for UDP plus KRN4884 (P < .01, n = 7). Essentially similar results were obtained with 3 µM KRN4884 (data not shown).

View larger version (19K): [in this window] [in a new window]   Fig. 7.   Synergistic action of KRN4884 and UDP on the KATP channel current. (A) After spontaneous rundown, KRN4884 (0.3 µM) did not reactivate KATP channels. Subsequently UDP (3 mM) partially restored the KATP channel activity, which was further enhanced by additional application of KRN4884 (0.3 µM). The protocols are shown at the top of the panel. The membrane potential was held at 50 mV. The arrow head indicates the zero current level. The data were low-pass filtered at 300 Hz for the trace reproduction. (B) Effects of KRN4884 (0.3 µM) in the absence (left panel) and presence (right panel) of UDP (3 mM). Each NPoi was calculated by integrating 5 to 15 s of continuous current record. Different symbols indicate different cells.

	Discussion

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In the present experiments, we demonstrated that KRN4884, a new K⁺ channel opener, activated cardiac K_ATP channels in ventricular cells of the guinea pig hearts. The unitary conductance of 96 pS (fig. 2C) and the gating properties (mean open time: approximately 2 ms at 60 mV) (fig. 3) were almost identical with those of K_ATP channels recorded in the ATP-free bath solution. These characteristics correspond to those of K_ATP channels described so far in cardiac myocytes (Tung and Kurachi, 1991; Shen et al, 1992). The EC₅₀ of KRN4884 (0.55 µM, fig. 1B) was much smaller than those of pinacidil (65 µM, Arena and Kass, 1989), cromakalim (>30 µM, A. Shinbo and T. Iijima, unpublished data), nicorandil (74 µM, Takano and Noma, 1990), KR-30450 (~10 µM, Kwak et al., 1995), HOE-234 (~1 µM, Terzic et al., 1994), but similar to ER-001533 (0.58 µM, Shen et al., 1992). Although the EC₅₀ can be significantly affected by the recording mode and/or intracellular ATP concentration (Terzic et al., 1994; Nakayama et al., 1990), we may conclude that KRN4884 is one of the potent K_ATP channel openers described so far.

At least two different mechanisms should be considered for the activation of K_ATP channels by KRN4884: decrease in the sensitivity of the K_ATP channel to ATP and activation of the channel independent of intracellular ATP. The former effect is characterized by a rightward shift of the ATP concentration-K_ATP channel activity relationship without a significant alteration of the slope (fig. 4C). On the other hand, the slight upward shift of the relationship (fig. 4C) may suggest the latter ATP-independent activation of the channel. Furthermore, more striking evidence for the ATP-independent mechanism is that KRN4884 (1 µM) reactivated K_ATP channels after rundown (figs. 5 and 6). It is well known that, after rundown of the channel, application of Mg-ATP into bath solution could restore the channel activity (Takano et al, 1990). Rundown has, therefore, been considered to be associated with dephosphorylation of the channel (Misler et al., 1986; Findlay, 1987) and/or depolymerization of actin cytoskeleton (Furukawa et al., 1996). If so, KRN4884 may activate K_ATP channels by producing a conformational change of the channel which is normally regulated by the ATP-dependent process. The upward shift of the relationship might be explained by assuming that even immediately after removing intracellular ATP some K_ATP channels had already been in the dephosphorylated state and/or actin cytoskeletons had been depolymerized.

Findlay (1994) classified various K⁺ channel openers into two categories according to their functional similarities. Type 1 drugs such as pinacidil and cromakalim possess ATP-dependent and independent activation properties, whereas type 2 drugs, such as HOE-234, ER-001533 and KR-30450, only decrease the sensitivity of the channel to ATP. The mode of action of these substances can be interpreted based on a functional model proposed by Tung and Kurachi (1991), where three different regulatory sites of the channel exist: the channel gate, the transducer unit and the ATP binding site. The transducer unit can send the signal to the gate only when it is phosphorylated or bound with NDP. Under such conditions, the removal of ATP from the ATP binding site enables the transducer unit to send the signal to open the gate whereas the binding of ATP closes the gate. According to this model, type 2 drugs act on the ATP binding site and antagonize the inhibitory effect of ATP on the channel in a competitive manner (Shen et al., 1992; Terzic, et al., 1994; Kwak et al., 1995). On the other hand, type 1 drugs are supposed to activate the channel by interacting with the transducer unit where it is either phosphorylated or bound with NDP. In support of this view, Shen et al. (1991) showed that pinacidil and lemakalim activated the channel which was inhibited by ATP before rundown, and were effective to reactivate the inactivated channel after rundown only in the presence of NDPs.

In the present study, KRN4884 activated K_ATP channels both before and after rundown irrespective of whether NDP was present or not (figs. 1, 4, 5, 6 and 7). The results might indicate that KRN4884 acts differently from either type 1 or type 2 drugs. However, this is not likely. Although KRN4884 reactivated the K_ATP channel more or less after rundown, the potency for reactivating the channel clearly depended on the extent of rundown, i.e., the reactivation was marked if the channel activity was still substantial (fig. 5A) but was insignificant against the little channel activity after prolonged exposure to ATP-free solution. The finding could be explained by assuming various degrees of rundown (Fan et al., 1990). Namely, KRN4884 could reactivate the channel during the partial rundown but not in the complete rundown. In this respect, KRN4884 might be classified in type 1 drugs. Furthermore, even after almost complete rundown where KRN4884 (0.3 µM) failed to activate K_ATP channels, the reactivation by KRN4884 was clearly produced in the presence of UDP (fig. 6). The results suggest that the reactivation by KRN4884 requires partial phosphorylation of the transducer unit or binding of NDP to its site, as indicated in pinacidil or lemakalim (Shen et al., 1991).

Recent studies have demonstrated that the pancreatic  cell K_ATP channel is a heteromultimer composed of at least two different subunits; K_ATP- and K_ATP- (Inagaki et al., 1995). K_ATP- is the inward rectifier subunit BIR (Kir6.2) and K_ATP- is sulfonylurea receptor (SUR), a member of the ATP binding cassette superfamily (Aguilar-Bryan et al., 1995). Then Inagaki et al. (1996) cloned an isoform of SUR (SUR2) from the rat brain and demonstrated that SUR2 mRNA was expressed at high levels in heart and skeletal muscle. Furthermore, the ATP sensitivity and pharmacological properties of K_ATP channels were determined by a family of structurally related but functionally distinct sulfonylurea receptors (Inagaki et al, 1995, 1996). It is thus likely that SUR2 represents a functional role of the transducer unit originally assumed in the functional model by Tung and Kurachi (1991). KRN4884 may interact with SUR2 to activate cardiac K_ATP channels.

KRN4884 was originally found to produce a profound hypotensive effect in anesthetized dogs and to have greater specificity in coronary arteries (Izumi et al., 1995). The differences in tissue affinities, which might be caused by different SUR isoforms, may help to establish the new therapeutic strategies for various diseases including hypertension, arrhythmia, congestive heart failure and ischemic heart disease. However, no data are available now to compare the activity of KRN4884 between native cardiac and smooth muscle K_ATP channels. Recent molecular analysis indicates that the channel composed of inward rectifier subunit Kir 6.1 (Inagaki et al., 1995.) and an isoform of SUR (SUR2B, Isomoto et al., 1996) resembles the K⁺ channel found in vascular smooth muscle cells (Yamada et al. 1997). Future molecular studies are clearly necessary to determine the tissue specificity as well as the nature of the interaction between the K_ATP channel and KRN4884.