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(Circulation Research. 2001;88:570.)
© 2001 American Heart Association, Inc.

Integrative Physiology

Functional Roles of Cardiac and Vascular ATP-Sensitive Potassium Channels Clarified by Kir6.2-Knockout Mice

Masashi Suzuki, Ronald A. Li, Takashi Miki, Hiroko Uemura, Naoya Sakamoto, Yuki Ohmoto-Sekine, Masaji Tamagawa, Takehiko Ogura, Susumu Seino, Eduardo Marbán, Haruaki Nakaya

From the Department of Pharmacology (M.S., H.U., N.S., Y.O.-S., M.T., T.O., H.N.), Chiba University School of Medicine, and Department of Molecular Medicine (T.M., S.S.), Chiba University Graduate School of Medicine, Chiba, Japan, and Institute of Molecular Cardiobiology (R.A.L., E.M.), The Johns Hopkins University School of Medicine, Baltimore, Md.

Correspondence to Haruaki Nakaya, M.D., Ph.D., Department of Pharmacology, Chiba University School of Medicine, Inohana 1-8-1, Chuo-Ku, Chiba 260-8670, Japan. E-mail nakaya{at}med.m.chiba-u.ac.jp

	Abstract

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Abstract—ATP-sensitive potassium (K_ATP) channels were discovered in ventricular cells, but their roles in the heart remain mysterious. K_ATP channels have also been found in numerous other tissues, including vascular smooth muscle. Two pore-forming subunits, Kir6.1 and Kir6.2, contribute to the diversity of K_ATP channels. To determine which subunits are operative in the cardiovascular system and their functional roles, we characterized the effects of pharmacological K⁺ channel openers (KCOs, ie, pinacidil, P-1075, and diazoxide) in Kir6.2-deficient mice. Sarcolemmal K_ATP channels could be recorded electrophysiologically in ventricular cells from Kir6.2^+/+ (wild-type [WT]) but not from Kir6.2^-/- (knockout [KO]) mice. In WT ventricular cells, pinacidil induced an outward current and action potential shortening, effects that were blocked by glibenclamide, a K_ATP channel blocker. KO ventricular cells exhibited no response to KCOs, but gene transfer of Kir6.2 into neonatal ventricular cells rescued the electrophysiological response to P-1075. In terms of contractile function, pinacidil decreased force generation in WT but not KO hearts. Pinacidil and diazoxide produced concentration-dependent relaxation in both WT and KO aortas precontracted with norepinephrine. In addition, pinacidil induced a glibenclamide-sensitive current of similar magnitude in WT and KO aortic smooth muscle cells and comparable levels of hypotension in anesthetized WT and KO mice. In both WT and KO aortas, only Kir6.1 mRNA was expressed. These findings indicate that the Kir6.2 subunit mediates the depression of cardiac excitability and contractility induced by KCOs; in contrast, Kir6.2 plays no discernible role in the arterial tree.

Key Words: gene targeting • heart • vascular smooth muscle • ATP-sensitive K⁺ current • action potential

	Introduction

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ATP-sensitive K⁺ (K_ATP) channels, originally discovered in cardiac myocytes,¹ have been described in many other tissues including pancreatic ß cells, skeletal and smooth muscle cells, and neurons.^{2 3 4 5} They play important roles in the physiology and pathophysiology of various tissues by coupling the metabolic state of the cell to its electrical activity.⁶ Recent progress in molecular biology and electrophysiology resulted in cloning of K_ATP channel genes and elucidation of their subunit composition.^{7 8} K_ATP channels are assembled with a hetero-octameric stoichiometry from the following 2 structurally distinct subunits: an inwardly rectifying potassium channel subunit forming the pore (Kir6.X) and a regulatory subunit, a sulfonylurea receptor (SUR) belonging to the ATP binding cassette superfamily.^{9 10 11} The pancreatic ß-cell K_ATP channels have been shown to consist of SUR1 and Kir6.2.¹⁰ In fact, our previous report indicated that insulin secretion in response to glucose or sulfonylurea was absent in pancreatic ß cells from K_ATP channel–deficient mice generated by genetic disruption of Kir6.2.¹² Cardiac K_ATP channels are thought to be formed by SUR2A and Kir6.2, because coexpression of these clones in a cell line reconstitutes the basic electrophysiological and pharmacological properties of the native cardiac channel.¹¹ In terms of vascular K_ATP channels, Isomoto et al¹³ showed that coexpression of Kir6.2 and SUR2B, a splice variant of SUR2A, reconstituted the pharmacological and electrophysiological properties of K_ATP channels described in some smooth muscle cells. Indeed, it has been suggested that K_ATP channels of smooth muscle cells of murine colon¹⁴ and guinea pig bladder¹⁵ comprise SUR2B and Kir6.2. However, Yamada et al¹⁶ have suggested that K⁺ channel composed of SUR2B and Kir6.1 closely resembles the NDP-dependent K⁺ (K_NDP) channel observed in vascular smooth muscle cells. To clarify the functional role(s) of a defined K_ATP-channel subunit in cardiac and vascular tissues, we studied K_ATP channel-deficient mice (Kir6.2^-/-) produced by knockout (KO) of the Kir6.2 gene. We find that Kir6.2 is essential for the depression of cardiac excitation and contraction, but not for vasodilation, in response to K⁺ channel openers (KCOs), highlighting major functional and genetic distinctions between cardiac and vascular K_ATP channels.

	Materials and Methods

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Kir6.2^-/- Mice
All procedures complied with the standards for the care and use of animal subjects as stated in the Guide for the Care and Use of Laboratory Animals (NIH publication No. 85-23, revised 1996). A mouse line deficient in K_ATP channel was generated by targeted disruption of the gene coding for Kir6.2, as described previously.¹² C57BL/6 mice were used as control because they had been backcrossed to a C57BL/6 strain for 5 generations.

Electrophysiology
Adult Ventricular Cells
Single ventricular cells of the adult mouse heart were isolated by conventional enzymatic digestion. Single-channel and whole-cell membrane currents were recorded by the patch-clamp method as previously described.^{17 18} For single-channel recordings from the open cell-attached patches, symmetrical high-K⁺ pipette solution (solution A) and internal solution (solution B) were used. After the gigaohm seal formation, part of the surface membrane of a rod-shaped cell was disrupted by penetrating the membrane with a glass pipette (1 to 2 µm in tip diameter) and the ruptured cell was exposed to the solution containing only a trace amount of ATP (solution B). The single-channel currents were recorded at room temperature and analyzed with pClamp software (version 5.5, Axon Instruments).

Whole-cell current recordings were performed at 36.0°C using glass patch pipettes filled with solution C (pCa 8.0). The external solution used was HEPES-Tyrode solution (solution D). A liquid junction potential between the internal solution and the bath solution of -8 mV was corrected. A ramp pulse protocol was used to record the quasi–steady-state membrane current as previously described.¹⁸ Current-clamp experiments were also performed in the whole-cell recording mode at 36.0°C.

Isolation of Neonatal Ventricular Myocytes and Viral Gene Transfer
Neonatal (1- to 2-day-old) ventricular myocytes were prepared by trypsin digestion as previously described.¹⁹ Briefly, ventricles of a single litter of mouse pups (10, 1 to 2 days old) were aseptically removed immediately after decapitation. Isolated hearts were pooled, minced, and digested at room temperature with trypsin. After digestion, cells were centrifuged, plated at low density (2x10⁵ cells mL^-1), and incubated at 37°C in a humidified atmosphere of 95%O₂/5%CO₂.

The ecdysone-inducible recombinant adenovirus (AdKir6.2) carrying the wild-type (WT) Kir6.2 channel and the reporter (enhanced green fluorescent protein) genes were generated by Cre-lox recombination.^{20 21 22} Neonatal myocytes were coinfected with the channel virus AdKir6.2 and the hormone receptor virus AdVgRXR (ratio=1:10) at a final concentration of 10 000 particles mL^-1. Protein expression was induced by adding 1 mmol/L ponasterone A (Invitrogen) to the culture medium.

Electrical recordings were performed at room temperature after 72 hours in culture using the whole-cell patch clamp technique with a pipette solution (solution E) and a bath solution (solution F). Only successfully infected cells, as identified by their green fluorescence using epifluorescent microscopy, were selected for experiments.

Vascular Smooth Muscle Cells
Single smooth muscle cells were enzymatically isolated from the adult mouse aorta, and the membrane currents were recorded at room temperature using the whole-cell patch clamp technique with high-K⁺ HEPES-buffered bath solution (solution G) and pipette solution (solution H).

Mechanical Function Study
Langendorff-Perfused Heart Preparation
The heart was quickly removed from the mouse anesthetized with urethane (1.5 mg/g, IP). The heart was retrogradely perfused at a constant flow (3 mL/min) with the Krebs-Henseleit solution (solution I, 37°C) gassed with 95% O₂/5% CO₂. A polyethylene film balloon was inserted into the cavity of the left ventricle through the left atrium. The balloon was filled with saline to adjust the baseline end-diastolic pressure to 5 to 10 mm Hg. Left ventricular pressure and its dp/dt were measured continuously.

Isolated Aortic Preparation
The aorta was carefully removed and cut into rings (4 mm in length). The endothelium was removed carefully by rubbing with a small steel pin, and the rings were mounted in a thermostatic organ bath for isometric tension recording under a resting tension of 0.5 grams. The bath was continuously perfused with the Krebs-Henseleit solution (solution H) gassed with 95% O₂/5% CO₂ at 37°C.

Hemodynamic Measurement
Blood pressure and heart rate (HR) were measured in mice anesthetized with urethane. Subcutaneous needle electrodes were inserted in the limbs for ECG recording. Femoral artery and vein were cannulated with small polyethylene catheters for the measurement of blood pressure and the injection of saline or drugs, respectively. Body temperature was monitored with a rectal probe and maintained at 37°C using a heating lamp.

Northern Blot Analysis
Total cellular RNA was isolated using the RNeasy Mini kit (QIAGEN). Total RNA (10 µg) from the hearts and aortas of the mice were denatured with formaldehyde, subjected to electrophoresis on a 1% agarose gel, and transferred to nylon membranes. Hybridization was carried out under high-stringency conditions with a ³²P-labeled full-length cDNA of Kir6.1 or Kir6.2. For autoradiography, the nylon membranes were exposed to x-ray film with an intensifying screen at -80°C for 1 day. Results from these analyses were quantified through densitometry.

Drugs
The following drugs were used: pinacidil (Research Biochemicals International), diazoxide and glibenclamide (Sigma), norepinephrine (Wako), P-1075 (Leo Pharmaceutical Products), and HMR1098 (Aventis Pharmaceuticals). Pinacidil was dissolved in 0.1N HCl+saline. Diazoxide and P-1075 were dissolved in DMSO (final concentration of solvent, <0.1%). L-Ascorbic acid (0.1 mmol/L) was added to the Krebs-Henseleit solutions to prevent the oxidation of norepinephrine.

Statistics
All data are presented as mean±SE. Statistical analyses of the data were performed using the Student t test or ANOVA. Probability values <0.05 were considered significant. EC₅₀ values were obtained by use of Delta Graph Professional (Delta Point).

A table listing the composition of the various solutions can be found in an online data supplement available at http://www.circresaha.org.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Electrophysiology in Cardiomyocytes
The open cell-attached patches of ventricular cells were held at -40 mV and exposed to a solution containing only a trace amount of ATP (1 µmol/L). Single K_ATP channel activity could be recorded from 32 of 32 patches of 6 WT mice (Figure 1a). The channel openings were inhibited by addition of 1 mmol/L ATP or 10 µmol/L glibenclamide to the solution. The linear slope conductance obtained from the current-voltage relationship from -80 to 0 mV for the single-channel current of WT ventricular cells was 79±1 pS (6 cells from 3 animals) (Figures 1c and 1d). In contrast, openings of K_ATP channels could not be recorded from any open cell-attached patches of KO mouse (22 cells from 4 animals) (Figure 1b). The channel activity could not be activated even by addition of Mg-UDP, Mg-ATP, and/or pinacidil.

View larger version (24K): [in this window] [in a new window]   Figure 1. Single-channel current recordings in the open-cell attached mode from ventricular cells of Kir6.2+/+ and Kir6.2-/- mice. KATP channel activity that was sensitive to ATP and glibenclamide was observed in membrane patches of Kir6.2+/+ (a) but not Kir6.2-/- (b) ventricular cells. c, Unitary K+ currents in a membrane patch held at various voltage levels. d, Current-voltage relationship for the single-channel current; slope conductance at 0 to -80 mV was 80 pS.

Whole-cell membrane currents were recorded using a ramp-pulse protocol. The reversal potential was close to the potassium equilibrium potential in ventricular cells of both WT and KO mice (Figures 2a and 2b). There was no significant difference in the density of the outward current at 0 mV between ventricular cells isolated from WT (2.67±0.83 pA/pF) and KO (3.47±0.91 pA/pF) mice under control conditions. Pinacidil produced concentration-dependent increases of an outward current, which, by virtue of its blockade by 1 µmol/L glibenclamide, was confirmed as the ATP-sensitive K⁺ current (I_K,ATP); such a current was observed in WT ventricular cells but not in KO cells (Figure 2c). There were no significant differences in the basal parameter values of the action potentials recorded from the ventricular cells stimulated at 0.2 Hz. Exposure to pinacidil revealed dramatic differences. The action potential duration (APD) was shortened by pinacidil in WT but not in KO ventricular cells (Figures 2d and 2e). APD at 90% repolarization level (APD₉₀) of WT cells was significantly decreased from 34.0±1.5 to 10.8±3.1 ms by 100 µmol/L pinacidil and reversed to 37.9±2.5 ms after the addition of 1 µmol/L glibenclamide (Figure 2f). However, the APD₉₀ of the KO ventricular cells was not significantly changed from 35.6±2.4 ms after exposure to pinacidil (Figure 2f).

View larger version (36K): [in this window] [in a new window]   Figure 2. Effects of the KCO pinacidil (PIN) and coapplication of glibenclamide (GLB) on whole-cell membrane currents (a and b) and action potentials (d and e) recorded from ventricular cells of WT (a and d) and KO (b and e) mice. Current densities at 0 mV in Kir6.2+/+ (12 cells from 11 mice) and Kir6.2-/- (7 cells from 5 mice) ventricular cells are summarized in panel c. f, Summary of changes in APD90 after (in µmol/L) PIN 100 and PIN 100 plus GLB 1 in Kir6.2+/+ (8 cells from 8 mice) and Kir6.2-/- ventricular cells (8 cells from 5 mice). *P<0.05, **P<0.01 vs control (CON); #P<0.01 vs 100 µmol/L pinacidil.

Restoration of I_K,ATP by Adenoviral Gene Transfer of Kir6.2
As anticipated from our data in adult mouse cells, application of 100 µmol/L P-1075, a pinacidil derivative and a potent specific opener of surface I_K,ATP,²³ induced sarcolemmal I_K,ATP only in neonatal myocytes isolated from WT but not from KO mice (Figure 3a). If this absence of the surface current in KO is solely due to the absence of functional Kir6.2 channels, we should be able to restore the missing current by expressing Kir6.2. Therefore, we attempted to rescue the KO phenotype by delivering Kir6.2 to cells via adenoviral gene transfer. Indeed, I_K,ATP was observed in KO cells infected with AdKir6.2 on addition of 100 µmol/L P-1075 (Figures 3a and 3b). The identity of this virally induced and P-1075–induced current as I_K,ATP was confirmed by its complete suppression by coapplication with 100 µmol/L P-1075 of the specific surface channel blocker HMR1098.²³

View larger version (23K): [in this window] [in a new window]   Figure 3. Restoration of IK,ATP by adenoviral gene transfer of Kir6.2. a, Summary of current densities of IK,ATP of uninfected WT, uninfected KO, and Kir6.2-infected KO cells in the absence and presence of 100 µmol/L P-1075 and/or 100 µmol/L HMR1098 measured at 0 mV for 300 ms from a holding potential of -80 mV preceded by a 100-ms prepulse to -10 mV. Numbers in parentheses are numbers of individual determinations. Data were pooled from 3 litters of neonatal mice. *P<0.05 using the paired Student t test. b, Time course of development of surface IK,ATP of Kir6.2-infected KO neonatal myocytes by 100 µmol/L P-1075 measured at 0 mV from -80 mV. Horizontal bars indicate periods of exposure to drugs. Current elicited by P-1075 was completely blocked by coapplication of 100 µmol/L HMR1098. Inset shows representative current traces of the same cell (A) before and (B) after application of P-1075 and (C) after coapplication of HMR1098. Same protocol as in panel a was used.

Electrophysiology in Aortic Smooth Muscle Cells
Effects of pinacidil on the whole-cell membrane current were examined in aortic smooth muscle cells held at -40 mV in the high-K⁺ solution. Pinacidil activated a glibenclamide-sensitive inward current in smooth muscle cells from both WT and KO mice in a concentration-dependent manner (Figures 4a and 4b). The concentration-response curves for the stimulatory effects of pinacidil in WT and KO smooth muscle cells were almost superimposable (Figure 4c). The calculated EC₅₀ values of pinacidil for activating the I_K,ATP were 0.21 and 0.16 µmol/L in aortic smooth muscle cells of WT and KO mice, respectively.

View larger version (18K): [in this window] [in a new window]   Figure 4. Pinacidil-induced current in aortic smooth muscle cells of Kir6.2+/+ (a) and Kir6.2-/- mice (b). Note that pinacidil induced an inward current that was sensitive to glibenclamide in a concentration-dependent manner in both Kir6.2+/+ and Kir6.2-/- aortic smooth muscle cells held at -40 mV in high-K+ (140 mmol/L) solution. c, Summary of the densities of the glibenclamide-sensitive current after various concentrations of pinacidil in aortic smooth muscle cells of Kir6.2+/+ () and Kir6.2-/- mice (). Data are mean±SE of WT (5 to 11 cells from 22 mice) and KO cells (7 to 13 cells from 24 mice).

Functional Experiments in Isolated Cardiac and Vascular Tissues
Cardiac function of the hearts isolated from WT and KO mice was evaluated by measuring the left ventricular developed pressure (LVDP) and its derivative. In WT hearts, pinacidil at a concentration of 100 µmol/L significantly decreased LVDP and maximal dp/dt to 70.7±2.0% (n=10, P<0.05) and 71.5±2.1% (n=10, P<0.05) of the control, respectively (Figure 5a). In WT hearts, the addition of 1 µmol/L glibenclamide reversed the pinacidil-induced decreases in LVDP and maximal dp/dt to 93.4±2.4% and 92.4±2.6% of the control, respectively. In contrast, the LVDP and maximal dp/dt of KO hearts were 99.1±3.2% (n=10, NS) and 98.4±3.1% (n=10, NS) of the control after 100 µmol/L pinacidil in KO hearts (Figure 5b). To assess vascular reactivity, effects of KCOs on aortic preparations precontracted with 0.1 µmol/L norepinephrine were examined. In both the aortic rings isolated from WT and KO mice, pinacidil and diazoxide produced a concentration-dependent vasorelaxing effect (Figure 6). The EC₅₀ values of pinacidil for the vasorelaxing effect were 1.29 and 1.66 µmol/L in aortic preparations isolated from WT and KO mice, respectively. The EC₅₀ values of diazoxide for vasorelaxing effects were 3.42 and 4.61 µmol/L in WT and KO aortas, respectively (Figure 6d). There were no significant differences in these EC₅₀ values of pinacidil or diazoxide between aortic preparations of WT and KO mice. Thus, KCOs produced vasodilation in vascular tissues isolated from WT and KO mice with similar potency.

View larger version (51K): [in this window] [in a new window]   Figure 5. Effects of pinacidil on mechanical function of Langendorff-perfused hearts of Kir6.2+/+ (a) and Kir6.2-/- (b) mice. Actual records of changes in left ventricular pressure (LVP) and its derivative (dp/dt) after pinacidil and coapplication of glibenclamide are shown. Lower traces of left ventricular pressure and dp/dt are depicted on an expanded time scale. Note that pinacidil produced a negative inotropic response in Kir6.2+/+ but not Kir6.2-/- heart.

View larger version (23K): [in this window] [in a new window]   Figure 6. Relaxing effects of pinacidil and diazoxide on aortic preparations of Kir6.2+/+ and Kir6.2-/- mice. Aortic rings were precontracted with 0.1 µmol/L norepinephrine (NE), and pinacidil or diazoxide was added in a cumulative manner. Representative changes of norepinephrine-induced contraction after various concentrations of pinacidil in aortic preparations of Kir6.2+/+ and Kir6.2-/- mouse are shown in panels a and b, respectively. c and d, Concentration-relaxation curves for pinacidil (c) and diazoxide (d) in aortic rings contracted with 0.1 µmol/L norepinephrine. and indicate data obtained from Kir6.2+/+ and Kir6.2-/- aortic preparations, respectively. Points are mean±SE of WT (8 to 9 preparations from 5 animals for each drug) and KO preparations (7 to 9 preparations from 4 to 5 animals for each drug).

Hemodynamic Measurements
There were no significant differences in the basal hemodynamic values between WT and KO mice anesthetized with urethane. The basal values of HR in WT and KO mice were 642±24 and 638±12 bpm, respectively. The basal values of mean arterial pressure (MAP) in WT and KO mice were 65.1±2.9 mm Hg and 73.5±4.0 mm Hg, respectively. Intravenous injection of pinacidil (0.3 mg/kg) decreased MAP and increased HR in WT and KO to a similar extent (Figure 7). There were no significant differences in the magnitude of MAP decrease and HR increase in response to pinacidil between WT and KO mice.

View larger version (20K): [in this window] [in a new window]   Figure 7. Effects of pinacidil on MAP and HR in anesthetized mice. Changes of MAP and HR after intravenous injection of 0.3 mg/kg pinacidil are indicated in panels a and b, respectively. and indicate data obtained from Kir6.2+/+ and Kir6.2-/- mice, respectively. Ordinates indicate changes in MAP and HR from baseline values at the time point of 0 minutes. Points are mean±SE of 6 animals for both groups.

Northern Blot Analysis
Kir6.2 mRNA was expressed in hearts of WT mice (n=4) but not in those of KO mice (n=4), whereas Kir6.1 mRNA was expressed in hearts of both WT and KO mice (Figure 8a). Kir6.1 mRNA was expressed in both aortic preparations of WT (n=4) and KO mice (n=4), whereas Kir6.2 mRNA was not detectable in either preparation. Expressed levels of Kir6.1 mRNA in KO hearts (Figure 8b) and aortas (Figure 8c) were similar to those observed in respective preparations of WT mice.

View larger version (49K): [in this window] [in a new window]   Figure 8. Northern blot analyses of Kir6.1 and Kir6.2 mRNA. a, Comparison of expressed levels in WT (lanes 1 through 4) and KO mice (lanes 5 through 8). Left and right panels show data obtained from 8 individual animals. b and c, Quantitative analyses of Kir6.1 mRNA expression. Expressed levels of Kir6.1 mRNA in hearts (b) and aortas (c) were normalized by those of GAPDH mRNA, respectively.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Coexpression of SUR2A and Kir6.2 produces a 80 pS K_ATP channel resembling that found in native cardiac cells.¹¹ The channel activity is inhibited by ATP with an IC₅₀ value of 100 µmol/L and by glibenclamide with less sensitivity compared with pancreatic-type K_ATP channel (ie, SUR1/Kir6.2). SUR2A, which showed 68% identity of amino acids with SUR1, was expressed at high levels in heart and skeletal muscle. Such pharmacological data, conductances, and tissue distributions suggested that the cardiac K_ATP channel may be composed of SUR2A and Kir6.2. The present study has confirmed directly that Kir6.2 is essential for the function of sarcolemmal K_ATP channels in cardiac cells. In open-cell attached patches of ventricular cells of Kir6.2^+/+ mice, we could record the K_ATP channel activity, which showed a slope conductance of 79 pS and was inhibited by application of ATP or glibenclamide. However, we did not observe any K_ATP channel activity in membrane patches of Kir6.2^-/- ventricular cells even in the presence of Mg-UDP and/or pinacidil. In addition, the KCO pinacidil activated the glibenclamide-sensitive outward current in ventricular cells of WT but not of KO mice. These findings indicate that Kir6.2 forms the pore of cardiac K_ATP channels. This concept was further strengthened by Kir6.2 rescue experiments. In neonatal myocytes of Kir6.2^-/- mice, adenoviral gene transfer of Kir6.2 restored the surface K_ATP current that was activated by P-1075 and blocked by HMR1098. P-1075 and HMR1098 have been shown to be a specific activator and a blocker of sarcolemmal K_ATP channel, respectively.²³ Therefore, Kir6.2 is essential for the function of sarcolemmal K_ATP channel in cardiac cells.

The present study has also demonstrated that sarcolemmal K_ATP channels mediate the depression of cardiac function by KCO. Pinacidil markedly shortened APD and decreased cardiac contractile function assessed by LVDP and dp/dt in WT but not in KO hearts. In Kir6.2^+/+ hearts, the action potential shortening induced by pinacidil might lead to a decrease in Ca²⁺ influx via Ca²⁺ channels and/or Na⁺-Ca²⁺ exchange system, resulting in a negative inotropic response. Similar pinacidil-induced negative inotropy associated with action potential shortening was observed with guinea pig papillary muscles.¹⁷ However, such electromechanical response to pinacidil was absent in Kir6.2^-/- hearts. We also found that the action potential shortening under metabolic inhibition was absent in Kir6.2^-/- ventricular cells (M.S., N.S., H.N., unpublished observations, 1999). The action potential shortening in hypoxic and ischemic conditions is expected to decrease Ca²⁺ influx, resulting in reduction of mechanical contraction, amelioration of intracellular Ca²⁺ overload, and energy sparing. Administration of KCO would accelerate the functional alterations and thereby protect ischemic myocardium. However, there is recent evidence that mitochondria may harbor another type of K_ATP channel, an additional site of action of KCOs, and that the mitochondrial K_ATP channel rather than the sarcolemmal K_ATP channel may be important for cardioprotection by KCOs and ischemic preconditioning.^{24 25} Because action potential shortening would not be observed during myocardial ischemia or administration of KCO, the Kir6.2-deficient mouse may be a useful model to define the underlying mechanism(s) of cardioprotection afforded by KCOs or ischemic preconditioning.

Currents attributed to K_ATP channels have been observed in a number of vascular and visceral smooth muscle preparations, such as rabbit mesenteric artery,²⁶ portal vein,²⁷ guinea pig trachealis,²⁸ and urinary bladder.²⁹ In terms of SUR of smooth muscle cells, Isomoto et al¹³ have proposed that SUR2B, a splice variant of SUR2A having different carboxyl-terminal amino acids, is the subunit constituting the smooth muscle–type K_ATP channel as inferred from pharmacological properties and tissue distribution. In smooth muscle cells of murine colon¹⁴ and guinea pig bladder,¹⁵ the pore region of the K_ATP channels was reported to comprise SUR2B and Kir6.2.

There is still controversy as to physiological properties of K_ATP channels in vascular smooth muscle cells. K_ATP channels of large conductance (120 to 258 pS) were reported in vascular smooth muscle cells from rabbit mesenteric artery^{30 31} and rat tail artery.³² Edwards and Weston³³ postulated that the large-conductance K⁺ channel might be a Ca²⁺-activated K⁺ (K_Ca) channel. K_ATP channels having small or intermediate conductance (15 to 50 pS) were observed in various vascular smooth muscle cells.^{33 34 35} They were inhibited by glibenclamide and activated by KCOs and NDPs. Because these characteristics were closely similar to the "K_NDP channel" composed of SUR2B and Kir6.1, Yamada et al¹⁶ suggested that vascular K_ATP channel may be composed of Kir6.1 rather than Kir6.2. The present study has demonstrated that KCOs relaxed the WT and KO aortic preparations precontracted by norepinephrine with similar potency and pinacidil induced the glibenclamide-sensitive current to a similar extent in vascular smooth muscle cells isolated from WT and KO aortas. These findings indicate that Kir6.2 is not necessary for the vasodilation by KCOs in mouse aorta. Strong support for this concept is the finding that Kir6.1 mRNA, but not Kir6.2 mRNA, was detected in both WT and KO aortas. In this study, not only pinacidil but also diazoxide relaxed the precontracted aortic preparations. Because the benzothiadiazine KCO, diazoxide, activated the K_ATP channels composed of Kir6.2/SUR2B¹³ but not Kir6.2/SUR2A,¹¹ the SUR subunit in mouse aorta might be SUR2B.

It may be of importance to examine the effect of KCO on the resistance vessels rather than conductance vessels such as thoracic aortas. Therefore, we examined the effect of pinacidil on systemic blood pressure in anesthetized WT and KO mice. There were no significant differences in baseline values and decrease of MAP in response to pinacidil between WT and KO mice. The target of pinacidil in resistance vessels might not be the K_ATP channel having Kir6.2 as a pore subunit. It has been suggested that K_ATP channels in vascular smooth muscle cells may be activated by metabolic inhibition and endogenous substances such as adenosine, calcitonin gene-related peptide, and prostacyclin.²⁶ Further studies may be needed to identify the pore subunit of K_ATP channels involved in the regulation of vascular tones under metabolic changes and released endogenous substances using the gene-targeted animal.

In conclusion, we provide direct evidence that Kir6.2 forms the pore region of cardiac K_ATP channel but not of vascular K_ATP channel and that Kir6.2 is essential for the action potential shortening and depressed cardiac contractility in response to KCOs.

	Acknowledgments

 
This work was supported by a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan; Mitsui Life Insurance Research Foundation; and K. Watanabe Research Fund. This work was also supported by grants from the NIH (Grant R37 HL36957 to E.M.) and the Heart and Stroke Foundation of Canada (fellowship to R.A.L.). E.M. holds the Michel Mirowski, MD Professorship of Cardiology at The Johns Hopkins University.

	Footnotes

 
Original received December 8, 2000; revision received January 29, 2001; accepted February 2, 2001.

This manuscript was sent to James T. Willerson, Consulting Editor, for review by expert referees, editorial decision, and final disposition.

	References

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