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(Circulation Research. 1998;83:1132-1143.)
© 1998 American Heart Association, Inc.

Original Contributions

Reconstituted Human Cardiac K_ATP Channels

Functional Identity With the Native Channels From the Sarcolemma of Human Ventricular Cells

Andrey P. Babenko, Gabriela Gonzalez, Lydia Aguilar-Bryan, , Joseph Bryan

From the Departments of Cell Biology (A.P.B., G.G., J.B.) and Medicine (L.A.-B.), Baylor College of Medicine, Houston, Tex.

Correspondence to Andrey P. Babenko, MD, PhD, Department of Cell Biology, Baylor College of Medicine, 112C, 1 Baylor Plaza, Houston, TX 77030. E-mail ababenko{at}bcm.tmc.edu

	Abstract

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Abstract—ATP-sensitive potassium (K_ATP) channels in striated myocytes are heteromultimers of K_IR6.2, a weak potassium inward rectifier, plus SUR2A, a low-affinity sulfonylurea receptor. We have cloned human K_IR6.2 (huK_IR6.2) and a huSUR2A that corresponds to the major, full-length splice variant identified by polymerase chain reaction analysis of human cardiac poly A⁺ mRNA. ATP- and glibenclamide-sensitive K⁺ channels were produced when both subunits were coexpressed in COSm6 and Chinese hamster ovary cells lacking endogenous K_ATP channels, but not when huSUR2A or huK_IR6.2 were transfected alone. Recombinant channels activated by metabolic inhibition in cell-attached configuration or in inside-out patches with ATP-free internal solution were compared with sarcolemmal K_ATP channels in human ventricular cells. The single-channel conductance of 80 pS measured at -40 mV in quasi-symmetrical 150 mmol/L K⁺ solutions, the intraburst kinetics that were dependent on K⁺ driving force, and the weak inward rectification were indistinguishable for both channels. Similar to the native channels, huSUR2A/huK_IR6.2 recombinant channels were inhibited by ATP at quasi-physiological free Mg²⁺ (0.7 mmol/L) or in the absence of Mg²⁺, with an apparent IC₅₀ of 20 µmol/L and a pseudo-Hill coefficient of 1. They were "refreshed" by MgATP and stimulated by ADP in the presence of Mg²⁺ when inhibited by ATP. The huSUR2A/huK_IR6.2 channels were stimulated by cromakalim and pinacidil in the presence of ATP and Mg²⁺ but were insensitive to diazoxide. The results suggest that reconstituted huSUR2A/huK_IR6.2 channels represent K_ATP channels in sarcolemma of human cardiomyocytes and are an adequate experimental model with which to examine structure-function relationships, molecular physiology, and pharmacology of these channels from human heart.

Key Words: ATP-sensitive K⁺ channel • K_IR6.2 • SUR2A • isoform • splice variant

	Introduction

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ATP-sensitive potassium (K_ATP channels), originally defined as weak potassium inward rectifiers (K_IR) specifically inhibited by ATP in inside-out patches from cardiomyocyte sarcolemma,^{1 2} directly link changes in cellular metabolism to plasma membrane excitability. These channels are abundant in mammalian cardiomyocytes, including adult human ventricular³ and atrial⁴ cells. Cardiac K_ATP channels exhibit a rich regulatory phenomenology^{5 6} whose underlying structure-function relationships have been difficult to investigate in the absence of a well-defined recombinant channel. The physiological role of sarcolemmal K_ATP channels in the heart remains a puzzle. Their activation is suggested to serve a cardioprotective function under ischemia.⁷ However, the significance of sarcolemmal K_ATP channels as the target for potassium channel openers (PCOs) is in dispute, given the absence of PCOs selective for these channels in cardiomyocytes, and the equivocal "cardioplegic" and antiarrhythmic effects of these drugs.⁸ The recombinant channel from human heart is desirable for the design and screening of cardioselective PCOs and is required to examine structure-function relationships.

Reconstitution of "ATP-regulated" potassium channels from cloned K_IR subunits, including ROMK1 (K_AB-1, K_IR1.1a),⁹ cK_ATP-1 (CIR, GIRK4, K_IR3.4),¹⁰ BIR9 (K_IR5.1),¹¹ BIR10 (K_AB-2, K_IR4.1),^{11 12} uK_ATP-1 (K_IR6.1),¹³ or IRK3 (similar to BIR11, K_IR2.3),¹⁴ has been reported. None of these channels has the properties of cardiac K_ATP channels, which, at one stage, actually were proposed to be a dephosphorylated form of the K⁺ delayed rectifier.¹⁵ Reconstitution of pancreatic beta cell K_ATP channels from 2 structurally distinct proteins, the high-affinity sulfonylurea receptor (SUR1)¹⁶ and K_IR6.2,¹⁷ led to the demonstration that K_ATP channels are heteromultimers (SUR1/K_IR6.2)₄.¹⁸ The finding that the low-affinity sulfonylurea receptor isoforms, rat SUR2A¹⁹ and mouse SUR2B,²⁰ coexpressed with K_IR6.2 generate currents with the pharmacological properties of K_ATP currents in striated myocytes and vascular smooth muscle cells, respectively, indicates that sarcolemmal K_ATP channels in cardiomyocytes have a similar structural basis.

The data on K_ATP channels from human cardiomyocytes are limited. The properties of K_ATP channels in cardiomyocytes from different species have been found to be quite variable, particularly the IC₅₀ values for inhibition by ATP, which are reported from micromolar to half-millimolar with Hill coefficients ranging from 1 to 5 (reviewed, for example, by Takano and Noma²¹ and Findlay and Faivre²² ). The reasons for this variability are not certain but could result from differences in methodology and/or true molecular heterogeneity. We²³ have described splice variants in the human SUR2 gene, and a preliminary report indicates that functional K_ATP channels are assembled from a splice variant in mouse SUR2A.²⁴ Thus, expression of a single species of these splice variants with wild-type K_IR6.2 is needed to determine if they can account for some of the observed variability. To begin to address this issue, we have cloned the full-length human SUR2A (huSUR2A) and human K_IR6.2 (huK_IR6.2) subunits.²³ Comparison of huSUR2A/huK_IR6.2 channels, reconstituted in mammalian cell lines, with K_ATP channels in sarcolemma of adult human ventricular cells, using single-channel current analysis under similar experimental conditions, demonstrates that all of the essential functional properties of the native and recombinant channels are identical. More specifically, using a protocol designed to estimate steady-state ATP inhibition, we found that K_ATP channels assembled from the predominant, full-length huSUR2A and huK_IR6.2 show an IC₅₀ for total ATP of 20 µmol/L, independent of the concentration of free Mg²⁺, with a Hill coefficient of 1. These values are at the low end of those reported for sarcolemmal K_ATP channels in mammalian cardiomyocytes.^{4 25 26 27 28}

	Materials and Methods

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Molecular Biology
Human SUR2B cDNA was cloned from a cardiac library prepared in phage (kindly provided by Dr M.M. Tamkun, Vanderbilt University, Nashville, Tenn) by homology screening using human SUR1 probes. The human SUR2 gene was obtained by screening P1 and PAC libraries to obtain overlapping clones. SUR2A was constructed using polymerase chain reaction (PCR) to amplify the 2A exon, identified by similarity to the last 42 amino acids of rat SUR2A.¹⁹ The SUR2A exon then was used to replace the 2B exon. The sequences for human SUR2A and SUR2B have been deposited in GenBank along with the intron-exon boundaries. The structure of the SUR2 gene, the order of the exons, and their relationship to SUR1 have been described.²³ The cDNAs were subcloned into the pECE vector used to express SUR1.¹⁶ HuK_IR6.2 was obtained by PCR from human genomic DNA and subcloned into a CMV expression vector¹⁹ (kindly supplied by Dr Susumu Seino, Chiba University, Chuo-ku, Chiba, Japan). Rat K_IR6.1 in pCMV was obtained from Dr Susumu Seino and mouse K_IR3.1 and 3.4 in CMV vectors were obtained from Dr David Clapham (Harvard University, Cambridge, Mass).

Human cardiac poly A⁺ RNA was purchased from Clontech (Palo Alto, Calif). First strand cDNA synthesis for RT-PCR was done using an Advantage RT-for-PCR kit (Clontech) according to the manufacturer's directions. Forward and reverse primers (below) were designed to amplify, and subclone, a 554-bp fragment based on the full length human SUR2A sequence and to check for splice variants in this region. The forward primer, with an EcoRI site, was 5'-GCGGAATTC-ACCATGATTGTGGGCCAAGT-3'; the reverse primer, with a HindIII site was 5'-GCGAAGCTT-GCATGCGTC-AGATACTGT-3'. The PCR products were cleaved with EcoRI and HindIII, separated by electrophoresis on low–melting point agarose, and subcloned into the Stratagene (La Jolla, Calif) Bluescript plasmid for sequencing. For estimation of the relative abundance of the PCR fragments, PCR reactions were done with [³²P]-dATP added. The reactions were terminated after 20 or 30 cycles and the products separated by electrophoresis. The gel was dried and radioactivity quantitated using a PhosphorImager (Storm 560, Molecular Dynamics). The values obtained were essentially the same for 20 and 30 cycles of amplification.

Cell Transfection and Cultivation
COSm6 or Chinese hamster ovary (CHO) cells were transfected with a mixture of plasmids; 1 µg of the SUR2A plasmid, 1 µg of the K_IR6.2 plasmid, and 0.5 µg of a green fluorescence protein (Green Lantern, Clontech, Palo Alto, Calif) plasmid. Transfections were done using FuGene6^TM following the manufacturers directions (Boehringer Mannheim). Transfected cells were cultured overnight, trypsinized, and replated on glass cover slips. Cells expressing green fluorescence protein as a marker were selected for analysis.

Isolation of Cardiomyocytes
Adult human ventricular myocytes were isolated from myocardium samples taken from 8 patients, aged 6 to 45 years, undergoing surgical treatment for interventricular septal defect, mitral valve defect, etc, as described previously.³ Rat ventricular cells were prepared as described previously.²⁹ Clearly striated, single cardiomyocytes with a length-to-width ratio>4, shown to have a zero-current potential of -69±2 mV (measured in 4 cells using the whole-cell configuration under quasi-physiological ionic conditions) were selected for patch-clamp experiments.

Patch-Clamp Recording and Single-Channel Current Analysis
Functional properties of reconstituted and native K_ATP channels were analyzed in cell-attached or inside-out configurations of the patch-clamp method at 23°C to 24°C, as described previously.²⁹ Pipettes filled with the quasi-physiological external solution containing (in mmol/L) NaCl 140, KCl 5, MgCl₂ 1, CaCl₂ 1, and HEPES 10 (pH 7.4) adjusted with 1 mol/L NaOH., or the K⁺-rich external solution containing (in mmol/L) KCl 145, MgCl₂ 1, CaCl₂ 1, and HEPES 10 (pH 7.4) adjusted with 1 mol/L KOH had resistance of 4 to 8 M. The internal, nominally Mg²⁺-free, solution containing (in mmol/L) KCl 140, EDTA 5, HEPES 5, and KOH 10, (pH 7.2) adjusted with 1 mol/L KOH, or the internal quasi-physiological solution with Mg²⁺ containing (in mmol/L) KCl 140, MgCl₂ 1, EGTA 5, HEPES 5, and KOH 10 (pH 7.2) adjusted with 1 mol/L KOH were used as control bath solutions. The free Mg²⁺ concentration in the internal test solutions was kept constant at a quasi-physiological level (0.7 mmol/L) by adjusting the concentration of MgCl₂ for the Mg²⁺-binding properties of nucleotides.³⁰ PCOs were added from stock solutions in dimethyl sulfoxide, which at a 2-fold higher concentration did not affect K_ATP channel currents. Pinacidil was obtained from Lilly Research Laboratories; other compounds were from Sigma. Bathing solutions were applied and ATP dose responses taken automatically, using a RSC-200 rapid solution changer (Biologic Inc) with a flow rate of 200 µL/min and a tube switching time of 2 to 5 ms. This time was much shorter than the diffusion-limited K_ATP current transients in inside-out patches observed on switching between ATP-containing and ATP-free solutions (Figure 1). At 0 mV, with quasi-physiological external solution in the pipette, outward currents through non-K_ATP channels were undetectable in inside-out membrane fragments. ATP inhibition of channel activity was determined using a protocol designed to estimate inhibition after correction for both channel "run-down" and reactivation by ATP in the presence of Mg²⁺. Briefly, quasi–steady-state inhibition of channel activity was determined from NPo_[ATP]/0.5(NPo_before+NPo_after), where N is the apparent number of channels in the patch, and Po_[ATP], Po_before, and Po_after are the mean open probabilities (Po) during, before, and after ATP application, respectively. The NPo values were calculated from the mean K_ATP channel current during ATP application or wash-out, without the current transients, divided by the mean unitary current amplitude (Figure 1). The zero–K_ATP channel current and the mean unitary current amplitude were estimated from peaks in a multiple Gaussian fit to current amplitude histograms constructed from record segments containing intervals with all K_ATP channels closed. NPo values for record segments that have only a few simultaneous, or single channel openings, were estimated from all-points current amplitude histograms. ATP-inhibitory curves were obtained by fitting a conventional pseudo-Hill function to the experimental data using a nonlinear least-squares method. Dwell-time distribution histograms were constructed from single-channel current records (low-pass Bessel filtered at 1 to 3 KHz [-3dB] and sampled at 10 to 20 KHz using a PCL-718 or LabMaster analog-to-digital converter with PClamp 6 drivers) with BioQuest software developed by Drs A.E. Alekseev and Yu M. Kokoz (Institute of Theoretical and Experimental Biophysics, Russian Academy of Science, Pushino, Russia). Single- or multi-exponential fits of the open or closed time distribution histograms were done using the Marquardt-Levenberg or Nelder-Mead algorithms (Origin 5.0 Professional, Microcal, Inc). Bins, corresponding to the time of underestimated events assumed equal to twice the digitization time, were excluded from fitting.

View larger version (22K): [in this window] [in a new window]   Figure 1. A segment from the current trace presented in Figure 5A, lower line, recorded in an inside-out patch from a COSm6 cell transfected with huSUR2A and huKIR6.2, showing a typical response of the reconstituted KATP channel current after rapid switches between the ATP-free and ATP-containing quasi-physiological internal solution, with the quasi-physiological external solution in the pipette at 0 mV holding. Diffusion-limited KATP channel current transients on application and washout of ATP are shown at an expanded time scale. The thick dashed lines indicate the mean currents. IKATP before and IKATP after indicate the mean KATP channel current before and after application of ATP respectively; dotted horizontal lines, zero KATP channel current level; upward deflection on the current trace, outward currents; bold solid lines, application of the bathing solutions supplemented with test compounds.

Averaged data were expressed as mean±SEM for n5 with error bars equal to SE unless otherwise noted. Significance was evaluated using the Student t test; differences with values of P<0.05 were considered significant.

	Results

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To clone SUR2 cDNAs from a human heart library, we used human SUR1 cDNA. These cDNAs, the encoded proteins, and the structure of the SUR2 gene have been described.²³ Comparison of the sequences of SUR1 and SUR2A indicated that the equivalent of exon 17 in SUR1 was missing in SUR2A.²³ We amplified this region to determine if there was a splice variant of SUR2 containing this sequence but failed to find a larger fragment. Instead, we observed 2 smaller fragments. Sequencing demonstrated these correspond to spliced mRNAs missing the 17th, and 17th plus 18th exons, respectively (Figure 2A). We estimated the relative abundance of the PCR products by incorporation of [³²P]-dATP. The studies presented below were performed with the major (80%) full-length species of human SUR2A, which corresponds to the rat SUR2A¹⁹ and mouse SUR2A²⁰ described previously.

View larger version (33K): [in this window] [in a new window]   Figure 2. Coexpression of the predominant, full-length huSUR2A and huKIR6.2 in mammalian cells produces ATP- and glibenclamide-sensitive K+ channels. A, Identification of SUR2A splice variants using PCR. The picture is a PhosphorImager scan of the [32P]-dATP–labeled PCR products obtained as described in Materials and Methods. The individual products were cloned and sequenced. The diagram summarizes the sequence data and shows that the lower bands are the result of deletions of the 17th and 17th plus 18th exons of SUR2, respectively. The percentage was estimated by dividing the sum of the intensity in each band by the total intensity of the 3 bands. B, A representative record of K+ current in an inside-out membrane fragment isolated (i-O and arrow) from a COSm6 cell cotransfected with the full-length huSUR2A and huKIR6.2. See Figure 1 legend for description of experimental conditions and labels.

Potassium currents were observed in almost every inside-out patch from COSm6 or CHO cells cotransfected with huSUR2A and huK_IR6.2 isolated into the nucleotide-free internal solution (Figure 2B). These currents ran down spontaneously, were reversibly inhibited and "refreshed" by ATP in the Mg²⁺-containing solution, and were not inhibited completely by 3 µmol/L glibenclamide, a concentration >100-fold higher than needed to completely inhibit ⁸⁶Rb⁺ fluxes through the hamster SUR1/mouse K_IR6.2 beta cell K_ATP channel,¹⁷ which corresponds to the IC₅₀ for glibenclamide-inhibition of native cardiac K_ATP channels.³¹ Similar currents were never observed in >50 patches from COSm6 and CHO cells transfected with either huSUR2A or huK_IR6.2 alone. The results suggest that coexpression of huSUR2A and huK_IR6.2 generates ATP- and sulfonylurea-sensitive K⁺ channels. Similar channels were not seen in >80 patches from cells coexpressing human or rat SUR2A plus K_IR6.1. To check for coupling to other K_IR, we examined >80 patches from cells transfected with human or rat SUR2A plus mouse K_IR3.1 and K_IR3.4 but never observed similar channels (not illustrated).

The activation of single-reconstituted and native channels by metabolic inhibitors is shown in Figure 3A. Channel activity was undetectable in intact cells, just visible after several minutes of inhibition of glycolysis by rotenone, and gradually increased as oxidative phosphorylation was uncoupled by carbonyl cyanide p-(trifluoro-methoxy)phenylhydrazone (FCCP). The metabolic inhibitors did not activate K_ATP channels in inside-out patches in the presence of millimolar ATP (not illustrated). The subsequent traces of the single-channel currents recorded at different voltages show the bursting behavior, nearly voltage- and time-independent gating, and mild inward rectification in the cell-attached configuration for the recombinant and native channels. After excision of the same patch into a nucleotide-free, Mg²⁺-containing solution, channel activity increased, ran down, and could be inhibited completely and reversibly and restored to the initial level by millimolar ATP (bottom trace). Comparative analysis of the single-channel current-voltage relationships under various conditions is given in Figure 3B. The 80 mV shift in reversal potential with a 30-fold increase in [K⁺]_o, close to that predicted by the Nernst equation for a K⁺ electrode, demonstrated the K⁺ selectivity of both channels. The unitary slope conductances, roughly proportional to the root of [K⁺]_o, were identical and showed a tendency to saturate at high positive voltages in the absence of Na⁺ and divalent cations. Both channels displayed obvious Mg²⁺-dependent inward rectification.

View larger version (68K): [in this window] [in a new window]   Figure 3. The huSUR2A/huKIR6.2 and KATP channels in human ventricular cardiomyocyte activated by metabolic inhibition display identical electrophysiological properties. A, Activation of a single recombinant channel in a CHO cell (upper left trace) or a KATP channel in a human ventricular myocyte (upper right trace) poisoned with 5 µmol/L rotenone and 0.5 µmol/L FCCP. Cell-attached patches were held at the voltages indicated, with the K+-rich external solution in the pipette and Mg2+-containing internal solution in the bath. The increase in noise in the left trace is due to switching from 0.3 to 1 KHz filtering. The activity of KATP channels in the cardiomyocyte was monitored at the positive membrane potential to prevent superposition with strong KIR openings. Subsequent current traces from the same cell-attached patches are shown at an expanded time scale and different voltages. Traces of the inward currents through sarcolemma, with no strong KIR openings were selected for illustrative purposes. The ATP-sensitivity of the observed channels was confirmed in inside-out patches (bottom trace). B, Recombinant (left) and native (right) single channel current-voltage relationships under different experimental conditions. In the cell-attached configuration, the cell resting potential was zeroed by the K+-rich bath solution, thus holding VVm, the membrane potential in the inside-out configuration. SEM bars are smaller than the symbol size for the majority of the data points. The i-V relationship for quasi-physiological [K+]pip=5 mmol/L and [K+]bath=155 mmol/L shown on the right panel was taken using a slow (note the 100-ms bar) voltage-ramp. The slope conductance given on the graphs was estimated in the inside-out configuration with quasi-symmetrical K+ solutions at -40 mV (within -60- to 20-mV "window").

The intraburst kinetics of cardiac K_ATP channels were used as a "signature" for intrinsic channel gating, because they are tolerant to run-down, changes in nucleotide concentration, and show a characteristic dependence on the K⁺ driving force.³² As can be seen in Figure 4A, the open times shorten, and the "gaps" or intraburst closed times are prolonged with a decrease in membrane potential from -40 to -70 mV. The single-exponential open time density probability functions, with time constant _O, and the double-exponential closed time density probability functions, with time constant _Cf for the fast exponent (see Discussion), are nearly identical for the 2 channels (Figure 4B). A more extended analysis (Figure 4C) confirms the opposite dependence of _O and _Cf on the K⁺ driving force³² with both relationships similar for the 2 channels.

View larger version (43K): [in this window] [in a new window]   Figure 4. Reconstituted and native KATP channels have indistinguishable kinetics. A, Segments of recombinant (left) and native (right) single KATP channel currents recorded at different voltages with their corresponding dwell-time distributions, and the fitted single- (for open time) or double- (for closed time) exponential probability density functions (B). The dwell-time histograms were constructed from continuous segments of 3-KHz–filtered records giving Po=0.85 and 10000 events. The time constant of the single-exponential open time probability density function, O, and the time constant of the fastest component in the multi-exponential fit to the closed time histogram, Cf, which corresponds to the distribution of intraburst gaps for cardiac KATP channels, were determined by the K+ driving force (the difference between Vm and VK, the reversal potential for K+). The closed time distributions are plotted semi-logarithmically. C, Dependence of O and Cf on V, equal to the K+ driving force with VK=0 mV. Spline interpolations were drawn for illustrative purpose.

The ATP inhibition curves of huSUR2A/huK_IR6.2 channels obtained from the same inside-out patch in either Mg²⁺-free (Figure 5A, upper trace) or Mg²⁺-containing (lower trace) internal solutions show a minor leftward shift of questionable physiological significance (Figure 5B). This minor shift was confirmed in a representative number of patches (Figure 5C; IC₅₀ decreased from 23±3 to 17±1 µmol/L at a pseudo-Hill coefficient [h] of 1). The estimated IC₅₀ for quasi–steady-state ATP inhibition of recombinant channels, with and without Mg²⁺, varied from 12 to 36 µmol/L, while h varied from 0.87 to 1.63 in 20 different patches. Native channels were half-maximally inhibited between 10 and 30 µmol/L ATP, ie, fell within the SD for the huSUR2A/huK_IR6.2 data. Averaging all the data gives a mean IC₅₀ of 20 µmol/L with a SD of 3 µmol/L and h of 1. In a preliminary analysis, Inagaki et al¹⁹ reported a considerably higher value (100 µmol/L ATP) for the rat SUR2A/mouse K_IR6.2 channel in the presence of Mg²⁺. To determine if this difference between the human and rodent channels was due to species factors or methodology, we reexamined the ATP inhibition of the rat SUR2A/mouse K_IR6.2 channels in the presence of quasi-physiological free Mg²⁺ using our protocol. We determined an IC₅₀ of 19±3.1 µmol/L at h=1 for ATP and half-maximal inhibition for 5'-adenylylimidodiphosphate (AMP-PNP) between 30 and 100 µmol/L (n=4). The higher [ATP]₅₀ values reported initially¹⁹ may have resulted from an uncontrolled stimulatory effect of Mg nucleotides. Native rat cardiac K_ATP channels were inhibited by ATP with an IC₅₀ of 18.8±3.8 µmol/L and h of 1.1 and half-maximal inhibition by AMP-PNP between 30 and 100 µmol/L (4 patches). Therefore, the statistically representative IC₅₀ for quasi–steady-state ATP-inhibition of these channels is 20 µmol/L with an h of 1.

View larger version (36K): [in this window] [in a new window]   Figure 5. Comparison of ATP inhibition of recombinant versus KATP channels from human ventricular cells. A, The responses of reconstituted channels to different concentrations of ATP in Mg2+-free (upper trace) and 0.7 mmol/L free Mg2+-containing (lower trace) internal solutions. The records are from an inside-out patch from a COSm6 cell held at 0 mV with the quasi-physiological external solution in the pipette. The lower trace was recorded using the capacitance, less noisy mode of a Biologic RK-400 amplifier. B, The quasi–steady-state ATP-inhibitory curves obtained as described in Materials and Methods for the experiment shown in (A). The thin and thick lines are the best fits of a pseudo-Hill curve to the experimental data for Mg2+-free and Mg2+-containing conditions, respectively. C, Averaged data from 20 different patches with corresponding ATP-inhibitory curves. Averaged data from 4 similar experiments for native KATP channels in the quasi-physiological internal solution are presented with SD bars.

ADP in the presence of Mg²⁺ stimulates native cardiac K_ATP channels partially inhibited by ATP.³³ Recombinant channel currents, preinhibited by submillimolar ATP in the presence of 0.7 mmol/L free Mg²⁺, were reversibly increased by ADP (Figure 6). To exclude the possibility that the observed increase was only due to MgADP reactivating different channels in multi-channel patches (upper trace), we confirmed the apparent antagonism of ATP-inhibition by MgADP on a single reconstituted channel (bottom trace).

View larger version (28K): [in this window] [in a new window]   Figure 6. ADP, in the presence of Mg2+, stimulates recombinant cardiac KATP channels inhibited by ATP. Records of currents through multi- (upper trace) and single- (lower trace) channel inside-out patches from a COSm6 and a CHO cell, respectively. Patches were held at 0 mV in the quasi-physiological internal and external solution in the bath and pipette, respectively (upper trace), or at –40 mV with the quasi-physiological internal and the K+-rich external solution in the bath and pipette, respectively (bottom trace).

K_ATP channels in striated myocytes may be distinguished from other non–ß cell SUR/K_IR6.2 channels by their pharmacology³⁴ : cromakalim and pinacidil stimulate K_ATP channels in cardiomyocyte sarcolemma, whereas the effect of diazoxide is negligible.^{8 35 36 37} The parameters of stimulation, particularly the efficacy of PCOs, are variable, being dependent on temperature, nucleotide concentration, configuration of patch-clamp recording, and other factors.⁸ We chose to verify qualitatively the sensitivity profile of huSUR2A/K_IR6.2 channels to different PCOs under conservative experimental conditions, using inside-out patches at 0.5 mmol/L ATP.³⁶ A representative experiment in which 100 µmol/L cromakalim and 100 µmol/L pinacidil stimulated recombinant channels, while diazoxide did not at a 2-fold higher concentration, is shown in Figure 7A. The same diazoxide test solution stimulated huSUR1/huK_IR6.2 channels expressed in the same cells (not shown). To check the low sensitivity of the huSUR2A/huK_IR6.2 channels to sulfonylureas, we estimated the concentration of tolbutamide required to half-maximally inhibit channel activity. Figure 7B indicates a value of 1 mmol/L, similar to that reported for K_ATP channels in ventricular cells.³¹ This result, and the result with glibenclamide (Figure 2), confirms that huSUR2A is a low-affinity sulfonylurea receptor and that the reconstituted human sarcolemmal cardiac K_ATP channel is weakly sensitive to these compounds.

View larger version (37K): [in this window] [in a new window]   Figure 7. The pharmacological profile of huSUR2A/huKIR6.2 channels. A, The "cardio-specific" sensitivity of reconstituted channels to different PCOs: CRML indicates cromacalim; PNCD, pinacidil; DZXD, diazoxide. B, The recombinant channels show low sensitivity to the sulfonylurea tolbutamide (TLB). The inhibitory curve is a pseudo-Hill function fitted to averaged experimental data from 4 different patches; error bars represent SD. Experimental conditions are described for the upper trace in the Figure 6 legend.

	Discussion

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The principal result of this study is a demonstration that the predominant form of SUR2A and K_IR6.2, cloned from human heart, assembles channels equivalent to the sarcolemmal K_ATP channels found in human ventricular cells. In a side-by-side comparison, under similar experimental conditions using single-channel current analysis, the channel conductance, K⁺ selectivity, apparent "intrinsic" inward rectification, weak Mg²⁺-dependent inward rectification, time-independent gating with nearly voltage-independent Po, and kinetics were indistinguishable for these channels. The sensitivity of the channels to inhibitory ATP was equivalent. The apparent antagonism of ATP-inhibition by MgADP and the pharmacological properties of the recombinant channels confirm that human SUR2A is a low-affinity sulfonylurea receptor that determines the MgADP-stimulation and selective response of the cardiac channels to PCOs.¹⁹ The results constitute an internally consistent set of data showing that full-length huSUR2A and huK_IR6.2 assemble channels that are an adequate model for human cardiac sarcolemmal K_ATP channels that have been traditionally difficult to study directly.

The results extend our original limited analysis¹⁹ using subunits from different rodent species. In the present report, we expressed the major form of human SUR2A with huK_IR6.2, thus eliminating possible species differences and the possibility that our observations result from a minor SUR2A splice variant. To determine if K_IR6.2 is the correct K_IR subunit, we tested whether, in addition to the expected permeation properties, the intraburst kinetics of the reconstituted channel were equivalent to those of the native channel. The ATP-inhibitory data for the rat SUR2A/mouse K_IR6.2 channels did not address the question of potency of ATP^4-- versus Mg-bound ATP, a subject of debate in the K_ATP channel field. The estimated IC₅₀ of 0.1 mmol/L for both ATP and AMP-PNP were controversial, as the latter usually is considered to be less potent that ATP. Therefore, we determined the parameters for steady-state ATP-inhibition in Mg²⁺-free versus quasi-physiological conditions. Finally, we verified the stimulation of ATP-inhibited huSUR2A/huK_IR6.2 channels by MgADP, examined the cardio-specific response of the reconstituted channel to structurally different PCOs, and demonstrated low sensitivity to sulfonylureas.

Given the diversity of K⁺ channel subunits in the heart,³⁸ it could be argued that cardiac K_ATP channels are assembled from SUR2A and another K_IR rather than K_IR6.2. We previously have demonstrated that the intraburst kinetics depend mainly on the K_IR subunit.²³ To address the question of whether K_IR6.2 forms a pore with the intrinsic fast gating seen in the ventricular K_ATP channel, we analyzed the intraburst kinetics of the native and reconstituted channels. Our kinetic analysis, restricted to estimation of _O and _Cf and their voltage dependence in nucleotide-free conditions, was done assuming a minimal model (1 open state plus 2 closed states). Similar and more complex schemes have been used to describe cardiac K_ATP channel kinetics, but no consensus exists on the number of exponential components required to "adequately" fit open and closed time histograms for ATP-free conditions.^{1 3 32 39 40 41 42 43} For example, multi-exponential open time distributions, constructed from outward K_ATP channel currents, have suggested that several cations can produce open channel block, which would affect the dwell-time histograms. Closed time distributions, distorted by prolongation of interburst gaps due to channel run-down, might be "better" fit with a multi-exponential function having an additional slow component, which would be interpreted as "inter-cluster" or long-lasting closed states. We analyzed records from highly operational native or recombinant single channels (Po>0.8) in different patches immediately after isolation. Using a 3-exponential fit, the relative area under the slowest exponent with a time constant >e³ ms was negligible; the open time distribution was single-exponential. Therefore, the minimal 3-state model was adequate for comparison, and _O and _Cf and their dependence on K⁺ driving force were indistinguishable for the 2 channels. Therefore, based on our comparison of the channel kinetics, together with the equivalence of the permeation properties, ie, the single channel conductance, selectivity, and rectification, described above, we conclude that K_IR6.2 forms the channel pore. This conclusion is supported further by data showing that C-terminally truncated K_IR6.2 with and without SUR1 form channels with similar conductance^{44 45} and by the observation that the hydrophobic core of K_IR6.2 determines the conductance properties of chimeric K_IR assembled with mouse SUR2A.⁴⁶

The ATP modulation of the kinetics of native cardiac K_ATP channels has been simulated using different models with the general consensus that the inter-burst transition rates are affected the most by ATP.^{28 40 41 42} However, examination of the transition rates versus [ATP] by analysis of steady-state single-channel activity is difficult, and the data are fragmentary. Therefore, we have compared only the inhibitory effect of ATP on NPo for recombinant versus native K_ATP channels. The apparent IC₅₀ values reported for ATP inhibition of K_ATP channels in cardiomyocytes vary from 0.01 to 0.5 mmol/L with pseudo-Hill coefficients from 1 to 5 (discussed by Takano and Noma²¹ and Findlay and Faivre²² ). It is not clear how much of this large variability reflects true molecular heterogeneity versus differences in experimental conditions and protocols. K_IR6.2 is known to be encoded by an intronless gene, eliminating splice variation, and there are no known posttranslational modifications of this subunit. The properties of channels assembled from splice variants of SUR2 have not been examined yet, and the possibility of heterogenous expression cannot be ruled out in cardiomyocytes. To eliminate this potential source of molecular variability, we used the major SUR2 variant in these studies, which accounts for 80% of the SUR2 mRNA in human heart (Figure 2A) and corresponds, for example, to SUR2A identified in rats¹⁹ and mice.²⁰ Several methodological factors can affect the estimation of the parameters of ATP inhibition from patch-clamp experiments, and in most cases, these errors will increase the apparent IC₅₀.

One source of variability is that the different protocols used for isolation of cardiomyocytes can give cells that are metabolically inhibited to varying degrees and have varying intracellular free Ca²⁺ level reported to mediate a decrease in ATP sensitivity of K_ATP channels.⁴⁷ We selected clearly striated cardiomyocytes with a length/width ratio of >4 and a resting membrane potential of -70 mV (see Materials and Methods). Patches with channels displaying the mean Po<0.8 after patch isolation and/or reactivation by mmol/L MgATP, expected to refresh the channel maximally,⁴⁸ were discarded. For our expression studies, we used COSm6 cells that we have observed to have a negligible number of endogenous K_IR, when compared with COS7, COS1, and HEK293 cells. The patch clamp technique itself is a potential source of variability. Macro or "giant" patches and the "open-cell" configuration can present accessibility problems due to diffusion. Gradients of nucleotides resulting from ATPase, adenylate kinase, and other ATP-utilizing enzymes activity, or limited diffusion and glycolysis in the vicinity of the channel^{21 29 49 50 51 52 53} are not well-controlled in these configurations. Estimation of K_ATP channel activity from inward macrocurrents at negative membrane potentials can be contaminated with other currents through, for example, stretch-activated cationic channels in Xenopus oocytes, strong K⁺ inward rectifiers, or Cl^- channels regulated by nucleotides. These currents make the estimation of the zero K_ATP channel current level difficult because they may vary with time, and subtraction of current or slope conductance, assumed to be constant over the course of the dose-response protocol, is not accurate. Determination of the zero K_ATP channel current level is preferable to fitting the experimental data with a pseudo-Hill function plus an offset. To reduce these problems, we used conventional inside-out patches formed without suction to minimize patch curvature and facilitate diffusional access. This produced patches with <100 ms half-time for transients after application of ATP using a rapid solution changer and excluded transients from our calculation of quasi–steady-state currents. Finally, application of different methods of data analysis can contribute to the reported variability of IC₅₀ and h values. Inadequate corrections for channel run-down and reactivation may affect the estimation of these parameters. For example, the common use of a low concentration of ATP (0.1 to 1 µmol/L) as the "uninhibited control," simply because it reduces the rate of channel run-down, leads to a rightward shift in the ATP-inhibitory curve. Similarly, a 15% to 20% decrease in the "initial" K_ATP current level, accepted in many studies as "relatively small," will also cause a significant distortion in the ATP-inhibitory curve. Our experimental protocol, described in Materials and Methods, was designed to minimize the problems associated with recording and analysis. Briefly, asymmetric K⁺, symmetric Cl^-, and symmetric total monovalent cation solutions at zero mV were used to set the driving force for Cl^- and monovalent cations to 0, thus eliminating contributions from other channels. This protocol, together with the nearly undetectable level of endogenous K_IR in COSm6 cells allowed direct estimate of the zero K_ATP channel current level and low NPo values from fitting of amplitude histograms. Millimolar ATP was applied repetitively over the course of the dose-response protocol to check for shifts in the zero current level and to reduce rundown of K_ATP channels in Mg²⁺-containing solutions. Because the presumably exponential time course(s) of rundown and reactivation vary with [ATP]⁴⁸ during application of the test solutions, we separated each application of test solution of ATP with pre- and postwash periods and estimated the NPo during these periods. The mean of these 2 values was used to normalize the NPo values for each [ATP]. These relative values were then fit with a pseudo-Hill function without offset.

Using this protocol, the estimated IC₅₀ for quasi–steady-state inhibition of recombinant and native channels by total ATP, with and without Mg²⁺, was 20 µmol/L at h=1. Practically identical IC₅₀ values (21 µmol/L²⁵ and 25 µmol/L²⁷) have been reported for K_ATP channels from rat ventricular cardiomyocytes under similar Mg²⁺-free conditions. We detect a small leftward shift in the ATP inhibition curves after addition of magnesium consistent with the original observation²⁶ that ATP^4- and MgATP inhibit rat ventricular K_ATP channels with a slightly higher K_i for total ATP in the absence of magnesium. Because ATP^4- was reported to be a more potent inhibitor and "an important physiological regulator of the beta cell channel,"⁵⁴ Findlay²⁶ suggested that cardiac K_ATP channels might "fall into the group of `total-ATP'-sensitive channels," whereas the beta-cell channel might be an ATP^4--sensitive channel. The present results argue that ATP^4-- and the Mg-bound forms of ATP are equally effective inhibitors of cardiac K_ATP channels and are effective at low micromolar levels. Although in a preliminary analysis,¹⁹ a considerably higher and similar [ATP] and [AMP-PNP] (0.1 mmol/L) producing half-maximal inhibition of the rat SUR2A/mouse K_IR6.2 channel in the presence of Mg²⁺ were reported, we found an IC₅₀ of 20 µmol/L for ATP and confirmed a lower potency of AMP-PNP at the inhibition^{26 27} in the presence of quasi-physiological free Mg²⁺ using the same K_ATP channels subunits and our protocol.

We estimate an h of 1, implying that binding of 1 ATP molecule can close a cardiac K_ATP channel. However, the ATP-inhibitory site of the channel has not been identified. SURs are ATP-binding proteins with 2 conserved nucleotide binding folds,^{16 19 20} but no mutations have been identified that alter ATP inhibition. At the same time, C-terminus truncated versions of mouse and huK_IR6.2 alone can generate K⁺ channels that are weakly inhibited (IC₅₀>0.1 mmol/L) by ATP,^{44 45} suggesting that the inhibitory locus may reside on K_IR6.2. The mechanism of the apparent increase in affinity for inhibitory ATP of channels assembled from the truncated K_IR6.2 and SUR1 is unknown. The present data indicate that SUR2A, like SUR1, increases the apparent affinity for ATP, contrary to the speculation of Tucker et al.⁴⁴

Finally, we confirmed the apparent antagonizing ATP inhibition of the huSUR2A/huK_IR6.2 channels by MgADP and "cardioselective" PCOs and verified that the activity of the recombinant channels is inhibited by sulfonylureas with an 100-fold higher IC₅₀ than for SUR1.^{17 31} In conclusion, our data demonstrate that coexpression of full-length huSUR2A and huK_IR6.2 is sufficient to assemble channels with the nucleotide sensitivity, maximal Po, temporal patterns of busting activity,²³ and pharmacology, ie, all of the channel-defining properties expected for human cardiac K_ATP channels.

	Acknowledgments

 
This work was supported in part by a Russian Foundation for Basic Research grant and a Juvenile Diabetes Foundation International Award to A.P.B., and NIH grant DK44311 to J.B. The authors wish to thank members of the Bryan laboratory, particularly L.-Z. Song for technical assistance and J.P. Clement IV for discussion. We thank S.K. Churina (I.P. Pavlov Institute of Physiology, Russian Academy of Science, Pushino, Russia) and S.T. Kazantseva (Cytomed, St Petersburg, Russia) for help with isolation of cardiomyocytes. We especially acknowledge V.O. Samoilov for encouragement and comprehensive support of A.P.B. in performing experiments on human ventricular cells in the Laboratory of Cell Biophysics (St Petersburg, Russia).

Received March 4, 1998; accepted September 8, 1998.

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