1Institute for Medicine and Engineering and Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania; 2Department of Medicine, Montreal Heart Institute, and University of Montreal, Montreal, Quebec, Canada; 3Department of Molecular, Cellular and Developmental Biology and Neuroscience Research Institute, University of California, Santa Barbara, California
Submitted 23 February 2005 ; accepted in final form 4 June 2005
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ABSTRACT |
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potassium channels; inward rectifier potassium channel
To date, four key Kir2 subunits have been identified (Kir2.1Kir2.4) in a variety of tissues. Three of these subunits (Kir2.1Kir2.3) are expressed in cardiac cells (24, 25, 52), vascular smooth muscle cells (14, 51), and neurons (27), whereas expression of Kir2.4 has been reported only in neuronal cells (35, 43). To date, only Kir2.1 mRNA has been identified in vascular endothelial cells (7, 9, 13, 50). Herein we show that all four Kir2.x channels are expressed in human aortic endothelial cells (HAECs) at the transcript level but provide evidence suggesting that only Kir2.1 and Kir2.2 contribute significantly to native endothelial IK. Furthermore, our results suggest that Kir2.2 provides the dominant K+ conductance in HAECs under resting conditions.
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MATERIALS AND METHODS |
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Electrophysiology.
Ionic currents were measured using the whole cell and cell-attached configurations of the standard patch-clamp technique. Pipettes were pulled (SG10 glass; Richland Glass, Richland, NJ) to produce a final resistance of 35 M and generated high-resistance seals without fire polishing. A saturated salt agar bridge was used as a reference electrode. Currents were recorded with an EPC9 amplifier (HEKA Electronik, Lambrecht, Germany) and accompanying acquisition and analysis software (Pulse and PulseFit; HEKA Electronik) running on a PowerCenter 150 (MacOS) computer. Pipette and whole cell capacitance was automatically compensated. Whole cell capacitance and series resistance were compensated and monitored throughout each recording. Whole cell current was recorded during 500-ms linear voltage ramps or a series of voltage steps from 160 or 110 mV to +60 mV at an interpulse interval of 5 s. The standard external solution contained (in mM) 150 NaCl, 6 KCl, 10 HEPES, 1.5 CaCl2, 1 MgCl2, and 1 EGTA, pH 7.3. In some experiments, 156, 96, or 60 mM extracellular KCl was used with equimolar substitution of KCl for NaCl to maintain osmolarity. The pipette contained (in mM) 145 KCl, 10 HEPES, 1 MgCl2, 1 EGTA, and 4 ATP, pH 7.3. For the cell-attached configuration, single-channel recordings were obtained in 1.6-s sweeps with a 0.1-ms sampling interval and were filtered at 500 Hz. Bath and pipette solutions for single-channel recordings contained (in mM) 156 KCl, 10 HEPES, 1.5 CaCl2, 1 MgCl2, and 1 EGTA, pH 7.3. All experiments were performed at room temperature (2225°C).
RNA isolation and RT-PCR. Total RNA was extracted using the Absolutely RNA Miniprep kit (Stratagene, La Jolla, CA). Highly purified total RNA was treated with DNAse I to remove traces of genomic DNA. The integrity and quantity of RNA were evaluated using an Agilent 2100 Bioanalyzer and the RNA 600 Nano Chips assay kit (Agilent Technologies, Waldbronn, Germany). cDNA was generated using SuperScript II reverse transcription reagents (Invitrogen, Carlsbad, CA) with oligo(dT) primers. PCR primers (Table 1) were designed on the basis of known human Kir2.1 (GenBank accession no. U12507), Kir2.2 (GenBank accession no. AB074970), Kir2.3 (GenBank accession nos. U07364 and U24056), and Kir2.4 (GenBank accession no. AF081466) sequences with oligo primer analysis software (Molecular Biology Insights, Cascade, CO). Primer specificity was confirmed by performing a BLAST search. PCR was performed for 35 cycles consisting of denaturation (98°C), annealing (60°C), and extension (72°C).
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Immunoblotting. For preparation of total membrane (TM) samples, cells were scraped into buffer A, composed of (in mM) 150 NaCl, 20 HEPES, 5 EDTA, pH 7.4, protease inhibitor cocktail (PIC), and 1 µg/ml pepstatin; homogenized in a Dounce tissue grinder; and centrifuged for 10 min at 1,000 g. The pellet was resuspended in buffer A, dounced, and recentrifuged for 10 min at 1,000 g. The combined supernatant was centrifuged for 1 h at 200,000 g (SW40Ti rotor; Beckman). The pellet was resuspended in Laemmli buffer and sonicated. Sample protein was measured using a bicinchoninic acid protein assay kit (Bio-Rad). Proteins were resolved with 12% SDS-PAGE at reducing conditions, followed by transfer to polyvinylidene difluoride membranes (Amersham). Channel-specific rabbit anti-peptide antibodies to Kir2.1 (rat amino acids 390411), Kir2.2 (rat amino acids 390410), and Kir2.3 (human amino acids 219) were prepared and purified by performing affinity chromatography or protein A chromatography as previously described for Kir2.2 (36). The membranes were probed with anti-Kir2.1 (1:1,000 dilution), anti-Kir2.2 (1:250 dilution), and anti-Kir2.3 (1:1,000 dilution), with dilutions optimized by probing Chinese hamster ovary (CHO) cells overexpressing Kir2.x subunits. Kir2.x-specific bands were detected using horseradish peroxidase-conjugated secondary antibodies (The Jackson Laboratory, Bar Harbor, ME). Finally, immunoreactivity was visualized using ECL Plus reagent (Amersham). The specificity of the antibodies was tested by transfecting COS-1 cells with Kir2.x constructs and harvesting the cells 2 days after transfection as previously described (19). Samples were run on 10% SDS-PAGE, transferred onto nitrocellulose membrane, and probed with affinity- or protein A-purified antibodies (1:250 dilution) to Kir2.1, Kir2.2, or Kir2.3, followed by probing with secondary antibodies conjugated to horseradish peroxidase and visualized using SuperSignal West Dura (Pierce).
Construction and functional assessment of dnKir2.x constructs. Dominant-negative (dn)Kir2.x subunits were engineered by replacing the GYG motif of the selectivity filter with three alanine residues (AAA). 5' and 3' fragments were generated by performing PCR (Elongase amplification system; Invitrogen) and combined by overlap extension. The resulting constructs were cloned into pCRII-TOPO (Invitrogen) and verified by performing sequencing analysis. dnKir2.x constructs were subcloned into the bicistronic mammalian cell expression vector pIRES2-EGFP (Clontech Laboratories/University College London, London, UK). Kir2.2/pCRII and Kir2.4/pSGEM were kind gifts from Barbara Wible (Case Western Reserve University, Cleveland, OH) and Andreas Karschin (University of Göttingen, Göttingen, Germany), respectively. Kir2.3/BluescriptSK was previously described (33). Kir2.x and dnKir2.x cRNA were generated using the mMESSAGE mMACHINE kit (Ambion, Austin, TX). The functionality of dnKir2.x constructs was verified by coexpression of wild-type (WT) Kir2.x-WT with the respective dnKir2.x-WT in Xenopus oocytes as previously described in detail (40).
Transfection. CHO cells and HAECs were transfected with Kir2.x-WT or dnKir2.x constructs using LipofectAMINE (GIBCO-BRL) according to the manufacturer's instructions. Electrophysiological recording and Western blot analysis were performed 24 h after transfection.
Data analysis.
Statistical analyses of the data were performed using a standard two-sample Student's t-test assuming unequal variances of the two data sets. Statistical significance was determined using a two-tailed distribution assumption and was set at 5% (P < 0.05). The time constants of voltage-dependent inactivation were measured by fitting a single exponential function, Vt = Aet/, where A is the current amplitude and
is the time constant. The fits were obtained using the Levenberg-Marquardt algorithm with PulseFit software (HEKA Electronik, Lambrecht, Germany). Single-channel events were analyzed using TAC software (Braxton, Seattle, WA). The frequency distribution of single-channel conductance was fitted using a weighted sum of two Gaussians (bimodal distribution), assuming unequal means and equal variance, with Origin 6.0 software (Microcal Software, Northampton, MA).
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RESULTS |
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Distribution of IK unitary conductance.
To further assess which Kir2.x channels constitute the endogenous IK conductance in HAECs, unitary conductance was evaluated using single-channel analysis. The typical values of Kir2.x unitary conductance are 2030 pS for Kir2.1, 3540 pS for Kir2.2, 1015 pS for Kir2.3, and 1415 pS for Kir2.4 (16, 22, 26, 33, 42, 43). In HAECs, 90% of the channels had unitary conductance of between 20 and 45 pS (Fig. 7) as would be expected if only Kir2.1 and Kir2.2 channels contributed significantly to the whole cell endogenous IK in these cells. Furthermore, there appear to be two distinct peaks in the distribution of the unitary conductances, one with a mean at 25 pS and another at 35 pS (Fig. 7B), supporting the notion that both Kir2.1 and Kir2.2 contribute to the endogenous IK in HAECs. It is also noteworthy that the peak at 35 pS is more prominent, suggesting that Kir2.2 is the major Kir2.x channel in HAECs. The ratio between the integrals for the major and minor histogram peaks is 1.58, suggesting that Kir2.1 channels constitute
40% and Kir2.2 constitute
60% of the channel population. A double-peak histogram of unitary conductance, however, does not exclude the possibility that multiple channel populations contribute to the endogenous IK in HAECs. This possibility is addressed further using dnKir2.x constructs.
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DISCUSSION |
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Consideration of the system.
The low-passage HAECs used in the present study appear to be the best available cell model to investigate IK in human aortic endothelium. Although it was reported earlier that aortic endothelial cells freshly isolated from mouse or rabbit aortas lack Kir channels (39), we have shown in the present study that Kir are clearly active in freshly isolated PAECs. Furthermore, IK in low-passage PAECs were similar to those in freshly isolated cells. Thus the fraction (80%) of cells that express Kir and the average current density of the current are similar in low-passage HAECs, low-passage PAECs, and freshly isolated PAECs, supporting the relevance of the low-passage HAEC system. The difference between the ECs isolated from mouse, rabbit, or pig aorta could be due to interspecies differences in Kir expression or to different isolation protocols. Interspecies differences in cardiac Kir subunit expression are well recognized (5, 24). It is not surprising that IK in HAECs and in PAECs are more similar than IK in mouse or rabbit endothelium, because it is generally accepted that the pig vasculature is more similar to the human vasculature than to that of mice or rabbits. Kir expression appears to vary in different types of ECs. Nilius and Schwarz (30) showed that only a fraction of ECs freshly isolated from the human umbilical vein expressed IK, and Himmel et al. (10) showed that microvascular ECs from human omentum lacked IK. In contrast, freshly isolated coronary ECs show pronounced IK with a unitary conductance similar to that observed in aortic endothelium (45). It is also possible that the expression profiles of Kir channels are modified under different physiological or pathological conditions. Thus it is important to take into account both the differences among species and between ECs isolated from different vascular beds when comparing the properties of endothelial IK.
Molecular diversity of Kir2-based native currents. In HAECs, the molecular diversity of Kir2 subunits at the transcript level is higher than the diversity of functional Kir. While for Kir2.3 this discrepancy could be explained by undetectable levels of protein expression due to very low transcription, the transcript level of Kir2.4 is similar to that of Kir2.1, suggesting that Kir2.4 functional expression is regulated at a posttranscriptional level. A discrepancy between the heterogeneity of K+ channels at the transcript and functional levels was reported previously for Kir2.x channels in human myoblasts (8) and for voltage-gated K+ channels in rat cardiomyocytes (2, 49), and it has been proposed that translational-posttranslational steps may contribute a rate-limiting step to channel expression (38). Protein expression of Kir2.x subunits in HAECs is consistent with the functional expression of the channels.
The peak IK unitary conductance levels in HAECs (25 and 35 pS) are similar to previously reported values in human umbilical vein endothelial cells (29 pS) and in bovine aortic endothelial cells and PAECs (3042 pS) (13, 29, 30, 37). We observed a dual-peak distribution of IK unitary conductances in HAECs. A similar distribution with an additional peak at a lower value (34 pS, 24 pS, and 11 pS) was reported in guinea pig cardiac myocytes (22). These peak values are consistent with the unitary conductances of Kir2.2, Kir2.1, and Kir2.3 expressed in Xenopus oocytes (3436 pS, 21 pS, and 815 pS, respectively) (16, 33, 42). The exact values of Kir2.x unitary conductances can vary among cell types, but an 10-pS difference between Kir2.1 and Kir2.2 is maintained. For example, Kir2.1 and Kir2.2 have conductances of 21 and 28 pS, respectively, when expressed in human myoblasts (8) and conductances of 31 and 42 pS, respectively, when expressed in human embryonic kidney HEK-293 cells (22). These variations have been attributed to the possible binding of an intracellular ligand (22). We propose that the observed bimodal distribution of unitary conductances suggests that both Kir2.1 and Kir2.2 contribute to the endogenous IK in HAECs. Taking into account the difference in unitary conductances between both channels, Kir2.2 appears to be the dominant conductance, contributing
70% of the whole cell K+ current. These ideas are supported by the observation that dnKir2.1 suppressed only the lower-conductance portion of the single-channel current histogram.
Dissecting the contributions of different Kir2.x subunits to the endogenous IK with dnKir2.x constructs further supports the hypothesis that both Kir2.1 and Kir2.2 are the main constituents of endothelial IK, with Kir2.2 having the dominant role. Earlier studies showed that coexpression of wild-type and dnKir2.x subunits resulted in the formation of Kir2.x heterotetramers (34, 40). Furthermore, Zobel et al. (52) demonstrated that both dnKir2.1 and dnKir2.2 inhibited endogenous IK in myocytes by >50%, suggesting the formation of heterotetramers between dnKir2.x and endogenous Kir2.x subunits. Consistent with these findings, our data show that the sum of current inhibition by dnKir2.x in HAECs (dnKir2.2, 85%; dnKir2.1,
50%) produced a value >1, implying possible heteromultimerization of native Kir2.x subunits. It is important to note, however, that heteromultimerization between overexpressed dnKir and native Kir does not necessarily mean that native Kir form heterotetramers. As Zobel et al. (52) pointed out, if a significant proportion of endogenous Kir channels were Kir2.1 and Kir2.2 heterotetramers, then dnKir2.1 and dnKir2.2 would be expected to inhibit endogenous IK equally. That was indeed the case for rabbit cardiomyocytes, but in HAECs, dnKir2.1 had a weaker effect than dnKir2.2.
Potential significance of our findings. Multiple Kir2.x subunits are expressed in individual cells of several types, including cardiomyocytes (22, 46) and smooth muscle cells (14). It is noteworthy that because Kir2.x subunits have differential sensitivities to several modulatory systems, such as those related to protein kinase C (15), G protein-coupled receptors (6), and chaperone molecules (18, 19), the expression pattern of multiple Kir2.x subunits may underlie differences in tissue electrophysiological properties. Indeed, differential expression of multiple Kir2.x in atrial vs. ventricular myocytes was suggested to account for different resting potentials and excitability properties between the two heart tissues (15), and it was suggested that changes in Kir2.x expression profile may be important for plasticity of electrophysiological responses in arterial smooth muscles (14). In summary, this study is the first to demonstrate the expression of multiple Kir2.x subunits in ECs and to identify the relative roles of specific subunits of endogenous endothelial Kir.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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