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Endothelial K⁺ channel lacks the Ca²⁺ sensitivity-regulating ß subunit

J. PAPASSOTIRIOU^*, R. KÖHLER^*, J. PRENEN, H. KRAUSE, M. AKBAR^*, J. EGGERMONT, M. PAUL^§, A. DISTLER^*, B. NILIUS and J. HOYER^*1

^* Abteilung für Nephrologie,
Abteilung für Urologie,
^§ Institut für Klinische Pharmakologie, UKBF, Freie Universität Berlin, 12200 Berlin, Germany; and
Laboratorium voor Fysiologie, Campus Gasthuisberg, KU Leuven, 3000 Leuven, Belgium

¹Correspondence: Abt. für Allg. Innere Medizin und Nephrologie, Med. Klinik und Poliklinik, Universitätsklinikum Benjamin Franklin, Freie Universität Berlin, Hindenburgdamm 30, 12200 Berlin, Germany. E-mail: hoy{at}zedat.fu-berlin.de

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Hyperpolarizing large-conductance, Ca²⁺-activated K⁺ channels (BK) are important modulators of vascular smooth muscle and endothelial cell function. In vascular smooth muscle cells, BK are composed of pore-forming subunits and modulatory ß subunits. However, expression, composition, and function of BK subunits in endothelium have not been studied so far. In patch-clamp experiments we identified BK (283 pS) in intact endothelium of porcine aortic tissue slices. The BK opener DHS-I (0.05–0.3 µmol/l), stimulating BK activity only in the presence of ß subunits, had no effect on BK in endothelium whereas the subunit selective BK opener NS1619 (20 µmol/l) markedly increased channel activity. Correspondingly, mRNA expression of the ß subunit was undetectable in endothelium, whereas subunit expression was demonstrated. To investigate the functional role of ß subunits, we transfected the ß subunit into a human endothelial cell line (EA.hy 926). ß subunit expression resulted in an increased Ca²⁺ sensitivity of BK activity: the potential of half-maximal activation (V_1/2) shifted from 73.4 mV to 49.6 mV at 1 µmol/l [Ca²⁺]_i and an decrease of the EC₅₀ value for [Ca²⁺]_i by 1 µM at +60 mV was observed. This study demonstrates that BK channels in endothelium are composed of subunits without association to ß subunits. The lack of the ß subunit indicates a substantially different channel regulation in endothelial cells compared to vascular smooth muscle cells.—Papassotiriou, J., Köhler, R., Prenen, J., Krause, H., Akbar, M., Eggermont, J., Paul, M., Distler, A., Nilius, B., Hoyer, J. Endothelial K⁺ channel lacks the Ca²⁺ sensitivity-regulating ß subunit.

Key Words: endothelium • Slo • maxi K • channel subunit • patch-clamp • beta subunit expression

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
LARGE-CONDUCTANCE, Ca²⁺-activated K⁺ channels (BK) represent a member of the family of Ca²⁺-activated K⁺ channels (K_ca). They are activated by increases in the intracellular Ca²⁺ concentration [Ca²⁺]_i and by membrane depolarization. BK channels have been identified in the vascular system where they play a key role in vasoregulation. They are present in endothelial cells (EC; refs 1 , 2 ) as well as in vascular smooth muscle cells (VSMC; refs 3 , 4 ). In endothelial cells, BK channels are implicated in mediating endothelial responses to humoral and hemodynamic stimuli (5) . This is demonstrated by the observation that endothelium-dependent vasodilation in blood vessels due to bradykinin or flow is reduced or completely blocked by pharmacological inhibition of endothelial BK channels (6 , 7) .

BK is formed by tetrameres of the pore-forming subunit, which was cloned from Drosophila melanogaster (8) and later from murine (9) , and human (10 , 11) tissues. A modulatory ß subunit has also been detected and cloned (12 , 13) . Smooth muscle cells, including VSMC, contain BK channels composed of both subunits (12 , 14) . There is strong evidence that the ß subunit plays a crucial role in modulating Ca²⁺ sensitivity of BK in VSMC (14) .

The subunit composition of the BK channel in endothelial cells is so far unknown. To characterize the regulation of BK in endothelial cells, we studied electrophysiological and pharmacological channel properties in intact endothelium of tissue slices from porcine aorta and in a human endothelial cell line. We performed patch-clamp experiments with subunit-specific activators of the BK channel (15 , 16) and investigated the expression of and ß subunit mRNA in EC. To further characterize the function of BK subunits, we transfected cells of the human endothelial cell line with plasmids expressing the ß subunit and compared the properties of the ion channel in transfected cells with those in nontransfected control cells.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Endothelial cells
Intact porcine aortic endothelium and freshly isolated porcine aortic endothelial cells (PAEC) as well as the human endothelial cell line EA.hy 926, derived from human umbilical vein (17) , were used.

Porcine aorta was obtained from the local abattoir and rinsed free of blood immediately after excision and stored on ice-cold phosphate-buffered saline (PBS) (Biochrom KG, Berlin, Germany) until preparation. Segments of the aorta were cleaned of connective tissue and cut open longitudinally. Tissue slices of ~0.5 cm² were prepared and used directly for patch-clamp experiments. PAEC were isolated from porcine vessels as described previously (18) . Briefly, tissue slices of ~5 cm² were incubated in PBS containing 0.25% trypsin for 25 min at 37°C. PAEC were harvested by gentle scraping of the luminal surface. The identity of EC was verified by uptake of acetylated low-density lipoprotein using indirect immunofluorescence as described previously (19) .

EA.hy 926 were grown in Dulbecco’s modified Eagle’s medium with high glucose containing 10% fetal calf serum (FCS) and supplemented with one part 50x HAT, nonessential amino acids, sodium-pyruvat, and penicillin/streptomycin (Biochrom KG, Berlin, Germany). Cells were cultured on coverslips in a fully humidified atmosphere of 5% CO₂.

Patch-clamp experiments
For patch-clamp experiments, tissue slices of porcine aorta were placed in a chamber on stage of an inverted microscope (Axiovert 100; Zeiss, Deisenhofen, Germany) with the luminal surface facing the bath solution. For investigation of EA.hy 926, coverslips with cultured cells were used.

Patch-clamp experiments were carried out as described (20 , 1 , 2) . Currents were monitored with an EPC-9 patch-clamp amplifier (HEKA Elektronik, Lambrecht, Germany). Single-channel membrane currents were low-pass filtered at a sample time of 0.5 or 1 ms. Single-channel open probability (P_o) was calculated as described previously (21) .

Whole-cell currents were measured using ruptured patches and sampled at 2 ms. A standardized voltage-clamp protocol was repeated every 15 s: from a holding potential of -80 mV the voltage was stepped to -150 mV, followed by a 2.6 s linear voltage ramp to +100 mV.

Patch pipettes were pulled from borosilicate glass capillaries with 0.3 mm wall thickness and had a tip resistance of 3 to 5 M in symmetric KCl solutions. Seal resistance in cell-attached patches ranged from 2 to 30 G. If not otherwise stated, experiments were performed at 37 ± 1°C.

Solutions and drugs
In single-channel patch-clamp experiments, two different pipette solutions were used. A KCl solution contained (in mmol/l): 140 KCl, 1 CaCl₂, 1 MgCl₂, 10 HEPES, pH 7.4. A NaCl solution contained (in mmol/l): 150 NaCl, 5 KCl, 1.5 CaCl₂, 10 HEPES, pH 7.4. MEM-EARLE (Biochrom KG, Berlin, Germany) was used as a bath NaCl solution in single-channel patch-clamp experiments (in mmol/l): 117 NaCl, 30 KCl, 1.8 CaCl₂, 0.81 MgSO₄, 5.5 glucose, 20 HEPES, pH 7.4. If not otherwise stated, the bath KCl solution used for single-channel recordings contained (in mmol/l): 140 KCl, 0.96 CaCl₂, 1 EGTA, 10 HEPES, pH 7.2 (corresponding to 3 µmol/l free Ca²⁺). In experiments with KCl bath solutions containing 0.1 to 20 µmol/l free Ca²⁺, the free Ca²⁺ concentration was adjusted with appropriate amounts of Ca²⁺ and EGTA or HEDTA calculated according to Fabiato and Fabiato (22) .

In whole-cell experiments, pipettes were filled with (in mmol/l): 40 KCl, 100 K-aspartate, 1 MgCl₂, 4 Na₂ ATP, 10 HEPES, 4.36 CaCl₂, 5 EGTA, pH 7.2 (corresponding to 1 µmol/l free Ca²⁺). As external solution Krebs was used (in mmol/l): 150 NaCl, 6 KCl, 1.5 CaCl₂, 1 MgCl₂, 10 glucose, 10 HEPES, pH 7.4.

NS1619 was purchased from Research Biochemicals International (Natick, Mass.). Iberiotoxin was obtained from Sigma. Dehydrosoyasaponin I (DHS-I) was a kind gift from O. B. McManus (Merck Research Laboratories, Rahway, N.J.).

RT-PCR analysis and sequencing
Reverse transcription-polymerase chain reaction (RT-PCR) was performed with primers for the BK and ß subunits on total RNA of PAEC and EA.hy 926. As a control, RNA of VSMC from porcine aorta, porcine renal artery, human umbilical artery, and vein was used. VSMC are known to express both subunits. RNA was purified using TRIzol (Life Technologies, Eggenstein, Germany), following the manufacturer’s instructions. Total RNA (1.5 µg) was reverse transcribed using random hexamers (Boehringer, Mannheim, Germany) and M-MLV reverse transcriptase (Life Technologies) in a 50 µl reaction. Subsequently, 3 µl of the cDNA was amplified in a standard 50 µl PCR reaction (35 cycles at 94°C for 30 s, 65°C for 60 s, and 72°C for 30 s) containing 0.2 µmol/l of each primer (Life Technologies).

Since the sequence of the porcine ß subunit is not known and the sequence of the porcine subunit had not been published when the experiments were performed, we used primers derived from the published human sequences (GenBank accession numbers: U11717, U25138) for all investigated tissues. For amplification of the subunit, the primers 5'-CTGTGTTTTGTGAAGCTCAAGC-3' and 5'-AGATGCATTCCTTGTCCTGC-3' were used; for amplification of the ß subunit, the oligonucleotides 5'-CCAGAAGCGGGGAGAGACAC-3' and 5'-CAGAAGAGGGAGAAGAGGAG-3' were used. In addition, primers derived from the sequence of the ß subunit of the rat (GenBank accession number: U79661) were designed for amplification in porcine tissues: 5'-AAAGAAGCTGGTGATGGCC-3' and 5'-TGGAAAGGGACTGGTTGATC-3'.

Identity of PCR products with the target sequence was verified by sequencing. Therefore, PCR-amplified fragments were gel purified (Quiagen, Hilden, Germany) and sequenced according to the cycle sequencing protocol, using primers from the respective PCR reaction. The products were analyzed on an ABI 377 automatic sequencer (ABI Prism, Weiterstadt, Germany).

Cloning of the human ß subunit, vector construction, and transfection
For cloning of the ß subunit of BK channels, two oligonucleotides, 5'-CCCAGTGAATATGGTGAAGAA-3' and 5'-ATGGATGGCTCTACTTCTGGG-3', adjacent to the start and stop codon of the human ß subunit sequence (GenBank accession number: U25138), were synthesized and used for RT-PCR to amplify ß subunit cDNA from human umbilical vein total RNA as described previously (23) . The amplified 596 bp fragment was excised, gel purified, and cloned via TA cloning technique into the mammalian expression vector pCR^TM3 (Invitrogen, Groningen, Netherlands) according to the manufacturer’s instructions. The orientation of the insert in the resulting plasmid pCR3/BKß was determined by cleavage with ApaI, which gives rise to asymmetrical cleavage products. In addition, authenticity of the cloned fragment was revealed by sequencing.

For stable transfection, 5 x 10⁶ EA.hy 926 cells were transfected with 20 µg of pCR3/BKß by electroporation using a Gene-pulser II (Bio-Rad Laboratories GmbH, Munich, Germany) at 200 V and 950 µF. Stable clones were selected after 2 wk in medium containing 200 µg/ml G418 (Sigma). Expression of BK ß subunit was verified by RT-PCR, using the respective ß subunit primers.

For transient transfections, the insert coding for the ß subunit was excised from pCR3/BKß by EcoRI and subcloned into the pCINeo/IRES-GFP vector (24) , resulting in the vector pCINeo/IRES-GFP/BKß. Insertion of the ß subunit in pCINeo/IRES-GFP allowed coexpression of the channel subunit and the green fluorescent protein (GFP).

EA.hy 926 (15x10⁴ cells) were transiently transfected with 3 µg of pCINeo/IRES-GFP/BKß using the polycationic SuperFect Transfection Reagent (Quiagen, Hilden, Germany) according to the manufacturer’s instructions. Cells were transferred to coverslips 24 h after transfection and electrophysiological measurements were performed 2–3 days after transfection. Transfected cells were visualized in a combined fluorescence and patch-clamp set up as described previously (24) . Current measurements in non-green, nontransfected cells served as control.

Immunoblotting
Western blot analysis was performed to confirm the presence of the ß subunit in transiently transfected EA.hy 926 cells. Three days after transient transfection, proteins were extracted as described previously (25) . Nontransfected cells served as control. Protein extracts (125 µg) were separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (12% polyacrylamide) and transferred to a 0.45 µm nitrocellulose membrane (Hybond-ECL, Amersham, Buckinghamshire, England) by semi-dry electroblotting. Rabbit polyclonal serum raised against amino acid residues 116–131 (RADVEKVRAKFQEQQV; SWISS-PROT accession number: Q16558) of the human ß subunit (BioGenes, Berlin, Germany) was used in a 1:250 dilution. As control, the preimmune serum was tested. After incubation with peroxidase labeled anti-rabbit immunoglobulins (Amersham, Buckinghamshire, England) diluted 1:2000, proteins were detected using chemiluminescence (Amersham, Buckinghamshire, England).

Statistics
If not otherwise stated, data are given as mean ± SE (n), where n refers to the number of experiments. A Mann Whitney U/Wilcoxon rank sum test was used to compare mean values within an experimental series. To compare whole-cell current (I)/voltage (V) curves, a Kruskal-Wallis test was used. A P value of < 0.05 was accepted to indicate statistical significance.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
BK in intact endothelium of porcine aorta and in freshly isolated porcine EC
In intact endothelium of porcine aorta, a large conductance, Ca²⁺-activated K⁺ channel (BK) was identified in excised inside-out patches (Fig. 1A ). Channel conductance was 282.6 pS ± 12.9 SD (n=16) in symmetrical KCl solutions (Fig. 1B ). Using a KCl pipette solution and a NaCl bath solution, mean channel conductance was 155.9 pS ± 8.9 SD (n=6); the channel proved to be highly selective for K⁺. By fitting the I/V data to the Goldman-Hodgkin-Katz equation (Fig. 1B ), a reversal potential of 38 mV and a corresponding permeability ratio for K⁺:Na⁺ of 1:0.032 were calculated. The mean reversal potential was 36.8 mV± 1.5 SD (n=6).

View larger version (24K): [in this window] [in a new window]   Figure 1. Large conductance Ca2+-activated K+ channel (BK) in intact endothelium of porcine aorta. A) Original current traces in excised inside-out patches at different holding potentials. Symmetrical KCl solutions. Pipette: KCl solution. Bath solution (in mmol/l): 140 KCl, 0.84 CaCl2, 1 mM HEDTA, 10 mM HEPES, pH 7.4 (corresponding to 6 µmol/l free Ca2+). The letter ‘c’ denotes closed state of the channel. B) Current (I)/voltage (V) relationship of BK channels in inside-out patches. Pipette was filled with KCl solution; bath contained NaCl solution (117 mmol/l NaCl, 30 mmol/l KCl •) or KCl solution (). Values are given as mean ± SD. Data were fitted to the Goldman-Hodgkin-Katz equation. C) Effect of [Ca2+]i on BK activity. Relation between BK channel Po and [Ca2+]i at a holding potential of -20 mV. Data were obtained in inside-out patches in symmetrical KCl solutions. Bath solutions contained (in mmol/l): 140 KCl, 10 HEPES, pH 7.2; free Ca2+ concentrations from 0.4 µmol/l to 1 mmol/l were adjusted with appropriate amounts of Ca2+ and EGTA or HEDTA. Values are given as mean ± SE. Data were fitted to Boltzmann equation. Half-maximal activation of the channel activity occurred at 4.0 µmol/l [Ca2+]i.

Channel activity measured as open probability (P_o) was strongly potential dependent (Fig. 1A ). Depolarization resulted in an increased channel activity. In symmetrical KCl solutions at 6 µmol/l [Ca²⁺]_i, the P_o was 0.92 ± 0.02 (n=5), 0.59 ± 0.04 (n=5), 0.29 ± 0.04 (n=5), and 0.09 ± 0.02 (n=5) at +10 mV, -30 mV, -50 mV, and -70 mV, respectively.

BK activity measured in inside-out patches of intact endothelium of porcine aorta strongly depended on [Ca²⁺]_i (Fig. 1C ). At [Ca²⁺]_i of < 0.1 µmol/l, the BK displayed no channel activity. A stepwise increase of [Ca²⁺]_i from 0.4 µmol/l to 1 mmol/l resulted in an increase in channel activity from a basal P_o of 0.001 ± 0.001 (n=5) to a maximal P_o of 0.94 ± 0.009 (n=6; P<0.01). Figure 1C shows the relation between BK channel P_o and [Ca²⁺]_i. The data were fitted with the following equation: P_o = P_{o min} + (P_{o max}-P_{o min})/(1 + exp((EC₅₀-[Ca²⁺]_i)/k)), where P_{o min} is the minimal P_o, P_{o max} is the maximal P_o, EC₅₀ is the [Ca²⁺]_i where P_o is half-maximal, and k is the slope factor. At a membrane potential of -20 mV, an EC₅₀ value of 4.0 µmol/l [Ca²⁺]_i was calculated for half-maximal channel activation (Fig. 1C ).

Channel activity was completely inhibited in inside-out patches with patch pipettes backfilled with Iberiotoxin (0.1 µmol/l), a specific blocker of BK, within 1–2 min after seal formation (n=4).

Application of the benzimidazolone derivate NS1619, a selective opener of BK (16) , to the cytosolic face of the channel in inside-out membrane patches from porcine aortic endothelium induced a rapid increase in BK activity (Fig. 2A, B ). In patches held at a membrane potential of -40 mV in the presence of 3 µmol/l [Ca²⁺]_i, NS1619 (20 µmol/l) increased P_o from 0.25 ± 0.07 (n=7) to P_o 0.68 ± 0.05 (n=7; P<0.01; Fig. 2B ). Another BK selective channel opener, DHS-I, is known to stimulate BK activity only in the presence of the ß subunit of the channel (26) . In intact endothelium of porcine aorta, no effect of DHS-I (0.05–0.3 µmol/l) on BK activity was detected (n=7; Fig. 2A, B ), suggesting the lack of the ß subunit in porcine endothelial cells.

View larger version (28K): [in this window] [in a new window]   Figure 2. A) Effects of DHS-I and NS1619 on activity of BK in intact endothelium of porcine aorta. Currents were recorded in inside-out patches in symmetrical KCl solutions (Pipette: KCl solution, KCl bath solution) at a holding potential of -40 mV. NS1619 (20 µmol/l) increased channel activity when applied to the intracellular surface, whereas 0.3 µmol/l DHS-I had no effect on BK channel in endothelium. B) In a series of experiments, the effect of NS1619 (n=7) and DHS-I (n=7) was compared to control (con; n=7). Solutions as described in panel A. *P<0.01. C) RT-PCR analysis of BK and ß subunit expression in porcine endothelial cells (EC) and porcine vascular smooth muscle cells (VSMC). The left part of the gel shows the expression of the subunit in freshly isolated porcine aortic endothelial cells (PAEC), and in VSMC from porcine aorta (PAo) and porcine renal artery (PRA). The left arrow indicates the 799 bp fragment. An alternatively spliced message is visible above the . The right part of the gel shows the expression of the ß subunit in porcine tissues. No fragment of 465 bp (right arrow) could be amplified in PAEC. Molecular weight marker: 100 bp ladder; ±, with and without reverse transcription. H2O: water control

Therefore, we investigated the composition of BK subunits in porcine endothelium by use of RT-PCR on total RNA of freshly isolated PAEC (Fig. 2C ). As control, RT-PCR was performed with cDNA derived from porcine aortic and renal artery VSMC, which are known to express both subunits. As shown in Fig. 2C , the BK subunit (799 bp) was expressed in all investigated tissues in PAEC and VSMC. In contrast, expression of BK ß subunit (465 bp) was detected in VSMC but not in PAEC (n=6). This finding was confirmed by use of two different primer pairs that both amplified the ß subunit from vascular smooth muscle tissues. Authenticity of PCR products for the and ß subunit was verified by sequencing. Porcine ß fragments showed 92% base pair identity to human BK ß subunits (base pair sequences not shown).

BK in cultured human endothelial cells
Properties of BK in the human endothelial cell line EA.hy 926 were similar to those of porcine endothelial cells. BK of EA.hy 926 had a single-channel conductance of 270 pS ± 16 SD (n=10) in symmetrical 140 mM KCl solutions and was highly selective for K⁺. Also, channel activity strongly depended on membrane potential and [Ca²⁺]_i. At a holding potential of -20 mV EC₅₀ for [Ca²⁺]_i was 7.2 µmol/l.

In analogy to intact porcine aortic endothelium, the BK opener DHS-I (0.3 µmol/l) showed no effect on BK in EA.hy 926 (n=7), whereas NS1619 markedly increased channel activity (Fig. 3A ). Application of 20 µmol/l NS1619 to inside-out patches in the presence of 3 µmol/l [Ca²⁺]_i increased P_o from 0.08 ± 0.02 (n=6) to P_o 0.64 ± 0.03 (n=6; P<0.01).

View larger version (45K): [in this window] [in a new window]   Figure 3. Electrophysiological and molecular biological properties of BK in human EA.hy 926. A) Effects of DHS-I and NS1619 on activity of BK in EA.hy 926. Currents were recorded in inside-out patches in symmetrical KCl solutions (pipette: KCl solution, KCl bath solution) at a holding potential of -40 mV. 20 µmol/l NS1619 increased channel activity when applied to the intracellular surface, whereas 0.3 µmol/l had no effect on BK channel in human endothelial cells. B) Expression of BK subunits in human endothelial cells (EC) and vascular smooth muscle cells (VSMC). The left part of the gel shows the expression of the subunit in human EA.hy 926, and in VSMC from human umbilical vein (HUV) and artery (HUA). The left arrow indicates the 799 bp fragment. An alternatively spliced message is visible above the . The right part of the gel shows the expression of the ß subunit in human tissues. No fragment of 465 bp (right arrow) could be amplified in EA.hy 926. Molecular weight marker: 100 bp ladder; ±, with and without reverse transcription.

The mRNA expression of the BK subunit (799 bp) was detected in EA.hy 926 and control tissues (VSMC from human umbilical vein and artery, Fig. 3B ). In contrast to the expression of BK mRNA in EA.hy 926, the BK ß mRNA (465 bp) was undetectable in human endothelial EA.hy 926 (n=5) but present in VSMC (Fig. 3B ).

Transfection experiments
To investigate the functional role of the BK ß subunit, ß subunit cDNA was amplified by RT-PCR from VSMC of umbilical vein vessel slices and cloned into two different expression vectors. pCINeo/IRES-GFP/BKß was used for transient transfection, pCR3/BKß for stable transfection of human endothelial EA.hy 926. Stably transfected cell clones as well as transiently transfected EA.hy 926 were tested for the presence of ß subunit expression by use of RT-PCR or Western blot, respectively (Fig. 4A, B ). Figure 4A demonstrates the expression of BK and ß subunit in an isolated cell clone. In nontransfected control cells, no ß subunit mRNA could be amplified. Figure 4B shows the presence of the ß subunit (22 kDa) in pCINeo/IRES-GFP/BKß transfected EA.hy 926. Control experiments with the preimmune serum were performed to confirm the specitivity of the protein band. In nontransfected control cells, no ß subunit could be detected by Western blot analysis. Furthermore, the presence of the ß subunit in pCINeo/IRES-GFP/BKß transfected EA.hy 926 was demonstrated in single-channel patch clamp experiments by use of the ß subunit-specific BK opener, DHS-I (Fig. 4C ). When applied to the cytosolic face of BK in an inside-out patch, DHS-I (0.3 µmol/l) increased open probability of BK in ß subunit transfected EA.hy 926.

View larger version (27K): [in this window] [in a new window]   Figure 4. Confirmation of BK ß subunit expression in ß subunit transfected EA.hy 926. A) RT-PCR analysis of EA.hy 926 stably transfected with ß subunit (EA.ß/stable) and nontransfected control cells (con). PCR was performed with primers specific for the subunit (799 bp, left arrow) and the ß subunit (465 bp, right arrow). Molecular weight marker: 100 bp ladder. B) Western blot analysis of pCINeo/IRES-GFP/BKß transfected EA.hy 926 (EA.ß/trans) and nontransfected control cells (con). Protein extracts were applied to a SDS-PAGE, blotted, and probed with rabbit polyclonal serum raised against residues 116–131 of the human ß subunit (left part of the figure). As control the preimmune serum was tested (right part of the figure). The arrow indicates the protein size of 22 kDa. C) Effect of DHS-I on BK activity in EA.hy 926 transiently transfected with the ß subunit. Po before (control) and after addition of 0.3 µmol/l DHS-I were compared. Currents were recorded in inside-out patches at a holding potential of -25 mV. Pipette: NaCl solution. Bath solution (in mmol/l): 150 KCl, 10 HEPES, 5 EGTA, 4.35 CaCl2, pH 7.2 (corresponding to 1 µmol/l free Ca2+). The letter ‘c’ denotes closed state of the channel; o1 and o2 represent open level 1 and 2, respectively. Experiments were performed at room temperature.

Inside-out patch clamp experiments were performed to compare BK properties in EA.hy 926 transiently expressing the ß subunit with BK properties in nontransfected control cells. ß subunit expression resulted in an increased Ca²⁺ sensitivity of BK activity (Fig. 5A-C ). Figure 5A illustrates a representative experiment comparing currents in control cells with only an subunit of BK vs. ß subunit transfected cells, with both subunits at 1 µmol/l [Ca²⁺]_i. At every membrane potential shown, the channel was more active (increased channel P_o) in ß subunit transfected cells compared to controls.

View larger version (36K): [in this window] [in a new window]   Figure 5. Increased Ca2+ sensitivity of BK by transient expression of the ß subunit in EA.hy 926. A) Representative single-channel recordings comparing BK currents in control cells () with those in ß subunit transfected cells (+ß). Currents were recorded in inside-out patches at different holding potentials. Solutions were as described for Fig. 4C . The letter ‘c’ denotes closed state of the channel. B) Po-voltage relationship derived from current/voltage data. Voltage was ramped from -100 mV to +100 mV over 0.9 s and currents were recorded at three different [Ca2+]i comparing BK in control (; open symbols; 0.1 µmol/l: n=9, 1 µmol/l: n=7, 10 µmol/l: n=10) and ß subunit transfected cells (+ß; closed symbols; 0.1 µmol/l: n=7, 1 µmol/l: n=8, 10 µmol/l: n=8). The protocol was repeated at least 30 times in each case, and the currents elicited were averaged before analysis. For each experiment, open probability was calculated from the average current devided by the single-channel current amplitude at every given voltage. Values are given as mean ± SE. Data were fitted to Boltzmann equation: Po = Po min + (Po max-Po min)/(1 + exp((V1/2-V)/k)), where Po min is the minimal, Po max is the maximal Po, V1/2 is the membrane potential where Po is half-maximal, and k is the slope factor. In the presence of 1 µmol/l [Ca2+]i, half-maximal activation of channel activity shifted from 73.4 mV ± 5.2 (n=7) to 49.6 mV ± 1.4 (n=8; P<0.01) for and +ß, respectively. Pipette was filled with NaCl solution; bath solutions contained (in mmol/l): 150 KCl, 10 HEPES, 5 EGTA, pH 7.2. Free Ca2+ concentrations of 0.1, 1, and 10 µmol/l were adjusted with 2.00, 4.35, and 4.94 mmol/l CaCl2, respectively. C) Po-[Ca2+]i relationship at a holding potential of + 60 mV. Experiments were performed as described for Fig. 5B . **P<0.001. Experiments were performed at room temperature.

To quantitate Ca²⁺-activated currents in inside-out patches the P_o vs. voltage relationship derived from current/voltage data was used (Fig. 5B ). Three different [Ca²⁺]_i (0.1, 1, 10 µmol/l) were tested to compare BK in control and ß subunit transfected cells. Data points were fitted by use of the Boltzmann equation. As shown in Fig. 5B , ß subunit expression resulted in a shift of the curves toward more negative membrane potentials for the physiologically relevant [Ca²⁺]_i of 0.1 and 1 µmol/l. This effect was reflected by a shift in the membrane potential where P_o is half-maximal (V_1/2). At 1 µmol/l [Ca²⁺]_i, V_1/2 values were 73.4 mV ± 5.2 (n=7) and 49.6 mV ± 1.4 (n=8; P<0.01) for BK in control and ß subunit transfected cells, respectively. Expression of the ß subunit thus resulted in a 24 mV shift of V_1/2. At 10 µmol/l [Ca²⁺]_i, the curves for BK in control and ß subunit transfected cells converged. Figure 5C shows the relation between P_o and [Ca²⁺]_i at a holding potential of +60 mV comparing BK activity in control and ß subunit transfected cells. ß subunit expression resulted in an increased activity of the channel. In the presence of the physiologically relevant [Ca²⁺]_i of 1 µmol/l, P_o values were 0.26 ± 0.05 (n=7) and 0.61 ± 0.02 (n=8; P<0.001) for BK in control and ß subunit transfected cells, respectively. By fitting the data using a one-parameter dose-response model, we estimated EC₅₀ values at +60 mV of 1.5 and 0.5 µmol/l [Ca²⁺]_i in control and ß subunit transfected cells, respectively.

We also performed whole-cell experiments to investigate BK function in EA.hy 926 transiently transfected with the ß subunit and control cells. Figure 6A shows whole-cell currents obtained from repetitive voltage ramps from -150 to +100 mV in the presence of 1 µmol/l [Ca²⁺]_i. In ß subunit expressing EA.hy 926 increased outward currents were observed compared to controls. These outward currents could be inhibited by 50 nmol/l Iberiotoxin and activated by 20 µmol/l NS1619, indicating BK selective K⁺ currents (data not shown). The I/V curves were significantly different from each other (P<0.01).

View larger version (31K): [in this window] [in a new window]   Figure 6. Effect of ß subunit expression on whole-cell currents and reversal potential in EA.hy 926. A) Whole-cell currents in transiently transfected (+ß; n=17) and nontransfected cells (; n=12). Currents were recorded using a ramp protocol from -150 to 100 mV. The I/V curves were significantly different (P<0.01 tested with the Kruskal-Wallis test). B) In a series of experiments the effect of ß subunit expression on reversal potential (+ß; n=17) was compared to control (; n=12). The reversal potential was measured in whole-cell experiments voltage ramped from -150 to 100 mV. *P<0.01 (tested with the Mann Whitney U test). Experiments were performed at room temperature.

In addition, reversal potentials were determined. In the presence of the ß subunit the reversal potential was significantly more negative (-40.7 mV±3.3; n=17) than control (-22.6 mV±3.5; n=12; P<0.01; Fig. 6B ).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In the present study we have identified and characterized BK in intact endothelium of porcine aorta and in an endothelial cell line (EA.hy 926). We investigated the BK subunit composition and the BK function in endothelium by electrophysiological, pharmacological, and molecular biological means.

Channel conductance, ion selectivity, toxin sensitivity, and voltage dependence of the endothelial BK in porcine aorta and in EA.hy 926 described here were comparable to those values reported for various other tissues (27) . BK have been observed in a number of endothelial cells of different origin, e.g., in intact endothelium of the human umbilical vein (28) and of rat aorta (2) and in cultured endothelial cells of human umbilical vein (1) and porcine coronary artery (29) . In isolated porcine coronary artery endothelial cells (29) , BK displayed a similar slope conductance of 285 pS in symmetrical K⁺-solutions compared to the endothelial BK of intact porcine aorta and EA.hy 926.

Ca²⁺ sensitivity of BK varies markedly between different tissues (27) . In this report, a [Ca²⁺]_i of 4.0 µmol/l and 7.2 µmol/l was calculated for half-maximal activation of BK at -20 mV in porcine aorta and EA.hy 926, respectively. Similar results were obtained in porcine (29) and human (28) endothelial cells, with a half-maximal activation at 4.5 µmol/l [Ca²⁺]_i and 5.9 µmol/l [Ca²⁺]_i, respectively. Thus, the endothelial BK proved to be less Ca²⁺ sensitive than BK in vascular smooth muscle cells (30 , 31) . In bovine mesenteric vascular smooth muscle cells (31) , BK channels have been described that were half-maximally activated by 0.2 µmol/l [Ca²⁺]_i at 0 mV.

BK subunit-specific openers were used for the pharmacological study of the endothelial subunit composition. NS1619 is an subunit-specific BK opener. This was demonstrated by NS1619 activation of BK subunits expressed in Xenopus oocytes, although the ß subunit was absent (32) . DHS-I is a ß subunit-specific BK opener, as demonstrated in experiments where ß subunit expression was needed to activate BK channels by 0.1 µmol/l DHS-I (26) . In the present study, NS1619 produced rapid activation of BK in inside-out patches from porcine aortic endothelium and EA.hy 926. A similar rapid activation of BK has also been described in other tissues (33 , 16) . In contrast, DHS-I in concentrations of up to 0.3 µmol/l failed to activate BK in both investigated endothelial cell types, suggesting a lack of the ß subunit in endothelial cells.

Subsequent analysis of BK and ß subunit RNA by RT-PCR has revealed expression of the subunit in porcine aortic and EA.hy 926 endothelial cells. In contrast, no expression of the ß subunit was detectable in both endothelial cells types investigated. Detection of the ß subunit in porcine and human vascular smooth muscle cells proved sufficient function of primers. Thus, subunit expression seems to be independent of the expression of the ß subunit in endothelial cells. The composition of BK channels and the expression levels of and ß subunits are not identical in all tissues. BK present in different smooth muscle tissues (e.g., aorta; ref 34 and this report), trachea (12) , umbilical vessels (this report) coronary artery (14) , colon (35) , and lens epithelium (36) appear to be composed of both and ß subunits. ß subunit expression was also shown in the corpus callosum and hippocampal formation of human brain, but expression of the ß subunit was virtually undetectable in some other brain areas (13) .

To demonstrate the effect of the ß subunit on endothelial BK function, we expressed the BK ß subunit in EA.hy 926. The ß subunit was found to increase Ca²⁺ sensitivity of endothelial BK subunits in single-channel and whole-cell experiments. ß subunit expression resulted in a decrease of the EC₅₀ value for [Ca²⁺]_i of ~1 µM. Accordingly, the potential of half-maximal activation (V_1/2) shifted to more negative potentials. V_1/2 shifts caused by and ß subunit coexpression have also been described for other expression systems (13 , 37 , 38 , 26 , 39) . Although the exact size of the V_1/2 shift varies between the different reports, our results are comparable with results obtained in transfection experiments with HEK cells (13 , 37 , 39) . In addition, the V_1/2 value of 49.6 mV at 1 µmol/l [Ca²⁺]_i in ß subunit transfected EA.hy 926 reported here corresponds well with V_1/2 of 47.3 mV at 1 µmol/l [Ca²⁺]_i in smooth muscle cells isolated from mesenteric artery (4) , which should be composed of and ß subunits.

The present study demonstrates for the first time that BK channels in endothelial cells are composed of subunits without association to ß subunits. This has been demonstrated by the low Ca²⁺ sensitivity of the endothelial BK compared to VSMC, pharmacological studies with specific openers, RT-PCR studies, and transfection experiments in which ectopic expression of the ß subunit increased Ca²⁺ sensitivity of native subunits. The lack of the ß subunit indicates a substantially different channel regulation in endothelial cells compared to vascular smooth muscle cells. Functional properties of BK can be tailored to the specific physiological requirements of a cell type not only by controlling the expression of different splice variants of the subunit (10) , but also by regulating ß subunit expression. The different composition of BK in VSMC and EC might become important for the development of tissue-specific therapeutics.

	ACKNOWLEDGMENTS

 
We thank Dr. C.-J. S. Edgell (University of North Carolina) for providing us the EA.hy 926 cell line. DHS-I was a kind gift from Dr. O. B. McManus (Merck Research Laboratories). This work was supported by the Deutsche Forschungsgemeinschaft (GRK 276/1) and the European Community BM H4-CT96–0602.

	FOOTNOTES

 
Received for publication June 29, 1999. Revised for publication December 14, 1999.

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