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(Circulation Research. 2000;87:e53.)
© 2000 American Heart Association, Inc.

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Mice With Disrupted BK Channel ß1 Subunit Gene Feature Abnormal Ca²⁺ Spark/STOC Coupling and Elevated Blood Pressure

Saskia Plüger, Jörg Faulhaber, Michael Fürstenau, Matthias Löhn, Ralph Waldschütz, Maik Gollasch, Hermann Haller, Friedrich C. Luft, Heimo Ehmke, Olaf Pongs

From the Institut für Neurale Signalverarbeitung (S.P., R.W., O.P.), ZMNH, Universität Hamburg, Hamburg, Germany; Franz-Volhard-Klinik am Max-Delbrück-Centrum für Molekulare Medizin (M.F., M.L., M.G., H.H., F.C.L.), Humboldt Universität zu Berlin, Berlin, Germany; and Institut für Physiologie (J.F., H.E.), Universität Hamburg, Hamburg, Germany.

Correspondence to Prof Dr O. Pongs, Zentrum für Molekulare Neurobiologie der Universität Hamburg, Institut für Neurale Signalverarbeitung, Martinistrasse 52, D-20246 Hamburg, Germany. E-mail pointuri{at}uke.uni-hamburg.de

	Abstract

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Abstract—Large-conductance potassium (BK) channels in vascular smooth muscle cells (VSMCs) sense both changes in membrane potential and in intracellular Ca²⁺ concentration. BK channels may serve as negative feedback regulators of vascular tone by linking membrane depolarization and local increases in intracellular Ca²⁺ concentration (Ca²⁺ sparks) to repolarizing spontaneous transient outward K⁺ currents (STOCs). BK channels are composed of channel-forming BK and auxiliary BKß1 subunits, which confer to BK channels an increased sensitivity for changes in membrane potential and Ca²⁺. To assess the in vivo functions of this ß subunit, mice with a disrupted BKß1 gene were generated. Cerebral artery VSMCs from BKß1 -/- mice generated Ca²⁺ sparks of normal amplitude and frequency, but STOC frequencies were largely reduced at physiological membrane potentials. Our results indicate that BKß1 -/- mice have an abnormal Ca²⁺ spark/STOC coupling that is shifted to more depolarized potentials. Thoracic aortic rings from BKß1 -/- mice responded to agonist and elevated KCl with an increased contractility. BKß1 -/- mice had higher systemic blood pressure than BKß1 +/+ mice but responded normally to ₁-adrenergic vasoconstriction and nitric oxide–mediated vasodilation. We propose that the elevated blood pressure in BKß1 -/- mice serves to normalize Ca²⁺ spark/STOC coupling for regulating myogenic tone. The full text of this article is available at http://www.circresaha.org.

Key Words: hypertension • potassium channels • spontaneous transient outward K⁺ currents • vasoconstriction

	Introduction

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Large-conductance, voltage, and Ca²⁺-sensitive potassium (BK) channels are very abundant in smooth muscle cells,¹ where they may play a key role in regulating the size of the lumen in blood vessels,² airways,³ the gastrointestinal tract,⁴ uterus,⁵ and bladder.⁶ BK channel activity in vascular smooth muscle cells (VSMCs) is correlated with the occurrence of spontaneous transient outward K⁺ currents (STOCs).⁷ The currents produce a K⁺ efflux, which causes membrane hyperpolarization and thus diminishes VSMC contraction. BK channel opening depends on local increases in Ca²⁺ in the VSMC cytoplasm, termed Ca²⁺ sparks.² Ca²⁺ sparks and STOCs are tightly coupled in that the appearance of a Ca²⁺ spark almost always transiently activates a group of BK channels.⁸ Interference with spark formation or BK channel opening prevents membrane repolarization and causes VSMC contraction. Thus, attenuation of the local release of Ca²⁺ from internal stores by agents such as ryanodine and thapsigargin induces vessel constriction.⁹ Similarly, iberiotoxin, which selectively blocks the opening of BK channels, causes membrane depolarization and vasoconstriction in pressurized isolated vessels.¹⁰ Because BK channels are near the sites where Ca²⁺ sparks are generated, they may function as endogenous Ca²⁺ detectors, which regulate vascular tone.^{8 11}

In VSMCs, BK channels are composed of and ß subunits.¹² The pore-forming BK subunits have a membrane topology similar to the superfamily of Shaker-related six-transmembrane domain potassium channel subunits.¹ The accessory BKß1 subunits are relatively small membrane proteins with two membrane spanning segments.¹² BKß subunits have important effects on the kinetics, voltage, and apparent Ca²⁺ sensitivity of BK currents.^{13 14 15 16 17} In the absence of BKß1 subunits, in vitro–expressed BK channels have a dramatically reduced Ca²⁺ sensitivity and are open only to a significant extent at very positive membrane potentials.¹⁸ On the other hand, coexpression of BK subunits with BKß1 subunits in vitro generates BK channels with an apparent Ca²⁺ sensitivity that can account for the properties of BK channels observed in VSMCs.^{15 19 20} Thus, formation of heteromultimeric BK channels from BK and BKß1 subunits may be an essential requirement for their proposed role as negative feedback regulators of vascular tone. To test this hypothesis, we investigated how a loss of BKß1 gene function in VSMCs of BKß1 -/- mice affected BK channel physiology related to the coupling of Ca²⁺ sparks and STOCs, the generation of vascular tone in vitro, and the regulation of blood pressure in vivo.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cloning of the Mouse BKß1 Gene
We screened a 129/SvJ mouse genomic library (Stratagene) using the rat BKß1 cDNA as a probe and obtained four positive clones.²¹ One clone (msloß1,6) contained the complete mouse BKß1 open reading frame. Restriction mapping, hybridization with BKß1 exon-specific probes, and exon and exon/intron border sequencing showed that the mouse BKß1 gene consisted of three exons like the human BKß1 (KCNMB1) gene.²² The 7-kb BamHI/NotI restriction fragment of the genomic msloß1,6 clone, which contained the three exons, was subcloned into pBluescriptKS⁺ (pKSmsloß1BN). In addition, the 3.2-kb KpnI restriction fragment upstream of the BamHI restriction site was separately cloned into pBluescriptKS⁺ (pKSmsloß1KK).

Gene Targeting
PKSmsloß1BN DNA was cut with NdeI and ligated with a blunt-ended SmaI/KpnI restriction fragment containing the neomycin resistance gene (neo) under the control of the phosphoglycerate kinase promoter flanked by two loxP elements (pKSmsloß1neo and ploxPneo-1).²³ In parallel, the 1.4-kb SacI/ApaI restriction fragment of pKSmsloß1KK DNA was cloned next to a loxP element into SacI/NotI-digested ploxPneo-1 DNA. Then, the msloß1 DNA together with the loxP element was isolated as a SacI/ApaI restriction fragment. This fragment was ligated back into SacI/ApaI-digested pKSmsloß1KK DNA (pKSmsloß1KKloxP). The KpnI restriction fragment of this clone together with the 1.3-kb KpnI/BamHI restriction fragment of msloß1 was ligated into KpnI-cut pKSmsloß1neo to yield the targeting vector pKSmsloß1neoloxP (Figure 1). The linearized targeting vector was electroporated into 129 (R1) embryonic stem (ES) cells,²⁴ which were subjected to selection by geneticin (G418, Life Technologies). Southern blotting was performed on 178 resistant ES cell clones, two of which were positive for the targeting event. Genomic DNA was digested with BamHI, electrophoresed on 0.8% agarose gel, transferred, and hybridized with a 0.21-kb 3' probe derived from DNA distal to the msloß1 NotI site (Figure 1). The wild-type 8.5-kb BamHI restriction fragment had been extended by 2 kb to 10.5 kb in case of a homologous recombination event. One of the two positive ES clones (msloß1,c34) was expanded and then transiently transfected by electroporation with the Cre recombinase–expressing clone pCrePac²⁵ to delete the sequences from the first loxP element to the last, such that the disrupted msloß1 locus missed the first sloß1 exon and had the neo selection cassette removed (Figure 1). Accordingly, BamHI digestion of DNA with sloß1 exon1 deleted gave rise to a 6.5-kb fragment. BamHI digestion of the genomic DNA of 120 clones showed that the DNA of eight clones generated the 8.5-kb and the 6.5-kb restriction fragments for the wild-type and the properly disrupted allele, respectively. One of the positive ES clones was expanded and microinjected into C57BL/6J mouse blastocysts,²⁶ which then were transferred into pseudopregnant CBAxC57BL/6J females. Three of seven chimeric mice that were mated gave rise to germ-line transmission of the disrupted allele. Males and females with different genotypes from different litters were randomly intercrossed to obtain sloß1 +/+, +/-, and -/- progeny. Mouse-tail genomic DNA was screened by Southern analysis following standard protocols.²⁷ The studies described below were performed on mice belonging to generations F4 to F7.

View larger version (48K): [in this window] [in a new window]   Figure 1. Figure 1. Targeted disruption of the mouse BKß1 gene. A, Structure of the mouse BKß1 locus before the targeting event (top), after the targeting event (middle), and after deletion of the first BKß1 exon (bottom). The coding exons are depicted by filled boxes, the open arrow denotes the mouse phosphoglycerate kinase (PGK) promoter, and the open box is the coding region of the neomycin transferase (neo) gene. Filled triangles denote the loxP sequences used for Cre-mediated deletion. A genomic fragment used as a probe for Southern blot analysis (B) is shown as a black line, and the expected sizes of the BamHI fragments that hybridize with the probe are indicated. B, Southern blot analysis of genomic DNA extracted from tail biopsies. The DNA was digested with BamHI and subjected to hybridization with the probe shown in panel A. C, PCR analysis of representative ear punches. PCR was performed with 3 primers as described in Materials and Methods. The sizes of the expected amplicons are indicated. D, Northern analysis of total RNA from intestinal tissue. Hybridization was performed with BKß1 (top) and GAPDH (bottom) probes, respectively. E, Top, Ethidium bromide staining of RT-PCR fragments. The BKß1 mRNA transcript was detected as a 471-bp fragment. RT-PCR assay was controlled by simultaneous detection of a 282-bp fragment of the BK transcript. Bottom, Southern blot of the RT-PCR fragments shown in the top panel. The identity of amplified BKß1 fragments was assessed using a 32P-labeled BKß1 probe. The different genotypes are denoted as +/+ (BKß1 wild type), -/- (BKß1 knockout), and +/- (heterozygous).

Northern and Reverse Transcriptase–Polymerase Chain Reaction (RT-PCR) Analysis
Total RNA from different mouse tissues was isolated using the S.N.A.P. Total RNA Isolation kit (Invitrogen). Five micrograms of total RNA was reverse-transcribed for 60 minutes at 42°C in a 20-µL reaction mixture containing 40 mmol/L Tris-HCl (pH 8.3), 100 mmol/L KCl, 5 mmol/L MgCl₂, 5 µmol/L oligo(dT)_12–18 (Life Technologies), 10 mmol/L DTT, 20 U of RNasin (Promega), 0.5 mmol/L of each dNTP, and 400 U of Superscript II reverse transcriptase (Life Technologies). One tenth of each cDNA sample was amplified by PCR with BKß1-specific primers slob7 and slob9 together with BK-specific primers BKMA-1s and mslo-4a. Primer pairs were complementary to adjacent exon sequences of both genes to avoid amplification of potential genomic DNA contamination. Samples contained 50 pmol of each primer, 0.25 mmol/L of each dNTP, 50 mmol/L KCl, 20 mmol/L Tris-HCl at pH 8.4, 1.2 mmol/L MgCl₂, and 1.25 U of Taq DNA polymerase (Life Technologies). Thermal cycling was performed for 2 minutes at 94°C, 1 minute at 58°C, and 30 seconds at 72°C for 35 cycles. The upstream and downstream primers were (5'-3' direction) as follows: slob7 5'-GGACCTGTTGAGCTTCACC-3'; slob9 5'-TGCT-GCCATCACCTACTACG-3'; BKMA-1s 5'-TTACGTACTGG-GATGTGTCTAC-3'; and mslo-4a 5'-TGTGCAGAAAGTC-CTTCAGG-3'. The specificity of the amplified BKß1 DNA fragment was determined by Southern blotting using a ³²P-labeled BKß1-specific probe.

For Northern analysis, 10 micrograms of each total RNA preparation was separated on denaturing 1.5% agarose gels. Blotting and hybridization were performed using standard methods.²⁸ Hybridization probes were generated by PCR, for BKß1 a 471-bp fragment (slob7/slob9 primer combination) and as a control a 511-bp GAPDH fragment amplified using primers GAPDH-1s (5'-TTGTCAGCAATGCATCCTTGC-3') and GAPDH-2as (5'-AACAGTATGGTCCTTTACTCG-3'). Both fragments were verified by sequencing before use.

Cerebral Artery Experiments
VSMCs were enzymatically isolated from cerebral arteries from BKß1 +/+ and BKß1 -/- mice as previously described.^{29 30} Briefly, arteries were placed in a Ca²⁺-free Hanks solution containing 55 mmol/L NaCl, 80 mmol/L sodium glutamate, 5.4 mmol/L KCl, 2 mmol/L MgCl₂, 1 mg/mL BSA (Sigma), 10 mmol/L glucose, and 10 mmol/L HEPES (pH 7.4 with NaOH) containing 1.0 mg/mL papain (Sigma), and 1 mg/mL DTT for 15 minutes at 36°C. The segments were then placed in Hanks solution containing 1 mg/mL collagenase (Sigma type F and H; ratio 30% and 70%) and 0.1 mmol/L CaCl₂ for 8 minutes at 36°C. After several washes in Ca²⁺-free solution, single cells were dispersed from artery segments by gentle trituration. Cells were stored in the same solution at 4°C. From these cells, STOCs were recorded by means of the perforated patch-clamp technique as previously described.²⁹ Holding potential was -60 mV. Depolarizing test potentials were gradually increased in 10-mV increments from -50 to 0 mV. External solution contained (in mmol/L) NaCl 134, KCl 6, MgCl₂ 1, CaCl₂ 2, HEPES 10, and glucose 10 (pH 7.4). Pipette solution contained 30 mmol/L KCl, 110 mmol/L potassium aspartate, 10 mmol/L NaCl, 1 mmol/L MgCl₂, 0.05 mmol/L EGTA (pH 7.2), and 250 µg/mL amphotericin. Confocal line-scan images of fluo-3–loaded smooth muscle cells were taken to measure Ca²⁺ sparks. Amplitudes of Ca²⁺ sparks were measured as local fractional fluorescence increases (F/F₀).² All measurements were done by experimenters blinded to the study conditions.

Data Analysis, Statistics, and Presentation
All values are given as mean±SEM. Data were statistically compared using Student’s t test. P<0.05 was regarded as significant. The term n represents the numbers of cells tested.

Aortic Ring Experiments
Anesthetized mice of comparable weight and age were killed. The thoracic aorta was excised and placed in cold Tyrode’s solution (in mmol/L: NaCl 140, KCl 5, MgSO₄ 1.2, CaCl₂ 2, glucose 10, and HEPES 5). The aorta was carefully cleaned of adhering paravascular tissue and cut into 3-mm-long rings. Usually, two to three rings of comparable size and properties could be isolated from one mouse. The rings were suspended in isolated tissue baths filled with 25 mL of Krebs-Henseleit bicarbonate buffer containing 70 µmol/L ascorbate and 5 µmol/L indomethacin (Sigma). Baths were continuously bubbled with a mixture of 5% CO₂-95% O₂ (pH 7.4) at 37°C. Aortic rings were mounted in organ chambers for isometric tension recording with two parallel wires inserted into the lumen of the segment. The signal of the isometric force transducer (8TRN001, Kent Scientific Corporation) was transmitted into a computerized system for data acquisition and signal analysis (ML500/M PowerLab/8e, AD Instruments). Analysis of the generated curves was performed using Chart 3.6 software (AD Instruments). The sensitivity of the system was 5±1 mg of tension generated. Rings were equilibrated for 90 minutes, and the buffer was replaced every 20 minutes. The length of the smooth muscle was increased stepwise during the equilibration period to adjust passive wall tension to 0.8 g. Once basal tension was established, the length of the rings was not modified. Functional integrity of endothelial tissue was controlled with 10^–5 mol/L acetylcholine.

Data Analysis, Statistics, and Presentation
Data are expressed as mean±SEM except where indicated otherwise. Data were statistically evaluated using Student’s t test, where applicable with Student’s test for paired comparisons. Data points with P<0.05 were considered to be significant. In all experiments, n equals the number of mice from which rings were taken.

Cardiovascular Studies
Arterial blood pressure and heart rate were measured in conscious, unrestrained mice (body weight 25 to 42 g; age 4 to 8 months) in accordance with national guidelines for the care and use of research animals. Forty-eight hours before the recordings, animals were anesthetized with ketamine and xylazine-HCl (Rompun, Bayer, 100 µg/g body weight and 4 µg/g body weight IP), and chronic catheters (manufactured as described by Mattson³¹ ) were implanted aseptically into the left femoral artery and vein. After implantation, the catheters were tunneled subcutaneously and exteriorized through a spring, sewn to the animal’s back. Cefazolin (10 mg IV) was given for antibiotic prophylaxis. The spring was connected to a swivel device at the top of the cage. The catheters were filled with heparin solution (50 IU/mL saline) and sealed until use. After surgery, the mice were housed individually in plastic cages with free access to water and standard mouse chow. On days 2 and 3 after surgery, baseline values of arterial blood pressure and heart rate were recorded for 1 hour in each mouse in its own cage. Blood pressure was measured in the abdominal aorta via the femoral artery catheter (transducer PRC-21K; amplifier MIO-0501; FMI) and was continuously recorded on a computer (80586; DAS-16, Keithley-Metrabyte; Labtech-Note-Book 10.2.1) at 500 Hz.

For determination of agonist-induced vasoconstriction, phenylephrine (5 ng/g body weight per minute; Merck) was infused after a control period of 5 minutes at a constant rate (250 nL/g body weight per minute) for 35 minutes. For determination of agonist-induced vasodilation, nitroprusside (5 ng/g body weight per minute; Merck) was infused after a control period of 5 minutes at a constant rate (250 nL/g body weight per minute) for 35 minutes. Both drugs were administered via the femoral vein catheter using a precalibrated infusion pump (Precidor 5003, Infors AG). The order of nitroprusside and phenylephrine was randomly assigned. Between both protocols, a recovery period of at least 45 minutes was allowed after flushing and reloading of the venous catheter.

Dose-response curves for phenylephrine were established as follows: BKß1 +/+ (n=8) and BKß1 -/- (n=5) mice were infused with increasing doses of phenylephrine (1.25, 2.5, 5, 10, and 20 ng/g body weight per minute). After a baseline recording of 5 minutes, each dose was infused for 10 minutes. Arterial blood pressure and heart rate responses were averaged over the last 5 minutes of each infusion period.

Data are mean±SEM, and n represents the number of animals. Statistical analysis was done by the unpaired or paired Student’s t test or ANOVA followed by the Newman-Keuls test (dose-response curve). An error level of P<0.05 was considered significant.

	Results

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Targeted Disruption of the Mouse BKß1 Gene
A targeting vector to disrupt the mouse BKß1 gene was constructed. We screened a 129 SV/J mouse genomic library and isolated a single clone containing the BKß1 open-reading frame (ORF). Restriction and sequence analysis of the clone revealed that the BKß1 ORF is encoded in three exons (Figure 1A). The targeted disruption strategy for deleting the first exon of the BKß1 gene in ES cells is described in Figure 1A. First, we flanked the first exon with loxP elements. Homologous recombinants were identified by Southern blot analysis of genomic DNA. We then transiently transfected the recombinant ES cells with a Cre recombinase–expressing clone to delete the first exon. ES cells with a successfully deleted BKß1 exon were microinjected into C57Bl/J6 blastocyst stage embryos. Three of seven chimeric mice that were mated gave rise to germ-line transmission of the disrupted allele. Males and females with different genotypes from different litters were randomly intercrossed to obtain BKß1 +/+, +/-, and -/- progeny. Genotypes were analyzed by genomic Southern blotting (Figure 1B) and/or PCR with appropriate primer pairs (Figure 1C). Analysis of the BKß1 -/- genotype frequencies after intercrosses of heterozygous mutant mice did not reveal any deviation from the mendelian expectations (results not shown). Also, the body weight of mice of different BKß1 genotypes did not show differences throughout adult life from days 3 to 7 to up to 12 months. Apparently, the disruption of the BKß1 gene did not have adverse effects on mouse development, reproductive behavior, growth, and feeding behavior. Furthermore, homozygous BKß1 -/- mice had a normal appearance, were fertile, and showed no abnormalities and no differences in exploratory behavior, compared with their normal littermates (data not shown).

BK and BKß1 mRNA Expression in BKß1 +/+ and BKß1 -/- Mice
We purified mRNA from isolated intestinal tissue of BKß1 +/+ and BKß1 -/- mice to assess the expression of BKß1 mRNA in Northern blots. The blots were hybridized with a ³²P-labeled probe specific for BKß1 mRNA and, respectively, GAPDH mRNA for control (Figure 1D). The control hybridizations showed that GAPDH mRNA was present in equal amounts in BKß1 +/+ and BKß1 -/- tissue. BKß1 hybridization signals were only obtained for mRNA of BKß1 +/+ tissue. The signals corresponded to a 1.6- and a 4.5-kb BKß1 mRNA species, comparable to the ones reported for rat²¹ and human BKß1 mRNAs.²² BKß1 -/- tissue expressed neither wild-type nor aberrant forms of BKß1 mRNA in detectable amounts. In addition, we used RT-PCR to investigate the expression of BKß1 mRNAs in intestine, kidney, testis, and stomach (Figure 1E). The results confirmed that the deletion of the first BKß1 exon abolished BKß1 mRNA expression in tissue of BKß1 -/- mice. As an additional control, we simultaneously assayed in the RT-PCR experiments BK mRNA expression in BKß1 +/+ and BKß1 -/- mouse tissue (Figure 1E). The results indicated that BK mRNA expression levels were similar in the investigated BKß1 +/+ and BKß1 -/- mouse tissues.

Ca²⁺ Sparks and STOCs
Figures 2A and 2B illustrates confocal line-scan images of fluo-3–loaded smooth muscle cells freshly isolated from cerebral arteries. BKß1 +/+ and BKß1 -/- cells generated Ca²⁺ sparks with a rise time of 20 ms, a half-time of decay of 50 to 60 ms, a spatial spread (full width at half-maximum amplitude) of 2.4 µm, and an apparent frequency of 0.04 to 0.05·s ^–1·µm^–1 (Figures 2A through 2E). The absence of BKß1 subunits neither affected the generation of Ca²⁺ sparks nor their apparent properties, which were similar to those reported previously.⁸ Also, the results did not reveal a statistically significant difference between the properties of Ca²⁺ sparks generated in BKß1 +/+ or BKß1 -/- smooth muscle cells (n=52 for each genotype; P>0.33).

View larger version (37K): [in this window] [in a new window]   Figure 2. Figure 2. Spatiotemporal characteristics of Ca2+ sparks in freshly isolated smooth muscle cells from male mouse cerebral arteries. A, Confocal line-scan images of a fluo-3–loaded BKß1 +/+ (top) and a BKß1 -/- (bottom) cerebral artery smooth muscle cell, with the time course of the respective Ca2+ sparks indicated below. The fluorescence time course of the Ca2+ sparks was determined over the line indicated by the two arrows. Each line-scan image is a plot of fluorescence along a scanned line (ie, position) on the ordinate versus time (on the abscissa). The line-scan image duration was 1 second, and each line was 2 ms. Cells were isolated from a total of 7 BKß1 +/+ and BKß1 -/- male littermates each. The total number of cells examined for Ca2+ spark frequency was 52 for each genotype. Each cell was scanned for a total time of 30 seconds. For comparison, Ca2+ spark frequencies were normalized and shown in panel E as sparks · s–1 · µm–1. B, Amplitudes of Ca2+ sparks were measured as local fractional fluorescence increases (F/F0). C, Duration of Ca2+ sparks was estimated at half-maximal amplitude. D, Width of Ca2+ sparks was determined at half-maximal amplitude. Error bars refer to SEMs. Ca2+ spark amplitudes (B), durations (C), widths (D), or frequencies (E) were not significantly different in BKß1 +/+ (open bars) and BKß1 -/- (filled bars) smooth muscle cells. Data are mean±SEM.

STOCs were recorded from the isolated smooth muscle cells by means of the perforated patch-clamp technique (Figure 3A). Test potentials were stepwise increased from a holding potential of -60 mV by 10-mV increments to 0 mV. STOCs were observed in BKß1 +/+ cells already at test potentials as negative as -50 mV. In contrast, the occurrence of STOCs in BKß1 -/- cells was shifted to more positive membrane potentials. As a result, STOC frequency in BKß1 -/- smooth muscle cells of cerebral arteries was greatly reduced near normal resting membrane potential at -40 mV. At this membrane potential, the STOC frequency was only 10% of the one seen in BKß1+/+ cells (P<0.01) (Figure 3B). Apparently, the Ca²⁺ spark/STOC relationship had been uncoupled in BKß1 -/- VSMCs at physiological membrane potentials. At more depolarized membrane potentials, eg, at -20 mV, the STOC frequency in BKß1 -/- smooth muscle cell had reached a level comparable to the one seen in BKß1 +/+ cells at -40 mV (Figure 3B). Thus, in comparison to BKß1 +/+, the generation of STOCs of similar frequencies had been shifted in BKß1 -/- smooth muscle cells by 20 mV, to more positive membrane potentials.

View larger version (31K): [in this window] [in a new window]   Figure 3. Figure 3. Measurement of STOC activity (A) in freshly isolated cerebral artery VSMCs of male BKß1 +/+ (top) and BKß1 -/- (bottom) mice. STOCs were recorded by means of the perforated patch-clamp technique at stepwise elevated holding potentials. B, Summary of STOC frequency at membrane potentials of -40 mV. Mean±SEM is given for BKß1 +/+ (n=11; open bar) and BKß1 -/- (n=9; filled bar) cells (P<0.01). Hatched bar shows STOC frequency at membrane potential of -20 mV for BKß1 -/- (n=9). P=0.35 vs BKß1 +/+ at -40 mV.

Aorta Contractility in BKß1 +/+ and BKß1 -/- Mice
Thoracic aortic ring preparations from BKß1 +/+ and BKß1 -/- mice showed a similar concentration dependence in their responses to norepinephrine. Half-maximal contractile responses were obtained at 5.7 · 10^–9 mol/L for BKß1 +/+ (n=9) and at 5.9 · 10^–9 mol/L for BKß1 -/- thoracic aortic rings (n=11) (Figures 4A and 4B). However, at 10^–6 mol/L norepinephrine, the contractile response of the thoracic aortic rings from BKß1 -/- mice generated a significantly greater tension than thoracic aortic rings from BKß1 +/+ mice (5.10±0.59 versus 3.24±0.29 mN above basal levels; P<0.001; Figure 4A). At saturating concentrations of norepinephrine, the contractile response was 65% stronger in BKß1 -/- mice than in BKß1 +/+ mice.

View larger version (31K): [in this window] [in a new window]   Figure 4. Figure 4. Different contractile response of thoracic aortic rings prepared from BKß1 +/+ and BKß1 -/- mice. A, Aorta contractility in BKß1 +/+ and BKß1 -/- mice in response to increasing concentrations of norepinephrine. Aortic rings were prepared from 16 BKß1 +/+ and 20 BKß1 -/- male littermates. The results are the mean±SD for each genotype (P<0.05). B, Dose-response curve of norepinephrine-induced contraction of BKß1 +/+ and BKß1 -/- aortic rings. The results are the mean±SEM of 9 (+/+) and, respectively, 11 (-/-) concentration-response curves for each genotype (P<0.001). C, Aortic ring constriction was induced by adding 30 mmol/L KCl to the bath in the absence (-IbTX) or presence (+IbTX) of 10–7 mol/L iberiotoxin. Data are mean±SEM (P<0.05) for each genotype (6 +/+ and 9 -/- male littermates). D, Relaxation-induced by acetylcholine (10–5 mol/L) in aortic ring preparations of BKß1 +/+ (n=5; open bars) and BKß1 -/- (n=3; filled bars) mice. Aortic rings were preconstricted with norepinephrine (10–7 mol/L). Results are mean±SD for each genotype (P<0.001).

In other experiments, we induced a contractile response in the thoracic aortic ring preparations by elevating the KCl concentration in the bath to 30 mmol/L. As for agonist-induced vasoconstriction, application of 30 mmol/L KCl produced an increased contractile response in BKß1 -/- (n=31) in comparison to BKß1 +/+ thoracic aortic rings (n=25) (Figure 4C). Then, we applied 30 mmol/L KCl together with 10^–7 mol/L iberiotoxin to inhibit BK channels. The contractile responses of BKß1 +/+ thoracic aortic ring preparations were augmented, reaching levels (9.0±1.2 mN, n=25) similar to those observed with BKß1 -/- preparations in the absence of iberiotoxin (8.8±0.9 mN, n=31). In contrast, the contractile responses of BKß1 -/- preparations (8.8±0.9 mN, n=31) were not significantly increased in the presence of iberiotoxin (9.2±1.1 mN, n=31) paired. Thus, block of BK channels in BKß1 +/+ VSMCs by iberiotoxin eliminated the difference in contractile responses between BKß1 +/+ and BKß1 -/- thoracic aortic ring preparations (Figure 4C). These experiments showed that the increased agonist-induced vasoconstriction of BKß1 -/- thoracic aortic rings was linked to a reduced BK channel activity, most likely due to absence of BKß1 subunits. Contractility experiments were terminated by adding acetylcholine to measure the relaxation response of the constricted thoracic aortic ring preparations and to assess their functional integrity. The relaxation response of preconstricted BKß1 +/+ and BKß1 -/- preparations to 10^–5 mol/L acetylcholine was similar (Figure 4D).

Blood Pressure Regulation
The effects of disrupting the BKß1 subunit on blood pressure regulation were investigated in conscious, unrestrained mice with chronically implanted catheters. Baseline values of heart rate and blood pressure were recorded in each mouse twice over 1 hour on two separate days. While heart rates were similar in both groups of mice (608±14 bpm in BKß1 +/+, n=9; 642±15 bpm in BKß1 -/-, n=8), blood pressure was significantly elevated in BKß1 -/- mice in comparison to BKß1 +/+ mice (116±2 versus 103±1 mm Hg; P<0.001). To determine whether this blood pressure difference resulted from an enhanced sensitivity to norepinephrine in BKß1 -/- mice, both groups of animals were treated with exogenous infusions of the selective ₁-adrenergic agonist phenylephrine (5 ng/g body weight per minute), which mimics the vasoconstrictor effects of norepinephrine. As summarized in Table 1, phenylephrine induced significant increases in blood pressure that were accompanied by pronounced reflex decreases in heart rate. The time course and magnitude of both responses were nearly identical in BKß1 +/+ and BKß1 -/- mice. Similar results were obtained with different doses of phenylephrine ranging from 1.25 to 20 ng/g body weight per minute (data not shown). In separate experiments, the capacity for blood pressure reductions in response to the potent vasodilator nitric oxide was assessed by infusing the nitric oxide donor nitroprusside (5 ng/g body weight per minute). In both strains, blood pressure was significantly lowered to a similar degree, whereas heart rate increased to the same level (Table 2). Again, the kinetics of the blood pressure and heart rate changes were nearly identical. Collectively, these results showed that the responses to ₁-adrenergic–mediated vasoconstriction or nitric oxide–induced vasodilation were not altered in BKß1 -/- mice, and that the difference in basal blood pressures between BKß1 -/- and BKß1 +/+ mice was maintained over a wide range of pressure.

View this table: [in this window] [in a new window]   Table 1. Cardiovascular Responses to Phenylephrine in BKß1 +/+ and BKß1 -/- Mice

View this table: [in this window] [in a new window]   Table 2. Cardiovascular Responses to Nitroprusside in BKß1 +/+ and BKß1 -/- Mice

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Recent studies have suggested that myogenic tone in VSMCs is regulated by a feedback mechanism that links focal increases in subsarcolemmal Ca²⁺ (ie, Ca²⁺ sparks) to hyperpolarizing transient outward currents (ie, STOCs).^{2 9 32} We showed that BKß1 -/- VSMCs produced, at physiological membrane potentials (-40 mV), STOCs at much lower frequency than BKß1 +/+ VSMCs. Most likely, this is a direct consequence of the absence of the BKß1 channel subunit. In in vitro expression systems, BK channels open in the absence of Bkß1 subunits at more positive potentials than in their presence because the association of BKß1 with BK subunits increases the Ca²⁺ sensitivity of BK channels.^{15 19 33} Apparently, the absence of BKß1 in BKß1 -/- mice also shifts in vivo the threshold of BK channel activation to more positive membrane potentials. As a result, we observed markedly reduced STOC frequencies in BKß1 -/- VSMCs at membrane potentials near -40 mV. By contrast, STOC frequencies at more positive potentials appeared to be normal in BKß1 -/- VSMCs. Again, this supports our idea that the absence of BKß1 subunits in BKß1 -/- mice did not eliminate BK channel function but shifted the threshold of BK channel activation to more positive membrane potentials.

Ca²⁺ sparks, on the other hand, appeared not to differ between BKß1 +/+ and BKß1 -/- VSMCs in both frequencies and properties. In the proposed feedback mechanism, Ca²⁺ sparks and STOCs are coupled in that the appearance of a Ca²⁺ spark is almost always associated with the appearance of a STOC.⁸ Obviously, the observed normal frequency of Ca²⁺ sparks and the largely reduced frequency of STOCs at -40 mV, ie, in the range of physiological membrane potentials, indicates that Ca²⁺ sparks and STOCs in BKß1 -/- VSMCs are not coupled normally as would be expected for BKß1 -/- channels with a reduced Ca²⁺ sensitivity. Alternatively, the observed dysfunction in Ca²⁺ spark/STOC coupling could be due to an inappropriate localization of BKß1 -/- channels to the cell surface because of the missing BKß1 subunits. It is possible that BKß1 subunits may be necessary to position BK channels in the plasma membrane in close approximation to the internal Ca²⁺ release sites that produce the Ca²⁺ sparks. Then a focal rise in Ca²⁺ concentration, ie, a Ca²⁺ spark, would dissipate by diffusion before reaching mislocated BK channels as Ca²⁺-sensitive target sites.¹¹ Our observation, however, that BK channel–mediated STOCs and Ca²⁺ sparks may couple normally at more positive membrane potentials might argue against this alternative. Because at more positive membrane potentials STOC frequencies in BKß1 -/- VSMCs appear to be normal, our data suggest that the feedback mechanism related to Ca²⁺ spark/STOC coupling may be normal at more positive membrane potentials. Consequently, the voltage range would be shifted in BKß1 -/- VSMCs for the feedback mechanism that limits vasoconstriction.

We observed in thoracic aortic ring preparations an abnormally increased vasoconstriction on agonist application and elevation of KCl, respectively. When we blocked the BK channels with iberiotoxin, the apparent differences between BKß1 +/+ and BKß1 -/- were eliminated. The results of these experiments demonstrated that BK channel activity is required to limit KCl-induced vasoconstriction and that a reduced BK channel activity in BKß1 -/- VSMCs evokes an increased contractile response. The data directly support, therefore, the previously proposed role of Ca²⁺ spark/STOC coupling to limit vasoconstriction in response to agents, such as norepinephrine, and elevated oxygen tension.^{2 8 9}

The increased contractile responses in the thoracic aortic ring experiments suggested that BKß1 -/- mice might be hypertensive in comparison to BKß1 +/+ littermates. Indeed, mice lacking the BKß1 subunit consistently had elevated blood pressure levels. The data showed that a normal function of BK channels is important for resting blood pressure homeostasis. Heart rate responses to exogenously induced changes in blood pressure were normal in BKß1 -/- mice, indicating that central nervous reflex mechanisms of blood pressure remained unaffected by the BKß1 disruption. However the absolute increase in blood pressure in BKß1 -/- mice was rather modest. Furthermore, the responses to ₁-adrenergic stimulation as well as to nitric oxide–induced vasodilation were identical in BKß1 +/+ and BKß1 -/- mice. This unexpected observation seems to argue against an important role of BK channels in VSMCs to limit and, respectively, to regulate vasoconstriction. Reports that BK channels are expressed at higher levels in conduction vessels compared with resistance levels and that administration of BK channel blockers had no effect³⁴ or relatively modest effects^{35 36} on the microcirculation under baseline conditions may support the idea that BK channels are not a major determinant for regulating vascular tone. Systemic vascular resistance is primarily determined by the VSMC tone of small arterioles. Therefore, our results obtained with isolated thoracic aortic rings might not be relevant for systemic blood pressure regulation in intact BKß1 -/- mice. Yet there is an important alternative interpretation for our blood pressure measurements in support of a significant role of the Ca²⁺ spark/STOC-linked feedback mechanism in vasoconstriction. Because intravasal pressure is a major determinant of membrane potential in small arteries and arterioles,^{9 37} the elevated blood pressure in BKß1 -/- mice may lead to a significant positive shift in the VSMC BKß1 -/- membrane potential. Consequently, BKß1 -/- mice with elevated blood pressure may have regained an efficient Ca²⁺ spark/STOC coupling and the associated feedback mechanism to limit vasoconstriction on exposure to agonists such as phenylephrine. In this view, the BKß1 -/- mice may have acquired an elevated basal blood pressure to match the shifted voltage range in Ca²⁺ spark/STOC coupling, which we observed in the in vitro experiments.

Our conclusions about the role of the BKß1 subunit in vasoregulation are in good agreement with recent data obtained with another null allele of the BKß1 subunit locus.³⁸ In this case, the second exon of the BKß1 subunit gene was replaced in 129 svj mice. Electrophysiological recordings on inside-out patches of cerebral artery myocytes showed that BK channels in 129 svj BKß1 -/- preparations did not open at -40 mV but had at +40 mV an open probability comparable to the one in wild-type 129 svj preparations. A direct measurement of the coupling of Ca²⁺ sparks and BK currents showed that Ca²⁺ sparks in 129 svj BKß1 -/- preparations often failed to evoke a detectable BK current. The results fit nicely to our observation that C57BL6 BKß1 -/- cerebral artery myocytes had a normal Ca²⁺ spark frequency but a much-reduced STOC frequency. Likewise, cerebral arteries of BKß1 -/- 129 svj mice were significantly more constricted at a given pressure than were control arteries.³⁸ Interestingly, 129 svj mice have a higher mean arterial blood pressure than any other mouse strain.³⁹ Nevertheless, 129 svj BKß1 -/- mice also had an elevated blood pressure in comparison to controls (134±5.1 mm Hg; n=6 versus 114±6.0 mm Hg; n=6).³⁸ This result supports our conclusion that differences in blood pressure between BKß1 +/+ and BKß1 -/- mice were maintained over a wide range of blood pressure levels. Furthermore, the results demonstrate that the different genetic backgrounds in the BKß1 -/- mice did not influence the observed increases in blood pressure.

	Acknowledgments

 

This work was supported by a grant of the European community (Biomed2-PL962118) and the Deutsche Forschungsgemeinschaft.

Received November 1, 2000; revision received November 3, 2000; accepted November 6, 2000.

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