Smooth muscle BK channel activity influences blood pressure independent of vascular tone in mice

Abstract

The large conductance voltage- and Ca(2+)-activated K(+) (BK) channel is an important determinant of vascular tone and contributes to blood pressure regulation. Both activities depend on the ancillary BKβ1 subunit. To determine the significance of smooth muscle BK channel activity for blood pressure regulation, we investigated the potential link between changes in arterial tone and altered blood pressure in BKβ1 knockout (BKβ1(-/-)) mice from three different genetically defined strains. While vascular tone was consistently increased in all BKβ1(-/-) mice independent of genetic background, BKβ1(-/-) strains exhibited increased (strain A), unaltered (strain B) or decreased (strain C) mean arterial blood pressures compared to their corresponding BKβ1(+/+) controls. In agreement with previous data on aldosterone regulation by renal/adrenal BK channel function, BKβ1(-/-) strain A mice have increased plasma aldosterone and increased blood pressure. Consistently, blockade of mineralocorticoid receptors by spironolactone treatment reversibly restored the elevated blood pressure to the BKβ1(+/+) strain A level. In contrast, loss of BKβ1 did not affect plasma aldosterone in strain C mice. Smooth muscle-restricted restoration of BKβ1 expression increased blood pressure in BKβ1(-/-) strain C mice, implying that impaired smooth muscle BK channel activity lowers blood pressure in these animals. We conclude that BK channel activity directly affects vascular tone but influences blood pressure independent of this effect via different pathways.

Figure 1. Recordings from BK channels with different subunit composition

BKα, BKαβ1 and BKαβ1-E channels were recorded in the patch-clamp inside-out configuration after cRNA injection into oocytes of Xenopus laevis. Recordings were conducted under symmetrical potassium concentrations and at an intracellular free calcium concentration of 10 μmol l⁻¹. A, representative recordings of BK channels consisting of BKα subunits, alone or after co-injection with sixfold molar excess of BKβ1 or BKβ1-E cRNA. The pulse protocol is shown at the upper left. Scale bars: 2 nA/50 ms. B, normalized tail currents, illustrating the effect of BKβ1 and BKβ1-E on BK channel inactivation kinetics. Tail traces were recorded at a membrane potential (V_M) of −80 mV after a V_M = +80 mV depolarization step as shown in A (n = 4 per group). C, V_M for half-maximal conductance (V_½) of BKα, BKαβ1 and BKαβ1-E channels. G–V curves were plotted from the recordings depicted in A, V_½ was calculated from best Boltzmann sigmoidal fits (G/G_max = 1/[1 + e^{(V½ − V)/dV}]) and then averaged (n = 4). Error bars: SEM.

Figure 2. Expression profile of transgenic BK channel subunit β1-E

A, representative mRNA expression pattern of the β1-E transgene for two founder lines (R9 and R13) and wild-type controls (WT) measured by real-time PCR. No RT, negative control without reverse transcription. HT, heart; AO, aorta; LU, lung; SP, spleen; KI, kidney; BL, bladder; LI, liver; BR, brain; SK, skeletal muscle. Total RNA was prepared from organs and used as template for real-time PCR as described in the Methods. B, β1-E mRNA compared to endogenous BKβ1 mRNA expression strength by quantitative real-time PCR. Both transcripts were amplified from the same samples. Samples were AO, KI, BL and colon from three BKβ1^+/+ β1-E transgenic mice of founder line R9. C, β1-E protein expression in bladder of BKβ1 R9 and BKβ1 R13 mice. Detection was by densitometry of chemoluminescence on anti-GFP Western blots normalized to anti-β-actin signals. n, number of animals. R9, BKβ1^−/− β1-E mice (founder 9); R13, BKβ1^−/− β1-E mice (founder 13); n.s., difference not statistically significant. Welch's paired t test was used for B and Mann–Whitney U test was used for C.

Figure 3. Smooth muscle-specific expression of BKβ1 in BKβ1 R mice

A, scheme showing BKβ1^+/+, BKβ1^−/− and BKβ1 R genotype. BKβ1^−/− mice have two knockout (ko) alleles of the BKβ1 coding gene Kcnmb1 that lack exon 1 and exon 2 of the wild-type (wt) allele. BKβ1 R mice have two ko alleles and a BKβ1 rescue transgene coding for a BKβ1-EGFP fusion protein under control of a 5.4 kb SM-specific Acta2 promoter fragment. Not indicated are introns in the promoter and poly A untranslated region. Schemes are drawn to the same scale (Acta2 promoter shortened for clarity). B, coronal kidney sections of BKβ1 R9 mice. Smaller (top) and larger (bottom) blood vessels, tubules and glomeruli are shown. Left: anti-GFP staining (green, γ = 0.5) and nuclei visualized by DNA stain (blue). Scale bars = 100 μm. Middle: magnifications of boxed areas (100 × 100 μm). Right: bright-field images. *Periglomerular myofibroblast. Kidney sections of BKβ1 R13 mice showed the same expression pattern (C). D, kidney sections of BKβ1^+/+ control mice, prepared and depicted as in B and C. Images are representative of n = 3–6 animals.

Figure 4. Rescue of smooth muscle BK channel function in BKβ1 R mice

A, representative inside-out patch clamp recordings of BK channels from vascular myocytes. Single channels were recorded under symmetrical potassium at membrane potentials (V_M) of −40 and +40 mV and 1, 3 and 10 μmol l⁻¹ free internal Ca²⁺. Arrowheads mark baseline. For statistical data and fits see Table 1. B, the BKβ1^−/− strain A is an inbred strain with a different genetic background (129S1/129X1/C57BL/6) than that of the BKβ1 R strain (DBA/C57BL/6). Therefore, BKβ1^−/− strain A and BKβ1 R mice were backcrossed repeatedly to the inbred strain C57BL/6J (strain B). C, aortic tone under α₁-adrenergic stimulation in BKβ1^−/− strain B mice and controls measured in perfused organ baths. Shown is tone above basal (8 mN) of 3.0 ± 0.1 mm long aortic rings in response to increasing concentrations of phenylephrine. n, number of animals. Mouse genotypes: +/+, BKβ1^+/+; −/−: BKβ1^−/−; R9, BKβ1 R9; R13, BKβ1 R13. Error bars: SEM. Two to three rings were measured per animal. D, endothelium-dependent vasorelaxation in strain B mice. Shown is relative relaxation back to basal from maximal tone at 100 μmol l⁻¹ phenylephrine in response to 10 μmol l⁻¹ acetylcholine.

Figure 5. Phenotypic parameters of BKβ1^−/− mice on two different genetic backgrounds

BKβ1^−/− and BKβ1^+/+ mice with strain A or strain B genetic background were investigated. For experimental details see Methods. A, telemetric recordings of mean arterial pressure (MAP). B, heart weight (HW) normalized to body weight (BW). C, plasma aldosterone concentration. D, serum K⁺ concentration. E, serum Na⁺ concentration. F, telemetric MAP recordings of BKβ1^−/− strain A mice (n = 7) and BKβ1^+/+ strain A controls (n = 4). Arrow denotes s.c. implantation of spironolactone 14-day release pellets. n, number of animals. +/+, BKβ1^+/+ mice; −/−, BKβ1^−/− mice. *Statistically significant difference; n.s., difference not statistically significant (ANOVA, post hoc: Bonferroni-corrected). Error bars: SEM.

Figure 6. Phenotype of BKβ1^−/− strain C mice

Strain C is an inbred C57BL/6J × strain A hybrid strain. For experimental details see Methods. A, telemetric recordings of mean arterial pressure (MAP). B, aortic tone under maximal α₁-adrenergic stimulation (100 μmol l⁻¹ PE). C, heart weight (HW) normalized to body weight (BW). D, plasma aldosterone concentration. E, serum K⁺ concentration. F, serum Na⁺ concentration. n, number of animals. G, BK channel open probabilities in vascular myocytes of BKβ1^−/− mice, BKβ1^+/+ controls and BKβ1 R9 mice backcrossed to strain C. Measurements were performed as described for Fig. 4A. Data points denote open probabilities (P_O) calculated from peak area integrals of current histograms of single channel recordings. Curves denote best free Boltzmann sigmoidal fits (P_O = 1/[1 + e^{([Cai]½ − [Cai])/dCa}] ). Mouse genotypes: +/+, BKβ1^+/+; −/−, BKβ1^−/−; R9, BKβ1 R9. *Statistically significant difference (ANOVA, post hoc: Bonferroni-corrected). Error bars: SEM.

Figure 7. Scheme integrating the blood pressure phenotypes of BKβ1^−/− strain A, B and C mice

BKβ1^+/+ control mice displayed increasing mean arterial blood pressure and plasma aldosterone levels from strain A over strain B to strain C. Loss of BKβ1 expression resulted in different phenotypic outcomes depending on strain. In BKβ1^−/− strain A mice, plasma aldosterone concentration was increased relative to BKβ1^+/+ controls, associated with a hypertensive phenotype that was completely reversible by mineralocorticoid receptor block, indicating a predominantly renal effect. Conversely, BKβ1^−/− strain C mice showed a hypotensive phenotype that was alleviated in conjunction with rescue of vascular BK channel activity, indicating this effect originates in smooth muscle. Arrows do not imply direct effects.