Function of BKCa Channels Is Reduced in Human Vascular Smooth Muscle Cells From Han Chinese Patients With HypertensionNovelty and Significance
Chronic hypertension is associated with an impaired vascular relaxation caused by an increased vascular tone; however, the underlying mechanisms are not fully understood in human patients. The present study was to investigate whether large-conductance Ca2+- and voltage-activated K+ (BKCa) channels are involved in dysfunctional relaxation of artery in Han Chinese patients with hypertension using the perforated patch clamp, inside-out single-channel, and macromembrane patch recording techniques to determine whole-cell current, spontaneous transient outward current, open probability, and Ca2+ sensitivity and the reverse transcription polymerase chain reaction and Western blot analysis to examine the gene and protein expression of α-subunit (KCa1.1) and β1-subunit (KCNMB1) of BKCa channels in isolated human vascular smooth muscle cells and mesenteric arteries from normotensive and hypertensive patients. It was found that whole-cell current density, spontaneous transient outward current, and Ca2+ sensitivity, but not single-channel open probability and slope conductance, were significantly decreased in vascular smooth muscle cells from patients with hypertension. Interestingly, mRNA and protein levels of KCNMB1, but not KCa1.1, were reduced in the arterial tissue from patients with hypertension. These results demonstrate for the first time that whole-cell current, spontaneous transient outward current, and Ca2+ sensitivity of BKCa channels are reduced in human vascular smooth muscle cells, which resulted from downregulation of β1-subunit of the channel. This may account, at least in part, for the dysfunction of artery relaxation in Han Chinese patients with primary hypertension.
It is well known that chronic hypertension is associated with increased morbidity and mortality from stroke, coronary artery disease, congestive heart failure, renal disease, and so on.1,2 Arterial tone is persistently increased as a result of malfunction of vessel relaxation in chronic hypertension.2 It is well recognized that arterial tone is regulated by functional balance of the ion channels responsible for cellular depolarization and hyperpolarization. The increased arterial tone is mainly related to depolarization of smooth muscle, which may have resulted from dysfunction of ion channels responsible for cell membrane hyperpolarization.3 The membrane depolarization activates voltage-dependent l-type Ca2+ channels, induces an increase in Ca2+ influx and global intracellular Ca2+ level, and causes vessel constriction.4,5 It is believed that large-conductance Ca2+-activated K+ (BKCa) channels play an important role in hyperpolarization of vascular smooth muscle cells (VSMCs).6 BKCa channels are activated by intracellular local Ca2+ release events through ryanodine receptors (Ca2+ sparks) from the sarcoplasmic reticulum and subsequently induce a hyperpolarization that opposes vasoconstriction.6–8
The Ca2+ sparks are highly localized and short-lived Ca2+ transients, which is a local Ca2+ signaling to induce spontaneous transient outward currents (STOCs) in VSMCs and neurons, and mediate different physiological functions.8,9 In VSMCs, functional coupling of sparks to STOCs hyperpolarizes the membrane potential, which in turn closes the voltage-dependent l-type Ca2+ channels, decreases global [Ca2+]i, and induces vascular relaxation.7 Studies from animal models demonstrated that a reduced activity of Ca2+ sparks and STOCs is involved in an increase of vascular tone in hypertension.10–12 However, little information is available in literature regarding BKCa channel activity in human arterial cells/tissue from patients with hypertension. The present study was therefore to investigate whether BKCa activity is altered in VSMCs isolated from mesentery arterial tissues of Han Chinese patients with hypertension using approaches of electrophysiology and molecular biology.
Materials and Methods
Human Arterial Tissue Collection and Cell Preparation
Mesenteric arterial tissues were collected from the removed tissues by abdominal operations in Han Chinese normotensive and primary hypertensive patients. The human tissue collection protocol was approved by the Ethics Committee of Luzhou Medical College. The characterization of the patients is shown in Table S1 in the online-only Data Supplement. Hypertension was defined as systolic blood pressure ≥140 mm Hg and systolic blood pressure ≥90 mm Hg according to the Chinese and international diagnostic criteria of hypertension guidelines.13,14 In the present study, informed consent was obtained from the patients for the use of vascular tissue (which is usually discarded).
Preparation of Human VSMCs
Signal VSMCs were enzymatically isolated using a procedure as described previously and in the online-only Data Supplement Methods.
Whole-cell current, STOCs, single-channel current, and macromembrane current of BKCa channels were recorded in human VSMCs with the techniques as described previously15,16 and in the online-only Data Supplement Methods.
Real-Time Reverse Transcription Polymerase Chain Reaction and Western Blot Analysis
Gene and protein expression of the BKCa α-subunit KCa1.1 and the β1-subunit KCNMB1 were determined using the procedure as described in the online-only Data Supplement Methods.
Data were expressed as means±SEM. The term n represented the number of cells or patients. Student t tests for independent test were used for statistical analysis. P<0.05 was considered statistically significant.
Macroscopic Current of BKCa Channels in Human VSMCs
Figure 1 shows the whole-cell macroscopic current recorded in representative cells with 400-ms voltage steps to between −50 and +60 mV from a holding potential of −60 mV. The oscillatory current was inhibited by the BKCa channel blocker iberiotoxin (200 nmol/L, Figure 1A), indicating typical BKCa current. BKCa current was greater in the cell from normotensive patient than that from hypertensive patients (Figure 1B). The amplitude of whole-cell BKCa current in response to the step-voltage depolarization was variable in individual cells, and we therefore normalized the current with cell membrane capacitance. Figure 1C illustrates the voltage-current (I-V) relationships of normalized BKCa current in VSMCs from normotensive and hypertensive patients. The current density (at 0 to +60 mV) was lower in VSMCs from hypertensive patients than that from normotensive patients (P<0.05 or P<0.01, hypertensive versus normotensive: n=90 cells/38 normotensive patients; n=51 cells/27 hypertensive patients). The cell membrane capacitance was 32.4±1.1 pF in cells from normotensive patients (n=90) and 34.0±1.5 pF in cells from hypertensive patients (n=51, P=NS). These results suggest that BKCa currents are downregulated in VSMCs from hypertensive patients.
Single-Channel Activity of BKCa Channels in Human VSMCs
To examine whether the decreased BKCa current is related to the reduced single-channel activity in human VSMCs from hypertensive patients, the single-channel activity was recorded in cell-attached or inside-out recording mode. Figure 2A and 2B shows the single-channel current of BKCa recorded at potentials from 0 to +60 mV in cell-attached mode and inside-out mode, respectively, in cells from normotensive or hypertensive patients. No difference was observed in single-channel activity, open probability (Figure 2C), or single-channel conductance (Figure 2D) in VSMCs from normotensive patients and hypertensive patients, either in cell-attached recording or inside-out recording. These results indicate that single-channel activity is not involved in the decreased BKCa current in hypertensive patients.
STOCs in Human VSMCs
It is well recognized that STOCs, as Ca2+ sparks, represent a local Ca2+ signaling in activation of BKCa channels mediating smooth muscle relaxation.7,8 We recorded STOCs in whole-cell voltage clamp mode to determine whether alteration in STOCs is involved in the decreased whole-cell BKCa current in VSMCs from hypertensive patients.
Figure 3 displays the STOCs recorded in human VSMCs from normotensive or hypertensive patients. STOCs were remarkably inhibited by 200 nmol/L iberiotoxin (Figure 3A). The activity of STOCs at potentials of 0 to −50 mV was lower in a cell from hypertensive patient than that from normotensive patient (Figure 3B). Mean values of voltage-dependent STOC frequency and amplitude were reduced in cells from hypertensive patients than those from normotensive patients. At 0 mV, STOC frequency was 5.0±0.4 Hz in cells from normotensive patients (n=64 cells/35 patients) and 2.6±0.3 Hz in cells from hypertensive patients (n=37 cells/25 patients, P<0.01), whereas the amplitude of STOCs was 23.5±2.3 pA in cells from normotensive patients (n=64 cells/35 patients) and 16.7±2.3 pA in cells from hypertensive patients (n=37 cells/25 patients, P<0.05). These results suggest that the decreased whole-cell current may be at least in part attributable to the reduced STOC activity of BKCa channels.
Ca2+ Sensitivity of BKCa Channels in Human VSMCs
Inside-out macropatch recording was used to determine whether alteration of Ca2+ sensitivity is involved in the reduced whole-cell BKCa current. Figure 4A illustrates the representative current traces recorded with 100 nmol/L Ca2+ in bath solution in a cell from normotensive patient and a cell from hypertensive patient. The current was elicited by 200-ms voltage steps to between −100 and +140 mV from a holding potential of −150 mV, then to −80 mV (to record the tail current). The inward tail current was measured in cells from normotensive and hypertensive patients. The normalized tail current (G/Gmax) was plotted against testing potentials (Figure 4B) and fitted to a Boltzmann function. The half activation potential (V1/2) of BKCa channels was clearly different in the cell from hypertensive patient than that from normotensive patient: the V1/2 of G/Gmax is positively shifted in the cell from hypertensive patient, indicating a reduced Ca2+ sensitivity. The effect of intracellular free Ca2+ on the G/Gmax of BKCa channels was determined using variable concentrations of free Ca2+ from 0.05, 0.1, 1.0 to 17.0 μmol/L CaCl2 in bath solution, respectively.
The variables of G/Gmax were fitted to a Boltzmann function in individual inside-out macropatch recordings with different concentrations of free Ca2+ concentrations. Figure 4C illustrates the mean values of V1/2 of BKCa activation conductance in response to variable concentrations of free Ca2+ in VSMCs from normotensive and hypertensive patients. The V1/2 of BKCa conductance was negatively shifted with an increase of free Ca2+ in bath solution in cells from normotensive patients or hypertensive patients. However, the V1/2 values of BKCa conductance were more positive at each concentration of free Ca2+ exposure in cells from hypertensive patients than those in cells from normotensive patients. These results indicate that the sensitivity of BKCa channels to Ca2+ is reduced in VSMCs from hypertensive patients.
mRNA and Proteins of BKCa α- and β1-Subunits
To investigate the molecular mechanisms underlying the reduced whole-cell current, STOCs, and Ca2+ sensitivity of BKCa channels in VSMCs from hypertensive patients, mRNA and proteins of the α-subunit KCa1.1 (KCNMA1 or Slo1) and the β1-subunit KCNMB1 of BKCa channels were determined using reverse transcription polymerase chain reaction and Western blot analysis in human mesenteric arterial tissues. The mRNA expression of KCNMB1, but not KCa1.1, was reduced in mesenteric arterial tissues from hypertensive patients (Figure 5A and 5B).
Figure 5C displays the Western blots of KCa1.1 and KCNMB1 proteins in human mesenteric arterial tissues from normotensive or hypertensive patients. As the observation in reverse transcription polymerase chain reaction for gene expression, the protein of KCa1.1 was not altered, whereas the KCNMB1 protein level was reduced in human mesenteric arterial tissues from hypertensive patients. Figure 5D illustrates the mean values of relative levels of mRNA to β-actin and protein levels to GAPDH of KCa1.1 and KCNMB1 in human mesenteric arterial tissues from normotensive or hypertensive patients. No difference in KCa1.1 mRNA or protein was observed in VSMCs from hypertensive and normotensive patients. However, KCNMB1 mRNA and protein were significantly reduced in the arterial tissues from hypertensive patients (P<0.05 or P<0.01 versus normotensive). These results indicate that the downregulation of KCNMB1 expression may be related to the reduced whole-cell current, STOCs, and Ca2+ sensitivity of BKCa channels in VSMCs from hypertensive patients.
In the present study, we demonstrate for the first time to our knowledge that whole-cell current of BKCa channels is decreased in VSMCs from hypertensive patients, which is associated with reduced STOCs and Ca2+ sensitivity of the channel, but not single-channel open probability. Biochemical analysis reveals that the reduced whole-cell current, STOCs, and Ca2+ sensitivity of BKCa channels likely result from the downregulation of the β1-subunit KCNMB1, but not the α-subunit KCa1.1 (or Slo) of the channel in Han Chinese patients with hypertension.
It is well recognized that BKCa channels are highly expressed in vascular smooth muscles and play a crucial role in vascular relaxation via an endogenous compensatory mechanism to buffer vasoconstriction, particularly in the intense myogenic constriction of resistance vessels exposed to high intraluminal pressures.17,18 The Ca2+-dependent relaxation is mediated by local Ca2+ release from the sarcoplasmic reticulum, termed as calcium sparks, which activates BKCa channels to induce STOCs. Therefore, BKCa channels in VSMCs are the key regulator in the vasoregulation by tuning vascular smooth muscle tone.17 BKCa channels may open in response to the depolarization of VSMCs and the rise in cytosolic Ca2+ concentration that occurs during arterial contraction, and the Ca2+ then enhances the open-state probability of the BKCa channel to produce VSMC relaxation19 by hyperpolarizing membrane potential, thereby preventing further Ca2+ influx. The physiological role of BKCa channels is protective against excessive vasoconstriction by a Ca2+-dependent relaxation mechanism.
The function of BKCa channels in VSMCs is finely tuned by its regulatory β1-subunit KCNMB1 via enhancing the channel sensitivity to intracellular local Ca2+. Animal experiments have demonstrated that dysfunction of KCNMB1 is associated with elevated blood pressure and left ventricular hypertrophy in mice.10,11 In spontaneously hypertensive rats, it has been observed that the β1-subunit of BKCa channels is reduced and the sensitivity of the channel to physiological changes in Ca2+ is decreased in VSMCs.12 These results support the notion that changes in the molecular composition of β1-subunit of BKCa channels may be a fundamental event contributing to the development of vascular dysfunction in hypertension.
Genetic epidemiological studies from a Spanish human population demonstrate that a gain of function of the β1-subunit KCNMB1 (E65K mutant) is associated with a low prevalence of diastolic hypertension.20 Their functional analysis of BK-β1E65K channel currents revealed a further negative shift in the G-V relationships, which becomes larger with progressive increases in intracellular Ca2+, presenting an increased activity of the channel at equivalent voltage and Ca2+ concentrations compared with the wild-type BKCa channel. However, interestingly, a recent study from Han Chinese population shows that a reduced function of BKCa channels with KCNMB1-rs11739136 is associated with essential hypertension susceptibility.21 The present study provides the novel direct evidence that function of BKCa channels is downregulated in VSMCs from Han Chinese patients with essential hypertension.
It is believed that Ca2+ sparks are tightly coupled to BKCa channels to create a hyperpolarization K+ current known as STOCs that oppose further vascular constriction.7 However, a sustained increase in arterial tone is always an essential component in the development of hypertension.2,22 Generally speaking, higher level of [Ca2+]i could induce a higher activity of BKCa channels so that the vascular dysfunction in hypertension might implicate a reduction in [Ca2+]i-BKCa channel coupling. Amberg et al12 found that the Ca2+ sparks-BKCa activation coupling was reduced in spontaneously hypertensive rats and that the sparks-STOC coupling efficiency was decreased as a result of the modulation of the subunit stoichiometry of BKCa channels in VSMCs in this animal model. This also happened in BKCa β1−/− mice11 and in insulin-resistant rats with hypertension.22 Similarly, in the present study we found that dysfunction of BKCa channels is related to reduced STOCs and the decreased sensitivity to physiologically relevant changes in [Ca2+]i in VSMCs from Han Chinese patients with hypertension, which resulted from the downregulation of the β1-subunit KCNMB1 of the channel. This result at least suggests that the decreased whole-cell current may be partly due to the reduced STOC activity of BKCa channels. However, it was obvious that the decrease of whole-cell BKCa current in hypertension does not just mean the reduction of STOCs.
Although whether loss of channel activity produces hypertension or whether hypertension causes the reduction in channel activity cannot be currently concluded, the present study provides the novel direct evidence that function of BKCa channels is downregulated in VSMCs from patients with essential hypertension. This study suggests that recovering the activity of BKCa channels by restoring β1 function may be a therapeutic approach to correcting vascular dysfunction in hypertension in humans.
A limitation of the present study was that we did not determine whether alteration of BKCa channel function in endothelial cells is involved in hypertension as a result of the shortage of human arterial specimens. It is interesting to make such observation in the future by collecting sufficient human vascular tissues.
The result of the present study demonstrated that BKCa activity is decreased in Han Chinese patients with hypertension. Our data indicate that the mechanism underlying dysfunction of BKCa channels in VSMCs is correlated to human hypertension. The decreased expression of the β1-subunit KCNMB1 is likely a common feature in hypertension. So this study suggests that recovering the activity of BKCa channels by restoring β1 function may be a therapeutic approach to correcting vascular dysfunction in hypertension in humans.
We thank Dr Gui-Rong Li and Dr Isao Inoue for manuscript writing and comments.
Sources of Funding
This work was supported by grants from the National Natural Science Foundation of China (No. 30670763 and No. 81173661 to Y. Yang and No. 30370527 to X.-R. Zeng).
The online-only Data Supplement is available with this article at http://hyper.ahajournals.org/lookup/suppl/doi:10.1161/HYPERTENSIONAHA.111.00211/-/DC1.
- Received September 19, 2012.
- Revision received November 13, 2012.
- Accepted November 14, 2012.
- © 2013 American Heart Association, Inc.
- Folkow B
- Rusch NJ
- Brayden JE,
- Nelson MT
- Nelson MT,
- Cheng H,
- Rubart M,
- Santana LF,
- Bonev AD,
- Knot HJ,
- Lederer WJ
- Cheng H,
- Lederer WJ
- Kim N,
- Chung J,
- Kim E,
- Han J
- Sobey CG
- Dimitropoulou C,
- Han G,
- Miller AW,
- Molero M,
- Fuchs LC,
- White RE,
- Carrier GO
Novelty and Significance
Whawt Is New?
The study demonstrated for the first time that whole-cell current, spontaneous transient outward currents, and Ca2+ sensitivity of BKCa channels are reduced in human vascular smooth muscle cells from hypertensive patients, which resulted from downregulation of β1-subunit of the channel.
What Is Relevant?
BKCa channels in vascular smooth muscle cells are the key regulator in the vasoregulation by tuning vascular smooth muscle tone. The function of BKCa channels in vascular smooth muscle cells is finely tuned by its regulatory β1-subunit via enhancing the channel sensitivity to intracellular local Ca2+. Changes in the molecular composition β1-subunit of BKCa channels may be a fundamental event contributing to the development of vascular dysfunction in hypertension.
The result of the present study demonstrated that BKCa activity is decreased in Han Chinese patients with hypertension. Our data indicate that the mechanism underlying dysfunction of BKCa channels in vascular smooth muscle cells is correlated to human hypertension. The decreased expression of the β1-subunit KCNMB1 is likely a common feature in hypertension.