BK Channels in Cardiovascular Diseases and Aging

Abstract

Aging is a major risk factor for cardiovascular diseases, one of the main world-wide causes of death. Several structural and functional changes occur in the cardiovascular system during the aging process and the mechanisms involved in such alterations are yet to be completely described. BK channels are transmembrane proteins that play a key role in many physiological processes, including regulation of vascular tone. In vascular smooth muscle cells, BK opening and the consequent efflux of potassium (K⁺) leads to membrane hyperpolarization, which is followed by the closure of voltage-dependent Ca²⁺ channels, reduction of Ca²⁺ entry and vasodilatation. BK regulates nitric oxide-mediated vasodilatation and thus is crucial for normal endothelial function. Herein we will briefly review general structural properties of BK and focus on their function in the cardiovascular system emphasizing their role in cardiovascular aging and diseases.

Aging of the cardiovascular system is intimately associated with cardiovascular diseases, one of the main world-wide causes of death (World Health Organization – http://www.who.int/en/). During the aging process, several structural and functional changes occur in the cardiovascular system including arterial stiffness, dilation of central elastic arteries and endothelial dysfunction [1]. These changes occur independently of other risk factors such as arterial hypertension, diabetes or hypercholesterolemia [2] and can be initially manifested either at the endothelium or at the smooth muscle cells layer. Alterations in signaling transduction pathways and abnormal communication between endothelial cells and smooth muscle cells have been reported during aging [3]. The knowledge of the mechanisms involved in cardiovascular aging is determinant to the development of therapeutic approaches aimed to decrease the incidence of age-related cardiovascular diseases. New therapeutic approaches would also diminish the impact of aging on overall quality of life, since cardiovascular diseases are implicated in increased risk of defective motor capacity and cognitive frailty [4]. Obviously, several key processes are involved in the functional changes of cardiovascular aging, including oxidative stress, inflammation and the before mentioned endothelial dysfunction. All of these processes can directly or indirectly influence and alter vascular relaxation and contraction.

Ion channels expressed in both endothelial and vascular smooth muscle cells are crucial elements setting the vascular tone status. More specifically, vascular smooth muscle cells express at least four different types of potassium (K⁺) channels, one or two types of voltage-gated calcium (Ca²⁺) channels, at least two types of chloride (Cl⁻) channels, store-operated Ca²⁺ (SOC) channels, and stretch-activated cation (SAC) channels in their plasma membranes, all of which may be involved in the regulation of vascular tone [5]. Experimental evidence indicates that at least one subtype of K⁺ channel, the BK channel, the large-conductance Ca²⁺-activated K⁺ channel, has its expression and function altered with aging. Accordingly, this review will focus on the mechanistic aspects by which BK operates in the cardiovascular system and the main changes undergone by BK during aging.

K⁺ channels

Potassium channels are transmembrane proteins that play important roles in several physiological processes including vascular tone [6–10], urinary bladder tone [11–14], neurotransmitter release [15–19], hormone secretion [20–22], respiratory tone [23–25] and cell excitability [26]. Potassium channels contribute to set the membrane voltage at resting or hyperpolarized potentials by increasing the efflux of K⁺ from the cytoplasm. Four main types of K⁺ channels have been described in vascular smooth muscle cells: voltage-dependent K⁺ channels (K_v), BK, ATP-sensitive K⁺ channels (K_ATP) and inward rectifier potassium channels (K_ir) [27, 28]. In the vascular smooth muscle, the opening of K⁺ channels leads to membrane hyperpolarization, which is followed by the closure of voltage-dependent Ca²⁺ channels, reduction of Ca²⁺ entry and vasodilatation [27]. The two most prevalent K⁺ channels expressed in vascular smooth muscle are the K_v channels and the BK [29]. Amongst these, BK has been shown to be age-regulated by not fully described mechanisms (please, refer to the BK in cardiovascular aging section).

Calcium-activated K⁺ channels

The history of Ca²⁺-activated K⁺ channels dates back to the 1970’s, when Meech first reported in 1972 that intracellular Ca²⁺ injections lead to an increase in K⁺ conductance in nerve cells [30]. Currently, three major categories of Ca²⁺-activated K⁺ channels have been described and classified according to their unitary conductance: large conductance, BK, with 100–300 Ps [31]; intermediate-conductance, IK, with 25–100 pS [32, 33], and small conductance, SK, with 2–25 pS [34, 35]. BK channels (also known as slo or maxiK) were the first ones in the family to be identified [31]. The open probability of these channels is determined by the membrane voltage, intracellular Ca²⁺ concentration ([Ca²⁺]_i) and also by the intracellular Mg²⁺ concentration [36, 37]. Activation of BK leads to fast repolarization of the membrane and therefore to the closure of voltage-dependent Ca²⁺ channels, resulting in decreased Ca²⁺ entry into the cell and also increased Ca²⁺ extrusion by the Na⁺-Ca²⁺-exchanger. Thus, its primary function is to exert a negative feedback on the membrane potential and on [Ca²⁺]_i[38]. As a result, BK are important participants in phenomena such as the setting of interspike intervals and spike frequency adaptation [39–44].

Structure of BK

The BK channel is formed by two interacting proteins, the α- and β-subunits. The α-subunit, coded by the gene slowpoke, constitutes the pore itself (four subunits are needed to form a functional channel) and it contains both the voltage sensor module and the Ca²⁺ binding domain. It is composed of eleven helical segments: S0–S6 (N-terminal, transmembrane spanning – constitute the “core” of the channel) and S7–S10 (C-terminal, cytoplasmic – constitute the tail of the channel) [29]. The S4 segment confers the voltage sensitivity to the channel. Depolarization causes the movement of the charged residues (arginines, gating charges), which induces conformational changes in the activation gate (S6) of each monomer, causing the movement of the gates and ultimately opening of the channel (when all the four gates are opened) [45].

The assembly of four α-subunits alone can form a functional channel pore [46], which contains the selectivity filter formed by the four P-loop segments (reentrant linker between S5 and S6 transmembrane segments). The selectivity filter is the narrowest part of the pore and despite this geometric configuration, K⁺ ions can flow in high flux rate, comparable to its diffusion rates in water. It is formed by a high conserved TVGYG sequence of aminoacids, which gives a 1000-fold K⁺ conductance over other monovalent cations [47].

The α-subunit has multiple regulatory sites in the C-terminal region (tail) including a sequence of aspartic amino acids that binds to Ca²⁺, known as the Ca²⁺ bowl, that confers the sensitivity to Ca²⁺[48]. The tail also contains phosphorylation sites for cAMP- and cGMP-dependent protein kinases [49], protein kinase C (PKC) and tyrosine kinases [50] (Figure 1).

Figure 1.

Schematic representation of BK channel structure. Adapted from J. Cui, H. Yang and U. S. Lee. Molecular mechanisms of BK channel activation. Cell. Mol. Life Sci. 66 (2009) 852 – 875.

The β-subunits are regulatory proteins that have the major function of enhancing the Ca²⁺ sensitivity of the channel [51–53]. The β-subunit is composed by two transmembrane segments with a long extracellular loop and cytoplasmic C- and N-termini [54, 55]. The extracellular loop contains cystein residues that form disulfide bonds, which permit the binding of toxins such as charybdotoxin [56]. There are four β-subunit isoforms (β1–β4) that are differentially expressed in tissues. Particularly in vascular smooth muscle cells, the β1 isoform is the most expressed [57, 58].

The association between the α- and β-subunits occurs in a 1:1 ratio [59] via the first transmembrane domain (S0) in the α-subunit and the extracellular N-terminal from the β-subunit. This association results in increased channel sensitivity to Ca²⁺and to voltage [52, 55, 56, 58, 60].

BK activation mechanisms

Under experimental conditions BK can be activated by voltage in the presence or absence of Ca²⁺ or Mg²⁺. It is currently known that the voltage- and Ca²⁺-dependence properties of the channels are independent of each other [61], meaning that both enhance the probability of the channel to open. As the [Ca²⁺]_i increases, the open probability is shifted to more negative potentials (Figure 2). In response to depolarization, the voltage sensors in BK move along the electric field of the membrane giving rise to gating currents [62, 63]. However gating currents are not mandatory for the channel to open [38].

Figure 2.

Schematic representation of the G–V (conductance-voltage) relationship curves representing the activation of BK channels in low and high intracellular [Ca²⁺]_i. The increase in [Ca²⁺]_i shifts the G–V curve towards negative voltage values. Adapted from J. Cui, H. Yang and U. S. Lee. Molecular mechanisms of BK channel activation. Cell. Mol. Life Sci. 66 (2009) 852 – 875.

Calcium can bind to two high-affinity binding sites in the cytoplasmic region of the α-subunit (K_d: 0.8∼11μmol/L) [64–68]. In contrast, Mg²⁺ binds to a low-affinity metal binding site in millimolar concentrations and it acts in the same manner as Ca²⁺ does, although with smaller affinity. The binding of Ca²⁺ first leads to gate opening with a small facilitating effect on voltage sensor activation [62, 63]. Calcium binding increases the open probability and shifts the conductance-voltage (GV) curve to more negative voltages. Similarly, millimolar concentrations of Mg²⁺ also increase the open probability and shift the G–V relationship to more negative voltages. However, experimental evidence shows that Mg²⁺ and Ca²⁺ act by different mechanisms in BK activation [69]. Whereas Ca²⁺ activates the channel in a voltage sensor-independent manner [70], Mg²⁺ does it in a voltage sensor-dependent way, by interacting electrostatically with the voltage sensor [71].

Regulatory mechanisms of BK

Regulation of BK can occur either by pre- or post-translational mechanisms. The α-subunit transcript undergoes extensive splicing resulting in diverse BK isoforms that differ in their sensitivity to both Ca²⁺ and voltage [6]. Moreover, splice variation can regulate BK targeting to different subcellular compartments [72], which suggests that there might be diverse trafficking partners for BK. On the other hand, BK can be the target of several post-translational modifications such as oxidation-reduction reactions [73], glycosylation [54] and phosphorylation [74,75]. In addition, the association of the α-subunit with different regulatory subunits might result in different pharmacological properties changing the voltage dependence, Ca²⁺ sensitivity and the kinetics of the assembled channels (reviewed elsewhere [26]).

Another important mechanism in the regulation of BK is protein-protein interaction. Increasing evidence indicates that BK, particularly in its intracellular domain, may serve as an anchor port where several proteins (such as protein kinases, L-type Ca²⁺ channels, β2-adrenergic receptor) bind in order to exert their function [76].

BK in the cardiovascular system

The expression of BK subunits in the cardiovascular system is heterogeneous. The α-subunit is found in many cell types (endothelial [77–79], vascular smooth muscle [80], cardiac fibroblast [81]), but it has not been described in its canonical form in cardiomyocytes. Actually, only an unique isoform of the α-subunit has been recently described in mouse-cardiomyocytes [82]. On the other hand, β-subunits are expressed in the heart and vascular smooth muscle [6]. However, they are probably not expressed in the endothelium since β1-subunits are not detected by RT-PCR or western blot analysis in this tissue [83].

In the vascular system, BK channels are considered key players in the maintenance of normal vasomotor tone by acting on the regulation of the excitation-contraction coupling mechanism [26]. Particularly, in vascular smooth muscle cells, BK are localized in close apposition to ryanodine receptors in the sarcoplasmatic reticulum [26]. The augmentation in [Ca²⁺]_i by Ca²⁺-induced Ca²⁺ release mechanisms leads to BK activation [84]. This phenomenon drives the membrane potential to more negative values, closing voltage-dependent Ca²⁺ channels that supply Ca²⁺ to initiate depolarization and contraction, and resulting in smooth muscle relaxation [26]. Accordingly, BK channels have been considered an interesting therapeutic target for the treatment of cardiovascular diseases.

Vasoactive substances can also regulate BK. Angiotensin II and endothelin-1 inhibit BK in coronary arteries [85–87] by PKC-independent mechanisms. In contrast, more recent data show that PKC phosphorylation inhibits BK in vascular smooth muscle [9, 88]. To note, activation of Src-kinases (effectors of angiotensin II effects) stimulate BK in HEK 293 cells [89], suggesting a possible complex regulation of BK by angiotensin II [86]. Likewise, potentiation of BK by α5β1 integrin is mediated by c-Src phosphorylation of the channel α-subunit at residue Tyr-766 [90].

Adenosine and β-adrenoceptor agonists activate BK through cAMP-dependent [76] and cAMP-independent [91] pathways. BK can also be activated by direct interaction of the α-subunit with G_αs subunits (reviewed elsewhere [92]). Particularly in vascular smooth muscle cells, increased cGMP levels and activation of protein kinase G, by nitric oxide (NO), result in BK phosphorylation and ultimately its activation [93].

In addition, a role for reactive oxygen species (ROS) in the regulation of BK has also been described. The open probability and Ca²⁺-sensing properties of the channel are changed by ROS in vascular smooth muscle cells [94]. In endothelial cells, hydrogen peroxide can open BK either by direct mechanisms or by cleavage of arachidonic acid [95]. In contrast, hydrogen peroxide inactivates BK [96]. Activation of BK by arachidonic acid metabolites occurs indirectly by activation of transient receptor potential (TRP) channels [97]. Particularly in cerebral arteries, Ca²⁺ influx via TRPV4 stimulates ryanodine receptors resulting in increased [Ca²⁺]_i and activation of BK [98] (Figure 3).

Figure 3.

Schematic representation of mechanisms involved in BK regulation in vascular smooth muscle cells.

BK in cardiovascular diseases

Altered properties of BK have been reported in different pathological conditions, such as arterial hypertension, hypoxia and diabetes [99–101]. Among cardiovascular diseases, BK functions have been most studied in arterial hypertension. The first evidences of a direct role of BK in the control of blood pressure were described by Brenner et al in 2000 [6], who reported that the mean arterial pressure of knockout animals for the β1-subunit was elevated by 21 mmHg. Later other studies showed that the β1 gene is involved in the manifestation of a hypertensive phenotype [102–106]. Contrasting evidences are also reported. Knockout mice for the BK β1-subunit have also been shown to present similar mean arterial pressure (recorded for 24 hours) and heart rate when compared to control [107]. Interestingly, a single point-mutation (E65K) of the human β1-subunit, which results in a gain of function of the channel, exerts a protective effect in diastolic hypertension [108]. Although a correlation between β1-subunit deficiency and hypertension has been described, an opposite correlation has been shown for the α-subunit. It has been reported augmented α-subunit protein expression in vascular smooth muscle cells isolated from spontaneously hypertensive rats [109]. In septic conditions (after lipopolysaccharide injection - 20 mg/Kg), knockout mice for the BK β-subunit present more rapid fall in heart rate and in blood pressure levels when compared to wild type [110], approximately six hours after injection. On the other hand, knockout mice for the BK α-subunit have similar hypotensive response when compared to wild type in a fecal slurry model of sepsis [111].

Non-genetic models of hypertension have also been associated to a dysfunction of the BK. Down-regulation of β1-subunits is observed in vascular smooth muscle cells from angiotensin II-induced hypertension animals [104]. It has also been recently demonstrated that in isolated mesenteric arteries from DOCA-salt hypertensive rats there is an increase of the α-subunit which is accompanied by a decrease in the β-subunit. In addition, there is increased expression of receptor for activated C kinase 1 (RACK1), known as a binding protein of BK and a negative modulator of BK activity [112]. Moreover, the vasodilator effects of NO are partly mediated by activation of vascular BK and, in hypertension, NO-mediated BK signaling is down-regulated [113]. Although BK mRNA transcripts are decreased in a whole body irradiation animal model of arterial hypertension, in vivo gene silencing of the same transcripts does not alter blood pressure levels which suggest that it is unlikely that BK play a major role in radiation-induced arterial hypertension [114].

In addition to high blood pressure, functional changes in BK have been described in other pathological conditions. Hypoxia does not seem to affect the expression of BK β1-subunit in cardiomyocytes but it leads to decreased glycosylation levels of this subunit [115]. BK function is diminished in hypertrophy [116], but is unaffected in ischemia [117]. However, mice lacking BK α-subunit present larger infarct volume when compared to wild types in a middle cerebral artery occlusion model of cerebral ischemia [118]. BK might play a role in preconditioning: treatment of mice with a BK activator (NS1619) leads to reduction on infarct size in a coronary artery occlusion model [119].

BK activity increases in endothelial and vascular smooth muscle cells with progressive hypercholesterolemia and atherosclerosis, probably by oxidized low-density proteins-dependent mechanisms [83]. Furthermore, vascular smooth muscle cells from human atherosclerotic aorta display two kinetically different single channel patterns [120]. One of these has a fourfold lower mean open time and decreased Ca²⁺ sensitivity. Interestingly, BK with these same characteristics are found in vascular smooth muscle cells from fetal human aorta [121].

Additionally, in streptozotocin-induced diabetic mice there is an increase in BK open probability accompanied by a decrease in BK single channel conductance [122]. BK has been show to mediate capsaicin-induced vasorelaxation in isolated pressurized mouse coronary microvessels which is impaired in diabetic db/db mice [123]. In insulin-resistance, while there are no alterations in BK expression or in its Ca²⁺ and voltage sensitivity, there is a clear reduction in the K⁺ current density through BK of the microvasculature [124].

Alterations in BK function have also been reported in subarachnoide hemorrhage (SAH). BK currents are attenuated in rats under SAH when compared to control which were accompanied by decreased BK activity and β1-subunit expression. However, inside-out patches did not reveal any differences in the conductance and voltage sensitivity of the BK channels [125]. Accordingly, it has been reported the reduction in Ca²⁺ spark-induced transient BK currents, without change in BK channel density or single channel properties in SAH model rabbits [126]. BK has been shown to mediate constriction in response to augmentation of external K⁺ in brain slices from SAH model rats [127], an effect reversed by inhibition of BK, leading to vasodilatation.

BK in cardiovascular aging

Aging is associated with changes in the structure and function of blood vessels leading to cardiovascular diseases. Hyperactivity of the coronary arteries, which can cause intense and sudden coronary spasm, is commonly observed in the elderly [3]. Studies on BK functionality in aging are still preliminary and little is known about BK role in cardiovascular aging. BK expression is diminished in coronary arteries from old rats. However, there are no changes in its biophysical and pharmacological properties [128]. Importantly, exercise training has been shown to attenuate the decreased BK expression in vascular smooth muscle cells from coronary arteries in old rats [129]. In contrast, even though aging decreases cerebral blood flow due to an increased arterial tone, the expression, kinetics or Ca²⁺ sensitivity of BK are not altered in cerebral vascular myocytes from old rats [130]. In small mesenteric arteries, BK contribute to acetylcholine-induced vasorelaxation and are at least partially responsible for the characteristic age-related endothelial dysfunction [131]. Inhibition of K⁺ channels impairs isoprenaline-induced vasorelaxation in a higher extension in aged rats (54 weeks-old) than in 8 weeks-old animals [132]. BK is also involved in the decreased 11,12-epoxyeicosatrienoic acid-mediated corpus cavernosum relaxation in old rats [133]. BK expression is decreased in arterioles of the soleus and gastrocnemius muscles from old rats, and it seems to play a role in the regulation of the vascular tone in soleus muscle [134]. In humans, particularly in women, increasing age upmodulates the protective effect of the E65K allele against moderate-to-severe diastolic hypertension [108].

Conclusions

BK channels can be regulated by several stimuli that allow them to work as integrators of cell signaling, excitability and metabolism. In the vascular system they play a crucial role in the maintenance of vascular tone. Accordingly, BK channels are potential targets for therapeutic approaches. Aging is a key factor in the development of cardiovascular diseases. Abnormal BK expression and activity observed in experimental animal models indicate that these channels are possible mediators of the functional changes seen in aged blood vessels. Thus, BK can be targeted to decrease the incidence of cardiovascular diseases in the elderly population. The mechanisms whereby dysfunction of BK contributes to the impairment of vascular function are still unclear and further investigation is required to determine the role of BK in aging and disease.