**2. Vascular function**

In the vascular system, transmembrane voltage regulates vascular function. The interactions between vascular conditions and membrane potential are complicated [10]. The hyperpolarization of the smooth muscle cell membrane potential is evoked by the activation of ion channels, which contributes to vasodilation. A decrease in Ca2+ influx resulting from a decrease in the open probability of voltage-dependent calcium channels (CaV) and the CaV-dependent activation of the sarcoplasmic reticulum are crucial factors contributing to this process [11]. The depolarization of vascular smooth muscle cells causes contraction by opening CaV and inducing calcium release. The Ca2+-activated K+ channels (KCa) are considered key elements that control vascular tone and blood pressure by modulating membrane hyperpolarization and relaxation. Ca2+-activated K+ channels, including large conductance Ca2+ and voltage-activated K+ (BK) channels, intermediate conductance Ca2+-activated K+ channels (IK), and small-conductance Ca2+-activated K+ (SK) channels, are widely expressed in the vascular system. Intercellular conduction of electric signals underlies the spread of vasodilation to resistance arteries [10, 12].

#### **3. Ca2+-activated K+ channel**

#### **3.1 The structure of BK channel**

BK channels (also known as MaxiK) are widely expressed in vascular smooth muscle cells. Vascular BK channels comprise four pore-forming subunits (BK-α) and four auxiliary subunits: β1 subunits (BK-β1) and/or γ1 subunits (BK-γ1). BK α, which is encoded by the KCNMA1 gene, has seven transmembrane domains (S0–S6). BK-α has an extra transmembrane segment, S0, and thus its N-terminus is located in the extracellular space. S1–S4 form the voltage-sensor domain (VSD), and S5 and S6 form the ion permeation domain that encompasses the conserved K+ filter (TVGYG) in the pore loop. The C-terminus of BK channels modulates the voltage sensor and affects the pore, thus influencing channel opening. The ability of specific BK channels to open as a function of Ca2+ concentration or as a function of voltage sensors is due to the use of alternative splice sites [13–15]. The C-terminus contains two homologous structural units termed "regulators of conductance for K+ ": the proximal portion RCK domain (RCK1) and the distal portion RCK domain (RCK2).

**29**

*Potassium Channels in the Vascular Diseases DOI: http://dx.doi.org/10.5772/intechopen.82474*

**Figure 1.**

*channels activity.*

functional BK channels [15, 22, 23].

**3.2 The physiological function of BK channel**

The RCK1 domain is related to the formation of the Ca2+-binding site, and the RCK2 contains a "high-affinity" Ca2+ bowl domain. The "gating ring" is a Ca2+-sensing apparatus composed of four pairs of RCK1 and RCK2 domains, and the function of gating ring is responsible for allosteric activation of BK channel by Ca2+ binding [15–19](**Figure 1**). Four types of β subunits (BK-β) and four types of γ subunits (BK-γ) modulate almost all aspects of the pharmacological actions and physiological processes mediated by BK channels. The functional mechanism of BK channels regulated by β and γ auxiliary subunits is extremely complicated but is crucial to our understanding of its implications in vascular diseases. In the vasculature, BK-β1, which is encoded by KCNMB1, is the dominant isoform in VSMCs, and the dysfunction of the β1 is associated with diabetes, hypertension, and other vascular diseases. Knockout of the BK-β1 gene produces a remarkable decrease in the Ca2+ sensitivity of the channel. In addition, coexpression of the BK-β1 subunit with the BK-α subunit dramatically alters the calcium sensitivity, similar to the results observed in native VSMCs [15, 20, 21]. As an auxiliary subunit of the BK channels, BK-γ also affects BK channel activity by modulating the voltage and Ca2+ sensitivity. The BK-γ subunit has the ability to regulate vascular tone, and knockdown of BK-γ subunits contributes to pressure-induced vasoconstriction and a decrease in the activity of

*Schematic structure of one BKα-subunit consisting of 7 transmembrane domains (S0–S6). S1–S4 constitute the voltage-sensing unit, and the S5-P loop-S6 form the ion permeation domain. The Ca2+ bowl is a high-affinity* 

*region is responsible for Ca2+ sensitivity. The presence of the β1 subunit increases Ca2+ sensitivity and thus* 

*) in the C-terminal* 

*divalent cation-binding domain, and the RCK domain (regulator of conductance for K+*

Hundreds of proteins (such as β-catenin and caveolins) are reported to interact

with BK channels in various systems *in vitro* and/or *in vivo*. The mutual effect between BK channel and these proteins regulates the BK channel functions and influences the biological pathways mediated by BK channels [24–26]. However, a key characteristic of BK channels is their ability to couple with calcium channels that mediate the increase of intracellular Ca2+. BK channels can prevent Ca2+ channels from further activation and limit Ca2+ influx. In smooth muscle cells, ryanodine receptors cause local, transient calcium release events from the endoplasmic reticulum. These spontaneous calcium release events lead to the activation of nearby BK channels, which induce membrane hyperpolarization. This kind of potassium current is called the spontaneous transient outward current (STOC), and by blocking, STOC contributes to the increased vascular muscle tone [24, 27, 28].

Accordingly, BK channel is a key regulator to induce vasodilatation.

#### **Figure 1.**

*Vascular Biology - Selection of Mechanisms and Clinical Applications*

and heterogeneous compartment of the vessel wall [9].

channels, and voltage-gated K+

sensitive K<sup>+</sup>

**2. Vascular function**

cium release. The Ca2+-activated K+

and voltage-activated K+

**3. Ca2+-activated K+**

**3.1 The structure of BK channel**

K+

tion and relaxation. Ca2+-activated K+

regulation, and pathological alterations in major classes of K<sup>+</sup>

channels (IK), and small-conductance Ca2+-activated K+

 **channel**

underlies the spread of vasodilation to resistance arteries [10, 12].

been detected in VSMCs and/or ECs, including Ca2+-activated K+

RNA, microRNA, and some other transcription factors jointly regulate the expression of smooth muscle α-actin (α-SMA) and the contraction of VSMCs [7, 8]. The outer most layer of the vessel wall is the adventitia. In vertebrates, the adventitia is important because the fibroelastic connective tissue stroma is an important structural component of all tissues. The adventitial stroma contains an extracellular matrix scaffold including fibroblasts, blood and lymphatic vessels, nerve endings, progenitor cells, and immune cells. In one sense, the adventitia is the most complex

Ion channels play an important role in the mechanism of action of vasodilators and vasoconstrictors that modulate vascular tone and the effects of disease states, such as hypertension, obesity, and diabetes, which depend on ion channel expression and function. We focus on the basic properties, physiological functions,

channels.

channels (KCa) are considered key elements that

channels, including large conductance Ca2+-

(SK) channels, are

filter

": the

(BK) channels, intermediate conductance Ca2+-activated

In the vascular system, transmembrane voltage regulates vascular function. The interactions between vascular conditions and membrane potential are complicated [10]. The hyperpolarization of the smooth muscle cell membrane potential is evoked by the activation of ion channels, which contributes to vasodilation. A decrease in Ca2+ influx resulting from a decrease in the open probability of voltage-dependent calcium channels (CaV) and the CaV-dependent activation of the sarcoplasmic reticulum are crucial factors contributing to this process [11]. The depolarization of vascular smooth muscle cells causes contraction by opening CaV and inducing cal-

control vascular tone and blood pressure by modulating membrane hyperpolariza-

widely expressed in the vascular system. Intercellular conduction of electric signals

BK channels (also known as MaxiK) are widely expressed in vascular smooth muscle cells. Vascular BK channels comprise four pore-forming subunits (BK-α) and four auxiliary subunits: β1 subunits (BK-β1) and/or γ1 subunits (BK-γ1). BK α, which is encoded by the KCNMA1 gene, has seven transmembrane domains (S0–S6). BK-α has an extra transmembrane segment, S0, and thus its N-terminus is located in the extracellular space. S1–S4 form the voltage-sensor domain (VSD), and S5 and S6 form the ion permeation domain that encompasses the conserved K+

(TVGYG) in the pore loop. The C-terminus of BK channels modulates the voltage sensor and affects the pore, thus influencing channel opening. The ability of specific BK channels to open as a function of Ca2+ concentration or as a function of voltage sensors is due to the use of alternative splice sites [13–15]. The C-terminus contains

proximal portion RCK domain (RCK1) and the distal portion RCK domain (RCK2).

two homologous structural units termed "regulators of conductance for K+

channels that have

channels, ATP-

**28**

*Schematic structure of one BKα-subunit consisting of 7 transmembrane domains (S0–S6). S1–S4 constitute the voltage-sensing unit, and the S5-P loop-S6 form the ion permeation domain. The Ca2+ bowl is a high-affinity divalent cation-binding domain, and the RCK domain (regulator of conductance for K+ ) in the C-terminal region is responsible for Ca2+ sensitivity. The presence of the β1 subunit increases Ca2+ sensitivity and thus channels activity.*

The RCK1 domain is related to the formation of the Ca2+-binding site, and the RCK2 contains a "high-affinity" Ca2+ bowl domain. The "gating ring" is a Ca2+-sensing apparatus composed of four pairs of RCK1 and RCK2 domains, and the function of gating ring is responsible for allosteric activation of BK channel by Ca2+ binding [15–19](**Figure 1**). Four types of β subunits (BK-β) and four types of γ subunits (BK-γ) modulate almost all aspects of the pharmacological actions and physiological processes mediated by BK channels. The functional mechanism of BK channels regulated by β and γ auxiliary subunits is extremely complicated but is crucial to our understanding of its implications in vascular diseases. In the vasculature, BK-β1, which is encoded by KCNMB1, is the dominant isoform in VSMCs, and the dysfunction of the β1 is associated with diabetes, hypertension, and other vascular diseases. Knockout of the BK-β1 gene produces a remarkable decrease in the Ca2+ sensitivity of the channel. In addition, coexpression of the BK-β1 subunit with the BK-α subunit dramatically alters the calcium sensitivity, similar to the results observed in native VSMCs [15, 20, 21]. As an auxiliary subunit of the BK channels, BK-γ also affects BK channel activity by modulating the voltage and Ca2+ sensitivity. The BK-γ subunit has the ability to regulate vascular tone, and knockdown of BK-γ subunits contributes to pressure-induced vasoconstriction and a decrease in the activity of functional BK channels [15, 22, 23].

#### **3.2 The physiological function of BK channel**

Hundreds of proteins (such as β-catenin and caveolins) are reported to interact with BK channels in various systems *in vitro* and/or *in vivo*. The mutual effect between BK channel and these proteins regulates the BK channel functions and influences the biological pathways mediated by BK channels [24–26]. However, a key characteristic of BK channels is their ability to couple with calcium channels that mediate the increase of intracellular Ca2+. BK channels can prevent Ca2+ channels from further activation and limit Ca2+ influx. In smooth muscle cells, ryanodine receptors cause local, transient calcium release events from the endoplasmic reticulum. These spontaneous calcium release events lead to the activation of nearby BK channels, which induce membrane hyperpolarization. This kind of potassium current is called the spontaneous transient outward current (STOC), and by blocking, STOC contributes to the increased vascular muscle tone [24, 27, 28]. Accordingly, BK channel is a key regulator to induce vasodilatation.

#### **3.3 The function of BK channel in diabetes and hypertension**

Diabetes is an independent risk factor for vascular diseases and is associated with increased risks of vascular complications, such as coronary artery disease, stroke, nephropathy, neuropathy, and retinopathy [29]. Vascular BK channel dysfunction is mainly due to a significant downregulation of BK-β1 subunit expression in vessels from subjects with T1DM and T2DM. The activity of BK channels is regulated by many factors, such as angiotensin II, reactive oxygen species (ROS), nitric oxide (NO), carbon monoxide (CO), and protein kinase A- and protein kinase C-mediated signaling pathways.

According to Lu et al., the ROS signaling cascade facilitates Forkhead box O subfamily transcription factor-3a (FOXO-3a)-dependent F-box-only protein (FBXO)-mediated BK-β1 degradation and leads to the dysfunction of diabetic BK channels. In diabetic mouse aortas and in high glucose-cultured human coronary arterial smooth muscle cells, p-Akt (S473) levels are decreased, and the level of protein kinase C (PKC) β, which stimulates ROS generation and contributes to diabetic cardiovascular complications in diabetic rats, is distinctly increased [29, 30]. This group also revealed that the nuclear factor erythroid-2-related factor 2 (Nrf2) signaling pathway plays a significant role in regulating coronary BK channel function and vasodilation in mice with high-fat diet (HFD)-induced obesity/diabetes [31].

Hypertension, which is characterized by increased arterial tone, is another risk factor for cardiovascular diseases. Substantial evidence shows decreased expression of the BK-β1 subunit that is considered to contribute to the development of vascular dysfunction during hypertension. Loss-of-function mutations in BK-β<sup>1</sup> decrease the prevalence of diastolic hypertension in humans [32]. Recently, the regulated trafficking of BK channel subunits (including α subunit and auxiliary β<sup>1</sup> and γ subunits) has been accepted as a functional mechanism to modulate arterial contractility. Endothelin-1 (ET-1) is a vasoconstrictor that activates protein kinase C (PKC) and stimulates PKC-mediated phosphorylation of Rab11A at serine 177. Subsequently, surface β1 trafficking is reduced, resulting in a decrease in BK channel currents and vasoconstriction [33, 34].

#### **3.4 BK channel in ECs**

BK channels are expressed in both VSMCs and endothelial cells [35, 36]. In the majority of the systemic vasculature, endothelial BK channels are electrically quiescent, but may be disinhibited under pathophysiological conditions [37]. Hydrogen sulfide (H2S) is an important, endogenously generated gaseous signaling molecule. H2S-mediated vasodilation involves the activation of endothelial BK channels, which depends on Ca2+ influx through endothelial transient receptor potential vanilloid-4 (TRPV4) channels [38].

Using the whole-cell recording technique, Dong et al. examined the effect of CO on the activity of BK channels. The application of exogenous CO-activated BK channels in endothelial cells and the stimulation of endogenous CO production increased BK channel activity in human umbilical vein endothelial cells (HUVECs). Stimulation of soluble guanylate cyclase (sGC) production is responsible for the early stage, but not the latter stage, of this process. The CO-induced activation of BK channels plays an essential role in modulating vascular function. In endothelial cells, BK channels are activated by CO and induce the hyperpolarization of the membrane potential. Afterwards, the driving force for Ca2+ influx increases, and the increase in the intracellular Ca2+ concentration stimulates NO generation, which diffuses into the smooth muscle cells to activate BK channels [35].

**31**

*Potassium Channels in the Vascular Diseases DOI: http://dx.doi.org/10.5772/intechopen.82474*

**3.5 Structures of SK and IK channel**

CaM interactions in a single complex [48].

Another key factor that interacts with BK channels and likely exerts a negative regulatory effect on channel activity is caveolin-1 (Cav-1). Under normal conditions, Cav-1 limits the contribution of the BK channels to EDHF-mediated arteriolar dilation. In obesity, the decreased expression of Cav-1 increases the contribution of the BK channels to EDHF-mediated arteriolar dilation, which seems essential for maintaining vascular homeostasis [39]. Chronic hypoxia (CH) enhances the activity of BK channels in ECs and alters vasoreactivity via the loss of an inhibitory effect of Cav-1. Under this condition, BK channels in ECs display a similar unitary conductance but greater Ca2+ sensitivity than BK channels from vascular smooth muscle cells [40]. Anandamide is an endogenous ligand for specific G-protein-coupled cannabinoid type 1 (CB1) and type 2 (CB2) receptors. In the cardiovascular system, anandamide acts as a direct BKCa opener, and vasodilatory responses to cannabinoids are thought to require a G-protein-coupled receptor (GPCR) located on endothelial cells, the activation of which results in the direct modification of BKCa channel activity and BKCadependent vasodilation. BKCa channels act as cellular sensors for cannabinoids in *in vitro* and *in situ* endothelial cells [40]. The mechanism of action of anandamide on endothelial cells was not previously believed to require CB1, CB2, or non-CB1/CB2 receptors, but was related to direct modulation of the BKCa channel gating without modification of unitary conductance [41]. However, the roles of BKca in endothelial cells observed in response to *in vitro* and *in situ* cannabinoid-induced vasodilation are undisputed.

SK and IK channels are two distinct types of voltage-independent KCa channels; these channels exhibit a close association between their calcium sensitivity and calmodulin [42]. In contrast to intestinal smooth muscle, little evidence is available suggesting a functional role for SK channels in vascular smooth muscle cells, although an unidentified apamin (a specific blocker of SK channel channels)-sensitive and voltage-dependent conductance has been reported [43]. In healthy and freshly isolated vascular smooth muscle cells, IK channels are expressed at very low levels. In contrast, the expression of IK channels increases when the vascular system is impaired, and this

The family of SK channels consists of three members: SK1 (also known as KCa2.1), which is encoded by the KCNN1 gene; SK2 (also known as KCa2.2), which is encoded by the KCNN2 gene; and SK3 (also known as KCa2.3), which is encoded by the KCNN3 gene. SK channels consist of six transmembrane regions (TMs) and a single pore loop, with four subunits located around a central pore. Both the N-terminus and C-terminus are oriented toward the cytoplasm. SK channels have no charged amino acids in the fourth TM domain, which is usually an important component of a voltage sensor. SK channels are activated and deactivated solely as a consequence of Ca2+ binding or release [45]. SK channels are heteromeric complexes that comprise pore-forming α subunits and the Ca2+-binding protein calmodulin (CaM) (**Figure 2**). CaM is not only necessary for Ca2+ sensitivity but also critical for the trafficking of SK channels. CaM binds to and activates its target proteins in both Ca2+-replete and Ca2+-depleted forms. CaM mutants affect the interaction of CaM with its target proteins [45, 46]. CaM binds to a highly conserved CaM-binding domain (CaMBD) residing within the C-terminus of the SK channels that is located immediately distal to the sixth transmembrane segment [47, 48]. Maria A. Schumacher et al. explored the structure of the CaMBD/Ca2+/CaM complex, and in this complex, CaM binds three α-helices instead of one, and the N-lobe and C-lobe of each CaM molecule contact different CaMBD monomers. The structure of the CaMBD/Ca2+/CaM complex provides detailed information about both Ca2+-dependent and Ca2+-independent

phenomenon also appears in proliferating smooth muscle cells [44].

#### *Potassium Channels in the Vascular Diseases DOI: http://dx.doi.org/10.5772/intechopen.82474*

*Vascular Biology - Selection of Mechanisms and Clinical Applications*

C-mediated signaling pathways.

(HFD)-induced obesity/diabetes [31].

currents and vasoconstriction [33, 34].

vanilloid-4 (TRPV4) channels [38].

**3.4 BK channel in ECs**

**3.3 The function of BK channel in diabetes and hypertension**

Diabetes is an independent risk factor for vascular diseases and is associated with increased risks of vascular complications, such as coronary artery disease, stroke, nephropathy, neuropathy, and retinopathy [29]. Vascular BK channel dysfunction is mainly due to a significant downregulation of BK-β1 subunit expression in vessels from subjects with T1DM and T2DM. The activity of BK channels is regulated by many factors, such as angiotensin II, reactive oxygen species (ROS), nitric oxide (NO), carbon monoxide (CO), and protein kinase A- and protein kinase

According to Lu et al., the ROS signaling cascade facilitates Forkhead box O subfamily transcription factor-3a (FOXO-3a)-dependent F-box-only protein (FBXO)-mediated BK-β1 degradation and leads to the dysfunction of diabetic BK channels. In diabetic mouse aortas and in high glucose-cultured human coronary arterial smooth muscle cells, p-Akt (S473) levels are decreased, and the level of protein kinase C (PKC) β, which stimulates ROS generation and contributes to diabetic cardiovascular complications in diabetic rats, is distinctly increased [29, 30]. This group also revealed that the nuclear factor erythroid-2-related factor 2 (Nrf2) signaling pathway plays a significant role in regulating coronary BK channel function and vasodilation in mice with high-fat diet

Hypertension, which is characterized by increased arterial tone, is another risk factor for cardiovascular diseases. Substantial evidence shows decreased expression of the BK-β1 subunit that is considered to contribute to the development of vascular dysfunction during hypertension. Loss-of-function mutations in BK-β<sup>1</sup> decrease the prevalence of diastolic hypertension in humans [32]. Recently, the regulated trafficking of BK channel subunits (including α subunit and auxiliary β<sup>1</sup> and γ subunits) has been accepted as a functional mechanism to modulate arterial contractility. Endothelin-1 (ET-1) is a vasoconstrictor that activates protein kinase C (PKC) and stimulates PKC-mediated phosphorylation of Rab11A at serine 177. Subsequently, surface β1 trafficking is reduced, resulting in a decrease in BK channel

BK channels are expressed in both VSMCs and endothelial cells [35, 36]. In the majority of the systemic vasculature, endothelial BK channels are electrically quiescent, but may be disinhibited under pathophysiological conditions [37]. Hydrogen sulfide (H2S) is an important, endogenously generated gaseous signaling molecule. H2S-mediated vasodilation involves the activation of endothelial BK channels, which depends on Ca2+ influx through endothelial transient receptor potential

Using the whole-cell recording technique, Dong et al. examined the effect of CO on the activity of BK channels. The application of exogenous CO-activated BK channels in endothelial cells and the stimulation of endogenous CO production increased BK channel activity in human umbilical vein endothelial cells (HUVECs). Stimulation of soluble guanylate cyclase (sGC) production is responsible for the early stage, but not the latter stage, of this process. The CO-induced activation of BK channels plays an essential role in modulating vascular function. In endothelial cells, BK channels are activated by CO and induce the hyperpolarization of the membrane potential. Afterwards, the driving force for Ca2+ influx increases, and the increase in the intracellular Ca2+ concentration stimulates NO generation, which

diffuses into the smooth muscle cells to activate BK channels [35].

**30**

Another key factor that interacts with BK channels and likely exerts a negative regulatory effect on channel activity is caveolin-1 (Cav-1). Under normal conditions, Cav-1 limits the contribution of the BK channels to EDHF-mediated arteriolar dilation. In obesity, the decreased expression of Cav-1 increases the contribution of the BK channels to EDHF-mediated arteriolar dilation, which seems essential for maintaining vascular homeostasis [39]. Chronic hypoxia (CH) enhances the activity of BK channels in ECs and alters vasoreactivity via the loss of an inhibitory effect of Cav-1. Under this condition, BK channels in ECs display a similar unitary conductance but greater Ca2+ sensitivity than BK channels from vascular smooth muscle cells [40].

Anandamide is an endogenous ligand for specific G-protein-coupled cannabinoid type 1 (CB1) and type 2 (CB2) receptors. In the cardiovascular system, anandamide acts as a direct BKCa opener, and vasodilatory responses to cannabinoids are thought to require a G-protein-coupled receptor (GPCR) located on endothelial cells, the activation of which results in the direct modification of BKCa channel activity and BKCadependent vasodilation. BKCa channels act as cellular sensors for cannabinoids in *in vitro* and *in situ* endothelial cells [40]. The mechanism of action of anandamide on endothelial cells was not previously believed to require CB1, CB2, or non-CB1/CB2 receptors, but was related to direct modulation of the BKCa channel gating without modification of unitary conductance [41]. However, the roles of BKca in endothelial cells observed in response to *in vitro* and *in situ* cannabinoid-induced vasodilation are undisputed.

### **3.5 Structures of SK and IK channel**

SK and IK channels are two distinct types of voltage-independent KCa channels; these channels exhibit a close association between their calcium sensitivity and calmodulin [42]. In contrast to intestinal smooth muscle, little evidence is available suggesting a functional role for SK channels in vascular smooth muscle cells, although an unidentified apamin (a specific blocker of SK channel channels)-sensitive and voltage-dependent conductance has been reported [43]. In healthy and freshly isolated vascular smooth muscle cells, IK channels are expressed at very low levels. In contrast, the expression of IK channels increases when the vascular system is impaired, and this phenomenon also appears in proliferating smooth muscle cells [44].

The family of SK channels consists of three members: SK1 (also known as KCa2.1), which is encoded by the KCNN1 gene; SK2 (also known as KCa2.2), which is encoded by the KCNN2 gene; and SK3 (also known as KCa2.3), which is encoded by the KCNN3 gene. SK channels consist of six transmembrane regions (TMs) and a single pore loop, with four subunits located around a central pore. Both the N-terminus and C-terminus are oriented toward the cytoplasm. SK channels have no charged amino acids in the fourth TM domain, which is usually an important component of a voltage sensor. SK channels are activated and deactivated solely as a consequence of Ca2+ binding or release [45]. SK channels are heteromeric complexes that comprise pore-forming α subunits and the Ca2+-binding protein calmodulin (CaM) (**Figure 2**). CaM is not only necessary for Ca2+ sensitivity but also critical for the trafficking of SK channels. CaM binds to and activates its target proteins in both Ca2+-replete and Ca2+-depleted forms. CaM mutants affect the interaction of CaM with its target proteins [45, 46]. CaM binds to a highly conserved CaM-binding domain (CaMBD) residing within the C-terminus of the SK channels that is located immediately distal to the sixth transmembrane segment [47, 48]. Maria A. Schumacher et al. explored the structure of the CaMBD/Ca2+/CaM complex, and in this complex, CaM binds three α-helices instead of one, and the N-lobe and C-lobe of each CaM molecule contact different CaMBD monomers. The structure of the CaMBD/Ca2+/CaM complex provides detailed information about both Ca2+-dependent and Ca2+-independent CaM interactions in a single complex [48].

#### **Figure 2.**

*Schematic of IK and three subtypes of SK (SK1, SK2, and SK3). SK3 and IK are thought to be the predominant KCa channels expressed in systemic vascular endothelia. The basic structure consisting of six transmembrane domains (S1–S6). The constitutively bound calmodulin (CaM), at the C-terminus. The founding member of Ca2+ binding proteins is CaM, a small, acidic, modular protein endowed with gymnastic-like flexibility that chelate Ca2+ ions.*

IK channel (also known as KCa 3.1) is widely expressed in cells of the immune system and red and white blood cells, where it plays an important role in cellular activation, migration, and cytokine production [49–51]. Moreover, KCa3.1 is also expressed in dedifferentiated vascular smooth muscle cells, fibroblasts, and the vascular endothelium, where the channel is involved in the EDH response [52, 53]. The KCa3.1 channel is a tetrameric membrane protein with each subunit (comprising 427 amino acids) organized in six transmembrane segments, S1–S6, with a pore motif between segments 5 (S5) and 6 (S6). The channel assembly and trafficking are regulated by the constitutively bound calmodulin (CaM) molecule, which also confers Ca2+ sensitivity [54, 55]. Ca2+ binds to the CaM-KCa3.1 complex in the C-terminus of KCa3.1. As the CaM-binding domain of KCa3.1 is directly connected to the S6 transmembrane helix, activation of the channel gate at the level of the selectivity filter might depend upon the coupling between each of the channel pore helices and the associated S6 transmembrane segment. The interactions of the KCa3.1 pore helix with the S5 and S6 transmembrane segments also contribute to setting POmax, which is one of the distinguishing features of the Ca2+-dependence of the KCa3.1 channel [55].

#### **3.6 SK and IK channel and EDH responses**

In small arterial and arteriolar ECs, KCa channels are activated by intrinsic spontaneous or receptor-mediated Ca2+ events, which contribute to the hyperpolarization of SMCs and vasodilation through a NO-independent process. This response is known as endothelium-dependent hyperpolarization (EDH), and it is the predominant mechanism in ECs [56]. SK and IK channels operate in parallel to generate EDH; they contribute to smooth muscle hyperpolarization and

**33**

of hydrogen peroxide (H2O2) [64].

*Potassium Channels in the Vascular Diseases DOI: http://dx.doi.org/10.5772/intechopen.82474*

of NOS activity [58].

emia (HHcy) [62].

vasorelaxation, and the hyperpolarization of the endothelial cells in turn increases calcium influx by increasing the driving force for this ion, but the channels can be activated independently. SK channels are distributed throughout the endothelial cell membrane, but cluster in the proximity of the large gap junctions between endothelial cells. In contrast, IK channels are only present in detectable amounts at endothelial cell projections toward adjacent smooth muscle cells, where they can form myoendothelial gap junctions [57]. EDHF-mediated responses play a physiological role in regulating vascular resistance. In rats, the hypotensive response to endothelium-dependent agonists, such as acetylcholine and bradykinin, is rapidly

arginine methyl ester (L-NAME). The compensatory relaxation is mediated by the activation of SK and IK channels. Endothelial dysfunction, measured as a reduced endothelium-dependent hypotensive response, does not develop after the inhibition

Over the past two decades, studies examining the physiological role of hydrogen

The activation of SK/IK channels may regulate electrical conduction along the endothelium of intact vessels, and some factors limit this process, such as myoendothelial coupling to SMCs, perivascular nerve activity, and circulating vasoactive agents. Using intact EC tubes produced after the dissociation of SMCs with mild enzymatic digestion, Behringer and his colleagues found that activation of SK/IK channels impairs the transmission between axial signals. This effect results from a decrease in membrane resistance (rm) that dissipates charge as current flows from cell to cell along the endothelium [63]. Another group verified these results and further assessed impairments in electric conduction along the endothelium of resistance arteries through the enhanced activation of SK/IK channels. Fresh EC tubes were isolated from resistance arteries in skeletal muscle from different groups of mice. Group 1 included young mice (approximately 4–6 month old), group 2 included middle-aged mice (approximately 12–14 month old), and group 3 included old mice (approximately 24–46 month old). The ability of the endothelium of skeletal muscle resistance arteries to conduct electric signals is impaired with aging. The dual function of SK/IK channels in initiating and modulating electric signaling along the endothelium is altered with aging. By increasing the activation of SK/IK channels (particularly the IK channel), aging promotes hyperpolarization of the endothelium while decreasing its ability to conduct electrical signals. Oxidative stress activates SK/IK channels in the resistance artery endothelium via the action

sulfide (H2S) have received increasing attention. Cystathionine γ-lyase (CSE) generates H2S under physiological conditions, and a CSE deletion in mice reduces H2S levels in some tissues, including the aorta. These mice lacking the CSE gene display pronounced hypertension, indicating that H2S is a physiological vasodilator and regulator of blood pressure [59]. In many ways, either H2S itself is an EDHF or H2S releases EDHF from the endothelium [60, 61]. The resting membrane potential of SMCs is increased in CSE knockout mice, and methacholine (a cholinergicmuscarinic agonist)-induced endothelium-dependent relaxation of mesenteric arteries was abolished. Methacholine hyperpolarizes SMCs in endothelium-intact mesenteric arteries from wild-type mice. The application of atropine (a muscarinic antagonist) or charybdotoxin and apamin, which block SK/IK channels, or knockout of the CSE gene in mice inhibited this effect. Simultaneously, the expression of SK2.3, but not the IK3.1 channel, in vascular tissues was increased by H2S and decreased by a CSE inhibitor or CSE gene knockout [51]. Moreover, insufficient H2S levels impair EDHF-induced vascular relaxation by increasing oxidative stress and IK inactivation in mice with type 2 diabetes mellitus (T2DM)/hyperhomocystein-


compensated within 1 day after treatment with the NOS inhibitor N<sup>ω</sup>

#### *Potassium Channels in the Vascular Diseases DOI: http://dx.doi.org/10.5772/intechopen.82474*

*Vascular Biology - Selection of Mechanisms and Clinical Applications*

IK channel (also known as KCa 3.1) is widely expressed in cells of the immune system and red and white blood cells, where it plays an important role in cellular activation, migration, and cytokine production [49–51]. Moreover, KCa3.1 is also expressed in dedifferentiated vascular smooth muscle cells, fibroblasts, and the vascular endothelium, where the channel is involved in the EDH response [52, 53]. The KCa3.1 channel is a tetrameric membrane protein with each subunit (comprising 427 amino acids) organized in six transmembrane segments, S1–S6, with a pore motif between segments 5 (S5) and 6 (S6). The channel assembly and trafficking are regulated by the constitutively bound calmodulin (CaM) molecule, which also confers Ca2+ sensitivity [54, 55]. Ca2+ binds to the CaM-KCa3.1 complex in the C-terminus of KCa3.1. As the CaM-binding domain of KCa3.1 is directly connected to the S6 transmembrane helix, activation of the channel gate at the level of the selectivity filter might depend upon the coupling between each of the channel pore helices and the associated S6 transmembrane segment. The interactions of the KCa3.1 pore helix with the S5 and S6 transmembrane segments also contribute to setting POmax, which is one of the distinguishing features of the Ca2+-dependence

*Schematic of IK and three subtypes of SK (SK1, SK2, and SK3). SK3 and IK are thought to be the predominant KCa channels expressed in systemic vascular endothelia. The basic structure consisting of six transmembrane domains (S1–S6). The constitutively bound calmodulin (CaM), at the C-terminus. The founding member of Ca2+ binding proteins is CaM, a small, acidic, modular protein endowed with gymnastic-like flexibility that* 

In small arterial and arteriolar ECs, KCa channels are activated by intrinsic spontaneous or receptor-mediated Ca2+ events, which contribute to the hyperpolarization of SMCs and vasodilation through a NO-independent process. This response is known as endothelium-dependent hyperpolarization (EDH), and it is the predominant mechanism in ECs [56]. SK and IK channels operate in parallel to generate EDH; they contribute to smooth muscle hyperpolarization and

**32**

**Figure 2.**

*chelate Ca2+ ions.*

of the KCa3.1 channel [55].

**3.6 SK and IK channel and EDH responses**

vasorelaxation, and the hyperpolarization of the endothelial cells in turn increases calcium influx by increasing the driving force for this ion, but the channels can be activated independently. SK channels are distributed throughout the endothelial cell membrane, but cluster in the proximity of the large gap junctions between endothelial cells. In contrast, IK channels are only present in detectable amounts at endothelial cell projections toward adjacent smooth muscle cells, where they can form myoendothelial gap junctions [57]. EDHF-mediated responses play a physiological role in regulating vascular resistance. In rats, the hypotensive response to endothelium-dependent agonists, such as acetylcholine and bradykinin, is rapidly compensated within 1 day after treatment with the NOS inhibitor N<sup>ω</sup> -nitro-Larginine methyl ester (L-NAME). The compensatory relaxation is mediated by the activation of SK and IK channels. Endothelial dysfunction, measured as a reduced endothelium-dependent hypotensive response, does not develop after the inhibition of NOS activity [58].

Over the past two decades, studies examining the physiological role of hydrogen sulfide (H2S) have received increasing attention. Cystathionine γ-lyase (CSE) generates H2S under physiological conditions, and a CSE deletion in mice reduces H2S levels in some tissues, including the aorta. These mice lacking the CSE gene display pronounced hypertension, indicating that H2S is a physiological vasodilator and regulator of blood pressure [59]. In many ways, either H2S itself is an EDHF or H2S releases EDHF from the endothelium [60, 61]. The resting membrane potential of SMCs is increased in CSE knockout mice, and methacholine (a cholinergicmuscarinic agonist)-induced endothelium-dependent relaxation of mesenteric arteries was abolished. Methacholine hyperpolarizes SMCs in endothelium-intact mesenteric arteries from wild-type mice. The application of atropine (a muscarinic antagonist) or charybdotoxin and apamin, which block SK/IK channels, or knockout of the CSE gene in mice inhibited this effect. Simultaneously, the expression of SK2.3, but not the IK3.1 channel, in vascular tissues was increased by H2S and decreased by a CSE inhibitor or CSE gene knockout [51]. Moreover, insufficient H2S levels impair EDHF-induced vascular relaxation by increasing oxidative stress and IK inactivation in mice with type 2 diabetes mellitus (T2DM)/hyperhomocysteinemia (HHcy) [62].

The activation of SK/IK channels may regulate electrical conduction along the endothelium of intact vessels, and some factors limit this process, such as myoendothelial coupling to SMCs, perivascular nerve activity, and circulating vasoactive agents. Using intact EC tubes produced after the dissociation of SMCs with mild enzymatic digestion, Behringer and his colleagues found that activation of SK/IK channels impairs the transmission between axial signals. This effect results from a decrease in membrane resistance (rm) that dissipates charge as current flows from cell to cell along the endothelium [63]. Another group verified these results and further assessed impairments in electric conduction along the endothelium of resistance arteries through the enhanced activation of SK/IK channels. Fresh EC tubes were isolated from resistance arteries in skeletal muscle from different groups of mice. Group 1 included young mice (approximately 4–6 month old), group 2 included middle-aged mice (approximately 12–14 month old), and group 3 included old mice (approximately 24–46 month old). The ability of the endothelium of skeletal muscle resistance arteries to conduct electric signals is impaired with aging. The dual function of SK/IK channels in initiating and modulating electric signaling along the endothelium is altered with aging. By increasing the activation of SK/IK channels (particularly the IK channel), aging promotes hyperpolarization of the endothelium while decreasing its ability to conduct electrical signals. Oxidative stress activates SK/IK channels in the resistance artery endothelium via the action of hydrogen peroxide (H2O2) [64].
