**4.1 L-type Ca2+ channel CaV1.2**

The L-type Ca2+ channel CaV1.2 (i.e., LTCCs) is essential for vascular smooth muscle contraction and vascular reactivity. Therefore, they play a key role in controlling blood flow and blood pressure [33, 73]. LTCCs are comprised of a pore-forming α1c subunit and auxiliary β, α2δ, and γ subunits that modulate channel function and trafficking [74]. The α1c subunit contains four homologous domains (I, II, III, IV). Each domain comprises of six membrane-spanning segments (S1– S6) with intracellular amino- and carboxyl termini, which contain many regions relevant for channel regulation and control of cell excitability. In vascular smooth muscle, expression of the α1c subunit is critical for pressure-induced constriction as evidenced by an absence of myogenic response after LTCC blockade and depletion of the CaV1.2-α1c subunit in mice (e.g., SMAKO mouse) [33, 73, 75]. The auxiliary subunits α2 and δ are the product of the same gene that gets proteolytically cleaved after translation but remains connected by disulfide bonds, which give rise to the mature subunit. The α2δ subunit has been linked to regulation of α1c subunit surface expression that controls Ca2+ influx in vascular smooth muscle and the level of myogenic constriction [76]. The β subunit, which remains cytoplasmic, also contributes to the α1c subunit surface expression and channel regulation and therefore can modulate vascular smooth muscle excitability in health and disease [77, 78]. Unlike the other subunits, the expression, regulation, and function of the γ subunit in vascular smooth muscle are unclear and likely the subject of further research.

LTCCs in vascular smooth muscle are distinctively regulated by the Gs/AC/ PKA, NO/sGC/PKG, and Gq/PLC/PKC axes [9, 79]. Accordingly, the NO/sGC/PKG signaling axis has been shown to inhibit vascular LTCCs [80]. This has been associated with a reduction in [Ca2+]i that may be part of the vasodilatory mechanism underlying the activation of this pathway [79]. Receptor-mediated signaling via the

**11**

*Ca2+ = calcium; ryanodine receptors = RyR.*

**Figure 3.**

*Ion Channels and Their Regulation in Vascular Smooth Muscle*

Gq/PLC/PKC axis typically results in potentiation of LTCC activity [9, 79, 81]. The functional effects of this Gq/PLC/PKC-mediated activation of LTCCs are vascular smooth muscle contraction and an increase in vascular tone. Intriguingly, activation of the Gs/AC/PKA axis has been shown to inhibit, activate, or produce no effect on vascular LTCC activity (**Figure 3**) [79]. Irrespectively of this however, PKA signaling has been generally linked with vasodilation, thus raising questions about the functional relevance, if any, of this kinase in the regulation of vascular LTCCs. Intriguingly, recent studies revealed that elevations in extracellular D-glucose (HG) potentiate LTCC activity via a Gs/AC/PKA pathway in vascular smooth muscle [82–85]. This HG-induced PKA-dependent activation of LTCCs resulted in increased global [Ca2+]i and vasoconstriction, thus providing the first example of a PKA-dependent pathway underlying vascular smooth muscle contraction. Future

studies should further examine the in vivo relevance of this pathway.

LTCC regulation by Gs/AC/PKA and Gq/PLC/PKC axes in vascular smooth muscle is mediated by AKAP5 (**Figure 3**) [85, 86]. The involvement of the scaffold in this regulation was initially speculated from total internal reflection fluorescence (TIRF) microscopy experiments that optically recorded the activity of single or clusters of LTCCs [87, 88]. From these experiments, it was clear that the activity and location of functional LTCCs were heterogeneous throughout the surface membrane of vascular smooth muscle cells [81, 87, 89]. Whereas some LTCCs showed stochastic activity with low Ca2+ flux and duration of events, others had persistent activity characterized by increased Ca2+ flux and events with prolonged open time that were produced by the opening of two or more channels [81, 87, 89–91]. The

*Regulation of vascular smooth muscle excitability by voltage-gated Ca***2+** *channels. Ca2+ influx via L-type Ca2+ channel CaV1.2 is essential for vascular smooth muscle contraction [33]. Their activity is regulated by Gs/PKA and Gq/PKC signaling pathways upon activation of a specific GPCR by a given stimulus (e.g., angII or HG). The association of CaV1.2 with these signaling pathways is orchestrated by AKAP5 [86]. PKA (and perhaps PKC) augments L-type Ca2+ channel CaV1.2 activity by increasing CaV1.2 phosphorylation at serine 1928 (pS1928) [82, 85]. The phosphatase PP2B suppresses enhancement of L-type Ca2+ channel CaV1.2 activity presumably by preventing/opposing channel phosphorylation in vascular smooth muscle cells (gray line). Two TTCC subtypes are expressed in vascular smooth muscle, with CaV3.1 contributing to contractile mechanisms and CaV3.2 forming a complex with RyR in the sarcoplasmic reticulum and BKCa channels in the surface membrane to foster relaxation [95, 100]. Although PKA has been shown to inhibit CaV3.2, whether this requires AKAP5 function is unclear (dotted red lines with perpendicular line at the end). It is also unclear whether AKAP5-anchored PKC and PKA regulate CaV3.1 channel activity (dotted light and dark red lines with star near CaV3.1). Finally, whether the GPCRs activated by angII and HG are targeted to specific complexes by AKAP5 is unclear (red dotted line with ?? symbols). The model was generated by taking in consideration studies cited and described above. GPCR = G-protein coupled receptors; angiotensin II = angII; high glucose = HG; AKAP5 = A kinase anchoring protein 5; protein kinase A= PKA; protein kinase* 

*C = PKC; protein phosphatase 2B; PP2B; phosphorylation at CaV1.2 serine 1928 = pS1928; K+*

 *= potassium;* 

*DOI: http://dx.doi.org/10.5772/intechopen.88962*

*Ion Channels and Their Regulation in Vascular Smooth Muscle DOI: http://dx.doi.org/10.5772/intechopen.88962*

Gq/PLC/PKC axis typically results in potentiation of LTCC activity [9, 79, 81]. The functional effects of this Gq/PLC/PKC-mediated activation of LTCCs are vascular smooth muscle contraction and an increase in vascular tone. Intriguingly, activation of the Gs/AC/PKA axis has been shown to inhibit, activate, or produce no effect on vascular LTCC activity (**Figure 3**) [79]. Irrespectively of this however, PKA signaling has been generally linked with vasodilation, thus raising questions about the functional relevance, if any, of this kinase in the regulation of vascular LTCCs. Intriguingly, recent studies revealed that elevations in extracellular D-glucose (HG) potentiate LTCC activity via a Gs/AC/PKA pathway in vascular smooth muscle [82–85]. This HG-induced PKA-dependent activation of LTCCs resulted in increased global [Ca2+]i and vasoconstriction, thus providing the first example of a PKA-dependent pathway underlying vascular smooth muscle contraction. Future studies should further examine the in vivo relevance of this pathway.

LTCC regulation by Gs/AC/PKA and Gq/PLC/PKC axes in vascular smooth muscle is mediated by AKAP5 (**Figure 3**) [85, 86]. The involvement of the scaffold in this regulation was initially speculated from total internal reflection fluorescence (TIRF) microscopy experiments that optically recorded the activity of single or clusters of LTCCs [87, 88]. From these experiments, it was clear that the activity and location of functional LTCCs were heterogeneous throughout the surface membrane of vascular smooth muscle cells [81, 87, 89]. Whereas some LTCCs showed stochastic activity with low Ca2+ flux and duration of events, others had persistent activity characterized by increased Ca2+ flux and events with prolonged open time that were produced by the opening of two or more channels [81, 87, 89–91]. The

#### **Figure 3.**

*Basic and Clinical Understanding of Microcirculation*

**4. Voltage-gated Ca2+ channels**

**4.1 L-type Ca2+ channel CaV1.2**

vascular smooth muscle [67], suggesting that these channels may be distinctively modulated by lipids depending on their tissue distribution. A functional Kir channel is formed when four pore-forming α subunits, each containing two membranespanning domains, come together. Two main α subunits (e.g., Kir2.1 and Kir2.2) have been identified in vascular smooth muscle from multiple species [68–71]. Intriguingly, the expression of these subunits in a specific vascular bed may be species-dependent. Accordingly, although Kir subunit expression and channel activity have been extensively reported in murine cerebral vascular smooth muscle [67–69, 71], minimal, if any, Kir subunit expression and channel activity were found in the human cerebral vascular smooth muscle [72]. The functional implication of the activation of these channels in vascular smooth muscle is relaxation. Kir channel activity can be modulated by vasoactive agents with those acting through the Gq/PLC/PKC axis, inducing channel inhibition, and those acting on the Gs/AC/ PKA pathway, promoting channel activity [1]. The physiological relevance of these regulatory mechanisms on Kir channels and their control of vascular function are

less well understood and therefore are in need of further evaluation.

Vascular smooth muscle cells express several subtypes of VGCCs [9]. These channels have been shown to be important for vascular smooth muscle contraction, and some subtypes have been implicated in relaxation mechanisms. In this section, we will focus on the role of two key subtypes of VGCCs, namely, LTCCs and T-type Ca2+ channels (TTCCs), in regulation of vascular smooth muscle excitability.

The L-type Ca2+ channel CaV1.2 (i.e., LTCCs) is essential for vascular smooth

muscle contraction and vascular reactivity. Therefore, they play a key role in controlling blood flow and blood pressure [33, 73]. LTCCs are comprised of a pore-forming α1c subunit and auxiliary β, α2δ, and γ subunits that modulate channel function and trafficking [74]. The α1c subunit contains four homologous domains (I, II, III, IV). Each domain comprises of six membrane-spanning segments (S1– S6) with intracellular amino- and carboxyl termini, which contain many regions relevant for channel regulation and control of cell excitability. In vascular smooth muscle, expression of the α1c subunit is critical for pressure-induced constriction as evidenced by an absence of myogenic response after LTCC blockade and depletion of the CaV1.2-α1c subunit in mice (e.g., SMAKO mouse) [33, 73, 75]. The auxiliary subunits α2 and δ are the product of the same gene that gets proteolytically cleaved after translation but remains connected by disulfide bonds, which give rise to the mature subunit. The α2δ subunit has been linked to regulation of α1c subunit surface expression that controls Ca2+ influx in vascular smooth muscle and the level of myogenic constriction [76]. The β subunit, which remains cytoplasmic, also contributes to the α1c subunit surface expression and channel regulation and therefore can modulate vascular smooth muscle excitability in health and disease [77, 78]. Unlike the other subunits, the expression, regulation, and function of the γ subunit in vascular smooth muscle are unclear and likely the subject of further research. LTCCs in vascular smooth muscle are distinctively regulated by the Gs/AC/ PKA, NO/sGC/PKG, and Gq/PLC/PKC axes [9, 79]. Accordingly, the NO/sGC/PKG signaling axis has been shown to inhibit vascular LTCCs [80]. This has been associated with a reduction in [Ca2+]i that may be part of the vasodilatory mechanism underlying the activation of this pathway [79]. Receptor-mediated signaling via the

**10**

*Regulation of vascular smooth muscle excitability by voltage-gated Ca***2+** *channels. Ca2+ influx via L-type Ca2+ channel CaV1.2 is essential for vascular smooth muscle contraction [33]. Their activity is regulated by Gs/PKA and Gq/PKC signaling pathways upon activation of a specific GPCR by a given stimulus (e.g., angII or HG). The association of CaV1.2 with these signaling pathways is orchestrated by AKAP5 [86]. PKA (and perhaps PKC) augments L-type Ca2+ channel CaV1.2 activity by increasing CaV1.2 phosphorylation at serine 1928 (pS1928) [82, 85]. The phosphatase PP2B suppresses enhancement of L-type Ca2+ channel CaV1.2 activity presumably by preventing/opposing channel phosphorylation in vascular smooth muscle cells (gray line). Two TTCC subtypes are expressed in vascular smooth muscle, with CaV3.1 contributing to contractile mechanisms and CaV3.2 forming a complex with RyR in the sarcoplasmic reticulum and BKCa channels in the surface membrane to foster relaxation [95, 100]. Although PKA has been shown to inhibit CaV3.2, whether this requires AKAP5 function is unclear (dotted red lines with perpendicular line at the end). It is also unclear whether AKAP5-anchored PKC and PKA regulate CaV3.1 channel activity (dotted light and dark red lines with star near CaV3.1). Finally, whether the GPCRs activated by angII and HG are targeted to specific complexes by AKAP5 is unclear (red dotted line with ?? symbols). The model was generated by taking in consideration studies cited and described above. GPCR = G-protein coupled receptors; angiotensin II = angII; high glucose = HG; AKAP5 = A kinase anchoring protein 5; protein kinase A= PKA; protein kinase C = PKC; protein phosphatase 2B; PP2B; phosphorylation at CaV1.2 serine 1928 = pS1928; K+ = potassium; Ca2+ = calcium; ryanodine receptors = RyR.*

stochastic and persistent activity of LTCCs was modulated by membrane potential [92]. However, the occurrence of LTCCs with persistent activity is limited to specific regions of the surface membrane and has been demonstrated to be highly dependent on PKC activity and AKAP5 expression [81, 86]. The activity of phosphatases, such as PP2B, that are targeted to the channel by AKAP5, counteracts anchored kinase activity and restricts persistent LTCC activity (**Figure 3**) [89]. Accordingly, in vascular smooth muscle in which PKC is inhibited or cells from mice with genetically depleted PKC or AKAP5, the frequency of persistent LTCC activity is minimal [86, 87, 93]. In addition, PP2B inhibition stimulates persistent LTCC events in cells from wild type but not AKAP5<sup>−</sup>/<sup>−</sup> mice, suggesting that removing this "brake" facilitates kinase-mediated potentiation of channel activity [86, 89]. These results suggest an important role for AKAP5-anchored PKC and PP2B activity in modulating basal persistent LTCC activity. The physiological significance of these findings is underscored by data indicating that persistent LTCC events account for 50% of the total dihydropyridine-sensitive (e.g., LTCCs) Ca2+ influx at physiological membrane potentials [92], which is critical for vascular smooth muscle contractility in health and disease [82, 84, 86, 93].
