Calcium-Dependent Ion Channels and the Regulation of Arteriolar Myogenic Tone

Arterioles in the peripheral microcirculation regulate blood flow to and within tissues and organs, control capillary blood pressure and microvascular fluid exchange, govern peripheral vascular resistance, and contribute to the regulation of blood pressure. These important microvessels display pressure-dependent myogenic tone, the steady state level of contractile activity of vascular smooth muscle cells (VSMCs) that sets resting arteriolar internal diameter such that arterioles can both dilate and constrict to meet the blood flow and pressure needs of the tissues and organs that they perfuse. This perspective will focus on the Ca2+-dependent ion channels in the plasma and endoplasmic reticulum membranes of arteriolar VSMCs and endothelial cells (ECs) that regulate arteriolar tone. In VSMCs, Ca2+-dependent negative feedback regulation of myogenic tone is mediated by Ca2+-activated K+ (BKCa) channels and also Ca2+-dependent inactivation of voltage-gated Ca2+ channels (VGCC). Transient receptor potential subfamily M, member 4 channels (TRPM4); Ca2+-activated Cl− channels (CaCCs; TMEM16A/ANO1), Ca2+-dependent inhibition of voltage-gated K+ (KV) and ATP-sensitive K+ (KATP) channels; and Ca2+-induced-Ca2+ release through inositol 1,4,5-trisphosphate receptors (IP3Rs) participate in Ca2+-dependent positive-feedback regulation of myogenic tone. Calcium release from VSMC ryanodine receptors (RyRs) provide negative-feedback through Ca2+-spark-mediated control of BKCa channel activity, or positive-feedback regulation in cooperation with IP3Rs or CaCCs. In some arterioles, VSMC RyRs are silent. In ECs, transient receptor potential vanilloid subfamily, member 4 (TRPV4) channels produce Ca2+ sparklets that activate IP3Rs and intermediate and small conductance Ca2+ activated K+ (IKCa and sKCa) channels causing membrane hyperpolarization that is conducted to overlying VSMCs producing endothelium-dependent hyperpolarization and vasodilation. Endothelial IP3Rs produce Ca2+ pulsars, Ca2+ wavelets, Ca2+ waves and increased global Ca2+ levels activating EC sKCa and IKCa channels and causing Ca2+-dependent production of endothelial vasodilator autacoids such as NO, prostaglandin I2 and epoxides of arachidonic acid that mediate negative-feedback regulation of myogenic tone. Thus, Ca2+-dependent ion channels importantly contribute to many aspects of the regulation of myogenic tone in arterioles in the microcirculation.


INTRODUCTION
Arterioles are prominent resistance vessels that regulate blood flow to and within tissues and organs; determine capillary blood pressure and fluid exchange in the microcirculation; and contribute to the regulation of systemic blood pressure (Renkin, 1984). A defining characteristic of arterioles is pressuredependent myogenic tone, the steady state vascular smooth muscle cell (VSMC) contractile activity that is induced and maintained by pressure-dependent mechanisms (Jackson, 2020(Jackson, , 2021. Myogenic tone sets resting arteriolar internal diameter such that these microvessels can dilate or constrict to maintain homeostasis by meeting the blood flow and pressure needs of the tissues and organs that they perfuse. Arterioles express numerous ion channels that are essential to their function (Figure 1). Plasma membrane and endoplasmic reticulum (ER) ion channels in VSMCs are a major source of Ca 2+ triggering contractile machinery activation and increased arteriolar tone (vasoconstriction). In endothelial cells (ECs), ion channels provide a key Ca 2+ source controlling EC autacoid production including prostacyclin (PGI 2 ), nitric oxide (NO) and epoxides of arachidonic acid (EETs; Jackson, 2016). Intracellular Ca 2+ also controls gene expression and cell proliferation in VSMCs (Cartin et al., 2000;Stevenson et al., 2001;Barlow et al., 2006) and in ECs (Quinlan et al., 1999;Nilius and Droogmans, 2001;Munaron, 2006;Minami, 2014). Ion channels play a major role in cell volume regulation in all cells (Hoffmann et al., 2009). Finally, ion channels help set and modulate VSMC and EC membrane potential (Jackson, 2016(Jackson, , 2020(Jackson, , 2021Tykocki et al., 2017). Membrane potential, in turn, regulates the open state probability of voltage-gated Ca 2+ channels (VGCCs) which provide a major source of activator Ca 2+ in VSMCs (Tykocki et al., 2017), but probably not most ECs (Jackson, 2016). The electrochemical gradient FIGURE 1 | Schematic representation of a cross section of one wall of an arteriole showing a myoendothelial projection (MEP) passing through a hole in the internal elastic lamina (IEL). Heterocellular gap junctions are present allowing electrical and chemical (Ca 2+ , IP 3 , etc.) communication between ECs and VSMCs. Also shown are homocellular (EC-EC and VSMC-VSMC) gap junctions that also allow electrical and chemical communication as shown. Only a few classes of ion channels expressed by arteriolar VSMCs and ECs are shown for clarity. TRPC6, transient receptor potential channel C family member 6; CaCC, Ca 2+ -activated Cl − channels; TRPM4, transient receptor potential channel melanostatin family member 4; VGCC, voltage-gated Ca 2+ channels, BK Ca , large-conductance Ca 2+ -activated K + channels; K V , voltage-gated K + channels; K ATP , ATP-sensitive K + channels; IP 3 R, inositol 1,4,5 trisphosphate receptor; RyR, ryanodine receptor; SERCA, smooth endoplasmic reticulum Ca 2+ ATPase; IK Ca , intermediate-conductance Ca 2+ -activated K + channel; TRPV4, Transient Receptor Potential Vanilloid-family 4 channels; TRPC3, transient receptor potential channel C family member 3; sK Ca , small-conductance Ca 2+ -activated K + channel.
Frontiers in Physiology | www.frontiersin.org 3 November 2021 | Volume 12 | Article 770450 for diffusion of Ca 2+ and other ions depends on membrane potential in all cells (Tykocki et al., 2017). Membrane potential also has been proposed to affect Ca 2+ release from ER Ca 2+ stores and may influence the Ca 2+ sensitivity of Ca 2+ -dependent processes [see (Tykocki et al., 2017) for references]. Lastly, membrane potential serves as an essential signal for cell-cell communication, because VSMCs and ECs express both homocellular and heterocellular gap junctions allowing electrical and chemical communication among cells in the arteriolar wall (de Wit and Griffith, 2010;Bagher and Segal, 2011;Dora and Garland, 2013;Garland and Dora, 2017;Schmidt and de Wit, 2020). Thus, arteriolar function critically depends on ion channels. Calcium-dependent ion channels in both VSMCs and ECs play a central role in the generation and modulation of myogenic tone and maintenance of homeostasis (Figure 1). These channels provide both positive-and negative-feedback control of intracellular Ca 2+ in VSMCs that allows fine tuning of arteriolar tone as will be outlined in Section VSMC Ca 2+ -Dependent Ion Channels, below.
The arteriolar endothelium provides negative-feedback signals to overlying VSMCs through Ca 2+ -dependent autacoid production and direct electrical communication via myoendothelial gap junctions (MEGJs; Lemmey et al., 2020). Endothelial Ca 2+dependent ion channels contribute to these processes (Figure 1) as outlined in Section Endothelial Ca 2+ -Dependent Ion Channels and Arteriolar Tone, below.
Section Integration of Ca 2+ -Dependent Ion Channels Into the Mechanisms Underlying Pressure-Induced Myogenic Tone then will integrate the VSMC and EC Ca 2+ -dependent ion channels into the mechanisms that establish, maintain, and modulate pressure-dependent myogenic tone in resistance arteries and arterioles.

VSMC BK Ca Channels and the Regulation of Arteriolar Tone
Arteriolar VSMCs express BK Ca channels that provide negativefeedback regulation of myogenic tone (Figure 1). Both membrane depolarization and increases in intracellular Ca 2+ activate BK Ca (Tykocki et al., 2017), and because of their large conductance (~200 pS), they powerfully dampen the excitation of VSMCs, preventing vasospasm. BK Ca channels consist of a tetramer of K Ca 1.1 α-pore-forming subunits (gene = KCNMA1) which have seven transmembrane spanning domains (Meera et al., 1997; Figure 2A). Voltage is sensed by positively charged amino acids in membrane spanning domains S2, S3, and S4 (Ma et al., 2006; Figure 2A), while Ca 2+ is sensed by two regulator of conductance of K + (RCK) domains (RCK1 and RCK2) in the long, cytosolic C-terminus of the α-subunit (see (Hoshi et al., 2013a) for references; Figure 2A).
Vascular smooth muscle cells express both β and γ subunits that modulate the function of the BK Ca channel α-pore-forming subunits (Figure 2A). The primary β subunits in VSMCs are β1 (KCNMB-1, K Ca β1; Tykocki et al., 2017; Figure 2A). These subunits modulate channel gating kinetics and increase the Ca 2+ sensitivity of the α-subunit (McCobb et al., 1995;McManus et al., 1995;Meera et al., 1996;Tseng-Crank et al., 1996). They also are dynamically trafficked to the cell membrane from Rab11Apositive recycling endosomes, providing the ability of VSMCs to tune BK Ca channel function (see (Leo et al., , 2017 for details). The expression of β1-subunits may be downregulated during disease states like hypertension (Amberg et al., 2003;Tajada et al., 2013) and diabetes (McGahon et al., 2007), decreasing the ability to activate VSMC BK Ca channels, increasing myogenic tone. The BK Ca channel agonists dehydrosoyasaponin I (McManus et al., 1995) and 17β-estradiol require expression of β1-subunits (Valverde et al., 1999). Thus, β1-subunits control the Ca 2+ sensitivity and the pharmacology of BK Ca channels in VSMCs.
Arteriolar VSMC BK Ca channels have a high Ca 2+ setpoint requiring >3 μM cytosolic Ca 2+ ([Ca 2+ ] in ) to open at negative, physiological membrane potentials (−30 to −40 mV; Jackson and Blair, 1998). For reference, global [Ca 2+ ] in measured with Fura-2 in arterioles with myogenic tone is on the order of 300-400 nM (Brekke et al., 2006). Patch clamp studies have shown that arteriolar BK Ca channels are silent when VSMCs are dialyzed with solutions containing 300 nM [Ca 2+ ] in (Jackson, 1998), consistent with a high [Ca 2+ ] in threshold for their activation. The high Ca 2+ setpoint (threshold) in arteriolar VSMCs may be due to lower expression of the β 1 -subunits (Yang et al., 2009(Yang et al., , 2013 and possible differences in expression of spliced variants of the α-pore-forming subunit (Nourian et al., 2014) compared to VSMCs in larger arteries.
There also are γ-subunits associated with BK Ca channels that are leucine-rich-repeat-containing proteins (LRRCs; Yan and Aldrich, 2010;Almassy and Begenisich, 2012;Evanson et al., 2014;Gonzalez-Perez et al., 2014; Figure 2A). LRRCs allow activation of BK Ca channels at negative membrane potentials, even in the absence of Ca 2+ , by shifting their voltage vs. activity relationships to the left (increasing their voltage-sensitivity), facilitating their negative feedback function (Yan and Aldrich, 2010;Gonzalez-Perez et al., 2014 to activation by docosahexaenoic acid (DHA) also is increased by LRRCs (Hoshi et al., 2013b). The role played by LRRCs in arteriolar VSMCs has not been studied.
BK Ca channels provide strong negative feedback regulation of both pressure-induced and agonist-induced tone in resistance arteries and arterioles [see (Tykocki et al., 2017) for numerous A B C D E F G FIGURE 2 | Membrane topology of Ca 2+ -dependent ion channels involved in the regulation of myogenic tone. (A) Components of VSMC BK Ca channels including a β 1 -subunit with two membrane-spanning domains, one pore-forming α-subunit with seven membrane-spanning domains and a γ-subunit (LRRC26, for example) with one membrane-spanning domain. (B) Shows one α-subunit of an RYR with a large cytosolic N-terminal domain, 6 membrane spanning domains and a short C-terminal sequence. (C) Shows one α-subunit of an IP 3 R with a large cytosolic N-terminal domain, 6 membrane spanning domains and a short C-terminal sequence. (D) Shows one α-subunit of a TRPM4 channel including an N-Terminal domain with a TRPM homology sequence, 6 membrane spanning domains, and a C-terminal domain containing a TRP sequence and binding sites for calmodulin. (E) Shows one α-subunit of ANO1 (TMEM16A) CaCC with 10 membrane spanning domains. (F) Shows α-subunit of either sK Ca or IK Ca channels with 6 membrane spanning domains and a C-terminal domain with bindings sites for calmodulin.
(G) Shows one α-subunit of a TRPV4 channel with N-terminal sequence containing ankyrin repeat domains (ARDs), 6 membrane spanning domains and a C-terminal domain with TRP sequence and calmodulin binding sites. See text for more information.
Frontiers in Physiology | www.frontiersin.org 5 November 2021 | Volume 12 | Article 770450 references]. However, there is regional heterogeneity in the source of Ca 2+ that activates BK Ca channels in resistance arteries versus arterioles. In most resistance arteries, BK Ca channels are controlled by Ca 2+ sparks which represent the simultaneous release of Ca 2+ from the ER through small, clustered groups of RyRs (Nelson et al., 1995). Vascular smooth muscle cells that utilize this mechanism of BK Ca channel activation display the so-called spontaneous-transient-outward currents (STOCs): bursts of activity of small groups of BK Ca channels coinciding with the RyR-based Ca 2+ sparks [ (Nelson et al., 1995), see (Tykocki et al., 2017) for additional references]. In VSMCs where this mechanism is active, pharmacological block of RyRs produces the same effect as block of BK Ca channels. In contrast to many larger resistance arteries, Ca 2+ influx through VGCCs directly activates BK Ca channels in skeletal muscle arteriolar VSMCs; RyRs are silent, at least under the conditions studied (Westcott and Jackson, 2011;Westcott et al., 2012). In resistance arteries immediately upstream from skeletal muscle arterioles, both RyR-dependent and VGCC-dependent control of BK Ca channels is apparent (Westcott and Jackson, 2011;Westcott et al., 2012). These data suggest that there may be a spectrum of control mechanisms that are involved in Ca 2+ -dependent control of BK Ca channels in the resistance vasculature. In cerebral penetrating arterioles, both RyRs and BK Ca channels are silent at rest, but both can be activated by low pH (Dabertrand et al., 2012). The molecular mechanisms underlying pH-sensitive recruitment of RyR-control of BK Ca channels has not been established. The mechanisms responsible for the differences in Ca 2+ sources that control BK Ca channels are not known, but likely relate to the number and location of BK Ca channels expressed relative to RyRs, VGCCs and other ion channels.
The elemental Ca 2+ signal generated by RyRs is the Ca 2+ spark which represents the simultaneous release of Ca 2+ from small clusters of RyRs as noted in Section VSMC BK Ca Channels and the Regulation of Arteriolar Tone. Calcium influx through VGCCs has been shown to indirectly regulate Ca 2+ spark frequency and amplitude by effects on global [Ca 2+ ] in and ER Ca 2+ store loading (Essin et al., 2007). Subsequent studies have shown that the magnitude of Ca 2+ influx through the persistent activity of membrane clusters of VGCCs, that can be recorded as VGCC Ca 2+ sparklets (Navedo et al., 2005;Amberg et al., 2007), controls the amplitude of Ca 2+ sparks (Tajada et al., 2013). These data suggest that local influx of Ca 2+ is a major determinant of RyR activity in VSMCs.
In skeletal and cardiac muscle, RyRs act in a positive-feedback manner through Ca 2+ -induced-Ca 2+ -release (CICR) to cause explosive release of Ca 2+ from the ER and subsequent muscle contraction. In both skeletal muscle and cardiac muscle, Ca 2+ sparks form the basis of this positive feedback process. A similar positive feedback role for Ca 2+ sparks has been proposed for some arteriolar VSMCs (Curtis et al., 2004(Curtis et al., , 2008Arendshorst, 2005, 2007;Balasubramanian et al., 2007;Tumelty et al., 2007;Kur et al., 2013). In addition to Ca 2+ sparks, RyRs can cooperate with IP 3 Rs and contribute to Ca 2+ waves and the positive regulation of myogenic tone in some resistance arteries (Jaggar, 2001;Mufti et al., 2010Mufti et al., , 2015Westcott and Jackson, 2011;Westcott et al., 2012). In other VSMCs, RyR-dependent Ca 2+ sparks may also act in an excitatory fashion by activating plasma membrane CaCCs producing the so-called spontaneous transient inward currents (STICs) that cause membrane depolarization, VGCC activation and an increase in tone (ZhuGe et al., 1998;Cheng and Lederer, 2008).
As outlined in Section VSMC BK Ca Channels and the Regulation of Arteriolar Tone, in many resistance arteries upstream from the microcirculation, RyRs function as part of a negative-feedback process limiting VSMC excitability. In these vessels, RyR-dependent Ca 2+ sparks are functionally coupled to BK Ca channels producing membrane hyperpolarization, VGCC deactivation and a decrease in tone (Nelson et al., 1995;Jaggar et al., 1998;Cheng and Lederer, 2008).
However, in skeletal muscle (Westcott and Jackson, 2011;Westcott et al., 2012), cerebral (Dabertrand et al., 2012), and ureteral (Borisova et al., 2009) arterioles downstream from resistance arteries, RyRs are not active and do regulate myogenic tone. Low pH has been shown to recruit RyR-dependent Ca 2+ sparks in cerebral arterioles, thereby activating BK Ca channels and mediating dilation (Dabertrand et al., 2012). Whether RyRs can be recruited by pH or other conditions in skeletal muscle or ureteral VSMCs has not been studied.
The mechanisms responsible for the heterogeneity in RyR function are not known but most likely result from the specific pattern and magnitude of RyR isoform expression, their cellular Frontiers in Physiology | www.frontiersin.org 6 November 2021 | Volume 12 | Article 770450 localization, and the expression and localization of other ion channels (for example, CaCC vs. BK Ca channels) in the plasma membrane over RyRs. This area of research should be explored in more detail in the future.
In contrast, myogenic tone in hamster cheek pouch arterioles  and in murine 4 th -order mesenteric arteries (Mauban et al., 2015) does not depend on IP 3 and activation of IP 3 Rs. Phospholipase-mediated hydrolysis of phosphatidylcholine and subsequent production of diacylglycerol was proposed to participate in the generation and maintenance of myogenic tone in murine 4 th -order mesenteric arteries (Mauban et al., 2015).
Myogenic tone in rat cerebral resistance arteries is accompanied by an increase in the frequency of Ca 2+ waves (Jaggar, 2001;Mufti et al., 2010Mufti et al., , 2015 that involve both IP 3 Rs (Mufti et al., 2015) and RyRs (Jaggar, 2001;Mufti et al., 2010Mufti et al., , 2015. Similarly, Ca 2+ waves in skeletal muscle resistance arteries depend on both RyRs and IP 3 Rs (Westcott and Jackson, 2011;Westcott et al., 2012). In contrast, Ca 2+ waves in downstream skeletal muscle arterioles depend only on Ca 2+ release from IP 3 Rs (Westcott and Jackson, 2011;Westcott et al., 2012) that may amplify Ca 2+ influx through VGCCs (Jackson and Boerman, 2018). However, in rat (Miriel et al., 1999) and mouse (Zacharia et al., 2007) mesenteric resistance arteries, Ca 2+ waves were inhibited as myogenic tone developed. Thus, there appears to be regional heterogeneity in the role played by IP 3 R in the development and maintenance of myogenic tone. The mechanisms responsible for the heterogeneity in function of IP 3 Rs among blood vessels has not been established but likely stems from differences in the IP 3 R isoforms that are expressed; their localization and interactions with other proteins; and their proximity to other ion channels.

VSMC Ca 2+ -Activated Cl − Channels and Arteriolar Tone
VSMCs also express CaCCs that may contribute to myogenic tone. The protein anoctamin-1 (gene = ANO1), also known as transmembrane member 16A (TMEM16A), appears to be the molecular basis of CaCCs in VSMCs (Ji et al., 2019). This protein exists as a homodimer with each monomer having 10 membrane spanning domains (S1-S10), with the pore being formed by S3-S7 helices which also contains a Ca 2+ binding domain (Ji et al., 2019; Figure 2E). TMEM16A demonstrates a synergistic dependence on voltage and Ca 2+ to control its activity, with depolarization and increases in [Ca 2+ ] in leading to opening of these channels (Ji et al., 2019). In vascular smooth muscle, [Cl − ] in is elevated due to intracellular Cl − accumulation from the activities of the Cl − /HCO 3 − exchanger and the Na + /K + /Cl − co-transporter (Matchkov et al., 2013). The elevated [Cl − ] in sets the equilibrium potential for Cl − [−40 to −25 mV, (Matchkov et al., 2013)] to be positive to the resting membrane potential [−45 to −30 mV, (Tykocki et al., 2017)] of VSMCs that develop myogenic tone. Therefore, opening of a Cl − channel results in an outward Cl − current (an inward current in electrophysiological terms), membrane depolarization, activation of VGCCs and an increase in tone (Matchkov et al., 2013).
Calcium-activated chloride channels contribute to agonistinduced tone in a variety of arteries (Bulley and Jaggar, 2014). In addition, STICs carried by Cl − and coupled to RyR-mediated Ca 2+ sparks or IP 3 -based Ca 2+ waves have been reported (Bulley and Jaggar, 2014). Cerebral resistance artery VSMCs express TMEM16A that are functionally coupled to transient receptor potential C-family member 6 (TRPC6) channels. Calcium influx Frontiers in Physiology | www.frontiersin.org through TRPC6 activates TMEM16A contributing to the membrane depolarization, VGCC activation and pressure-induced myogenic tone in these vessels (Bulley et al., 2012;Wang et al., 2016). In hamster cheek pouch arterioles, CaCCs appear to contribute to myogenic tone when VGCCs are active (Jackson, 2020), suggesting that CaCCs may be functionally coupled to VGCCs in those VSMCs. The molecular identity of CaCCs in hamster cheek pouch arteriolar VSMCs has not been established. Additional research on expression and function of CaCCs in resistance arteries and arterioles appears warranted.

VSMC TRPM4 Channels and Arteriolar Tone
VSMCs express many members of the transient receptor potential (TRP) family of ion channels that contribute to myogenic tone [see (Earley and Brayden, 2015;Tykocki et al., 2017) for more information; Figures 1, 3]. Of these, TRPM4 channels are Ca 2+ -activated and are essential for pressure-induced myogenic tone in cerebral resistance arteries (Gonzales et al., 2014). Like all TRP channels, the pore-forming subunit of TRPM4 channels has six transmembrane domains (S1-S6) which assemble as a tetramer to form a functional ion channel with residues in the intramembrane loop between S5 and S6 forming the channel's pore (Earley and Brayden, 2015; Figure 2D). A conserved TRP domain located distal to S6 and a TRPM homology region in the NH2 terminus (Earley and Brayden, 2015) distinguish all members of the TRPM family (Earley and Brayden, 2015; Figure 2D). TRPM4 channels selectively conduct monovalent cations such that opening of these channels produces membrane depolarization due primarily to the influx of Na + (Earley and Brayden, 2015). Calmodulin binding sites in the C-terminus of TRPM4 are essential for Ca 2+ -dependent activation and the Ca 2+ -sensitivity of these channels is increased by protein kinase C-dependent phosphorylation in their amino terminus (Earley, 2013). Rho kinase also has been reported to increase the Ca 2+sensitivity of TRPM4 channels in cerebral parenchymal arterioles (Li and Brayden, 2017).
In cerebral resistance arteries and arterioles, TRPM4 channels are part of the signal transduction pathway for pressure-dependent myogenic tone (Gonzales et al., 2014;Li et al., 2014;Li and Brayden, 2017; see Figure 3 and Section Integration of Ca 2+ -Dependent Ion Channels Into the Mechanisms Underlying Pressure-Induced Myogenic Tone for more details). In this scheme, TRPM4 channels are activated by release of Ca 2+ through IP 3 Rs into the subplasmalemmal space (Gonzales et al., 2010), with the IP 3 Rs being activated by IP 3 , formed by mechanosensitive G-protein coupled receptor-mediated stimulation of phospholipase C (PLC)γ 1 , and Ca 2+ entry through TRPC6 channels, likely activated by both pressure and PLCγ 1production of diacylglycerol (DAG; Gonzales et al., 2014; Figure 3). As noted above, in cerebral parenchymal arterioles, rho-kinase, which also is activated and contributes to myogenic tone, appears to modulate the Ca 2+ sensitivity of TRPM4 channels (Li and Brayden, 2017; Figure 3). The Na + entry through TRPM4 channels, along with the entry of Ca 2+ and Na + through TRPC6 channels produces membrane depolarization and activation of Ca 2+ entry through VGCCs, hallmark elements of pressure-dependent myogenic tone (see (Tykocki et al., 2017) for numerous references; Figure 3). The role of TRPM4 in myogenic tone of vessels in other vascular beds has been questioned because global knockout of TRPM4 has no effect on pressure-induced tone in hind limbs of mice (Mathar et al., 2010). However, the details of the mechanisms responsible for pressure-induced tone in the TRPM4 knockout animals was not determined, such that compensation for the global knockout of TRPM4 channels may have occurred. Additional research on TRPM4 and myogenic tone appears warranted.

VSMC TRPP1 (PKD2) Channels and Myogenic Tone
Another potentially Ca 2+ -activated ion channel that is involved in regulation of myogenic tone are TRPP1 (PKD2) channels. Similar to TRPM4 channels already described, TRPP1 channels are tetramers of 6 membrane spanning domains encoded by the PKD2 gene that have coiled-coil domains in their C-termini and a Ca 2+ -binding EF-hand motif that may be involved in Ca 2+ -dependent activation of these channels (Giamarchi and Delmas, 2007). The channels formed from TRPP1 are non-selective cation channels that conduct both Ca 2+ and Na + (Giamarchi and Delmas, 2007). The function of TRPP1 in regulation of myogenic tone is unclear. In murine mesenteric arteries, VSMC TRPP1 channels appear to inhibit myogenic tone (Sharif-Naeini et al., 2009), whereas in rat cerebral arteries VSMC TRPP1 channels significantly contribute to myogenic tone (Narayanan et al., 2013). Conditional knockout of TRPP1 from VSMCs decreases blood pressure and substantially reduces myogenic tone in murine skeletal muscle resistance arteries (Bulley et al., 2018). The plasma membrane expression of TRPP1 in VSMCs is controlled by recycling of sumoylated channels and SUMO1 modification of TRPP1 channels is required for pressure-induced myogenic tone (Hasan et al., 2019). How TRPP1 channels "fit" with other channels that have been shown to be involved in initiation and maintenance of myogenic tone (TRPC6 and TRPM4, for example) remains to be established. Nor has it been established that VSMC TRPP1 channels are activated by Ca 2+ or that Ca 2+ -dependent activation is part of their role in pressure-dependent myogenic tone. It is known that TRPP1 channels can heterodimerize with other members of the TRP family (Giamarchi and Delmas, 2007) such that it is feasible that TRPP1 channels may be part of a multi-channel complex. Additional research will be required to determine how TRPP1 channels and all of the other VSMC ion channels implicated in the generation and maintenance of myogenic tone fit together.

Inhibition of VSMC Ion Channels by Ca 2+
Voltage-gated Ca 2+ channels composed of CaV1.2 α-subunits (gene = CACNA1C) play a central role myogenic tone as these channels provide the main source of intracellular Ca 2+ , the primary trigger of VSMC contraction (Tykocki et al., 2017). Calcium-dependent inhibition of VGCCs is mediated by calmodulin that binds to the C-terminus of CaV1.2 channels Frontiers in Physiology | www.frontiersin.org 8 November 2021 | Volume 12 | Article 770450 that make up VSMC VGCCs (Shah et al., 2006). Thus, VGCCs themselves may contribute to the negative-feedback regulation of myogenic tone through this process (Figure 3). Vascular smooth muscle cells express a diverse array of K V channels that participate in the negative-feedback regulation of myogenic tone (Tykocki et al., 2017). Early studies showed Ca 2+ -dependent inhibition of K V channel currents in VSMCs from large arteries (Gelband et al., 1993;Ishikawa et al., 1993;Gelband and Hume, 1995;Post et al., 1995;Cox and Petrou, 1999). However, the molecular identity of the K V channel isoform that was inhibited was not identified: it was only suspected to be a channel inhibited by 4-amino pyridine (4-AP).
Frontiers in Physiology | www.frontiersin.org 9 November 2021 | Volume 12 | Article 770450 Block of K V channels by 4-AP appears to be Ca 2+ -dependent, making interpretation of 4-AP sensitivity difficult (Baeyens et al., 2014). It is well established that increased [Ca 2+ ] in inhibits K V 7.2-7.5 channels via binding to calmodulin associated with these channels (Alaimo and Villarroel, 2018). K V 7 channels contribute substantially to the regulation of myogenic tone in resistance arteries (Mackie et al., 2008;Greenwood and Ohya, 2009;Jepps et al., 2013;Cox and Fromme, 2016). Therefore, it is likely that at least some of the inhibitory effect of elevated [Ca 2+ ] in on whole-cell K V currents is through inhibition of K V 7 channels. Regardless, Ca 2+ -dependent inhibition of active K V channels will cause membrane depolarization, activation of VGCCs and a further increase in [Ca 2+ ] in contributing to the positive-feedback regulation of myogenic tone (Figure 3). It should be noted that the density of K V channels is such that Ca 2+ -dependent inhibition of these channels serves only to blunt the main, negative-feedback role that K V channels play in the regulation of myogenic tone (Tykocki et al., 2017;Jackson, 2018). Elevated [Ca 2+ ] in also inhibits ATP-sensitive K + (K ATP ) channels through Ca 2+ -dependent activation of the protein phosphatase, calcineurin (Wilson et al., 2000). These channels are active at rest in the microcirculation of a number of vascular beds (Tykocki et al., 2017). Closure of K ATP channels by increased Ca 2+ would contribute to membrane depolarization, activation of VGCCs, and a further increase in [Ca 2+ ] in , a positive-feedback process that would increase myogenic tone (Figure 3).

ENDOTHELIAL Ca 2+ -DEPENDENT ION CHANNELS AND ARTERIOLAR TONE
Numerous ion channels also contribute to EC function and to the modulation of myogenic tone (Jackson, 2016). Calciumdependent ion channels in ECs include IP 3 Rs, small conductance Ca 2+ -activated K + (sK Ca ) channels, intermediate conductance Ca 2+ -activated K + (IK Ca ) channels, CaCCs, transient receptor potential vanilloid-family member 4 (TRPV 4 ) channels and TRPP1 channels.

EC IP 3 Rs and Arteriolar Tone
Endothelial cells express IP 3 Rs that contribute to the negativefeedback regulation of arteriolar myogenic tone. Early EC studies demonstrated that the initial increase in [Ca 2+ ] in in response to agonists of EC Gα q -coupled receptors resulted from Ca 2+ release from ER stores (Hallam and Pearson, 1986;Colden-Stanfield et al., 1987;Busse et al., 1988;Schilling et al., 1992;Davis, 1994, 1995). Subsequent studies pinpointed IP 3 Rs as the primary Ca 2+ release channel involved in this response (Sharma and Davis, 1995;Cohen and Jackson, 2005).
Endothelial cells from arteries (Mountian et al., 1999(Mountian et al., , 2001Grayson et al., 2004;Ledoux et al., 2008) and arterioles (Jackson, 2016) appear to express all three isoforms of IP 3 R. However, the dominant isoform may display regional-or species-dependent heterogeneity. For example, IP 3 R2 is the dominant IP 3 R expressed in mouse mesenteric artery ECs (Ledoux et al., 2008), whereas IP 3 R3 is the dominant IP 3 R in mouse cremaster muscle arteriolar ECs (Jackson, 2016). There is little information about the specific localization of IP 3 R in native arteriolar ECs. In both EC-VSMC co-cultures and in intact mouse cremaster arterioles, IP 3 R1 localizes at sites of MEGJs (Isakson, 2008). Similarly, in mouse mesenteric resistance arteries, EC IP 3 Rs cluster near holes in the internal elastic lamina (Ledoux et al., 2008), that are sites of myoendothelial projections (MEPs) and MEGJs (Sandow and Hill, 2000; Figure 1). Although the IP 3 R isoform(s) expressed in these IP 3 R clusters has not been identified, they were demonstrated to be the sites of EC Ca 2+ pulsars, localized IP 3 -dependent Ca 2+ events arising from clusters of IP 3 Rs in the ER that extend into MEPs (Kansui et al., 2008;Ledoux et al., 2008 ; Figure 1).

Arteriolar ECs Do Not Express Functional RyRs
Early studies of ECs from large arteries provided evidence for expression of functional RyRs (Lesh et al., 1993;Graier et al., 1994Graier et al., , 1998Ziegelstein et al., 1994;Rusko et al., 1995;Kohler et al., 2001b). In contrast, there is a lack of evidence for expression of RyRs in resistance artery and arteriolar ECs. Mouse mesenteric resistance artery ECs do not express mRNA for the three RyR isoforms, whereas transcripts for IP 3 Rs are readily detected (Ledoux et al., 2008). In addition, resting Ca 2+ levels or acetylcholine-evoked Ca 2+ events in mouse (Ledoux et al., 2008) or rat (Kansui et al., 2008) mesenteric resistance artery ECs are unaffected by concentrations of ryanodine that block RyRs. Similarly, mouse cremaster arteriolar ECs do not express message for RyRs (Jackson, 2016), and the RyR agonist, caffeine (10 mM), has no effect on [Ca 2+ ] in in these ECs (Cohen and Jackson, 2005). These data do not support a role for RyRs in resistance artery or arteriolar EC Ca 2+ signals.

EC sK Ca and IK Ca Channels and Arteriolar Tone
Resistance artery and arteriolar ECs express both sK Ca (K Ca 2.3; gene = KCNN3) and IK Ca (K Ca 3.1; gene = KCNN4) channels (Kohler et al., 2001a;Eichler et al., 2003;Taylor et al., 2003;Sandow et al., 2006;Si et al., 2006;Grgic et al., 2009). These channels are a tetramer of six transmembrane domain subunits with cytosolic N-and C-termini (Adelman et al., 2012; Figure 2F). The ion conducting pore is formed from a pore loop between membrane spanning domains 5 and 6, as in voltage-gated K + channels (Adelman et al., 2012). Calmodulin interacts with the intracellular C-terminus to gate opening of both channels (Xia et al., 1998;Fanger et al., 1999;Adelman et al., 2012;Sforna et al., 2018). The Ca 2+ sensitivity of sK Ca and IK Ca channels is an order of magnitude higher than for BK Ca channels. The threshold for activation by Ca 2+ binding to calmodulin occurs at 100 nM, 50% of maximal activation at 300 nM and maximal activation at 1 μM for both sK Ca channels (Xia et al., 1998) and IK Ca channels (Ishii et al., 1997). The distinct pharmacology of sK Ca and IK Ca channels has helped to define their function in intact vessels (Jackson, 2016).
Endothelial cell sK Ca channels also exist in macromolecular signaling microdomains around the EC periphery. They are found in cholesterol-rich areas (caveolae or lipid rafts) and colocalize with caveolin-1 (Absi et al., 2007). Ca 2+ influx through TRPC3 channels selectively activates sK Ca channels in rat cerebral arteries (Kochukov et al., 2014), suggesting that TRPC3 and sK Ca channels exist in the same microdomain. In mouse carotid arteries, sK Ca channels are in caveolae adjacent to EC-EC gap junction plaques (Brahler et al., 2009). Conditional knockout of sK Ca channels attenuates shear-stressinduced vasodilation in these arteries, suggesting that sK Ca channel localization has functional consequences (Brahler et al., 2009). The respective EC localization of sK Ca and IK Ca channels and their signaling microdomains explain how these two channels mediate different facets of EC hyperpolarization and the regulation of myogenic tone (Crane et al., 2003;Si et al., 2006).
Because ECs are electrically coupled to VSMCs via MEGJs, resting membrane potential of ECs can impact myogenic tone. Resting EC membrane potential is determined, in part, by the activity of sK Ca and IK Ca channels. Overexpression of sK Ca channels (which hyperpolarizes ECs) reduces myogenic tone of mesenteric resistance arteries (Taylor et al., 2003). In contrast, conditional knockout of sK Ca channels has the opposite effect (EC depolarization and an increase in myogenic tone; Taylor et al., 2003). Consistent with these data, pharmacological inhibition of sK Ca and IK Ca channels, or both channels augment(s) myogenic tone in rat cerebral parenchymal arterioles (Cipolla et al., 2009;Hannah et al., 2011). Endothelial cell sK Ca and IK Ca channels seem to play a smaller role in modulating myogenic tone of larger cerebral resistance arteries, although they remain important in endothelium-dependent agonistinduced vasodilation (Cipolla et al., 2009). Nonetheless, sK Ca and IK Ca channels significantly contribute to EC-dependent negative-feedback regulation of myogenic tone.
Endothelium-dependent vasodilators that act through G qcoupled receptors also activate sK Ca and IK Ca channels. In some vessels, such as guinea-pig carotid artery (Corriu et al., 1996), rat mesenteric arteries preconstricted with phenylephrine (Crane et al., 2003) and porcine coronary arteries (Bychkov et al., 2002) both channels appear to be involved because block of both sK Ca and IK Ca channels is necessary to inhibit agonistinduced EC hyperpolarization. In contrast, IK Ca channels mediate endothelium-dependent hyperpolarization and vasodilation in rat cerebral arteries (Marrelli et al., 2003) and in murine arteries and arterioles (Brahler et al., 2009). The reason for this heterogeneity in the roles played by sK Ca and IK Ca channels between vascular beds is not apparent and will require further research.

EC BK Ca Channels and Arteriolar Tone
The expression and function of BK Ca channels in ECs remains debatable (Sandow and Grayson, 2009). As described for VSMCs, BK Ca channels are activated by both voltage and Ca 2+ , have a much larger conductance (~250 pS) than sK Ca and IK Ca channels, do not require association with calmodulin, and display pharmacology distinct from sK Ca and IK Ca channels (Hoshi et al., 2013a;Tykocki et al., 2017). Cultured large artery ECs have been reported to express BK Ca channels (see (Sandow and Grayson, 2009) for references). Native ECs isolated from hypoxic rats (Hughes et al., 2010;Riddle et al., 2011) or cholesterol depleted ECs (Riddle et al., 2011) express functional BK Ca channels. In cultured ECs, BK Ca channels are located in caveolae and caveolin inhibits their function . These studies open the possibility that EC BK Ca channels are normally inhibited. Conversely, chronic hypoxia, and potentially other stresses or pathologies, that alter membrane lipid domains may upregulate EC BK Ca channel function (Sandow and Grayson, 2009). Electrophysiological studies of freshly isolated bovine coronary artery (Gauthier et al., 2002), mouse carotid artery (Brahler et al., 2009), and rat cerebral parenchymal arteriolar (Hannah et al., 2011) ECs found only sK Ca channel and IK Ca channel currents; no BK Ca channel currents were detected. While it has been reported that ECs in freshly isolated rat cremaster arterioles express protein for BK Ca channels (Ungvari et al., 2002), neither mRNA nor protein for this channel were detected in bovine coronary artery ECs (Gauthier et al., 2002). Murine skeletal muscle resistance artery and arteriolar ECs lack BK Ca channel mRNA (Jackson, 2016). Thus, there may be regional or species heterogeneity in EC expression of BK Ca channels. Additional research appears to be warranted to define if and where EC BK Ca are expressed, how they are regulated and their function in the regulation of myogenic tone.

EC Ca 2+ -Activated Cl − Channels and Arteriolar Tone
Electrophysiological studies of bovine pulmonary artery and human umbilical vein ECs demonstrate the functional expression of CaCCs (Nilius et al., 1997;Zhong et al., 2000). Unlike VSMCs (see Section VSMC Ca 2+ -Activated Cl − Channels and Arteriolar Tone), initial studies did not report expression of TMEM16A in ECs in lung sections (Huang et al., 2009;Ferrera et al., 2011). However, more recent studies have identified TMEM16A expression and function in human pulmonary artery ECs and have shown that over expression of these channels leads to EC dysfunction (Skofic Maurer et al., 2020). In hypertension, EC TMEM16A also contributes to endothelial dysfunction (Ma et al., 2017). TMEM16A is expressed in murine cerebral capillary ECs where it regulates membrane potential, Ca 2+ signaling, proliferation, migration, and blood brain barrier permeability (Suzuki et al., 2020). Block of TMEM16A preserves blood brain barrier function after ischemic stroke (Liu et al., 2019). Hypoxia stimulates proliferation of brain capillary ECs via increased expression of TMEM16A (Suzuki et al., 2021).
Hypoxia also increases expression of TMEM16A in mouse cardiac ECs (Wu et al., 2014).
The function of TMEM16A in arteriolar ECs related to regulation of myogenic tone is not clear. In murine capillary ECs, block of TMEM16A results in membrane hyperpolarization suggesting that in ECs, like in VSMCs (see Section VSMC Ca 2+ -Activated Cl − Channels and Arteriolar Tone), activation of these CaCCs leads to membrane depolarization, counter to the effects of activation of EC sK Ca and IK Ca channels which produce robust EC hyperpolarization. Thus, it may be that CaCCs in ECs are part of a negative feedback mechanism to dampen membrane hyperpolarization induced by EC sK Ca and IK Ca channels when intracellular Ca 2+ is elevated.

EC TRPV4 and Regulation of Arteriolar Tone
Transient receptor potential vanilloid-family member 4 channels are another prominent Ca 2+ -modulated ion channel expressed in ECs (Sonkusare et al., 2012(Sonkusare et al., , 2014Hong et al., 2018;Chen and Sonkusare, 2020). These channels are formed from a tetramer of six membrane spanning domain subunits, with the pore of the channel formed by a pore-loop between domains 5 and 6 like many other ion channels ( Figure 2G). They conduct primarily Ca 2+ and are activated by a diverse array of chemicals including EETs (Nilius et al., 2004). In ECs, TRPV4 channels exist in signaling complexes near MEGJ's along with IK Ca channels, IP 3 Rs and other proteins (Sonkusare et al., 2012(Sonkusare et al., , 2014Hong et al., 2018;Chen and Sonkusare, 2020 ; Figures 1, 3). Intracellular Ca 2+ potentiates the activation of TRPV4 channels through calmodulin that binds to the C-terminal region of this channel (Strotmann et al., 2003).
Endothelial TRPV4 channels mediate agonist-induced, endothelium-dependent vasodilation, particularly in arterioles where activation of these receptors leads to activation of IK Ca channels, EC hyperpolarization and conduction of this hyperpolarization to overlying VSMCs to induce vasodilation (Marrelli et al., 2007;Earley et al., 2009b;Sonkusare et al., 2012Sonkusare et al., , 2014Zhang et al., 2013;Zheng et al., 2013;Du et al., 2016;Diaz-Otero et al., 2018; Figure 3). In addition, TRPV4 channels play a central role in myoendothelial negative-feedback that tempers vascular tone in the absence of an endothelial agonist. Agonist-induced activation of VSMC Gq-coupled receptors leads to a global increase in EC intracellular Ca 2+ (Dora et al., 1997;Schuster et al., 2001;Tuttle and Falcone, 2001;Jackson et al., 2008;Kansui et al., 2008) that contributes to the negative-feedback regulation of vascular tone (Lemmey et al., 2020). Studies in murine mesenteric resistance arteries have shown that endothelial TRPV4 channels are activated during this process through a mechanism involving Ca 2+ release through IP 3 Rs, resulting in activation of IK Ca channels blunting agonist-induced vasoconstriction (Hong et al., 2018 ; Figure 3). Similarly, studies in rat cremaster arterioles have shown that endothelial TRPV4 channels are activated at low intravascular pressure, leading to TRPV4 Ca 2+ sparklets (localized [Ca 2+ ] in signals through small groups of TRPV4 channels), activation of IK Ca channels and dampening of myogenic tone (Bagher et al., 2012). The precise signal that is communicated from VSMCs to ECs to initiate myoendothelial feedback remains in question, with data supporting Ca 2+ as the signal  and other findings supporting IP 3 as the signal (Tran et al., 2012;Hong et al., 2018). Additional research will be required to determine whether Ca 2+ or IP 3 mediates myoendothelial negative-feedback and whether there is heterogeneity among vessels in which signal (Ca 2+ or IP 3 ) is used.

EC TRPP1 Channels and Myogenic Tone
Endothelial cells also express TRPP1 channels where they function in shear-stress dependent vasodilation (MacKay et al., 2020). Shear-stress-induced increases in EC [Ca 2+ ] in that activate sK Ca channels, IK Ca channels and EC nitric oxide synthase were shown to be substantially impaired by conditional knockout of EC TRPP1 with no change in Ca 2+ signals activated by muscarinic receptor activation (MacKay et al., 2020). Calciumdependent activation of TRPP1 channels was not established in these studies, so [Ca 2+ ] in modulation of these channels in ECs and their role in regulating myogenic tone other than when activated by shear-stress remains to be established.

INTEGRATION OF Ca 2+ -DEPENDENT ION CHANNELS INTO THE MECHANISMS UNDERLYING PRESSURE-INDUCED MYOGENIC TONE
As outlined in Sections above, Ca 2+ -dependent ion channels in VSMCs and ECs are involved in the initiation, maintenance and modulation of pressure-induced myogenic tone. Figure 3 integrates this information into a working model with the function of VSMC and EC Ca 2+ -dependent ion channels highlighted.

Activation of Plasma Membrane Ion Channels Produces Membrane Depolarization
Pressure-and likely DAG-induced activation of plasma membrane TRPC6 channels results in Ca 2+ influx through these channels (Slish et al., 2002;Welsh et al., 2002). The resultant local [Ca 2+ ] in increase, along with IP 3 , activates IP 3 Rs to release Ca 2+ from the ER, amplifying the local [Ca 2+ ] in increase. This subplasmalemmal increase in [Ca 2+ ] in then activates overlying plasma membrane TRPM4 channels. Calcium influx through TRPC6 channels also activates plasma membrane Ca 2+ -activated Cl − channels (CaCCs; Bulley et al., 2012;Wang et al., 2016). The cation influx through TRPC6 and TRPM4 channels, and Cl − efflux through CaCCs causes membrane depolarization (Figure 3). As noted in Section Pressure-Dependent Activation of Mechanosensors Leads to Formation of IP 3 and DAG and shown in Figure 3, additional cation channels including TRPP1 channels may contribute to the pressure-induced depolarization.

K + Channels Provide Negative Feedback to Dampen Myogenic Tone
Membrane depolarization-induced activation of VGCCs is inherently a positive-feedback process because the Ca 2+ influx through these channels will itself lead to depolarization and further activation of VGCCs. This process is limited in VSMCs by activation of at least three negative-feedback processes. Membrane depolarization activates K V channels, and membrane depolarization along with increased [Ca 2+ ] in activates BK Ca channels. The K + efflux through these two K + channels (which by themselves would cause membrane hyperpolarization) blunts and limits depolarization-induced activation of VGCC (Figure 3; Jackson, 2017Jackson, , 2020. Additional negative feedback arises from Ca 2+ -dependent inactivation of VGCCs (Shah et al., 2006; Figure 3). Parallel Activation of Protein Kinase C and Rho-Kinase In addition to activating TRPC6 channels, the DAG formed from the activity of PLC along with elevated [Ca 2+ ] in activates protein kinase C (PKC) supporting the increase in tone by increasing the activity of TRPM4 channels (supporting depolarization) and VGCCs (promoting Ca 2+ influx) while blunting the activity of several K + channels (also supporting membrane depolarization; Jackson, 2020, 2021; Figure 3). The negative feedback involving K V channels is blunted by Ca 2+ -dependent inhibition of these channels (Gelband et al., 1993;Ishikawa et al., 1993;Gelband and Hume, 1995;Post et al., 1995;Cox and Petrou, 1999; Figure 3). Ca 2+ -dependent activation of the protein phosphatase, calcineurin, inhibits ATP-sensitive K + (K ATP ) channels, limiting their activity and promoting depolarization (Wilson et al., 2000; Figure 3). Stimulation of the mechano-sensors in vascular smooth muscle also activates the small G-protein rhoA, which, in turn, activates rho-kinase (Chennupati et al., 2019; Figure 3). Rho kinase phosphorylates a number of substrates that also support myogenic tone including inhibition of myosin light chain phosphatase (MLCPPT; Cole and Welsh, 2011), stimulation of actin cytoskeleton remodeling that accompanies activation of the contractile machinery (Loirand et al., 2006;Moreno-Dominguez et al., 2013), inhibition of K V channels as a consequence of actin remodeling (Luykenaar et al., 2009) and increasing the Ca 2+ sensitivity of TRPM4 channels (Li and Brayden, 2017; Figure 3). Activated PKC also may inhibit MLCPPT through phosphorylation of the inhibitory protein, CPI 17 (Cole and Welsh, 2011; Figure 3).

Endothelial Cells Contribute to the Negative-Feedback Regulation of Myogenic Tone
Endothelial cells lining resistance arteries and arterioles play a negative-feedback role, dampening myogenic tone both through the Ca 2+ -dependent production of vasodilator autacoids (PGI 2 , NO, EETS, etc.) and by conduction of Ca 2+ -dependent membrane hyperpolarization from the endothelium to overlying VSMCs via MEGJs (Figures 1, 3). Endothelial cells chemically and electrically converse with VSMCs through MEGJs that may form at myoendothelial projections that penetrate holes in the internal elastic lamina and contact the overlying VSMCs. Heterocellular gap junctions (MEGJs) between ECs and VSMCs form and allow small molecules (like IP 3 ) and ionic currents (including Ca 2+ ) to move between the cells. Pressure-induced increases in VSMC [Ca 2+ ] in or IP 3 can pass to endothelial cells leading to EC IP 3 R-induced Ca 2+ signals (Ca 2+ pulsars and wavelets) that can increase the production of Ca 2+ -dependent EC vasodilator autacoids that feedback to the VSMCs reducing myogenic tone (Figure 3). In addition, increased EC [Ca 2+ ] in will activate EC sK Ca and IK Ca channels causing EC membrane hyperpolarization. Myoendothelial gap junctions allow this hyperpolarization to be passed from ECs to VSMCs, producing VSMC hyperpolarization, deactivation of VSMC VGCCs and reduced myogenic tone (Figure 3). Thus, the production of EC autacoids and EC membrane potential are both strongly dependent on the activity of Ca 2+ -dependent ion channels in the endothelium including IP 3 Rs, TRPV4 channels, sK Ca channels and IK Ca channels (Lemmey et al., 2020).

FINAL PERSPECTIVE
As outlined in this perspective, Ca 2+ -activated ion channels in both VSMCs and ECs contribute to the regulation of myogenic tone. However, there appears to be considerable heterogeneity in the specific details of their roles in this process among vessels in different vascular beds around the body. The mechanisms responsible for this heterogeneity remains to be established. It is also clear that there is a paucity of information about the cellular and molecular details surrounding which channels are expressed, their localization and their regulation relative to myogenic tone in arterioles around the body. Mesenteric and cerebral resistance artery ion channel expression and function has been well studied. However, we know relatively little about ion channel expression and function in the downstream arterioles in microcirculation, which is really the business end of the cardiovascular system. Future studies directed specifically at understanding control of ion channel expression and function in the microcirculation and how they vary among vascular beds in different organs is warranted.

AUTHOR CONTRIBUTIONS
WJ conceived, wrote, and edited this manuscript.

FUNDING
Supported by National Heart, Lung and Blood Institute grants HL-137694 and PO1-HL-070687.