Ion Channels Expressed in Arterioles

Vascular smooth muscle and endothelial cells in the wall of arterioles express a large number of ion channels

(Table I). These include one or more varieties of voltage-gated Ca2+ channels, at least four different classes of K+ channels, several types of nonselective cation channels and at least two classes of Cl- channels. All of these channels arise from multigene families that also display splice variation. Table I shows the isoforms of channel proteins that are thought to underlie the ion channels present in arteriolar vascular smooth muscle and endothelium. In addition, blockers or inhibitors of the channels are shown. Please note that with the exception of a few substances, such as the scorpion toxin, iberiotoxin, that selectively blocks large-conductance Ca2+-activated K+ channels, most of the substances listed show only limited selectivity. For example, all of the inhibitors of Ca2+-activated and swelling-activated Cl- channels also inhibit voltage-gated Ca2+ or nonselective cation channels, such that their use in functional studies is very limited.

Voltage-Gated Ca2+ Channels

Voltage-gated Ca2+ channels in smooth muscle cells play a major role in the regulation of arteriolar tone. Importantly, they transduce changes in vascular smooth muscle membrane potential (produced by other ion channels and transporters) into changes in Ca2+ influx and intracellular Ca2+. Signals that depolarize smooth muscle open voltage-gated Ca2+ channels, allowing Ca2+ to diffuse into cells down their electrochemical gradients, raising intracellular Ca2+, leading to smooth muscle contraction and increased arteriolar tone. Conversely, hyperpolarization of smooth muscle closes these channels, reducing intracellular Ca2+ and leading to decreases in arteriolar tone.

The dominant voltage-gated Ca2+ channels expressed in vascular smooth muscle cells are nifedipine-sensitive, L-type Ca2+ channels that begin to activate at -50 mV under physiological conditions. Smooth muscle cells in some arte-rioles also express other types of voltage-gated Ca2+ channels. These include T-type channels that are insensitive to

Table I Ion Channels in Arteriolar Smooth Muscle and Endothelial Cells.

Ion channel3

VSM"

ENDOc Additional Conductance Primary stimuli subunits (pS)

Inhibitor(s)"

BKCa sKCa

IKCa kv katp kir

ClSW ClCa

TRPC 1

TRPC 6 or TRPM4 TRPC 6? Slo1

TRPC 1?

SK3 IK1

25-30

Slo b

25-30 250

Calmodulin 4-20 Calmodulin 20-80

50-100?

Release of Ca2+ from internal stores, PKC Membrane stretch, PKC

Depolarization, T[Ca2+]in, NO, CO, EETs T[Ca2+]m T[Ca2+]m

Depolarization 0ATP, TADP, PKA, PKG

Hyperpolarization, T[K+]out

TCell volume, membrane stretch

TCa2+

Nifedipine, diltiazem, verapamil,

Kurtoxin, mibefradil, pimozide, flunarizine Ni2+ > Cd2+ Nimodipine, amiloride, mibefradil,

G. spatulata venom, SKF 96365, amiloride, Gd3+, La3+ SKF 96365, amiloride, Gd3, La3+ Iberiotoxin, charybdotoxin, penitrem A, paxillin, 1 mM TEA Apamin, d-tubocurarine, TBA TRAM-39, TRAM-34, charybdotoxin, clotrimazole, TBA

4-aminopyridine, correolide, Agitoxin-2

Glibenclamide, tolbutamide, TPA, Ba2+

Ba2+, quinidine, phencyclidine

Tamoxifen, DIDS, niflumic acid, IAA-94, 9-AC, NPPB

Niflumic acid, flufenamic acid, 9-AC DIDS, NPPB

a See text for definitions of channel abbreviations. b Vascular smooth muscle. c Endothelium.

d Inhibitor abbreviations: 2-APB, 2-aminoethoxydiphenyl borate; TEA, tetraethylammonium; TBA, tetrabutylammonium; TPA, tetrapentylammonium; DIDS, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid; IAA-94, indanyloxyacetic acid; 9-AC, 9-anthracenecarboxylic acid; NPPB, 5-nitro-2-(3-phenyl-propylamino)benzoic acid.

e —, Not present; ?, present, but specific isoform unclear, or mechanism unclear.

nifedipine, activate at very negative membrane potentials (—70 mV), and inactivate rapidly, and nifedipine-insensitive, high voltage-activated Ca2+ channels (R-type Ca2+) with activation properties similar to those of L-type channels.

The presence of voltage-gated Ca2+ channels in endothelial cells remains controversial. Studies of endothelial cells from conduit arteries have uniformly failed to demonstrate their presence. However, a few studies of microvascular endothelial cells have demonstrated the presence of both L-type and T-type voltage-gated Ca2+ channel currents. The generality of these findings has not been established and the physiological function of endothelial cell voltage-gated Ca2+ channels in the regulation of arteriolar tone is unknown.

K+ Channels

Arteriolar smooth muscle cells express at least four different classes of K+ channels in their membranes. These include large conductance, Ca2+-activated K+ (BKCa)

channels, several types of voltage-gated K+ (KV) channels, ATP-sensitive K+ (KATP) channels, and inward rectifier K+ (Kir) channels. All appear to contribute to the regulation of arteriolar tone in the microcirculation. As in other cells in the body, K+ channels play a major role in determining membrane potential. Opening of K+ channels causes K+ ions to diffuse out of cells, down their electrochemical gradients. This loss of positive charge causes membrane hyper-polarization, closure of voltage-gated Ca2+ channels, and decreased arteriolar tone. Conversely, closure of open K+ channels leads to membrane depolarization and opening of voltage-gated Ca2+ channels, leading to increased arteriolar tone.

Microvascular endothelial cells also express multiple types of K+ channels including two types of Ca2+-activated K+ channels, KV channels, KATP channels, and KIR channels. These K+ channels also significantly contribute to the regulation of membrane potential in endothelial cells that, itself, can act as a signal to regulate arteriolar tone (see later discussion). In addition, membrane potential determines the electrochemical gradient for Ca2+ influx through nonselective cation channels, that contributes to the production of endothelium-derived vasodilators such as NO, prostacyclin, and epoxides of arachidonic which also participate in the regulation of arteriolar tone (see Figure 4). Potassium channels also may allow changes in membrane potential to be conducted, in a nondecremental fashion, along the length of arterioles, further contributing to the regulation of arteriolar tone.

BKCa Channels

Large-conductance, Ca2+-activated K+ channels represent one of the most abundant K+ channels expressed in smooth muscle membranes. These channels open with membrane depolarization and increases in intracellular Ca2+ and are modulated by a number of cell signaling pathways. They play a major role in the negative feedback regulation of smooth muscle membrane potential during vasoconstriction (see Figure 2) and participate in the mechanism of action of several vasodilators (see Figure 3). Like many ion channels, BKCa channels exist in signaling complexes with voltage-gated Ca2+ channels, protein kinases (protein kinase A, cGMP-dependent protein kinase, protein kinase C), phosphatases, and other signaling proteins (Figure 1). These macromolecular signaling complexes also appear to be located adjacent to smooth endoplasmic reticulum Ca2+-activated, Ca2+-release channels (ryanodine receptors, RYR) such that focal release of Ca2+ from RYR triggered by Ca2+ influx through voltage-gated Ca2+ channels (i.e., Ca2+ sparks) can activate BKCa channels in smooth muscle. Microvascular endothelial cells likely do not express BKCa channels in vivo.

sKCa and IKCa Channels

Native endothelial cells in arterioles express two types of Ca2+-activated K+ channels: small conductance, sKCa channels, and intermediate conductance, IKCa channels that are distinct from BKCa channels. These channels are responsible for agonist-induced hyperpolarization of endothelial cells and thus play an important role in regulation of arteriolar tone. In addition to having much smaller single channel conductances, both channels are insensitive to the highly selective BKCa channel blocker, iberiotoxin, and are not voltage-gated such that they are easily distinguished from BKCa channels. Both sKCa and IKCa channels require calmodulin to display Ca2+-dependent modulation, unlike BKCa channels that bind Ca2+ directly.

KV Channels

Arteriolar smooth muscle cells express a number of different KV channels and there are species and regional differences in the specific gene products that are expressed. As their name implies, KV channels are activated by membrane depolarization. These channels participate in the regulation of resting membrane potential, the negative feedback regulation of membrane potential during vasoconstriction (Figure 2), and may participate in the mechanism of action of

Figure 1 Ion channels form signaling complexes. Simplified schematic diagram of a signaling complex in a smooth muscle cell consisting of voltage-gated Ca2+ channels (VGCC), G-protein-coupled receptors, adenyl cyclase (AC), protein kinase A (PKA), protein phosphatase 2A (PP2A), and large-conductance Ca2+-activated K+ channels (BKCa). The VGCC and BKCa channels are functionally coupled to ryanodine receptors (RYR) in the membranes of smooth endoplasmic reticulum (sER) adjacent to the plasma membrane. 1, Ca2+ influx through VGCC increases local Ca2+ concentration in the sub-plasmalemmal space. 2, This local increase in Ca2+ triggers Ca2+-induced Ca2+ release from the SER through RYR. 3, This Ca2+ release results in a focal increase in subplasmalemmal Ca2+ (a Ca2+ spark). 4, The Ca2+ spark, along with Ca2+ entry through VGCC activates BKCa channels. 5, The resultant K+ efflux hyperpolarizes the membrane. 6, The hyperpolarization closes VGCC providing negative feedback control of the system.

Figure 1 Ion channels form signaling complexes. Simplified schematic diagram of a signaling complex in a smooth muscle cell consisting of voltage-gated Ca2+ channels (VGCC), G-protein-coupled receptors, adenyl cyclase (AC), protein kinase A (PKA), protein phosphatase 2A (PP2A), and large-conductance Ca2+-activated K+ channels (BKCa). The VGCC and BKCa channels are functionally coupled to ryanodine receptors (RYR) in the membranes of smooth endoplasmic reticulum (sER) adjacent to the plasma membrane. 1, Ca2+ influx through VGCC increases local Ca2+ concentration in the sub-plasmalemmal space. 2, This local increase in Ca2+ triggers Ca2+-induced Ca2+ release from the SER through RYR. 3, This Ca2+ release results in a focal increase in subplasmalemmal Ca2+ (a Ca2+ spark). 4, The Ca2+ spark, along with Ca2+ entry through VGCC activates BKCa channels. 5, The resultant K+ efflux hyperpolarizes the membrane. 6, The hyperpolarization closes VGCC providing negative feedback control of the system.

both vasoconstrictors (Figure 2) and vasodilators (Figure 3). As noted in Table I, most KV channels expressed in arteriolar smooth muscle can be blocked by aminopyridines such as 4-aminopyridine. In addition, KV 1 family members can be selectively inhibited by the triterpene correolide. Agi-toxin-2 also blocks KV 1 family channels with the notable exception of KV 1.5 and can be used to distinguish between KV 1.5 and other KV 1.X-mediated responses. Microvascular endothelial cells also may express KV channels that function in the negative feedback regulation of membrane potential to limit depolarization.

Katp Channels

Both arteriolar smooth muscle and endothelial cells express KATP channels. These channels open when intracellular ATP levels decrease; hence their name. However, they also may be activated by elevated nucleotide diphosphates, reduced intracellular pH, and phosphorylation by protein kinase A (PKA) and cGMP-dependent protein kinase (PKG). In both smooth muscle and endothelial cells, they may act as sensors of the metabolic status of cells, opening during ischemic or hypoxic conditions in response to

Figure 2 Ion channels and vasoconstriction. Simplified schematic diagram showing the role played by ion channels in contraction of arteriolar smooth muscle cells. Solid lines and arrows indicate stimulatory effects, whereas dashed lines represent inhibitory effects. 1, Vasoconstrictors such as norepinephrine, angiotensin II, vasopressin or endothelin, bind to G-protein-coupled receptors to activate effector proteins such as phospholipase C-b (PLC-b). 2, Activated PLC-b then acts on membrane phospholipids to form inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG). 3, IP3 binds to IP3 receptors (IP3R) on the smooth endoplasmic reticulum, releasing stored Ca2+, and raising intracellular Ca2+. 4, The DAG formed and the presence of increased [Ca2+]in activate protein kinase C, which then phosphorylates a number of ion channels including store-operated channels (SOC), stretch-activated channels (SAC), voltage-gated Ca2+ channels (VGCC), ATP-sensitive K+ channels (KATP), voltage-gated K+ channels (KV), and large-conductance Ca2+-activated K+ channels (BKCa). 5, In addition to activating PKC, DAG also can activate receptor-operated channels (ROC). 6, The release of Ca2+ from intracellular stores, through, or in addition to, activation by PKC, opens SOC channels. 7, The influx of Ca2+ and Na+ through SAC, SOC, and ROC, along with Cl- efflux through Ca2+-activated Cl- channels (ClCa),

Figure 2 Ion channels and vasoconstriction. Simplified schematic diagram showing the role played by ion channels in contraction of arteriolar smooth muscle cells. Solid lines and arrows indicate stimulatory effects, whereas dashed lines represent inhibitory effects. 1, Vasoconstrictors such as norepinephrine, angiotensin II, vasopressin or endothelin, bind to G-protein-coupled receptors to activate effector proteins such as phospholipase C-b (PLC-b). 2, Activated PLC-b then acts on membrane phospholipids to form inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG). 3, IP3 binds to IP3 receptors (IP3R) on the smooth endoplasmic reticulum, releasing stored Ca2+, and raising intracellular Ca2+. 4, The DAG formed and the presence of increased [Ca2+]in activate protein kinase C, which then phosphorylates a number of ion channels including store-operated channels (SOC), stretch-activated channels (SAC), voltage-gated Ca2+ channels (VGCC), ATP-sensitive K+ channels (KATP), voltage-gated K+ channels (KV), and large-conductance Ca2+-activated K+ channels (BKCa). 5, In addition to activating PKC, DAG also can activate receptor-operated channels (ROC). 6, The release of Ca2+ from intracellular stores, through, or in addition to, activation by PKC, opens SOC channels. 7, The influx of Ca2+ and Na+ through SAC, SOC, and ROC, along with Cl- efflux through Ca2+-activated Cl- channels (ClCa), channels, and BKCa channels, Rho-kinase-dependent inhibition of KV channels, and downregulation of BKCa channels by the tyrosine kinase cSRC. 9, Membrane depolarization along with stimulatory effects of PKC and cSRC on VGCC open these channels and provide a major source of steady-state Ca2+ influx. 10 and 11, The released Ca2+, along with Ca2+ influx through SAC, SOC, ROC, and VGCC, raise intracellular Ca2+ leading to smooth muscle contraction and vasoconstriction. 12, The increase in [Ca2+]in along with membrane depolarization activate BKCa channels, and the depolarization also activates KV channels. 13, The resulting efflux of K+ provides a negative feedback signal that limits membrane depolarization and appears to prevent overactivation of the muscle and vasospasm. Other abbreviations: PLB, phospholamban; SERCA, smooth endoplasmic reticulum Ca2+ ATPase.

depolarizes the smooth muscle cell membrane. 8, This depolarization is supported by PKC-dependent closure of K-r-p channels, KV

elevated ADP, reduced ATP, and reduced intracellular pH. Endogenous vasodilators such as adenosine, prostacyclin, epinephrine acting through b-adrenergic receptors, and calcitonin-gene-related peptide (CGRP) open these channels (Figure 3), whereas vasoconstrictors tend to close KATP channels (Figure 2). In skeletal muscle and coronary arteri-oles they play an important role in the regulation of smooth muscle resting membrane potential and tone, participate in functional hyperemia, and mediate, in part, hypoxia-induced vasodilation in these tissues.

Kir Channels

Arteriolar smooth muscle and endothelial cells also express KIR channels. These channels open with small, physiological increases in extracellular K+ concentration (Figure 3) and may participate in functional hyperemia in skeletal muscle, cardiac muscle, and the brain. As implied by their name, KIR channels show strong inward rectification due to intracellular block of the outward current flow through the ion-conductive pore by Mg2+ and polyamines at membrane potentials positive to the K+ equilibrium potential. More importantly, these channels may display an "N"-shaped current-voltage relationship such that between the potassium equilibrium potential and potentials 10 to 20mV more positive, KIR channels conduct small outward K+ currents. It is these small outward currents that allow KIR channels to contribute to the regulation of arteriolar tone. Endothelial KIR channels also are stimulated by increased

Figure 3 Ion channels and vasodilation. Simplified schematic diagram of the role played by ion channels in the mechanism of action of vasodilators on arteriolar smooth muscle cells. Solid lines and arrows indicate stimulatory effects, whereas dashed lines represent inhibitory effects. 1, Vasodilators such as adenosine, prostacyclin (PGI2), epinephrine, and calcitonin-gene-related peptide (CGRP) all bind to G-protein-coupled receptors and activate adenyl cyclase (AC) to form cAMP, which then activates protein kinase A (PKA). 2, PKA phosphorylates and activates KATP channels, KV channels, BKCa channels, and VGCC. The activation of the K+ channels tends to hyperpolarize smooth muscle, closing VGCC, reducing Ca2+ influx, lowering [Ca2+]in, and leading to smooth muscle relaxation and vasodilation. 3, Vasodilators such as NO, CO, and atrial natriuretic peptide (ANP) stimulate guanylyl cyclase (GC) to form cGMP, which then activates cGMP-dependent protein kinase (PKG). 4, PKG targets KATP, KV, and BKCa channels, leading to hyperpolarization and, ultimately, vasodilation. 5, In some instances NO, CO, and epoxides of arachidonic acid may directly activate BKCa channels. 6, Phosphorylation of VGCC by PKG is inhibitory and contributes to cGMP-PKG induced vasodilation. 7, Both PKA and PKG also phosphorylate other proteins such as phospholamban (PLB) and phospholipase C-b (not shown in figure). Phosphorylated PLB disinhibits the smooth endoplasmic reticulum Ca2+ ATPase (SERCA) reducing [Ca2+]in and refilling SER Ca2+ stores. 8, This latter effect closes store-operated channels (SOC) as indicated. Phosphorylation of phospholipase C-b reduces production of DAG and IP3, leading to reduced activity in both stretch-activated channels (SAC) and receptor-operated channels (ROC) contributing to the reduction in [Ca2+]in. 9, The reduced [Ca2+]in also closes Ca2+-activated Cl- channels, removing another depolarizing current, and tends to close BKCa channels, limiting their effect. 10, In some arterioles, smooth muscle cells are electrically coupled to endothelial cells such that hyperpolarization of endothelial cells (ENDO) can also hyperpolarize smooth muscle leading to vasodilation. 11, Inward-rectifier K+ channels (KIR) may be recruited during membrane hyperpolarization. These channels are also activated by increases in extracellular K+ ([K+]out).

shear stress (Figure 4). In both smooth muscle and endothelial cells, Kir channels are probably modulated by protein kinases, although this has not been studied in the microcirculation.

Nonselective Cation Channels

Vascular smooth muscle cells and endothelial cells express a number of nonselective cation channels that likely all arise from the transient receptor potential (TRP) family of ion channels originally described in the photoreceptors of the fruit fly, Drosophila. All of the TRPC isoforms expressed in smooth muscle and endothelial cells (see Table I) form nonselective cation channels that conduct Na+ and Ca2+ into cells, and none appear to be voltage-

gated. This large family of proteins forms channels that open when intracellular Ca2+ stores are depleted (store-operated channels, SOC), open with membrane stretch (stretch-activated channels, SAC), or open upon receptor activation (receptor-operated channels, ROC). They contribute to the regulation of arteriolar tone by providing a regulated pathway for entry of Ca2+ into both smooth muscle and endothelial cells. In smooth muscle, Ca2+ entry through SOC and ROC contributes to vasoconstrictor-induced increases in arteriolar tone, and Ca2+ influx through SAC importantly contributes to pressure-induced changes in arteriolar tone (Figure 2). In endothelial cells, SOC are the source of maintained increases in intracellular Ca2+ that contribute to the steady-state production of endothelium-derived autacoids (Figure 4).

Figure 4 Ion channels and endothelial hyperpolarization. Simplified schematic diagram of the role played by ion channels in the mechanism of action of endothelium-dependent vasodilators, shear-stress and elevated extracellular K+ (([K+]out). 1, Endothelium-dependent vasodilators such as acetylcholine, bradykinin, ATP, and histamine bind to G-protein-coupled receptors to activate phospholipase C-b (PLC-b) leading to the formation of inositol 1,4,5-triphosphate (IP3) and diacyl-glycerol (DAG, not shown). 2, IP3 binds to IP3-receptors (IP3R) on the smooth endoplasmic reticulum, releasing stored Ca2+. 3, This causes a rapid rise in intracellular Ca2+. 4, The increased [Ca2+]in then binds to calmodulin (CAM) associated with a number of proteins including intermediate (IKCa) and small (sKCa) conductance Ca2+-activated K+ channels, leading to membrane hyperpolarization. 5, The release of Ca2 from intracellular stores activates store-operated channels (SOC). 6, The membrane hyperpolarization caused by opening of IKCa and sKCa channels increases the electrochemical gradient for diffusion of Ca2+ into the cells through SOC, and amplifies this current. 7, This amplified Ca2+ influx yields a sustained elevation in [Ca2+]in. 8, The hyperpolarization induced by IKCa and sKCa channels can activate inward rectifier K+ channels, augmenting the hyperpolarization. 9, This effect may be enhanced by increases in shear-stress or elevated [K+]out, both of which activate KIR channels. 10, The elevated Ca2+, through Ca2+-CAM then activates proteins such as NO-synthase (e-NOS) and phospholipase A2 (PLA2). Activation of e-NOS leads to production of NO, while activation of PLA2 liberates arachidonic acid (AA) from membrane phospholipid stores. 11, Released arachidonic acid is then rapidly metabolized by cyclooxygenase (COX) to prostacyclin (PGI2) and other vasodilator prostaglandins, and to epoxides (EETs) by cytochrome P-450 (P450). 12, In some arterioles, smooth muscle cells (VSM) are electrically coupled to endothelial cells through myoendothelial gap junctions (MEGJ), such that endothelial hyperpolarization, per se, in the absence of another mediator, can lead to vasodilation. 13, In addition, endothelial cells in arterioles are electrically coupled so that hyperpolarization can be conducted for long distances along the length of arteriolar endothelium. 14, All of these signals (hyperpolarization, NO, PGI2, EETs) are integrated by overlying smooth muscle cells to yield vasodilation.

Cl Channels

Vascular smooth muscle and endothelial cells also express chloride channels. These include Ca2+-activated Cl-(ClCa) channels and swelling or volume-activated Cl- (ClSW channels). The molecular identity of the Cl- channels expressed in vascular smooth muscle and endothelial cells remains unclear as indicated in Table I. In both smooth muscle and endothelial cells, the intracellular concentration of Cl- is such that opening of channels conducting these anions leads to diffusion of Cl- out of the cells, and membrane depolarization. Calcium-activated Cl- channels are activated by increases in intracellular Ca2+. In smooth muscle, they contribute to vasoconstrictor-induced membrane depolarization (Figure 2). Their function in arteriolar endothelial cells has not been established. In endothelial cells and in smooth muscle cells, ClSW channels play an important role in the regulation of cell osmolarity and volume and likely participate in the regulation of resting membrane potential. Smooth-muscle ClSW channels may play a role in pressure (stretch)-induced membrane depolarization and increases in tone.

Was this article helpful?

0 0
Essentials of Human Physiology

Essentials of Human Physiology

This ebook provides an introductory explanation of the workings of the human body, with an effort to draw connections between the body systems and explain their interdependencies. A framework for the book is homeostasis and how the body maintains balance within each system. This is intended as a first introduction to physiology for a college-level course.

Get My Free Ebook


Post a comment