Ion channels in the regulation of smooth muscle tone Dr. Janos Pataricza – Dr. Andras Toth Department of Pharmacology and Pharmacotherapy University of Szeged 28th of November, 2017

Content Regulation of smooth muscle contraction Some important ion channels and the tone of the smooth muscle K+ channels and vascular smooth muscle tone Cl- channels and vascular smooth muscle tone -Possible relationships among voltage-dependent Ca2+ channels, ryanodine-sensitive Ca2+- release (RyR) channels, large-conductance Ca2+-sensitive K+ (BKCa) channels, and Ca2+- a activated Cl− (ClCa) channels to regulate smooth muscle contractility Ion channels in bronchial smooth muscle cells Involvement of ion channels in the regulation of cholinergic excitation in gastrointestinal tract Ion channels in the urinary tract Ion channels in the regulation of uterinal smooth muscle tone -Strech-activated TREK-1, a type of two-pore K+ channels (K2P) also regulates myometrial tone -Pregnancy, hypoxia and K+ ion channels -Subfamily members of voltage dependent K+ channels (Kv7 and Kv11) in the regulation of uterinal tone Transient receptor potential channels (TRPC) in smooth muscle cells - link to intracellular signaling A crosstalk between the plasma membrane and sarcoplasmic reticulum involving Ca2+ and K+ channels in smooth muscle cells -Surface coupling between junctional sarcoplasmic reticulum (SR) and plasma membrane: leaflets of SR and cell membranes are separated by an 12- to 20-nm gap -Possible mechanisms of action of PKA/PKG and PKC on Ca2+ sparks, BKCa channels, and SR Ca2+-ATPase in arterial smooth muscle cells Endothelial nitric oxide influence the tone through modulation of ion channels in smooth muscle cell of human umbilical artery Some basic properties of ionic regulation of smooth muscle tone Current research

Principle of regulation of smooth muscle contraction/relaxation

2 principal pathways of regulation of smooth muscle contraction MLCK dependent MLCK independent

Regulation of smooth muscle contraction Webb RC, Advan in Physiol Edu 2003;27:201-206 Regulation of smooth muscle contraction. Various agonists (neurotransmitters, hormones, etc.) bind to specific receptors to activate contraction in smooth muscle. Subsequent to this binding, the prototypical response of the cell is to increase phospholipase C activity via coupling through a G protein. Phospholipase C produces two potent second messengers from the membrane lipid phosphatidylinositol 4,5-bisphosphate: diacylglycerol (DG) and inositol 1,4,5-trisphosphate (IP3). IP3 binds to specific receptors on the sarcoplasmic reticulum, causing release of activator calcium (Ca2+). DG along with Ca2+ activates PKC, which phosphorylates specific target proteins. In most smooth muscles, PKC has contraction-promoting effects such as phosphorylation of Ca2+ channels or other proteins that regulate cross-bridge cycling. Activator Ca2+ binds to calmodulin, leading to activation of myosin light chain kinase (MLC kinase). This kinase phosphorylates the light chain of myosin, and, in conjunction with actin, cross-bridge cycling occurs, initiating shortening of the smooth muscle cell. However, the elevation in Ca2+ concentration within the cell is transient, and the contractile response is maintained by a Ca2+-sensitizing mechanism brought about by the inhibition of myosin phosphatase activity by Rho kinase. This Ca2+-sensitizing mechanism is initiated at the same time that phospholipase C is activated, and it involves the activation of the small GTP-binding protein RhoA. The precise nature of the activation of RhoA by the G protein-coupled receptor is not entirely clear but involves a guanine nucleotide exchange factor (RhoGEF) and migration of RhoA to the plasma membrane. Upon activation, RhoA increases Rho kinase activity, leading to inhibition of myosin phosphatase. This promotes the contractile state, since the light chain of myosin cannot be dephosphorylated.

Some important ion channels and vascular smooth muscle tone Jackson WF, Hypertension. 2000;35:173-178 Ion channels and vascular tone. Schematic of a cross section through part of a vascular muscle cell. Along the top membrane are shown KIR, KATP, KV, and BKCa channels. Also shown are voltage-gated Ca2+ channels, 2 types of Cl− channels (see text), SOC channels (SOCC), and SAC channels (SACC). Shown in the membranes of the sarcoplasmic reticulum (SR) are ryanodine receptors (RyR) and inositol 1,4,5-trisphosphate receptors (IP3R). Bottom, A few of the signals that are known to modulate the function of the ion channels depicted. AC indicates adenylate cyclase; PKA, cAMP-dependent protein kinase; sGC, soluble guanylate cyclase; PKG, cGMP-dependent protein kinase; EETs, epoxyeicostetraenoic acid (epoxides of arachidonic acid; see text); PLC,phospholipase C; DAG,diacylglycerol; PKC=protein kinase C; and 20-HETE, 20-OH-arachidonic acid. See text for details. Inward rectifier K+ channel (KIR) ATP-sensitive K+ channel, (KATP), voltage dependent K+ channel (KV), big conductance Ca2+ activated K+ channel (BKCa), store-operated Ca2+ channel (SOCC), stretch-activated K+ channel (SACC), sarcoplasmic reticulum ryanodine-sensitive Ca2+ channel (RyR)

K+ channels and vascular smooth muscle tone Jackson WF, Hypertension. 2000;35:173-178 K+ channels and vascular tone. Schematic of a vascular smooth muscle cell (top) and cross sections through an arteriole (bottom) that shows that opening K+ channels leads to diffusion of K+ ions out of the cell, membrane hyperpolarization, closure of voltage-gated Ca2+ channels, decreased intracellular Ca2+, etc (see text), which leads to vasodilatation. Closure of K+ channels has the opposite effect. Modified from Jackson.8

Cl− channels and vascular smooth muscle tone Jackson WF, Hypertension. 2000;35:173-178 Cl− channels and vascular tone. Schematic of a vascular smooth muscle cell (top) and cross sections through an arteriole (bottom) that shows that opening of Cl− channels leads to diffusion of Cl− ions out of the cell, membrane depolarization, opening of voltage-gated Ca2+ channels, increased intracellular Ca2+, etc (see text), which leads to vasoconstriction. Closure of Cl− channels has the opposite effect.

Possible relationships among voltage-dependent Ca2+ channels, ryanodine-sensitive Ca2+-release (RyR) channels, large-conductance Ca2+-sensitive K+(BKCa) channels, and Ca2+-activated Cl− (ClCa) channels to regulate smooth muscle contractility. JaggarJH et al., Am J Physiol Cell Physiol 2000;278:C235-C256 - + Possible relationships among voltage-dependent Ca2+channels, ryanodine-sensitive Ca2+-release (RyR) channels, large-conductance Ca2+-sensitive K+(BKCa) channels, and Ca2+-activated Cl− (ClCa) channels to regulate smooth muscle contractility. Voltage-dependent Ca2+ channels (VDCC) are the primary Ca2+ entry pathway in most types of smooth muscle. For tonic smooth muscle, such as resistance arteries, steady Ca2+entry through VDCC determines intracellular [Ca2+] ([Ca2+]i; indicated by thick arrow). Ca2+ entry through VDCCs also stimulates RyR channels as Ca2+ spark events or as non-Ca2+spark events. The type of Ca2+ communication from VDCCs to RyR channels (e.g., local Ca2+ control) in smooth muscle is not known. RyR channels can serve as negative- and positive-feedback transducers to VDCCs. Ca2+ release from RyR channels could inactivate VDCCs (negative-feedback element); this is not known for smooth muscle. Ca2+ release from RyR channels may also contribute to global [Ca2+]i for contraction [i.e., Ca2+-induced Ca2+release (CICR); positive feedback element]. Contribution of Ca2+ sparks to global [Ca2+]i may be significant in phasic smooth muscle and less important in tonic smooth muscle. Ca2+ sparks activate BKCa channels in virtually all types of smooth muscle to cause a very substantial membrane potential (V m) hyperpolarization (negative-feedback element). Sparks activate ClCa channels in some types of smooth muscle to cause membrane depolarization (positive-feedback element). Ca2+ release through RyR channels could also activate SKCa channels to cause membrane potential hyperpolarization in some types of smooth muscle (effect not illustrated). The final outcome of these various signaling elements depends on a number of factors, including proximity of the various elements, Ca2+ sensitivity, and phosphorylation state. MLCK, myosin light-chain kinase.

Ion channels in bronchial smooth muscle cells Perez-Zoghbi JF et al, Pulm Pharmacol Ther. 2009 ;22(5):388-97 Find: Receptor-operated Ca2+ influx or channels (ROC) Store-operated Ca2+ entry or channels (SOC) Ca2+-activated K+ channels (KCa1.1, KCa3.1) Voltage-dependent Ca2+ channels (VDC) Stretch-activated channels (SA) are directly gated by physical stimuli

Koh SD, Rhee PL J Neurogastroenterol Motil. 2013;19(4):426-32. Involvement of ion channels in the regulation of cholinergic excitation in gastrointestinal tract Koh SD, Rhee PL J Neurogastroenterol Motil. 2013;19(4):426-32. Possible post-junctional mechanisms responsible for cholinergic excitation. Acetylcholine (ACh) is coupled to Gq/11 protein and activates conductance(s) through inositol-1,4,5-triphosphate receptor (IP3R) in interstitial cells of Cajal (ICC) and smooth muscle cells (SMC). ACh might also be coupled to G12/13 protein and activate Rho-Kinase (RhoK) pathway to induce contraction in SMC. ER: endoplasmic reticulum; PLC: phospholipase C; DAG: diacyl glycerol; PKC: protein kinase C; CaCC: Ca2+-activated Cl- channels; NSCC: non-selective cation channels; GJ: gap junction; MLCP: myosine light chain phosphatase.

Ion channels in the urinary tract Kyle BD,Channels (Austin). 2014;8(5):393-401 urinary bladder urethra KATP, ATP-sensitive K+ channel; BKCa, large conductance, Ca2+-activated K+ channel; Kv, voltage-gated K+ channel; K2P, 2-pore domain K+ channel; IKCa, intermediate conductance K+ channel; SKCa, small conductance K+ channel; VGCC, voltage-gated Ca2+ channel; CaCC, Ca2+-activated Cl− channel.

Ion channels in the regulation of uterinal smooth muscle tone Brainard  AM, Semin Cell Dev Biol. 2007;18(3):332-9. Big conductance Ca2+ activated K+ channel (BKCa), small conductance Ca2+ activated K+ channel (SK3), ATP-sensitive K+ channel (KATP) as a subtype of inward rectifier K+ channels (Kir), voltage dependent K+ channel (Kv)

Strech-activated TREK-1, a type of two pore domain K+ channels (K2P) also regulates myometrial tone Buxton IL. et al, Acta Pharmacol Sin. 2011;32(6):758-64

- - - - + + Pregnancy, hypoxia and K+ ion channels Zhu R et al. Curr Vasc Pharmacol. 2013;11(5):737-47. - + - + - -

Subfamily members of voltage dependent K+ channels (Kv.7 and Kv.11) in the regulation of uterinal tone Greenwood IA, Tribe RM.Exp Physiol. 2014;99(3):503-9

Transient receptor potential channels (TRPC) in smooth muscle cells – link to intracellular signaling Gonzalez-Cobos JC1, Trebak M, Front Biosci (Landmark Ed). 2010 Jun 1;15:1023-3 Phospholipase C (PLC), phosphatidylinositol 4,5-bisphosphate (PIP2), inositol 1,4,5-trisphosphate (IP3), diacylglycerol (DAG), stromal interactive molecule (STIM1), store-operated channel (Orai1), transient receptor potential channel (TRPC), transcription factors: nuclear factor kappa B (NF-κB), activator protein-1 (AP-1), cAMP response element-binding protein (CREB)

Surface coupling between junctional sarcoplasmic reticulum (SR) and plasma membrane: leaflets of SR and cell membranes are separated by an 12- to 20-nm gap. Jaggar JH et al. Am J Physiol Cell Physiol 2000;278:C235-C256 A: surface coupling between junctional sarcoplasmic reticulum (SR) and plasma membrane: leaflets of SR and cell membranes are separated by an ∼12- to 20-nm gap. Electron-opaque material present between the two opposing membranes has a “periodicity” of ∼20–25 nm (arrows). Section is taken from rabbit portal anterior mesenteric vein. Scale bar = 0.1 μm. [From Devine et al. (45) by copyright permission of The Rockefeller University Press.]B: confocal image of an adult cerebral artery smooth muscle cell stained with a monoclonal anti-RyR2 antibody illustrating distinct staining along the cell membrane. [Modified from Gollasch et al. (61).]

A crosstalk between the plasma membrane and sarcoplasmic reticulum involving Ca2+ and K+ channels in smooth muscle cells Jaggar JH. et al. Am J Physiol Cell Physiol 2000;278:C235-C256

Possible mechanisms of action of PKA/PKG and PKC on Ca2+ sparks, BKCa channels, and SR Ca2+-ATPase in arterial smooth muscle cells. Jaggar JH. et al. Am J Physiol Cell Physiol 2000;278:C235-C256 Possible mechanisms of action of PKA/PKG and PKC on Ca2+sparks, BKCa channels, and SR Ca2+-ATPase in arterial smooth muscle cells. Activation of PKA or PKG increases Ca2+ spark frequency and increases Ca2+ load of the SR (probably through activation of the SR Ca2+-ATPase via disinhibition of phospholamban). Increased Ca2+ spark frequency could occur due to a direct effect on the RyR channel and/or be a secondary effect from increased SR Ca2+ load. PKA/PKG activation also increases the activity of BKCa channels, which could manifest itself by an elevation in STOC amplitude and steady-state BKCa channel activity. Synergistic effect of increased Ca2+ spark frequency and a direct effect on BKCa channels will result in a significant increase of BKCa channel activity. Membrane potential hyperpolarization closes L-type Ca2+ channels, which reduces Ca2+influx, lowering the cytoplasmic Ca2+ concentration, and leads to vasodilation (see Ref. 157). Activation of PKC reduces frequency of Ca2+ sparks and amplitude of STOCs, due to a direct inhibitory effect on RyR channels and BKCa channels, respectively. Additive effect results in decreased BKCachannel activity, a depolarization of the smooth muscle cell membrane potential, and activation of L-type Ca2+ channels. PKA, PKG, and PKC also modulate the voltage-dependent Ca2+channel, which would contribute to modulation of the Ca2+channel → RyR channel → BKCa channel pathways (not illustrated).

Endothelial nitric oxide influence the tone through modulation of ion channels in smooth muscle cell of human umbilical artery Martin P. et al., Reprod Sci. 2014 April; 21(4): 432–441. NO: nitric oxide; KV: voltage-dependent K+ channels (different subfamilies); BKCa: big conductance, voltage- and Ca2+-sensitive K+ channel; SKCa: small conductance Ca2+-sensitive K+ channels; K2P: 2-pore domains K+ channels; KIR, inward rectifier K+ channels. Intermediate conductance Ca2+-sensitive K+ channels (IKCa), and ATP-sensitive K+ channels (KATP) are not included because the evidence about their presence in HUA is either weak (KATP) or altogether not present in the literature (IKCa). 1/ basal tone, 2/ agonist-induced contraction 3/ regulation by endothelium

Some basic properties of ionic regulation of smooth muscle tone Smooth muscle function is mainly regulated by the voltage operated (or dependent, or gated) Ca2+ channels (VOCC or VDCC or VDC or VGCC) Typically, a large majority of other ion channels modulate membrane potential that regulates VOCC Most types of smooth muscle cells are „electrically silent”- in contrast to nerves or skeletal muscles-; no action potential is generated (resting membrane potential is about -50 mV) Ion channels maintain resting (basal) tension, modulate agonist- induced contractions and vasodilations by endothelial/ interstitial cell-derived factors

Current research Research focuses mainly on K+ channels in the regulation of smooth muscle tone of different organs (vascular, bronchial, uterinal) – an exception may be the gastrointestinal tract A typical ion channel is still considered to be a receptor without cellular effector, however, some ion channels reveal a complex interaction with intracellular signaling mechanisms. This ‘crosstalk’ may lead not only to immediate changes in their tone, but in addition to modifying the cellular phenotype of the smooth muscle cells (see TRPC channels)