1. Ion channels in the regulation of smooth muscle tone Dr. Janos Pataricza Department of Pharmacology and Pharmacotherapy University of Szeged 1st of December, 2015

2. 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  I on 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

3. Regulation of smooth muscle contraction Webb RC, Advan in Physiol Edu 2003;27:201-206

4. Some important ion channels and vascular smooth muscle tone Jackson WF, Hypertension. 2000;35:173-178 Inward rectifier K channel (K IR ) ATP-sensitive K channel, (K ATP ), voltage dependent K channel (K V ), big conductance calcium activated K channel (BK Ca ), store-operated calcium channel (SOCC), stretch-activated K channel (SACC)

5. K+ channels and vascular smooth muscle tone Jackson WF, Hypertension. 2000;35:173-178

6. Cl− channels and vascular smooth muscle tone Jackson WF, Hypertension. 2000;35:173-178

7. 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

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

9. Involvement of ion channels in the regulation of cholinergic excitation in gastrointestinal tract 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. Koh SDKoh SD, Rhee PL J Neurogastroenterol Motil. 2013;19(4):426-32.Rhee PLJ Neurogastroenterol Motil.

10. Ion channels in the urinary tract 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. Kyle BDKyle BD,Channels (Austin). 2014;8(5):393-401Channels (Austin). urinary bladder urethra

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

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

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

14. Subfamily members of voltage dependent K+ channels (Kv.7 and Kv.11) in the regulation of uterinal tone Left-hand panel shows that open K+ channels result in membrane hyperpolarization that indirectly limits the opening of voltage-dependent calcium channels shown in blue. This results in a less contracted smooth muscle. In the right-hand panel, the potassium channels are non-functional due to blockade, loss-of-function mutations or trafficking defects. This leads to membrane depolariziation, and the open probability of the calcium channels increases. The concomitant influx of calcium contributes to smooth muscle contraction. Greenwood IA, Tribe RM.Exp Physiol. 2014;99(3):503-9

15. Transient receptor potential channels (TRPC) in smooth muscle cells – link to intracellular signaling The engagement of a vasoactive compound/growth factor receptor in vascular smooth muscle cells leads to the activation of phospholipase C (PLC) which catalyzes the breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2) into two intracellular second messengers, the Inositol 1,4,5-trisphosphate (IP3) and Diacylglycerol (DAG). IP3-mediated Ca2+ store depletion activates store-operated Orai1 channels in a mechanism dependent on STIM1 aggregation and translocation into areas of close SR-PM contacts. The role of TRPC channels in mediating SOC channels remains to this day a highly contentious issue. All TRPC are Activated by mechanisms downstream of PLC; TRPC3/6/7 have been shown to be activated by DAG in a PKC independent manner while TRPC1/4/5 exact mechanisms of activation via membrane receptors is still unclear and seems to involve PIP2 breakdown and Ca2+. Na+entry through nonselective TRPC channels has been proposed to couple to activation of Ca2+ entry either through the Na+/Ca2+ exchanger (NCX) or via depolarization and subsequent activation of L-type Ca2+ channels. Increasing evidence supports a signaling paradigm in which Ca2+ signals mediated by specific TRPC isoforms are able to activate transcription factors in smooth muscle that act to increase the corresponding TRPC channel expression. Gonzalez-Cobos JCGonzalez-Cobos JC1, Trebak M, Front Biosci (Landmark Ed). 2010 Jun 1;15:1023-3Trebak M Front Biosci (Landmark Ed).

16. 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

17. 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

18. 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

19. Endothelial nitric oxide influence the tone through modulation of ion channels in smooth muscle cell of human umbilical artery 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 include 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 Martin P. et al., Reprod Sci. 2014 April; 21(4): 432–441.

20.  Smooth muscle function is mainly regulated by the voltage operated (or dependent, or gated) calcium 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 Some basic properties of ionic regulation of smooth muscle tone

21. 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 to changes of cellular phenotype in addition to modify an immediate change in tone of smooth muscle cells (see TRPC channels)

22. Questions 1/ Which ion is mainly responsible for the contraction of a smooth muscle cell? (Ca++) 2/ List some important ion channels – other than Ca2+ channel- that play roles in regulating the tone of smooth muscle cells! (K+ channels, Cl- channels, Store-operated channels, Stretch-activated channels) 3/ How K+ and Cl- channels regulate the tone of smooth muscle cells? (activation of K+ channels relaxes, that of Cl- channels contracts smooth muscles via modulation of membrane potential) 4/ List some known common ion channels that regulate the tone of bronchial, urinary and uterinal smooth muscles! (Ca2+ channels, K+ channels, Store-operated channels, Stretch-activated channels)

23. Questions 5/ Which ion channels appear to be important in cholinergic excitation of gastrointestinal smooth muscles? (Calcium- activated Cl- channels, Non-selective cation channels) 6/ What are K2P channels and which type of tissue is known to be regulated by these channels? (uterinal smooth muscle, in another word: myometrial tissue) 7/ How may pregnancy affect the function of K+ channels? (activates K+ channels– relaxation of the uterus) 8/ What kind of K+ ion channel is involved in a ‘crosstalk’ between plasma membrane and sarcoplasmic reticulum of smooth muscle cells? (BKCa) 9/ How do protein kinases influence K+ channels in smooth muscles according to our present knowledge? (PKA and PKG activate, PKC inhibits)