Ionchannels and channelopaties in the heart

1. Viktória Szűts Ionchannels and channelopaties in the heart

2. Action of membrane transport protein ATP-powered pumpIon chanels Transporters 101-103ions/s 107-108ions/s 102-104ions/s

4. Cardiac K+ channels control the resting membrane potentials and the amplitude, duration, refractoriness and automaticity of action potentials. K+ channels share a similar structure, composed by four pore-forming α-subunits assembled as tetramers or dimers forming K+ selective pores and modulated by accessory subunits. The main K+channel pore forming protein is not translated from a single gene as Na+ and Ca+channels, but is made up of four separate subunits, which assembly with ß-subunits to form the functional channel More than 80 different K+ channels are expressed in the heart, display considerable diversity of native K+channels. • Ca-independent transient outward potassium current (I to1)underliesby KCNAgenes encoded Kv3.x and Kv4.x proteins. • Delayed rectifier currents: the rapid (IKr) and slow (IKs) are encoded by different voltage-gated K+ channel genes. IKr is produced by the α-subunit ERG (KCNH2), in co-assemblance with the ß-subunit MiRP1 (KCNE2).IKsis produced by the α-subunit KvLQT1 (KCNQ) assembly with the accessories subunits of minK and MIPRs (KCNE1, KCNE2, KCNE3) • Inward rectifier current (IK1)carried by Kir 2.1, Kir 2.2 and Kir 2.3 (KCNJ2, KCNJ12 and KCNJ4)channel proteins.

5. Molecular composition of the cardiac K-ionchannels Selectivity filter Nerbonne et al . Circ Res. 2001;89:944-956

6. Membrane topology of the Kv and Kir2.x K-ionchannels Voltage gated K+channel Inward rectifier K+channel Kv channel CO2 CO2 CO2 H5 H5

7. MiRP N N  C C  KChAP PSD Kv complex

8. Gating moviIonchannels are open and close changing the permeability

9. Assembly of different ionchannel subunits Extracellular Intracellular Abott et al Neuropharm. 2004

10. Molecular assembly of ion channels Cavα Kvα Kir

11. Activation and Inactivation of The Sodium Channel Sodium channels are characterized by voltage-dependent activation, rapid inactivation, and selective ion conductance. Depolarization of the cell membrane opens the ion pore allowing sodium to passively enter the cell down its concentration gradient . The increase in sodium conductance further depolarizes the membrane to near the sodium equilibrium potential. Inactivation of the sodium channel occurs within milliseconds, initiating a brief refractory period during which the membrane is not excitable. The mechanism of inactivation has been modeled as a "hinged lid" or "ball and chain", where the intracellular loop connecting domains III and IV of the a subunit closes the pore and prevents passage of sodium ions.

12. Voltage-Gated Calcium Channels • Voltage-gated calcium channels are heteromultimers composed of an α1 subunit and three auxiliary subunits, 2-δ, β and γ. The α1 subunit forms the ion pore and possesses gating functions and, in some cases, drug binding sites. Ten α1 subunits have been identified, which, in turn, are associated with the activities of the six classes of calcium channels. L-type channels have α1C (cardiac), α1D (neuronal/endocrine), α1S (skeletal muscle), and α1F (retinal) subunits; The α1 subunits each have four homologous domains (I-IV) that are composed of six transmembrane helices. The fourth transmembrane helix of each domain contains the voltage-sensing function. The four α1domains cluster in the membrane to form the ion pore. The β-subunit is localized intracellularly and is involved in the membrane trafficking of α1subunits. The γ-subunit is a glycoprotein having four transmembrane segments. The α2 subunit is a highly glycosylated extracellular protein that is attached to the membrane-spanning d-subunit by means of disulfide bonds. The α2-domain provides structural support required for channel stimulation, while the δ domain modulates the voltage-dependent activation and steady-state inactivation of the channel.

13. Abriel H. et al., Swiss Med Wkly 2004, 685-694. www.sm w. ch Ionic currents and ion transporters responsible for cardiac action potential

14. The expression and properties of these K+ channels are altered in cardiac diseases (ie. cardiac arrhythmia, Long QT syndrome, hypertrophyc cardiomyopathy, Andersen syndrome, heart failure). These K+ channels still require further investigation because they are involved in the basic normal heart rhythm, and may be altered in cardiac diseases.

15. Proposed cellular mechanism for the development of Torsade de pointes in the long QT syndrome

16. Prolonged QT interval on ECG (reflects prolonged APD) • APD governed by a delicate balance between inward (Na+ or Ca+) and outward (K+) ionic current • Affecting the Na+ or Ca+ channel prolong APD via“gain-off-function”mechanism, while mutation in genes encoding K+ channel by “loss-off-function” mechanism

17. Risk factors for developing Torsade de pointes Genetic variants (polymorphysm or mutations) Abriel H. et al., Swiss Med Wkly 2004, 685-694.

18. Ionic current, proteins and genes associated with inherited arrhythmias Napolitano et al. Pharm. & ther. 2006,110:1-13

19. Congenital and aquired forms of long QT syndromes Abriel H. et al., Swiss Med Wkly 2004, 685-694. www.sm w. ch

20. Current Genes Disease ITo1 Kv4.3 LQT IKs KvLQT1(KCNQ1) Mink (KCNE1) LQT1, JLN1 LQT5, JLN2 IKr HERG (KCNH2) MiRP1 (KCNE2) LQT2 LQT6, FAF INa SCN5A LQT3 Brugada Syndrome, Cardiac conduction defect, Sick sinus syndrome Ik1 Kir2.1 (KCNJ2) LQT7 Andersen-Tawil Syndrome Ikur Kv1.7(KCNA7),Kv1.5 Progressziv familial heart Block1 IkAch Kir3.4 IkATP Kir6.2 ICaL Cav1.2 (CACNA1c) LQT8 Timothy Syndrome K+, Na+ channel LQT-associated genes and proteins AF

22. Gene mutations in LQT1 and LQT2 HERG KCNH2 KvLQT1 KCNQ1 LQT2 LQT1

24. Mutations in HERG channel Molecular structure and the membrane topology of the HERG channel

25. Atrial fibrillation (AF): • Rapid shortening of the AERP • Functional changes of ion channel • Reduction of ICaL and gene expression of L-type Ca channel • Increase in K+-ion channel activity of IkAch, Ik1 • Reduction in Ito and Isus • Reduced gene expression in Kv1.5, Kv4.3, Kir3.1, Kir3.4, Kir6.2

26. Pivotal role of Ser phosphorilation as a regulatory mechanism in Cav1.2 mode1/mode2 gating. Timothy’s syndrome

27. Current Genes Disease IKr IK1 IKs HERG (KCNH2) Kir2.x (KCNJ2) KvLQT1(KCNQ1) ShortQT Kv3.1, Kv3.4 CPVTcatecholamine-induced polymorphic ventricular tachycardia CASQ2(Calsequestrin2) ICa CPVT ICa CPVT RyR2 Risk factor, modify disease or influence progression of disease β1-adrenoceptor (β1-AR) Risk factor, modify disease or influence progression of disease β2-adrenoceptor (β2-AR) IkAch AF

28. Complexity of protein-protein interaction in cardiomyocytes

29. Missense mutation in calsequestrin2 (CASQ2) wild type Syncope Seizures or Sudden death In response to Physical activity or Emotional stress mutant Associated with autosomal recessive catecholamine- induced polymorphic ventricular tachycardia (CPVT)

30. Kir2.1 ionchannel has an autosomal dominant mutation in Andersen-Tawil Syndrome Cardiac arrhytmias Periodic paralysis Dysmorphic bone structure(scoliosis, low-set ears, small chin, broad forehead

31. Facial and sceletal features in Andersen-Tawil syndrome

32. Kir2.1 ion channel mutation GIRK mutation

33. ANP role

34. Gene-specific mutation study • Genexpression study • Microarray, qRT-PCR • Proteomica

35. Kir2.x analysis by RT-PCR

36. Expression of Kv1.5 protein in human and dog kDa 75 66 RV LV RA LA RV LV RA LA DOG HUMAN n=12 n= 6

37. 100 m Co-localization of Kv2 auxillary subunit with Kv1.5 in dog left ventricular myocytes Kv1.5-FITC Kv2-Texas red Kv1.5-FITC Kv2-Texas red