K + Channel

1. K+ Channel Sukhee Cho Greg Richard

2. K+ Channels • Found everywhere • Contribute to resting potential (neurons) • Major roles in cardiac tissue • Involved in hormone secretion Open Closed Slow to close Inactivated

3. K+ Channel Anatomy Senyon Choe (2002)

4. Gating Bezanilla 2004

5. Classes • Inwardly Rectifying • ROMK, GIRK, ATP-sensitive • Tandem Pore Domain • TWIK, TREK, TASK, TALK, THIK, TRESK • Voltage-Gated • hERG, KvLQT1 • Calcium Activated • BK, IK, SK

6. Inwardly Rectifying (Kir, IRK) • Subclasses: ROMK, GIRK, ATP-sensitive • 2 TMD, 1 P • Current flow into cell (“inward”) • Differ from delayed rectifier or A- type channels (outward current)

7. Tandem Pore Domain (K2P) • Subclasses: TWIK, TREK, TASK, TALK, THIK, TRESK • 4 TMD, 2 P (two 2 TMD, 1 P) • “Leak channels” – contribute to resting potential • Activated by mechanical stretch, pH, temperature

8. Voltage-Gated (Kv) • Subclasses: hERG, KvLQT1 • 6 TMD, 1 P • Sensitive to voltage changes • S4 domain • Return to resting state • Repolarization • Limits AP frequency (RRP)

9. Calcium Activated (KCa1 ) • Subclasses: BK, IK, SK • 6 TMD, 1 P • Activated by intracellular Ca2+ • Some activated by intracellular Na+ & Cl- • N-terminus extracellularly (Unlike Kv)

10. Paper #1

11. Amyloid β Hypothesis in Alzheimer’s disease Aβ1-40 Aβ1-42 Aβ1-40 Aβ1-42 Alzheimer's diseased brain Amyloid precursor protein http://en.wikipedia.org/wiki/Beta_amyloid

12. BK channel Large conductance Ca2+-activated K+ channels, Maxi-K, BK or Bkca, Kca1.1 VSD - voltage sensing domain PGD - pore-gating domain RCK - regulator of K conductance Controlling neurotransmitter release Fast after-hyperpolarization Spike frequency adaptation Lee et al., Trends Neurosci. 2010 Sep;33(9):415-23. Review.

13. Aβ1-40 Aβ1-42 Fura-2 500 ms 100-250 pA Figure 1. Intracellular infusion of Aβ1-42broadens spike width and augmemted Ca2+ influx in rat neocortical pyramidal neurons.

14. Charybdotoxin - Ca2+-activated K+ channel blocker 4-AP(4-Aminopyridine) – A-type potassium channel blocker Figure 3. Intracellular Aβ1-42enlarges spike width by suppressing BK channels, thereby increasing spike-induced Ca2+ entry.

15. Isopimaric acid Electroconvulsive shock Figure 5. ECS blocked Aβ1-42-mediated suppression of BK channels in rat neocortical neurons.

16. Figure 7. Blocking effects of ECS on Aβ1-42 was absent in H1aKO mice.

17. 4 months of age Juvenile Juvenile Figure 8. Spike broadening in 3xTG neurons.

18. Figure 9. Recovery of single BK current by ECS in 3xTG mice.

19. Conclusions Intracellular Aβ1-42 broadens spike width in neocortical pyramidal neurons by downregulation of BK channel activities. ECS counteracts Aβ1-42induced BK channel inhibition by expression of Homer 1a

20. Paper #2

21. Trek Channels • Two-pore domain K+ channels (K2P) • 4 TMD, 2 pore • Subfamilies: • Trek1 (Kcnk2) • Trek2 (Kcnk10) • Underlie “leak” and background K+ conductances • Sensitive to membrane stretch, temperature, & pH • Inhibited by PKC & PKA

22. Trek2 • Trek2b • Differs from Trek2a & Trek2c at N-terminus • Trek2-1p • C-terminal truncation (2 TMD & 1 pore) Does alternative splicing of Trek2 contribute to functional diversity of channel as seen with Trek1?

23. Trek2 Variants Trek2 Trek2-1p C-terminus Trek2b N-terminus

24. Immunoblotting Myc-tag : N-EQKLISEEDL-C (1202 Da)

25. +60mV 20mV -100mV Whole-cell Currents(Voltage-step)

26. +60mV Non-selective channel 1 s -100mV Reversal Potential (Erev)(Voltage-ramp)

27. Whole-cell Currents

28. Surface Trek2 Expression Total Protein Surface Protein

29. Conclusions • Trek2b exhibited larger currents than Trek2b & 2c; > # of Trek2b channels on membrane surface. • As [K+]o, Erev ; overexpression of K+-selective channels • Trek2-1p may require additional assembly to form functional channels. • N-terminal variation can influence current amplitude and surface level of Trek2 channels, as seen in Trek2b.

30. Sculpture by Julian Voss-Andreae How does nature accomplish high conduction rates and high selectivity at the same time?

31. Visualize a K+ channel and its selectivity filter Roderick MacKinnon - 2003 Nobel Prize in Chemistry

32. The signature sequence of the potassium channel

34. Carbonyl oxygens attract K+ ions Yellow : carbon, Red : oxygen

35. Electrostatic repulsion favors high conduction rates Yellow : carbon, Blue : nitrogen, Red : oxygen

36. Paper #3

37. The renin-angiotensin-aldosterone system regulating blood pressure http://radiographics.rsna.org

38. The angiotensin-renin-aldosterone system regulating blood pressure Adrenal glomerulosa cells in the zonaglomerulosa Choi et al., Science

39. Aldosterone-producing adenomas (Aka Conn’s syndrome) One of the most common types of the primary aldosteronism (the overproduction of aldosterone) Conn’s sydrome is caused by a discrete benign tumor of the adrenal gland (APA) Diagnosed between ages 30 and 70 Most of them are classified as idiopathic and a small number have mutations Resulting in hypertension and hypokalemia (low plasma K+ level) Surgical procedure can relieve symptoms Hereditary hypertension Mendelian form of primary aldosteronism Bilateral adrenal hyperplasia (increase in number of cells/proliferation of cells) Bilateral adrenalectomy in childhood

40. Protein-changing somatic mutations in aldosterone-producing adenomas

41. Mutations in KCNJ5 in aldosterone-producing adenoma and inherited aldosteronism The probability of seeing either of two somatic mutations recur by chance in 6 of 20 other tumors is <10-30

42. H.s., Homo sapiens Human M.m., MusmusculusRodent G.g., Gallus gallusChicken X.t., XenopustropicalisFrog D.r., DaniorerioZebrafish C.I., CionaintestinalisSea squirt

43. KCNJ5 channel Kir3.4, GIRK4 Subclasses: ROMK, GPCR, ATP-sensitive 2 TMD, 1 P Current flow into cell (“inward”) Differ from delayed rectifier or A-type channels (outward current) Magnesium ions, that plug the channel pore at positive potentials, resulting in a decrease in outward currents. A voltage-dependent block by external Cs+ and Ba2+

44. Location of human mutations in KCNJ5 mapped onto the crystal structure of chicken K+ channel KCNJ12

45. KCNJ5 mutations result in loss of channel selectivity and membrane depolarization

46. KCNJ5 mutations result in loss of channel selectivity and membrane depolarization

47. Membrane depolarization by either elevation of extracellular K+ or closure of K+ channels by angiotesin II activates voltage-gated Ca2+ channels, increasing intraceullular Ca2+ level. Channel containing KCNJ5 wit G151R, T158A, or L168R mutations conduct Na+, resulting in Na+ entry, chronic depolarization, constitutive aldosterone production, and cell proliferation.