K Channel Sukhee Cho Greg Richard K Channels

+ K Channel Sukhee Cho Greg Richard

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

K+ Channel Anatomy Senyon Choe (2002)

Gating Bezanilla 2004

Classes • Inwardly Rectifying – ROMK, GIRK, ATP-sensitive • Tandem Pore Domain – TWIK, TREK, TASK, TALK, THIK, TRESK • Voltage-Gated – h. ERG, Kv. LQT 1 • Calcium Activated – BK, IK, SK

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

Tandem Pore Domain (K 2 P) • 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, p. H, temperature

Voltage-Gated (Kv) • Subclasses: h. ERG, Kv. LQT 1 • 6 TMD, 1 P • Sensitive to voltage changes – S 4 domain • Return to resting state – Repolarization – Limits AP frequency (RRP)

Calcium Activated (KCa 1 ) • Subclasses: BK, IK, SK • 6 TMD, 1 P • Activated by intracellular Ca 2+ • Some activated by intracellular Na+ & Cl • N-terminus extracellularly (Unlike Kv)

Paper #1

Amyloid β Hypothesis in Alzheimer’s disease Aβ 1 -40 Aβ 1 -42 Alzheimer's diseased brain Amyloid precursor protein http: //en. wikipedia. org/wiki/Beta_amyloid

BK channel Large conductance Ca 2+-activated K+ channels, Maxi-K, BK or Bkca, Kca 1. 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.

Aβ 1 -40 Aβ 1 -42 Fura-2 500 ms 100 -250 p. A Figure 1. Intracellular infusion of Aβ 1 -42 broadens spike width and augmemted Ca 2+ influx in rat neocortical pyramidal neurons.

Charybdotoxin - Ca 2+-activated K+ channel blocker 4 -AP(4 -Aminopyridine) – A-type potassium channel blocker Figure 3. Intracellular Aβ 1 -42 enlarges spike width by suppressing BK channels, thereby increasing spike-induced Ca 2+ entry.

Isopimaric acid Electroconvulsive shock Figure 5. ECS blocked Aβ 1 -42 -mediated suppression of BK channels in rat neocortical neurons.

Figure 7. Blocking effects of ECS on Aβ 1 -42 was absent in H 1 a. KO mice.

4 months of age Juvenile Figure 8. Spike broadening in 3 x. TG neurons.

Figure 9. Recovery of single BK current by ECS in 3 x. TG mice.

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

Paper #2

Trek Channels • Two-pore domain K+ channels (K 2 P) – 4 TMD, 2 pore • Subfamilies: – Trek 1 (Kcnk 2) – Trek 2 (Kcnk 10) • Underlie “leak” and background K+ conductances • Sensitive to membrane stretch, temperature, & p. H • Inhibited by PKC & PKA

Trek 2 • Trek 2 b – Differs from Trek 2 a & Trek 2 c at N-terminus • Trek 2 -1 p – C-terminal truncation (2 TMD & 1 pore) Does alternative splicing of Trek 2 contribute to functional diversity of channel as seen with Trek 1?

Trek 2 Variants Trek 2 -1 p C-terminus Trek 2 b N-terminus

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

Whole-cell Currents (Voltage-step) +60 m. V 20 m. V -100 m. V

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

Whole-cell Currents

Surface Trek 2 Expression Total Protein Surface Protein

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

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

Visualize a K+ channel and its selectivity filter Roderick Mac. Kinnon 2003 Nobel Prize in Chemistry

The signature sequence of the potassium channel

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

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

Paper #3

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

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

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

Protein-changing somatic mutations in aldosterone-producing adenomas

Mutations in KCNJ 5 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

H. s. , Homo sapiens Human M. m. , Mus musculus Rodent G. g. , Gallus gallus Chicken X. t. , Xenopus tropicalis Frog D. r. , Danio rerio Zebrafish C. I. , Ciona intestinalis Sea squirt

KCNJ 5 channel Kir 3. 4, GIRK 4 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 Ba 2+

Location of human mutations in KCNJ 5 mapped onto the crystal structure of chicken K+ channel KCNJ 12

KCNJ 5 mutations result in loss of channel selectivity and membrane depolarization

KCNJ 5 mutations result in loss of channel selectivity and membrane depolarization

Membrane depolarization by either elevation of extracellular K+ or closure of K+ channels by angiotesin II activates voltage-gated Ca 2+ channels, increasing intraceullular Ca 2+ level. Channel containing KCNJ 5 wit G 151 R, T 158 A, or L 168 R mutations conduct Na+, resulting in Na+ entry, chronic depolarization, constitutive aldosterone production, and cell proliferation.