K+ Channel

Sukhee ChoGreg Richard

K+ Channels

• Found everywhere

• Contribute to resting potential (neurons)

• Major roles in cardiac tissue

• Involved in hormone secretion

ClosedOpen

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– hERG, KvLQT1

• 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 A- type channels (outward current)

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

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)

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)

Paper #1

Amyloid β Hypothesis in Alzheimer’s disease

http://en.wikipedia.org/wiki/Beta_amyloid

Alzheimer's diseased brain

Aβ1-40

Aβ1-42

Aβ1-40

Aβ1-42

Amyloid precursor protein

Controlling neurotransmitter releaseFast after-hyperpolarizationSpike frequency adaptation

VSD - voltage sensing domainPGD - pore-gating domainRCK - regulator of K conductance

Lee et al., Trends Neurosci. 2010 Sep;33(9):415-23. Review.

BK channelLarge conductance Ca2+-activated K+ channels, Maxi-K, BK or Bkca, Kca1.1

Figure 1. Intracellular infusion of Aβ1-42 broadens spike width and augmemted Ca2+ influx in rat neocortical pyramidal neurons.

Aβ1-40

Aβ1-42

Fura-2

100-250 pA500 ms

Figure 3. Intracellular Aβ1-42 enlarges spike width by suppressing BK channels, thereby increasing spike-induced Ca2+ entry.

Charybdotoxin - Ca2+-activated K+ channel blocker4-AP(4-Aminopyridine) – A-type potassium channel blocker

Figure 5. ECS blocked Aβ1-42-mediated suppression of BK channels in rat neocortical neurons.

Isopimaric acid

Electroconvulsive shock

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

Figure 8. Spike broadening in 3xTG neurons.

Juvenile

Juvenile4 months of age

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

Conclusions

Intracellular Aβ1-42 broadens spike width in neocortical pyramidal neurons by downregulation of BK channel activities.

ECS counteracts Aβ1-42 induced BK channel inhibition by expression of Homer 1a

Paper #2

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

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?

Trek2b

N-terminus

Trek2-1p

C-terminus

Trek2 Variants

Trek2

Immunoblotting

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

Whole-cell Currents(Voltage-step)

-100mV

+60mV

20mV

Reversal Potential (Erev)(Voltage-ramp)

-100mV

+60mV

1 s

Non-selective channel

Whole-cell Currents

Surface Trek2 Expression

Total Protein

Surface Protein

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.

How does nature accomplish high conduction rates and high selectivity at the same time?

Sculpture by Julian Voss-Andreae

Roderick MacKinnon - 2003 Nobel Prize in Chemistry

Visualize a K+ channel and its selectivity filter

The signature sequence of the potassium channel

Yellow : carbon, Red : oxygen

Carbonyl oxygens attract K+ ions

Yellow : carbon, Blue : nitrogen, Red : oxygen

Electrostatic repulsion favors high conduction rates

Paper #3

http://radiographics.rsna.org

The renin-angiotensin-aldosterone system regulating blood pressure

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 70Most of them are classified as idiopathic and a small number have mutationsResulting in hypertension and hypokalemia (low plasma K+ level)Surgical procedure can relieve symptoms

Hereditary hypertension

Mendelian form of primary aldosteronismBilateral adrenal hyperplasia (increase in number of cells/proliferation of cells)Bilateral adrenalectomy in childhood

Protein-changing somatic mutations in aldosterone-producing adenomas

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

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

KCNJ5 channelKir3.4, GIRK4

Subclasses: ROMK, GPCR, ATP-sensitive2 TMD, 1 PCurrent 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+

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

KCNJ5 mutations result in loss of channel selectivity and membrane depolarization

KCNJ5 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 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.