Bio2005 Minli Lectures Ii

LECTURE II

Inactivation: mechanisms and domains

Voltage-gated potassium channels

•Domain organization •Inactivation particle/ball Where is the inactivation particle and how was it identified? •Pore region •Assembly domain (T1) •Composition and stoichiometry

Hoshi et al Science (1990)

Hoshi et al Science (1990)

Hoshi et al Science (1990)

Modulation of channel activity by accessory subunits

Voltage-gated potassium channels

•Domain organization •Inactivation particle/ball •Pore region

What are the regions that form the ion permeation pathway? How was it identified? •Assembly domain (T1) •Composition and stoichiometry

Ion Permeation and Selectivity – I 1. TEA gains access to the internal binding site only when the channel is open. 2. When the gate is open, blockade by TEA is sensitive to the transmembrane in a fashion consistent with partial penetration of TEA into the pore. 3. Dissociation of the TEA analogs is enhanced by a high concentration of K on the opposite (external) side of the channel, as though K can enter the pore from the opposite side and expel the TEA analog docked on internal side.

Ion Permeation and Selectivity – I

Yellen et al Science 251:939 (1991)

Ion Permeation and Selectivity – I

Hartmenn et al Science 251:942 (1991)

Ion Permeation and Selectivity – I

Ion Permeation & Selectivity – II

Voltage-gated potassium channels

•Domain organization •Inactivation particle/ball •Pore region •Assembly domain (T1)

What region(s) mediate assembly, specificity? •Composition and stoichiometry

Voltage-gated sodium channels

Pore-forming subunits

Diversity by assembly: Heteromultimers

Subunit assembly of Shaker type potassium channels

Science 257:1225

Subunit assembly of Shaker type potassium channels

Subunit assembly of Shaker type potassium channels

Subunit assembly of Shaker type potassium channels

Subunit assembly of Shaker type potassium channels

Voltage-gated potassium channels

•Domain organization •Inactivation particle/ball •Pore region •Assembly domain (T1) •Composition and stoichiometry

What are the components, their roles? Whether and how the stoichiometric assembly to be achieved

“Accessory” subunits

Stoichiometric assembly – Many channels are multimeric complexes with specific stoichiometry, e.g. (alpha)4(beta)4. How would a cell know an assembled complex is in a correct stoichiometry?

Biogenesis of membrane proteins

ER retention – localized biological activities N

N

C C Jackson et al., 1990

ER retention – localized activities

Can we conclude… -KK is position-specific (?). -KKXX is necessary & sufficient for the ER retention (?). -KKXX retention activity is dominant (?). Jackson et al., 1990

Forward transport (trafficking)… … motifs and machinery which potentiate surface expression

Reduced Expression

High Expression

Low Expression

High Expression

Forward transport – “DXE”….

VSV-G

N

C

-18aa-YTDIEMNRLGK Sevier et al., 2000

Nishimura and Blach, 1997

Critical steps controlling the membrane protein expression …

Vesicular trafficking… Key issues (questions): • Entry of ER • Exit of ER • Where to be transported (or should be “ where to go.)

Retention and forward trafficking

Assembly of KATP channel – an example of interplay between proteinprotein interaction and vesicular trafficking

Stoichiometric assembly of KATP channel

Zerangue et al (1999) Neuron 22:537

Stoichiometric assembly of KATP channel

Zerangue et al (1999) Neuron 22:537

Stoichiometric assembly of KATP channel

Zerangue et al (1999) Neuron 22:537

Stoichiometric assembly of KATP channel

Zerangue et al (1999) Neuron 22:537

Stoichiometric assembly of KATP channel - what is the evidence that RKR is ERretention signal?

Zerangue et al (1999) Neuron 22:537

Ion channels, human diseases and therapeutics

Genetic approaches identified KCNQ2 & KCNQ3 channels involved in epilepsy • Benign familial neonatal convulsions (BFNC) – autosomal dominant idiopathic epilepsy • Cloning and identification of KCNQ2 K+ channel in BFNC - Biervert, C. et al., 1998 Science 279: 403-406 - Singh, N. A., et al., 1998, Nature Genet. 18: 25-29

• kcnq2 – chromosome 20 kcnq3 – chromosome 8

- From Jentsch, T. J., 2000 Nature Review Neurosci. (1):21-30

Molecular identities of the M-current • KCNQ2 and KCNQ3 potassium channel subunits: molecular correlates of the M-channel - Wang, H. S. et al., 1998, Science, 282:1890-3 • KCNQ2 and KCNQ3 mRNA injected into Xenopus oocytes, followed by electrophysiological recording. Compared with recordings in rat sympathetic neurons - Similar kinetic properties with native M-current - Similar drug sensitivity – linopirdine, XE991 • Functional M-channels contain KCNQ2 + KCNQ3 heteromers

Putative structures of the KCNQ channel

• S5-S6 linker - G(Y/F)G K+ selectivity signature motif • Four pore loop domains - K+ conducting pore - Tetrameric From Shieh, et al., Pharmacol Rev 2000 Dec;52(4):557-94

KCNQ Channels and Human Diseases

From Cooper & Jan, 1999 PNAS 96:4759-66

Functional role of ion channels …

Credit: Drs. C. Peters & D. Isbrandt

Human mutations of hERG A561V

N598Q

G601S N629Q

T65P ∆147

N S1 (397-425) S2 (452-472) S3 (497-513) S4 (521-538) S5 (549-572) S6 (638-666)

R752W

Human mutations of hERG

Outline Background • General biology of membrane proteins • Receptors, channels and transporters • Ionic gradients in animal cells

Molecular biology of ion channels • • • •

Architecture and functional domains of ion channels Classification of ion channels Regulation and gating of ion channels Methods for studying ion channels

Cell biology of ion channels • Subunit assembly of ion channels • Membrane trafficking and regulation • Molecular coupling of ion channels and signaling proteins

Ion channels, human diseases and therapeutics • CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) • KCNQ potassium channels (epilepsy) • hERG (cardiac safety pharmacology)

Discussion questions

Discussion questions