Midterm 1 Content

Question 1

Draw Kv membrane topology

Answer 1

See notes

Question 2

Draw Kir membrane topology

Answer 2

See notes

Question 3

Draw Two-pore K membrane topology

Answer 3

See notes

Question 4

What channel did Doyle et al study?

Answer 4

KcsA Shaker bacterial channel

Question 5

Draw membrane topology of KcsA

Answer 5

See notes
N -outer helix - pore region (pore helix w TVGYG) - inner helix - C
n = 4, four-fold symmetry

Question 6

What shape do the inner helices give KcsA?

Answer 6

“Upside down teepee” tilted 25 degrees with respect to membrane normal

Question 7

What interaction holds the pore of KcsA at its proper diameter and determines position of carbonyl oxygens?

Answer 7

Y78 of selectivity filter (tvgYg) and 2 tryptophans (W) of the inner helix.`

Question 8

3 ways K+ channel is highly selective for K+:

Answer 8

• Negative residues at entrance/exits of the pore
• Selectivity filter is narrow through Y and W interactions, so that ion must dehydrate to be let through and only favourable for K+ ions to be dehydrated.
• Selectivity filter is held at precise diameter: carbonyl oxygens act as surrogate water; distance is such that it is only favourable for K+ molecules to be dehydrated (too far apart for Na+)

Question 9

4 ways a K+ channel receives high throughput:

Answer 9

• Electrostatic repulsion of the two K+s in the pore: the energy of repulsion overcomes the energy of the K+ ions binding to the selectivity filter, pushing them through the channel.
• Central cavity diameter: allows water molecules to surround ion and stabilize it.
• Pore helices: direct cations toward the pore exits/entrances
• Hydrophobic inner pore lining: is hydrophobic up until selectivity filter; minimizes interaction of K+ with the channel.

Question 10

What channel did Long et al. study?

Answer 10

Kv1.2

Question 11

What is the structure of Kv1.2 like?

Answer 11

Tetramer, 4-fold symmetry
Associated with beta subunit (helps with translocation)
TM domain, T1 domain (specifies what subunits can associate with), Beta subunit.

S4 is voltage sensor
S3-S4 is voltage sensor paddle
Ion conduction pore is S5-pore helix-S6
S4-S5 linker helix runs parallel to intracellular membrane surface near inner helices
S6 is inner helix, contains Pro-Val-Pro (PXP) gating hinge; this bends S6 so it is almost parallel to intracellular surface.

Question 12

When Long et al. superimposed Kv1.2, KcsA, and KvAP, which were open/closed?

Answer 12

Kv1.2 and KvAP open, KcsA closed

Question 13

What are the purpose of alpha-helical linkers in Kv1.2?

Answer 13

Connect T1 and S1 domains..

1) Gives enough space for inner helices (S6) to radiate outward while gating
2) Provides diffusion pathway between pore and cytoplasm (side portals)
3) Attract cations and repel anions (negativly charged)

Question 14

How does N-type/Ball and Chain Inactivation work in Kv1.2 channels and what are the requirements?

Answer 14

First 10 residues of N-terminus inactivation gate should be hydrophobic, followed by hydrophilic and positively charged residues. Upon depolarization, positive residues bind to negatively-charged side portals of Kv1.2, allowing hydrophobic region to block pore and bind with hydrophobic region of Kv1.2. This blocks ion flow.

Question 15

What is the movement/stabilization of the Kv1.2 voltage sensor like?

Answer 15

Consists of 4 Rs; consists of an antiparallel S3-S4 helix-turn-helix arrangement into a voltage-sensor paddle.

Closed: lies parallel/horizontal to the membrane

Open: lies vertical/upright to the membrane
- R are stabilized by negatively charged external and internal clusters, whose Es and Ds interact with the Rs. Also Rs are stabilized by S1, S2, and S3 alpha helices

Moves large distance (15A)

Question 16

What is the structure of Kv1.2 S4-S5 linker like?

Answer 16

Amphipathic alpha helix running parallel to cell membrane; polar residues face cytoplasm, hydrophobic residues face lipid membrane.

Question 17

What is the gating mechanism of Kv1.2 like?

Answer 17

Closed:

• S4-S5 linker interacts with S6, locking it into place
• The voltage sensor S4 is horizontal in the cell membrane.

Open:

• Depolarization results in upward movement of the voltage sensor paddle (stabilized by S1-S3 and internal/external clusters)
• This upward movement moves S4-S5 linker up, resulting in S6 bending outward (via PVP or PXP) and splaying channel open.

Question 18

What is inward rectification due to in GIRK channels?

Answer 18

Intracellular Mg2+ or polyamine block that moves into the channel with depolarization.

Question 19

How do you calculate FRET efficiency? What is it?

Answer 19

Proportion of donor molecules that have transferreed excitation statge energy to acceptor moelcules

E = 1 - Ida/Id

Question 20

What are 2 conditions for FRET?

Answer 20

• Molecules must be close together

- Must be overlap of donor emission and acceptor absorbance

Question 21

What channel did Riven et al study?

Answer 21

GIRK1 and GIRK4 Kir.3 channels

Question 22

What kind of microscopy did Riven et al. use to measure FRET? What are two ways they activated GIRK channels and what was seen with this?

Answer 22

Total Internal Reflection Fluorescence Microscopy (TIRF)

Activated with adenosine and Gprotein beta-gamma subunit.
- Resulted in decrease in F ratio (FCFP/FYFP), suggesting increased FRET efficiency

Question 23

What kind of FRET efficiency changes did Riven et al observe with different fluorophore positioning on GIRK channels?

Answer 23

If both fluorophores on C termini or both on N termini: FRET efficiency increased

If both were on the same protein: FRET efficiency stayed the same

If one was on the C terminus of one protein and the other was on the N terminus of the other: FRET efficiency decrease.

Question 24

What did Riven et al discover about the movement of C and N termini of GIRK channels upon activation?

Answer 24

C termini of GIRK1 and GIRK4 rotate 10 degrees clockwise while the N-termini radiate outward

Question 25

What are two functions of Nav auxillary subunits?

Answer 25

Speed up inactivation

Increase magnitude of current

Question 26

Where are the activation and inactivation gates of Nav?

Answer 26

Activation gate on S6

Inactivation gate on S3-S4 loop

Question 27

What is a major Nav channel blocker?

Answer 27

Tetradotoxin (TTX) - from puffer fish!

Question 28

What is a major channelopathy for Na+ channels?

Answer 28

Epilepsy - mutation causing worse inactivation.

Question 29

What is a major channelopathy for K+ channels?

Answer 29

Ataxia - mutation in voltage sensor S4 results in diminished current.

Question 30

What kind of channel did Payandeh et al study?

Answer 30

NavAB (bacterial VG from Arcobacter)

Question 31

What are the four regions of a NavAB channel?

Answer 31

Extracellular funnel
Selectivity filter
Central cavity
Activation gate

Question 32

How does Na+ move through the selectivity filter of NavAB channels? Name specific residues.

Answer 32

E177 helps partially strip water molecules off hydrated Na+
–> hydrated Na+ has 4 water molecules; must strip off 2 before can pass through

Backbone carbonyl oxygens of L176 and T175 interact with fully hydrated Na+

L176 is the central site; Na+ cannot pass until 2 water molecules are stripped off.

Question 33

What is the structure of a NavAB voltage sensor like? What stabilizes it?

Answer 33

A 310 helix, consisting mostly of Rs.

Negatively charged internal and external clusters stabilize the Rs.

Question 34

What is the hydrophobic constriction site?

Answer 34

A site on NavAB that is made up of hydrophobic residues and seals against ion leakage.

Question 35

What is the gating mechanism of NavAb like?

Answer 35

Closed: S4-S5 linker interacts with the inner helix S6 and prevents it from moving. The S4 310 helix Rs are stabilized by negative internal and external clusters.

Open: The inner helix S6 bends via glycine and splays open as the S4-S5 linker moves out of the way through movement of S4. The channel opens.

Question 36

What is the structure and mechanism of the NavAb inactivation gate?

Answer 36

Located on the S3-S4 cytoplasmic loop.
Isoleucine, Phenylalanine, and Methionine are hydrophobic residues that plug up the pore from the cytoplasmic side to cause inactivation.

Question 37

What are similarities(1)/differences(5) between Kv1.2 and NavAB?

Answer 37

Differences:

• NavAb has 2 pore helices per subuint which Kv1.2 has 1
• Na+ is partially hydrated while Kv1.2 is not
• Maximal occupancy of NavAb is 3 while Kv1.2 is 4
• NavAb voltage sensor is 310 helix while Kv1.2. is alpha helix.
• NavAb has inactivation gate.

Similarites:
- Rs in the voltage sensor are stabilized by inner and outer negative cluster of amino acids.