W1L2 Molecular Diversity of Ion Channels (Dustin)

Question 1

Ion channels can be classified by what 2 aspects?

Answer 1

Their pore and/or their gate

Pore: refers to K+, Na+, etc.

Gate: ligand or voltage gated, but could be some others like signal transduction, phosphorylation

Question 2

All ligand-gated ion channels have what structure?

Answer 2

All are pentamers

Question 3

ATP-sensitive K+ channels have a common origin/ many similarities with what other type of channel?

Answer 3

CFTR Chloride Channel

(Cystic fibrosis transmembrane conductance regulator)

Question 4

Voltage-gated potassium channels, like VG Na+ channels, open up only ______ in response to depolarization

Answer 4

transiently

So activity has initial activation phase, then inactivation phase

Question 5

What is the implication of this statement, regarding V.G. potassium channels?

“open probability (P_o) is a steep function of membrane voltage (V_m)”

But what must be done to the channel to create this steep Po/Vm relationship?

Answer 5

These channels are an extremely good transistor, better than the silicon used in electronics

In the case of V.G. K+ channels, the membrane potential can change 10 mV and create a huge change in open probability

• This only occurs in channels that have had their inactivation artificially eliminated (by trypsin). Otherwise, the function would show steep decrease in Po at more depolarized Vm values.

Question 6

What do you call the charged part of the channel protein that moves in response to depolarization?

What provides this part of V.G. potassium channels, and where is it in the molecule?

Answer 6

“Gating Charge”

It’s in the 4th TM domain (S4 helix) of voltage-gated K+ channels.

It contains basic amino acid residues (Arg/Lys), with a total of 12-16 unitary charges.

(I guess a unitary charge = one (+) charge)

Question 7

In this V.G. K channel structure, what is the relationship between voltage sensor domains and pore domains?

Answer 7

the voltage sensor domain of each subunit contains the pore domain of the neighboring subunit

Question 8

Which subunits of the V.G. K+ channel are the “paddle?”

What does that mean?

Answer 8

The S3 & S4 subunits, which are more flexible. Remember the S4 is also the voltage-sensing unit.

Can undergo large movements in response to voltage changes.

Question 9

The image shows the paddle conformation when a V.G. K channel is open, how does the paddle change when the channel is closed?

Answer 9

The tip of the paddle moves closer to the intracellular surface, compressing the other subunits inwardly to form a “cup”

Question 10

With V.G. K+ channels, the voltage sensors are located on the _______ of the channel

Answer 10

periphery

(at the protein-lipid interface)

Question 11

What molecule can be used to remove the inactivation tendency of K channels?

Why does this work?

Answer 11

Trypsin, makes channels just stay open as long as the depolarizing voltage pulse is maintained

Trypsin cleaves the N terminal segment from the peptide, which otherwise forms a “ball” which will plug the pore.

This K channel feature is called a “ball and chain”

Question 12

Mutations of KCNQ1, HERG cause what problems?

Answer 12

Prolonged ventricular action potential (seen in a long QT segment)

With non-functioning V.G. K+ channels, the cell cannot repolarize

This can lead to ventricular fibrillation, causing sudden death

These channels repolarize ventricular myocytes after action potential

Question 13

ATP-sensitive K + channels have what structure/how many subunits?

What two types of subunits do they have?

Answer 13

ATP-sensitive K + channels are octamers
built from four pore-forming K + channel
(Kir6.2) subunits and four sulfonylurea
receptor (SUR)subunits

Question 14

In an ATP-sensitive K channel, The SUR (sulfonylurea receptor) contains how many transmembrane domains?

How many intracellular nucleotide binding domains?

What binds to them?

Answer 14

The SUR contains three TMDs and two
intracellular NBDs.

ADP binds to the nucleotide binding domains on the cytosolic

(ATP binds to the pore-forming Kir6.2 subunit C-terminal end, not part of the SUR subunits)

Question 15

What ER retention signals ensure correct
assembly of the octamer in ATP-sensitive K+ channels?

Answer 15

Arginine-Lysine-Arginine, the highly conserved sequence characteristic of these channels.

The quality control mechanisms of the cell will detect if these are immature and block them from going to the cell membrane until they are complete

Question 16

What does inward rectification mean?

How does it work?

Answer 16

In a symmetrical potassium environment, the open pore conducts smaller outward current at depolarized membrane potentials than it does inward current at hyperpolarized membrane potentials

In cytosol, there are several large cations like Mg2+ and polyamines that are small enough that they can enter central cavity of the channel when the gate is open but too large to fit in narrow selectivity filter, so they get stuck and block it. At depolarized potential, the voltage drives these cations into the pore so they impede the exit of K ions through the pore. At hyperpolarized potentials, the voltage drives the cations out of the pore, and the filter is then open.

Question 17

What is the normal cytosolic concentration of ATP?

How does this relate to ATP-sensitive K+ channels?

Answer 17

5-10 mM

This is much larger than the micromolar range for which the ATP-sensitive channels open, so in fact these channels are activated/opened by intracellular ADP and inhibited by ATP

So what these channels sense is the ATP/ADP ratio, but will still open with high ADP even in presence of ATP (“physiological activation”)

Question 18

What are some activators and inhibitors of ATP-sensitive K+ channels?

What is the overall effect, and when might you use them?

Answer 18

• K + Channel Openers (e.g., diazoxide) stimulate similarly to ADP
  
  • Activation of ATP-sensitive K+ channels leads to more hyperpolarization of pancreatic beta cells -> resistance to beta cell depolarization -> less insulin is released. Useful with hyperinsulinemia disorders
• Sulfonylureas (e.g., tolbutamide) inhibit the channel
  
  • Inhibition of these channels -> less hyperpolarization of channels -> easier depolarization of pancreatic beta cells -> more insulin is released. Useful in hyperglycemia disorders (the usual case for diabetes mellitus type II)

Question 19

In ATP-sensitive K channels:

ATP acts on the ____ subunit?

ADP, sulfonylureas and K+ channel openers act on the ____ subunit?

Answer 19

ATP acts on the Kir6.2 subunit

ADP, sulfonylureas and K+ channel openers act on the SUR subunit

Question 20

What are 3 important locations in the body of ATP-sensitive K+ channels, and what is their function?

Answer 20

1. Pancreatic beta cells -> insulin secretion
2. Cardiac myocytes -> in ischemia, reduce the ATP consumption
3. Vascular smooth muscle cells -> decrease vascular tone, reducing hypertension

(Only pancreatic cells were red-boxed on the lecture, so if you aren’t remembering the other two, you still deserve to mark this card green/blue. You’re the best.)

Question 21

For ATP-sensitive K channels,

Where is the most important place in the body are Kir6.2 + SUR1 found?

What are the important medical implications of this?

Answer 21

location: pancreatic β-cells
role: ATP/ADP ratio ↑ after a meal ⇒ K ATP channels shut
⇒ depolarization ⇒ insulin secretion

mutations: persistent hyperinsulinaemic hypoglycaemia (PHHI)
(some patients respond to diazoxide)

with type II diabetes mellitus, patients who have hyperglycemia can be treated with sulfonylureas (oral antidiabetics)

Question 22

What channel has a “double barrel shotgun” mechanism with a common lock?

Answer 22

Clc-0 Cl^- channels

Pores are in pairs, the smallest function unit has 2 pores.

The two pores are independently gated and so can fire at different times, but they have a common (slow gate) inactivation mechanism.

Question 23

What is the structure of Clc-0 Cl^- channels?

How was this determined?

(Include molecular weight numbers… unfortunately they were in the red box.)

Answer 23

Homodimer

• But both subunits individually form separate pores
• Cloning and biochemical characterization of the channel showed that…
  • the monomer weighed 90 kDt
  • the whole protein weighed 200 kDt

… so the channel is a dimer of 2 identical subunits.

(Not sure how to account for that extra 20 kDt, but whatever.)

Question 24

In the Clc channel pore, a negatively charged Cl- ion is stabilized by ______.

Answer 24

In the Clc channel pore, a negatively charged Cl- ion is stabilized by helix dipoles.

Each alpha helix acts as an elementary dipole

Question 25

Where are 3 important locations of Clc chloride channels? What is their function?

Answer 25

1. Skeletal muscle -> stabilizes resting membrane potential
2. Proximal tubule of kidney -> chloride transport
3. Thick ascending loop of kidney -> chloride reabsorption

(Again here, only #1 was red-boxed on the lecture. If it’s all your dumb ass can manage to remember that’s fine. Dummy.)

Question 26

Where is Clc-1 located, most importantly? (red box)

What does it do?

What are the medical implications?

Answer 26

location: skeletal muscle
role: stabilizes resting membrane potential (normally that’s from K channels in other tissues). In muscles this is bc of T-tubules forming the equivalent of extracellular space. K channels -> high “fractional change” that impacts mV. The Cl- equilibrium potential is more stable resting membrane potential for muscle cell than K+ equilibrium potential)
mutations: unstable resting potential, hyperactive muscle fiber ⇒ myotonia

(delayed relaxation of muscles after contraction)

Inheritability: if autosomal dominant, then have only 25% of normal chloride conductance and poor function. Autosomal recessive -> mostly asymptomatic, still have 75% of normal conductance

Question 27

The CFTR Cl- channel belongs to the _____ protein family, among which it is the only ion channel.

Answer 27

The CFTR Cl - channel belongs to the ABC (ATP-Binding Cassette) protein family

Question 28

What is the structure of CFTR?

CFTR consists of what 3 kinds of domains? How many of each?

Answer 28

CFTR consists of a single polipeptide chain (not really explained, but it should still be a dimer)

2 transmembrane domains (TMD),
2 intracellular nucleotide binding domains (NBDs)

1 regulatory (R-) domain

Question 29

What is the prerequisite for CFTR activation?

What about the requirement for gating?

Answer 29

cAMP-dependent Protein Kinase A

PKA phosphorylates and activates this channel, but gating will only be functional as long as there is cytosolic ATP available. CFTR activity is proportional to ATP concentration

Question 30

Describe the active conformation of ABC proteins.

How are their NBDs configured + what else is present?

Answer 30

The “active conformation” of ABC proteins is a head-to-tail NBD1-NBD2 heterodimer, with two ATP molecules sandwiched at the interface.

(seen in O1 on image)

Question 31

CFTR uses ___ to transport Cl- in what is essentially passive transport

Why?

Answer 31

1 ATP, it’s basically wasted

Probably evolved from a transporter (and not an ion channel)that needed to use ATP for uphill transport

Question 32

What is the major effect of mutations of the CFTR channel?

How is it inherited?

What are the symptoms?

Answer 32

Cystic Fibrosis

• autosomal recessive - the most common lethal inherited disease in white people
• protein folding/processing defects (70% of all cases), gating or permeation defects (< 30% of all cases)
• Symptoms:
  
  • viscous alveolar mucus (⇒ lung infections)
  • pancreatic insufficiency
  • meconium ileus
  • high-salt sweat (diagnostic)

Question 33

Where are CFTR channels located in the body?

What is their role?

Answer 33

Location: apical surfaces of epithelial cells

Role:

• lung, alveoli: salt balance of alveolar surface fluid
• pancreatic duct: Cl- secretion (⇒ HCO3- and H2O secretion)
• intestinal epithelium: Cl- secretion (⇒ Na+ and H2O secretion)
• sweat duct: Cl- reabsorption (⇒ Na+ reabsorption)

Question 34

How does the toxin produced by cholera affect CFTR?

Answer 34

cholera toxin ⇒ adenylate cyclase irreversibly activated ⇒ cAMP↑ ⇒ CFTR active ⇒ salt/fluid loss in diarrhea

Question 35

With nicotinic acetycholine receptors, how are the different subunits expressed in skeletal muscle vs neurons?

i.e. α, β, γ, δ, etc.

Answer 35

Skeletal muscle (neuromuscular junction)

• 2α, β, γ, δ (embryonic)
• 2α, β, ε, δ (adult)

Neuron (dendrites, axon terminals)

• 2α, 3β
• or 5α

Question 36

What is the structure of the nAchR?

What kind of ligand-binding sites are there and where are they?

(some detailed features are mentioned on answer card)

Answer 36

homo-pentamer,

perfect 5x rotational symmetry

aromatic ligand-binding pockets in intersubunit clefts

Question 37

the nAchR channel is activated by binding how many ligand molecules?

Answer 37

the nAchR channel is activated by binding of two ligand molecules

has 2 ligand binding sites and won’t open until both ligands are bound

Question 38

Prolonged stimulation (or high-affinity agonist) of nAchR leads to what?

What prevents this from happening under physio conditions?

Answer 38

prolonged stimulation (or high-affinity agonist) leads to desensitization

But this does not occur normally due to acetylcholinesterase, but other high-affinity ligands to nAch receptors can be used medically or as poisons

Question 39

Mutations of the skeletal muscle type nAchR lead to what?

Autoimmune attack of the skeletal muscle type of nAchR leads to what?

Answer 39

mutations -> congenital myasthenia (slow channel syndrome)

autoantibody against MIR (main immunogenic region of receptor) -> myasthenia gravis

Question 40

What are 2 inhibitors of skeletal muscle-type nAchR?

Answer 40

• *α-bungarotoxin** (banded krait venom)
• *d-tubocurarine** (plant alkaloid)

Question 41

How can inactivation be restored in VDK+ channels whose inactivation mechanism has been removed?

Answer 41

Trypsin cleaves the N-terminal “ball and chain” mechanism of the channel.

Exposure to the isolated N-terminal peptide can restore inactivation functions of the channel.

Question 42

What are the two mechanisms by which VDK+ channel inactivation can be removed?

Answer 42

1. Enzymatic cleavage (via trypsin)
2. Artificial deletion (via genetic means)

Question 43

What are the two names for the inactivation mechanism of voltage-gated K+ channels?

In most channels this mechanism is what part of the protein?

But in some channels it is another part… what part?

Answer 43

N-type inactivation -or- Ball and Chain mechanism

• most channels - N-terminus = ball and chain
• some channels - Associated β-subunit = ball and chain

Question 44

What is the state of ATP-sensitive K+ channels in resting living cells?

And in excised patches of cells?

(Why? Less important.)

Answer 44

They are inactive in resting living cells…

…and constitutively active in excised patches.

(Not totally sure here, but I guess the resting living cells have higher ATP/ADP ratio, thus inactivating the channel. Once excised, ATP creation in the patches ceases and ATP/ADP ratio decreases.)

Question 45

How many human ABC proteins are there?

List 3 examples other than CFTR and their general function.

Answer 45

48 ABC proteins in humans

1. SUR (sulfonylurea receptor subunit of K_ATP channel)
2. P-glycoprotein (multidrug resistance)
3. TAP (antigen presentation)

Question 46

Where, what and how many things does PKA phosphorylate on CFTR?

How does this affect channel activity?

Answer 46

PKA phosphorylates at least 10 individual serines in the regulatory (R-) domain.

Channel activity incrementally increases with each phosphorylation.

Question 47

Describe ATPs effect on CFTR.

Where does it bind? With what affinity?

Does it “regulate” CFTR?

Answer 47

It drives gating of already PKA-phosphorylated CFTR channels by binding to the 2 cytosolic NBDs.

This activates the channel with high affinity.

It is not considered the physiological regulator of CFTR, because the regulatory domain must first be P-ated by PKA before ATP can bind the NBD.

Question 48

What conformational change of which parts of CFTR triggers opening?

And what event triggers closing?

Answer 48

The CFTR channel pore opens upon dimerization of its NBDs. (top right in img)

The NBD2 “head” hydrolyzes ATP ⇒ the dimer dissociates ⇒ the pore closes. (bottom left in img)