Ion Channels

Question 1

Name 4 cell types where ion channels play an important role. What role do they play in these cells?

Answer 1

Muscle (triggering contraction/relaxation); neurons (sensory transduction/signalpropagation/neurotransmitter release/postsynaptic responses/plasticity); T lymphocytes (activation); pancreatic Beta cells (insulin relase)

Question 2

How many domains are there in the Kv; Nav and Cav ion channels? (The V means the channels are activated by voltage)

Answer 2

4 membrane-spanning domains

Question 3

How many alpha helices are there in each domain? What are their names?

Answer 3

6 alpha helices in each domain. S1 through S6

Question 4

Describe the relationship between polypeptides and domains in the Kv channel

Answer 4

Each domain is a separate polypeptide and four of these assemble to form the channel.

Question 5

Describe the relationship between polypeptides and domains in the Nav and Cav channels

Answer 5

In NaV and CaV the four domains (I; II; III; and IV) are linked into a single polypeptide

Question 6

Describe the function and role of the S4 helices

Answer 6

Have positively charged residues (lys or arg) at every third position and are the structures that “sense” voltage

Question 7

Describe the function and role of the S5 and S6 helices. What connects them?

Answer 7

The “P loop” connects them (P stands for pore). The S5; S6 and P loop assemble to form the ion conducting pathway. Important for selectivity

Question 8

Where are Kv; Nav and Cav essential?

Answer 8

Nerve and muscle cells

Question 9

What are ionotropic receptors?

Answer 9

1 of 2 types of neurotransmitter receptors. The receptor and channel are part of the same protein

Question 10

What are metabotropic receptors?

Answer 10

Another type of neurotransmitter receptor. The neurotransmitter receptor activates second messenger pathways which can affect physically separate ion channels

Question 11

What type of neurotransmitter receptor are the pentameric ligand gated channels? What are they AKA?

Answer 11

They are within the ionotropic category. AKA cys-loop family of neurotransmitter receptor channels

Question 12

What are some examples of pentameric ligand gated channels?

Answer 12

GABAaRs; GlyRs; nAChRs; 5-Ht3Rs. All named after ligand most important for controlling their gating

Question 13

How many alpha helices are there in each subunit of the heteropentamer pentameric ligand gated channels?

Answer 13

Each of the 5 subunit has four transmembrane alpha helices (M1 through M4)

Question 14

What does M2 do in the pentameric ligand gated channels?

Answer 14

Assembles around the central; ion-conducting pathway

Question 15

What are the pentameric ligand gated channels selective for?

Answer 15

Either selective for the permeation of chloride; or allow permeation of both sodium and potassium (with a slight preference for sodium).

Question 16

What are Ionotropic glutamate receptors?

Answer 16

tetrameric ligand gated channels (4 subunits w/ 3 alpha-helices each)

Question 17

What are NMDA receptors?

Answer 17

A type of ionotropic glutamate receptro; where two of the four subunits bind glutamate and the other two bind glycine. Studied as a molecular source for associative learning

Question 18

What is the structure of chloride channels? To which family do they belong?

Answer 18

Of the CLC family. Dimers in which each subunit has an ion permeation pathway.

Question 19

How do the gates work on chloride channels?

Answer 19

Each permeation pathway can gate open and closed independently of the other. There is also another gate which controls both pathways simultaneously. Some are H+ Cl- exchangers

Question 20

What are CLC chloride channels important for?

Answer 20

Stabilizing the resting membrane potential

Question 21

What mutations are seen in CLC chloride channels?

Answer 21

Dominant mutation affects gate that opens and closes both channels. Recessive only afffects 1 channel

Question 22

What is the structure of aquaporins?

Answer 22

Tetramers in which each of the subunits contains a permeation pathway for water molecules

Question 23

How do the four water channels work with respect to ions?

Answer 23

They are strictly speaking “anti-ion” channels. They exclude all ions including protons

Question 24

Where are aquaporins expressed?

Answer 24

In cells/tissues where rapid movement of water is important. Kidney

Question 25

How many channels are there in an aquaporin?

Answer 25

5 total. 4 water channels in each subunit + the assemblage of the four subunits also produces a central pore which may allow ion permeation and be gated between open and closed.

Question 26

How is selectivity categorized in channel selectivity? What are examples of each category?

Answer 26

Can be: highly selective (ex Kv channels - only 1:10000 ions is not K+. Cav channels select for Ca2+ over Na+ by 3000:1); moderately selective (Nav channels - 1 in 12 is not sodium); low selectivity (nicotinic AChRs show only a slight preference for Na+ over K+ (~1.3 fold).

Question 27

What are two main factors that influence selectivity?

Answer 27

Charge and size. Also dehydration and multiple binding sites make things more selective

Question 28

How does hydration/dehydration of ions work with selectivity?

Answer 28

Ions in solution are energentically stabilized; also makes the ions slightly larger. Ions must be substantially dehydrated before they pass through the channel pore. To compensate for this energentically unfavorable dehydration the ion is stabilized within the pore by energetic interactions with the amino acids forming the pore (but not too much or the ion would stay “stuck” in the pore).

Question 29

What are the charges of various amino acid side chains?

Answer 29

Positive/basic residues: lysine or arginine. Negative/acidic residues: glutamate or aspartate

Question 30

Are backbone carbonyls + or -?

Answer 30

Negative

Question 31

Which side of alpha helix dipoles are +/-?

Answer 31

N-terminal: positive. C-teminal: negative

Question 32

What is another way to increase selectivity?

Answer 32

Multiple binding sites. If an ion interacts with multiple sites while traversing the channel pore; even relatively slight differences in the strength of interaction between preferred and non-preferred ions at each site can result in a significant enhancement of overall selectivity for the preferred ion.

Question 33

How is gating controlled in Kv and Nav channels?

Answer 33

By membrane potential (Vm). Remember these two are important for generating action potential.

Question 34

What cellular conditions are there when the activation gate on Kv channels are closed?

Answer 34

When the inside of the cell has a negative potential (-60 mV) with respect to the outside; the gate is held in its closed position and the current is zero.

Question 35

What happens during Kv activation?

Answer 35

When the inside of the cell is made positive the activation gate rotates to its open position and K+ ions flow out of the cell (upward deflection in the current). Goes up to +20 mV.

Question 36

What happens during Kv deactivation?

Answer 36

When the inside of the cell is made negative again; the activation gate rotates back to the closed position and the current decays away

Question 37

How is Nav gating different than Kv gating?

Answer 37

Nav channels have both an activation gate and an inactivation gate

Question 38

How does the Nav activation gate work?

Answer 38

At negative potentials (-80mV) it is closed and making the inside of the cell positive causes the NaV activation gate to swing open (�activate�) and sodium ions to flow into the cell (INa).

Question 39

How does the Nav inactivation gate work?

Answer 39

It is open at resting potential (the activation gate block access to a site within the inner end of the por where the inactivation gate can bind). After the activation gate opens; the inactivation gate closes and causes the current to decay to zero during a maintained depolarization. **Note this is different than deactivation.**

Question 40

How is selectivity achieved in the Kv and Nav channels?

Answer 40

Occurs within a central; ion conducting pathway formed by the 4 Kv subunits or 4 repeats of Nav. This central pathway is surrounded by S5 and S6 helices and connecting P loop contributed by the each of the four subunits or repeats

Question 41

How is voltage sensing at the activation gate achieved? Which residues contribute?

Answer 41

Accomplished by S4 helices. These helices contain positively-charged Lys or Arg residues at every third position and translocate in response to changes in voltage across the membrane.

Question 42

How does the translocation of the S4 helices cause activation?

Answer 42

Not exactly known. The movement corresponding to the opening of the activation gate likely corresponds to a hinge-like motion of the S6 segments around a conserved glycine

Question 43

How is the inactivation gate of Nav channels formed?

Answer 43

Formed by the cytoplasmic loop which connects repeats III and IV. This cytoplasmic III-IV linker folds over the inner end of the central ion-conducting pathway when the channel is in a closed/inactivated state. IFM residues are important for this binding

Question 44

How does tetrodoxin (TTX) work? An example of sidedness of agents acting on Kv and Nav

Answer 44

TTX is a charged molecule that cannot cross the membrane. When it is added to the extracellular side it binds within the entrance of the pore (just above the selectivity filter of NaV). The binding of TTX is essentially independent of the position of the activation/inactivation gates; TTX has no effect when added intracellularly

Question 45

What kind of amine is Lidocaine? What is its charge? Can it cross the membrane?

Answer 45

Lidocaine is a tertiary amine; equilibrates between de-protonated (neutral) and protonated (+) forms. The protonated form is dominant at physiological pH and cannot cross the membrane; de-protonated form can.

Question 46

How does lidocaine work? An example of sidedness and state-dependence of agents acting on Kv and Nav.

Answer 46

Protonated lidocaine has no effect on NaV from the extracellular side but can block the channel from the intracellular side (thereby producing local anesthesia). The block from the intracellular side can only occur if the protonated lidocaine can access the vestibule (requires that the activation and inactivation gate be open). Note that if lidocaine is at its binding site within the vestibule; it can be trapped there if the activation or inactivation gates are closed.