W1L1 Ion Channels, General (Dustin)

Question 1

the permeability of the membrane for ions is a result of
opening of _____, the function of
these can be individually studied with the help of
the ______

Answer 1

the permeability of the membrane for ions is a result of
opening of discrete pores (ion channels), the function of
these pores can be individually studied with the help of
the patch-clamp

Question 2

What is the best studied example of a ligand-gated receptor for an ion channel?

Answer 2

Nicotinic acetycholine receptor

Question 3

How many subunits do nicotinic acetycholine receptors have?

What are the subunits?

Answer 3

5 subunits (pentamer)

2 alpha, 1 each of beta, gamma, epsilon

Question 4

How many transmembrane domains does the nAchR have?

Answer 4

4

Question 5

In a transmembrane loop, how many amino acid residues does it usually take to cross the lipid bilayer?

Answer 5

About 20

Question 6

What is “open probability?”

What aspect of ion channels determines the open probability?

Answer 6

The time an ion channel spends in the open state / total observation time

The rate constants of the ion channels determine the probability with which they move between stable open/closed conformations (and thus their open probability).

P_O = time open / time observed

Question 7

How do nicotinic acetycholine receptors and voltage-gated sodium channels differ in the mechanism for which they typically close their pore?

Answer 7

Acetycholine is usually degraded by acetycholinesterase which causes the pore to return to a closed confromation, while V.G. Na+ channels go into an inactive conformation for a temporary period regardless of depolarization signal

Question 8

How many subunits does the voltage-gated sodium channel have?

Answer 8

4 homologous subunits (tetramer)

Each one has a voltage sensor, and channels can only open when all 4 are in activated position

Question 9

How a pore conducts ions when it is open is called _____

Answer 9

permeation

Question 10

When an ion channel pore opens, what is the current?

How many ions per second moving through the pore does this current translate to?

How does this compare to a normal transporter?

Answer 10

Pore opening -> 10 picoAmperes (pA) current

= 10^8 ions per second or 100 millimoles/ second (very fast, approaches the theoretical limit of what is possible. only limited by diffusion)

Normal transporter = 10^3 ions/second (much slower!)

Question 11

If there is a simple pore in a lipid membrane that is just big enough for an ion to pass through (~3 Angstroms), what makes this process still energetically unfavorable?

Answer 11

Because the ion is dissolved in a polar solvent (mostly water), it has a hydration shell around it due to the attractive forces of water molecules

The ion cannot easily get rid of this water shell

Question 12

What do cation channels have in the pore to promote loss of the hydration shell and allow selectivity?

Answer 12

Negatively-charged binding sites, which lower the energy barrier to pass

(Also prevents anions from passing, but doesn’t account for selectivity between cations)

Question 13

What makes cation channels permeable to specific ions (either K+ or Na+, other than size and charge)

Answer 13

These channels have “P loops” (pore loops) that are transmembrane domains which are capable of allowing only specific ions through

Question 14

When ion channels pores simultaneously accommodate more
than one ion, what kind of ion movement is possible?

Answer 14

“Single file”

Question 15

In voltage-gated calcium channels, if there is an absence of calcium, what can sodium do? Why?

Answer 15

Sodium can then pass through, because at normal concentrations 1 calcium is inside of the channel and blocking Na from coming through. When calcium concentration increases, calcium can pass through.

Question 16

Why doesn’t a sodium ion pass through a potassium channel, even though sodium is a smaller element?

Answer 16

Because sodium is so small that it’s less energetically favorable to lose its hydration shell in a K+ channel.

The K channel has carbonyl oxygens around the inside of the pore which perfectly match the size of the K ion, thus K+ can flow through “without noticing they even lost their hydration shell”

Question 17

What do you call this type of shape?

(This is a potassium channel)

Answer 17

“inverted teepee”

has 4 fold rotational symmetry, a water-filled central cavity, and a short narrow selectivity filter (P loop)

C terminal ends point into cavity. results in partial negative charge internally, which further stabilizes channel for cations

Question 18

Once researchers suspected they knew which proteins had something to do with ion movement…

how did they prove they were right, using samples of the protein in question?

Answer 18

1. Purify suspected ion channel protein from native tissue.
2. Add the purified protein to a liposome membrane
3. Measure currents across liposome membrane using patch clamp technique
4. Observe unitary ionic currents as a result of this liposomal modifcation.

By this they knew that the proteins they suspected were indeed ion channels.

Question 19

What was the final proof of the hypothesis that ion channels were proteins?

Answer 19

Heterologous expression of cloned ion channel genes resulted (again) in unitary ionic currents.

(Heterologous expression = expression of a gene in a host organism that doesn’t usually have that gene. In this case it means they used recombinant DNA technology to insert ion channel genes into something that didn’t have them, the genes were expressed and the proteins created ionic currents.)

Question 20

How can the rate constant of an ion channel change?

Answer 20

Voltage and ligands affect rate constants (and thus open probabilities).

Question 21

What is the term for how an ion channel’s kinetics can be illustrated?

What else can these things illustrate?

Answer 21

Gating schemes are a way of describing ion channel kinetics.

They can also describe whole-cell ion currents.

(Gating schemes are figures showing the different open/closed active/inactive conformations of a channel with one or more domains involved in gating and activation, plus the rate constants showing likelihood of moving between any two of those conformations.)

Question 22

What happens to ion channels at high (as in above physiological) concentrations?

Answer 22

They become saturated.

This is not usually physiologically significant, but was an important experimental observation in the study of ion channels, leading to elucidation of the mechanism by which they carry ions through the membrane.

Question 23

What are the general steps of protein structure determination via x-ray crystallography?

Answer 23

1. Produce + purify large quantity of protein
2. Slowly condense the water-soluble protein
3. Ordered c__rystal lattic__e formation
4. X-ray diffraction pattern of the crystal is observed + protein structure is deduced.

Question 24

What makes x-ray crystallography of ion channels difficult?

Answer 24

The large hydrophobic surfaces of ion channel proteins (necessary for their location in the membrane) lead them to aggregate and precipitate from solution rather than forming ordered crystal lattices.

Question 25

On which side of the membrane is the K+ channel’s gate found?

And what is it called, in terms of the “inverted teepee” structure of the channel?

Answer 25

It is intracellular and is known as the smoke hole of the teepee.

Question 26

What stabilizes a K+ ion as it moves through the central cavity of a K+ channel?

(Not through the selectivity filter!)

Answer 26

Pore helix dipoles, with their negative sides facing the central cavity, stabilize the hydrated K+ ion before it enters the selectivity filter.

Question 27

Describe the positioning of K+ ions in the selectivity filter of a K+ channel.

What other molecules travel through with them + how?

( Not how they coordinate with P-loop atoms for energetic favorability, just their positioning as they pass through. )

Answer 27

• They can be found in four different positions (labeled 1-4) in the filter.
• They are either in the 1/3 or 2/4 positions, separated by water molecules occupying the two positions without K+ ions.

Question 28

What is the highly conserved AA sequence that allows for K+ selectivity in the K+ channel?

How exactly does it do this?

(Include numbers of atoms involved, etc.)

Answer 28

TXGYG seqeunces (Thr-X-Gly-Tyr-Gly)

• via their main chain carbonyl oxygens, these sequences coordinate with the K+
  
  • in stable 1/3 + 2/4 positions 8 carbonyl oxygens coordinate w/ K+
  • in transitions btwn stable states 6 oxygens (4 carbonyl + 2 water) coordinate w/ K+
• the oxygen rings are rigid and their diameter is too large to be energetically favorable for Na+, so the pore is selective.