lecture 3, 4, 5- active transport and an introduction to ion channels

Why is it crucial that pumps in the Jardetzky model are never open to both sides of the membrane simultaneously?

It prevents the transported molecules from flowing back down their concentration gradient (leaking).

According to the Jardetzky model, how does a pump's binding site affinity change when open to the low-concentration side?

It has a high affinity ('sticky') to effectively capture the scarce molecules available.

According to the Jardetzky model, how does a pump's binding site affinity change when it flips to the high-concentration side?

It switches to a low affinity ('less sticky'), forcing the bound molecules to detach.

What fundamental process is NOT performed by carrier proteins in secondary active transport?

They do not perform the hydrolysis of ATP.

What two types of molecular flow are coupled in secondary active transport?

A favorable flow of one substance down its gradient and an unfavorable flow of another substance against its gradient.

In secondary active transport, what is the term for when both substances move in the same direction across the membrane?

Symport (or co-transport).

In secondary active transport, what is the term for when substances move in opposite directions across the membrane?

Antiport (or exchange).

What specific function does Lactose permease (LacY) perform in E. coli?

It is responsible for the symport of lactose and a proton into the cell.

LacY is composed of 12 transmembrane alpha-helices organized into two distinct 6-helix bundles: the N-terminal bundle and the _ bundle.

C-terminal

What type of symmetry exists between the two 6-helix bundles of LacY?

Two-fold pseudosymmetry.

Where is the large interior hydrophilic cavity of LacY located?

It is located between the N-terminal and C-terminal 6-helix bundles.

On which helices is the sugar binding site of LacY located?

The sugar binding site is on helices IV, V, and VIII.

On which helices is the proton-translocation site of LacY located?

The proton-translocation site is on helices VII, IX, and X.

What is the effect of the C154G mutation in LacY?

It makes the protein more stable and locks it in its inward-facing conformation.

In the Lac permease pumping cycle, what is the first step that occurs on the outside of the cell?

A proton (H+) from the outside binds to the protein.

After a proton binds, what is the second step in the Lac permease pumping cycle?

Lactose from the outside binds to the protein.

What conformational change occurs in Lac permease after both the proton and lactose have bound?

The protein undergoes eversion, flipping its conformation to be open to the inside of the cell.

What happens after Lac permease releases lactose into the cell's interior?

The proton (H+) is released into the inside of the cell.

What is the final step of the Lac permease cycle after it has released both lactose and the proton inside the cell?

The empty protein everts, flipping back to its outward-facing state.

What is the function of the vestibule in the generic architecture of an ion channel?

It is the wider opening of the channel that funnels ions towards the narrow pore.

What key component within the narrow pore of an ion channel is responsible for discriminating between different ions?

The selectivity filter.

What is the common quaternary structure of a functional potassium channel?

It is a tetramer, composed of four identical protein subunits arranged in a ring.

What is the function of the hole in the middle of the tetrameric K+ channel structure?

It forms the ion conductance pathway, also known as the pore.

The core of a K+ channel domain consists of two transmembrane helices, a short pore helix, and a signature sequence in the _.

selectivity filter

What is the function of the outer helices in a K+ channel's structure?

They face the lipid bilayer and act as a structural anchor for the channel in the membrane.

The inner helices of a K+ channel line the lower part of the pore and can act as a _ by crossing to regulate ion flow.

gate (or activation gate)

How do the short pore helices in a K+ channel help stabilize potassium ions?

They point their negative dipole towards the center of the channel, stabilizing the positively charged K+ ions.

How many transmembrane helices do voltage-gated K+ (Kv) channels have in total?

They have six transmembrane helices (S1-S6).

In Kv channels, which helices form the voltage-sensing domain?

Helices S1-S4 form the voltage-sensing domain.

In Kv channels, which helices form the pore domain?

Helices S5-S6 form the pore.

What is the primary distinguishing feature of inward-rectifying K+ (Kir) channels?

They have a simpler 2-TM architecture and a large cytosolic domain used for regulation.

What activates calcium-activated K+ (KCa) channels?

The binding of Ca2+ ions to their extensive cytosolic tails.

Why was the bacterial KcsA channel a landmark in biology?

It was the first K+ channel to have its structure solved by X-ray crystallography.

What must an ion do to its hydration shell before it can enter the narrow selectivity filter of a K+ channel?

The ion must shed its surrounding water molecules (hydration shell).

What atoms line the K+ channel's selectivity filter to mimic the ion's hydration shell?

Carbonyl oxygen atoms.

Why are smaller Na+ ions rejected by the K+ channel's selectivity filter?

Sodium is too small to interact optimally with all the carbonyl oxygen atoms, so the energy gained does not compensate for the energy lost shedding its water shell.

Why does it take more energy to dehydrate a sodium ion compared to a potassium ion?

Sodium has a higher 'charge density', causing it to pull water molecules more tightly.

What prevents K+ ions from getting stuck in the selectivity filter, thus ensuring high throughput?

Electrostatic repulsion between multiple K+ ions moving in a queue pushes them through the filter.

What component of a voltage-gated channel acts as the primary voltage sensor?

The S4 helix, which contains positively charged arginine residues.

In the resting state of a cell, where is the positively charged S4 helix located?

The negative charge inside the cell pulls the S4 helix towards the inside, keeping the channel closed.

What happens to the S4 helix when the cell membrane depolarizes (becomes more positive inside)?

The positively charged S4 helix is pushed outwards, towards the outside of the cell.

What physical linkage transmits the movement of the S4 helix to the channel's gate?

The S4-S5 linker.

How does the movement of the S4-S5 linker open the pore in a voltage-gated K+ channel?

It exerts a mechanical force that causes the S6 helices to bend at their glycine hinges, widening the pore.

What molecule gives bacteriorhodopsin (bR) its characteristic purple color?

A covalently attached chromophore called retinal, a derivative of vitamin A.

How does bacteriorhodopsin convert sunlight into chemical energy?

It uses light energy to generate a transmembrane proton gradient, which is then used by ATP synthase.

To which amino acid is retinal covalently attached in bacteriorhodopsin?

It is attached to Lys216 on the 7th alpha helix.

What is the specific chemical linkage between retinal and Lys216 in bR?

A protonated Schiff base linkage.

What initial event happens to retinal when it absorbs light in bacteriorhodopsin?

It isomerizes from an all-trans configuration to a 13-cis configuration.

In bacteriorhodopsin, what is the natural, stable form of retinal in the dark/resting state?

The all-trans configuration.

How do three individual bacteriorhodopsin molecules organize in the cell membrane?

They cluster together to form a trimer.

What is the name of the highly ordered, crystalline lattice formed by bacteriorhodopsin trimers?

The purple membrane.

In the bR photocycle, the transition from the L to the M intermediate involves the release of a proton from the Schiff base to which amino acid?

Asp85.

During which transition in the bR photocycle does the Schiff base get reloaded with a proton from the cytoplasm?

During the M -> N transition, where Asp96 provides the proton.

What is the role of Asp96 in the bR proton pumping cycle?

It acts as the proton donor to reload the Schiff base from the cytoplasmic side.

What is the role of Asp85 in the bR proton pumping cycle?

It acts as the initial proton acceptor from the Schiff base before the proton is released to the extracellular side.

What is the role of Arg82 in the resting state of bacteriorhodopsin?

It is oriented to help prevent protons from the outside environment from flowing backward into the protein.

What is meant by a 'proton wire' in the context of bacteriorhodopsin?

It is a chain of strategically placed water molecules and amino acids that allows a proton to hop from one to the next across the protein.

How does bacteriorhodopsin achieve 'gating' to ensure the unidirectional flow of protons?

By switching the accessibility of the Schiff base between the extracellular and cytoplasmic half-channels.

What critical property of the Schiff base and aspartate residues changes throughout the photocycle to drive proton transfer?

Their pKa values change due to conformational shifts.

In the _ state of the bR photocycle, the Schiff base has a low pKa, making it eager to donate its proton.

L

In the _ state of the bR photocycle, the Schiff base has a high pKa, making it eager to accept a proton.

N

During the transition to the M-intermediate in bR, which helix tilts outwards on the cytoplasmic side?

Helix F (the 6th helix).

What is the consequence of Helix F tilting during the bR M-intermediate transition?

It allows water to enter the cytoplasmic half-channel, facilitating proton uptake from the cytoplasm.

How does the physical orientation of the Schiff base change after light absorption in bR?

It physically rotates, breaking its connection to the extracellular channel and opening a new one to the cytoplasmic channel.