Ch 8 Membrane Transport

Passive mediated transport

something helping across membrane

no E required, moving down a gradient

Active transport

requires E, moving against concentration gradient

such an endergonic process must be coupled to a sufficiently exergonic process to make it favorable

non-mediated transport

simple diffusion

O₂ from Hb → Mb

Mediated transport

needs carrier

can be passive or active

transport of acetate (b/c charged)

_________ and ___________ dictate exergonicity

concentrations and membrane potential

chemical potential

chemical potential difference

if concentration is greater outside, things flow in spontaneously

if concentration is greater inside, active mediation via ATP or another gradient is required

electrochemical potential

for charged species: Na⁺, Ca²⁺, K⁺, Cl^-, CO₃^2-, R-COO^-, R-NH₃⁺

passive-mediated transport

aka facilitated diffusion, molecules flow high→low

1. ionophores

2. porins

3. ion channels

4. aquaporins

5. transport proteins

ionophores

increase increase permeability of membranes to ions

there effect can only be to equilibrate concentrations on either side of membrane

• carrier ionophores - actually moves in membrane

• channel-forming ionophores - allows flow

channel-forming ionophores

form transmembrane channels or pores through which select ions can diffuse

Carrier ionophores

increase permeability of membranes to their selected ions by binding it, diffusing through membrane, and releasing ion on other side

their ionic complexes are soluble in nonpolar solvents

porins

H₂O, ions, small, molecules

beta-barrels (antiparallel beta-strands)

select solutes using diameter and a.a. residues

found in outer membrane of gram (+) mitochondria and chloroplast

ion channels

highly selective and may be gated

aquaporins

mediate transmembrane passage of H₂O but not protons

transport proteins

mediate uniport, symport (coupling), or antiport transport

Valinomycin

carrier ionophore

cyclic molecule of alternating D- and L-amino acid residues with hydroxy acids

repeating trimer of L-Val, D-Hydroxyisovaleric acid, D-Val, and L-Lactic acid

peptide bonds and ester linkages

all carbonyls point inward, coordinating a complex bound to K⁺

nonpolar side chains creates a hydrophobic exterior that allows it to diffuse freely across lipid bilayer membrane

the pre-organized ring size provides strict specificity for K⁺ ions

its cyclic structure makes it specific, generating a size-selective cavity

Maltoporin

channel-forming ionophore, large B-barrel structure spanning outer membrane

selectively permits transport of specific solutes based on diameter and amino acid side-chain properties lining interior

greasy slide mechanism: interior is full of aromatic a.a. side chains creating a greasy slide → this interact specifically with the flucose rings of maltodextrins

Low Energy Barrier: smooth surface provided by the hydrophobic aromatic residues ("greasy slide") minimizes friction

• Multiple polar groups located at the channel constriction allow for a continuous exchange of hydrogen bonds as the maltodextrin molecule glides smoothly through the constriction zone

K⁺ Channel KcsA

ion channel

potassium ions passively diffuse from the cytoplasm to extracellular space through transmembrane proteins known as K⁺ channels

diffusion of K⁺ is due to selectivity filter

• backbone carbonyls

• 3 angstrom diameter → remove solvation shell

entrance = 4 anionic residues attract cations and repel anions

Gated Passive-Mediated Transport

• Mechanosensitive

• ligand-gated

• signal-gated

• voltage-gated

Mechanosensitive

open in response to local deformations in lipid bilayer

physical stimuli - touch, sound changes in osmotic pressure

Ligand-gated

open in response to an extracellular chemical stimulus like a NT or in response to building an intracellular signal such as Ca²⁺ ion

Signal-gated

intracellular signals

Voltage-gated

open in response to changes in membrane potential

nerve impulses

Action potential

1. initiation & depolarization: a ligand/stimulus causes voltage-gated Na⁺ channels ot open, driving an influx of sodium into the cell

   1. spiking the membrane potential toward the Na⁺ equilibrium potential (+60mV)

2. repolarization: peak depolarization triggers voltage-gated K⁺ channels to open, driving repolarization and an overshoot into hyperpolarization near K⁺ equilibrium potential (-75mV)

3. resting recovery: ion gradients are eventually reset using ion pumps to restore

Nerve impulses - constant amplitude and coding stimulus intensity

constant amplitude: AP propagates along a nerve cell, continuously renewing itself so that the strength remains constant

coding stimulus intensity: because all AP share the same amplitude, the magnitude (strength) of stimulus is controlled by firing rate

Voltage-gating in Kv Channels

voltage-gated K+ channels = Kv channels

voltage gating is triggered by motion of a positively charged protein helix

S4 segment is packed with positive charges

Gate opens:

1. Normally, the inside of a resting cell is negative, which keeps this gate firmly shut

2. When a nerve impulse arrives, the inside of the cell suddenly becomes positive (this is called depolarization)

3. Because like charges repel, the positive electrical charge inside pushes against the positive charges on the S4 part, forcing it to slide outward

4. That outward physical movement pulls the door open, letting potassium rush out of the cell.

Gate stops:

1. channel has built in stopper, attached to the inside of the channel is a small piece (inactivation ball on flexible chain)

2. right after the channel opens, this ball swings up and physically plugs the hole, stopping flow of ions

Cl^- Channels are an example of anion channels

basic a.a. line the entrance, selectivity filter made of N-termini and hydroxyl groups

homodimer with each subunit forming an anion-selective pore

Aquaporins mediate…

AQP1 = homotetrameric glycoprotein

each of its subunits consists of 6 transmembrane alpha helixes plus 2 shorter helices in the bilayer

transmembrane water movement

found largely @ kidneys, salivary glands, sweat glands, and lacrimal glands which are tissues that rapidly transport of H₂O

How do aquaporins select for JUST H2O

lined with hydrophobic groups that lack strong interactions with H2O

for a H2O molecule to transmit the constriction region, it must be separate from other H2O molecules to which it is H-bonded

• this is facilitated by Arg and His

aquaporin forms hydrogen bonds from the side chain NH2 groups of 2 Asn residues in pore

Gap junctions

important intracellular communication channels

small molecules (not macromolecules) are allowed to pass through

GLUT-1 in RBC

1. glucose binds to the protein which is specific to glucose

2. conformational change closes the 1st binding site on other side of membrane

3. glucose dissociated from protein

4. transport cycle is completed by reversion of GLUT-1 to its initial conformation in absence of bound glucose

all known transport proteins appear to be ______ situated transmembrane proteins that alternated between __ conformational states…

asymmetrically situated transmembrane proteins that alternate between 2 conformational states in which ligand binding sites are exposed to opposite sides of membrane

mediated vs non-mediated

1. speed and specificity

2. saturation

   1. mediated = hyperbolic

   2. nonmediated = linear rate

3. mediated transport is competitive

4. protein transport can be inactivated

uniport translocation system

involves the movement of a single molecule at a time

ex: GLUT-1

Symport translocation system

simultaneously transports 2 different molecules in the same direction

Antiport translocation system

simultaneously transports 2 different molecules in opposite directions

5 general ATP-driven families for Active Transport

1. P-type ATPases

2. F-type ATPases

3. V-type ATPases

4. A-type ATPases

5. ABC transporters

P-type ATPases

undergo phosphorylation of a specific Asp residue within the P domain

as they transport cations like Ca²⁺, K⁺, Na⁺ across membrane

transport ions in opposite directions

Na⁺/K⁺-ATPase

E1 and E2 possess different structures, catalytic activities, and different ligand specificities…

1. In E1 state, ions to be exported and ATP bind from cytosolic side

2. ATP phosphorylates aspartate

3. complex undergoes conformational E1→E2 change

4. At E2, ADP is released and channel opens to exterior and ion binding affinity decreases, leading it to release the ions

5. exterior ions bind to E2 and aspartyl phosphate = hydrolyzed

6. P_i is released

7. Protein undergoes a conformational change back to E1

8. release of counterions completes cycle

Na+/K+-ATPase Transports Ions in Opposite Directions

maintains low [Na⁺] and high [K⁺] inside cells

3 Na⁺ out and 2K⁺ in

Ca2+ ATPase Pumps Ca2+ Out of Cytosol

Ca²⁺ pump actively pumps 2 Ca2+ ions out of cytosol at expense of ATP hydrolysis, while counter transporting 2-3 protons

after muscle contraction: pumps Ca²⁺ ions back to SR

F-type ATPases

found in mitochondrial and bacterial membranes

proton transport complexes

proton-gradient driven ATP synthesis

V-type ATPases

found in acidic vesicles and plant vacoules

similar to f-type

A-type ATPases

anion transport across membranes

ABC transporters

ATP Binding Cassette

bind ATP and hydrolyze ATP to move amphipathic substances across membrane

P-Glycoprotein

conformational change at transmembrane alpha-helices

ABCG2

found in human cells

ABC transporter

pumps out chemotherapeutics and maintains physiological barriers (blood-brain and maternal-fetal)

Glucose Transport in Intestinal Epithelium

oral rehydration solution: Na⁺ (lesser extent K⁺), glucose

Lactose Permease in E. coli

symport transporter: uses energy stored in H⁺ gradient to drive lactose across the bacterial cell membrane against its concentration gradient

2 main conformational states:

1. E-1 (Inward-facing): The binding site faces the cytoplasm and has low affinity for lactose

   1. large internal hydrophilic cavity is open to the cytoplasmic side

2. E-2 (Outward-facing): The binding site faces the periplasm (outside) and has high affinity for lactose.

Step-by-step cycle for Lactose Permease in E. coli

1. Outside binding

   1. proton binds to the outward-facing transporter (E2) followed by a molecule of lactose

   2. because it is facing outside, it has a high affinity for lactose

2. Transport - crossing the membrane

   1. The transporter changes shape, moving the bound H⁺ and lactose across the membrane toward the inside (cytoplasm)

   2. This movement is powered by the proton gradient (higher H⁺ concentration outside / low pH, moving toward higher pH/ lower H⁺ inside).

3. Release (inside)

   1. now facing the cytoplasm, the transporter shifts to the E1 state, dropping its affinity for lactose

   2. lactose and proton are released into cytoplasm

4. Recovery (reset pump)

   1. empty transporter is rest

   2. cell oxidative metabolism uses a proton pump to continuously pump H⁺ back out of the cell, maintaining the proton gradient needed to drive future transport