Section 3.1: Transporters

The Druggable Proteome

• 25% of the human genome codes for membrane proteins (eg. channels, transporters, ion pumps, etc.)

• 67% of known drug targets are membrane proteins

  • this is b/c they control essential physiological processes and are accessible to drugs (on outside of cells, drug doesn’t need to enter the cell first)

  • binding sites are often well-defined and modulating them produces large, fast effects

The Druggable Proteome: Examples of Membrane Proteins as Drug Targets

• stomach ulcers: drugs that target the gastric H⁺/K⁺ ATPase (proton pump in the stomach lining that acidifies the stomach) → reduces stomach acidity

• Type II diabetes: drugs targeting the K⁺ channel in β pancreatic cells → higher insulin secretion

• Hypertension: drugs acting on water channels in the kidney → reduce salts reabsorption

• Stress: drugs that target the β-adrenoreceptor (β-blockers) → less production of adrenaline

Permeability Properties of Synthetic Lipid Bilayer

• molecules can diffuse across the lipid bilayer down its concentration gradient 

• size: smaller molecules diffuse more easily

• polarity: the bilayer’s interior is hydrophobic (oil-like), so nonpolar/hydrophobic molecules cross easily and rapidly

• gases (O₂, CO₂) and small uncharged molecules (urea, ethanol) can move by diffusion

• charged molecules (ions and larger) are very impermeable, no matter how small (eg. Na⁺, K⁺, Cl^-)

  • hydration shells prevent them from entering the hydrophobic membrane 

Passive Transporters Part 1

• move molecules down their concentration gradient without using energy

• direction is determined by the concentration gradient (always high → low)

• there's a finite number of transporters, so increasing substrate beyond a certain point doesn’t increase rate indefinitely (saturable)

  • binding is typically 1:1, one substrate at a time

• transport is specific, each transporter recognizes particular substrates

• no chemical bonds are made or broken in the conversion

Passive Transporters Part 2

• affinity for substrate is the same on both sides; the transporter can work in either direction (process is fully reversible)

• cannot concentrate substrate inside, only moves it to balance the gradient 

• passive transporters are for large hydrophilic substrates (eg. sugars, amino acids, vitamins)

  • these molecules can’t diffuse, so they require transporters

• rate of transport is faster than diffusion 

• rate depends on concentration gradient (more substrate outside = faster), and conformational change speed (flip of transporter between outward and inward facing states)

Kinetics of Glucose Transport in RBCs: GLUT1

• different Km → different transports behaviors (Km reflects affinity)

• RBCs need a constant glucose uptake

• GLUT1 Km = 1.5 mM, blood glucose is 5mM 

• at 5mM, there is import of glucose close to Vmax

  • below 5mM, GLUT1 in RBCs still imports glucose efficiently

  • above 5mM, transport of glucose is limited 

• after import, glucose is phosphorylated to glucose-6-phosphate, which has no affinity for GLUT1 

  • this traps glucose inside, and makes import one-way/irreversible

• accounts for 2% of the protein in the PM of RBCs 

Km

• substrate concentration at which an enzyme-catalyzed reaction reaches half its maximum velocity (V_max)

  • measure of how efficiently an enzyme works

• low Km = high affinity → transporter works well even at low substrate conc.

• high Km = low affinity → transporter mainly active when substrate conc. is high

Kinetics of Glucose Transport in RBCs: GLUT2

• high Km, only active when glucose is high (eg. after a meal)

  • liver stores it as glycogen

  • the more glucose, the more transport into the liver

• transports glucose out of the hepatocytes (glucose storage) to replenish blood glucose

• import of glucose is quasi-linear (curve looks like simple diffusion)

• at low glucose (below 5mM), glucose is released from hepatocytes, and GLUT2 does not import 

• has high Km (~15mM) → low affinity

• in the liver and β-cells of pancreas

Kinetics of Glucose Transport in RBCs: Figure

Enzyme Kinetics

• even though transporters are not enzymes, their behavior follow Michaelis-Menten-like kinetics because:

  • the transporter binds a substrate, it undergoes a conformational change, and it can become saturated

• V_max: the maximum rate of transport, when all transporters are fully occupied with substrate

  • reflects how fast the transporter flips between outward-open and inward open states 

• Km: substrate conc. where the rate is ½ V_max

Enzyme Kinetics: Measuring Kinetics of Glucose Transport

• measure the initial rate of glucose uptake (V₀)

• V₀ depends on external glucose concentration

• at high glucose, transport reaches V_max; all transporters are occupied → saturation

• when the rate of uptake is ½ V_max, the transport if half-saturated

GLUT: Conformations

• the transporter exists in two conformations

• T1: the glucose binding site exposed on the outer surface of the PM

• T2: the binding site exposed on the inner surface

Glucose Transport Steps

• glucose binds to T1

• binding lowers the activation energy for T1 → T2 transition

  • the transporter flips its conformation

• in T2, glucose is released into the cytoplasm

• release allows the transporter to return to the T1 conformation

The Isoforms of the Glucose Transporter

• the human genome encodes at least 12 highly homologous GLUT proteins

• each transporter has a unique distribution

• the same function, but different affinity for glucose, gives different transport kinetics

• GLUT3 is expressed in neuronal cells, which depend on a constant influx of glucose (5-fold higher affinity than GLUT1)

• GLUT4 in skeletal (Km ~5 mM): presence in the membrane is regulated by insulin

Increase of Glucose Transport with Insulin: What Normally Happens

Between meals: GLUT4 transporters are stored in intracellular vesicles in muscle cells and adipocytes

• very little glucose uptake at the cell surface

After a carbohydrate rich-meal: blood glucose rises above ~5mM

• pancreas releases insulin, triggering GLUT4-containing vesicles to move to and fuse with the PM

• more GLTU4 on surface → 15x increase in glucose uptake

• excess glucose is stored as glycogen in muscle or TAG in adipocytes

• when insulin levels drop, GLUT4 is endocytosed back into vesicles

Increase of Glucose Transport with Insulin: Type 1 Diabetes

• in type 1 (insulin-dependent) diabetes mellitus, insulin is not released and GLUT4 remains in vesicles

• autoimmune destruction of β-cells → no insulin production

• cells do not take up glucose → blood sugar stays high

• insulin injections restore the signal → GLUT4 moves to the surface

Increase of Glucose Transport with Insulin: Type 2

• insulin is produced, often in high levels

• but cells do not respond → signaling pathway fails

• GLUT4 does not efficiently move to the surface

• insulin injections are less effective because the signaling pathway is the issue, not the insulin level

Increase of Glucose Transport with Insulin FIGURE

SGLT Transporter

• Sodium Glucose Transporter (Symporter)

• uses energy stored in sodium gradient to import glucose against its concentration gradient

• outside the cell: High Na⁺, inside the cell: low Na⁺

  • since Na⁺ strongly wants to enter the cell (downhill energetically), SGLT couples this downhill movement of Na⁺ to the uphill transport of glucose

  • this means SGLT can import glucose even when extracellular glucose is low

• Na⁺ gradient was created by the Na⁺/K⁺ ATPase

• binding of Na⁺ and glucose is cooperative. If Na⁺ isn’t bound first to increase the glucose affinity of the transporter, glucose won’t bind well

SGLT Transporter: Stoichiometry

• 2 Na⁺ ions + 1 glucose transported inward together

• the binding of Na⁺ increases the affinity for glucose → ensures glucose gets captured even at low concentrations

SGLT Transporter: Transport Cycle

1) Outward facing conformation (open to outside)

• binding sites for Na⁺ and glucose are exposed to exterior

2) Occluded conformation: both gates are closed, no access to either side

• transporter flips to face inside

3) Inward-facing conformation (open to inside)

• binding sites now face the cytoplasm

4) Once transporter is empty, the transporter resets back to outward-facing

SGLT Transporter: Transport Cycle Figure

Energy Stored in an Ion Gradient

• for ions: ∆G = ∆G_m + ∆G_c

  • ∆G is how much energy is available when Na⁺ moves into the cell

• ∆G_c = concentration gradient contribution, this is the energy due to the difference in concentration of Na⁺ across the membrane

• ∆G_m = electrical gradient concentration, this is the energy due to the membrane potential

  • inside of the cell is typically negative relative to the outside

Energy Derived from Na⁺ Gradient

• the movement of Na⁺ across the membrane is driven by the concentration gradient and the membrane electric potential

• both point forces inward, so the total free energy change for Na⁺ is highly favorable

• ∆G_c = RT ln( [Na_in] / [Na_out]

• ∆G_m = FE

  • E: membrane electric potential = -70mV

  • F: Faraday constant: 23062 cal/molV

LacY Transporter

• LacY is a proton-driven symporter in bacteria , where it uses an ion (H⁺ gradient) gradient as the energy source

  • 417 amino acids long, but only 3 are essential

  • some residues serve for lactose binding, some for proton transport

• moves a sugar (lactose) against its concentration gradient

• has 12 TM helices forming a central cavity in the middle of the membrane (where lactose binds)

• switches between outward facing and inward facing states

  • the 6 N-ter and 6 C-ter produce a structure with a rough two-fold symmetry

• requires cooperative binding of ion and sugar

• in the cytosol, β-galactosidase cleaves lactose into glucose and galactose

LacY Figure

LacY Inhibition: Cyanide

• when cyanide is added in the middle of the experiment, lactose goes back out

  • blocks transport of lactose

• cyanide blocks ETC which makes the proton gradient

The PMF drives Transport of Lactose

• essential a.a’s: E269 (Glu), R144 (Arg)

• H⁺ loading is favored b/c E269 attracts H⁺

• H⁺ binding increases lactose affinity

• the presence of both substrates favors the electrostatic interaction of R144 with E269

• the electrostatic bond triggers conformational change, tilting the transporter from the outward facing → inward facing

• inside the cell, the cytosol is more basic and E269 deprotonates

  • the loss of H⁺ weakens lactose binding → lactose is released

• once H⁺ is released, the salt bridge breaks and the transporter resets to outward facing

• the process is fully reversible, lactose can escape the cell

The PMF drives Transport of Lactose FIGURE

Antiporter The Cl^- / HCO₃^- (AE1): Background

• enables CO₂ transport between tissues and lungs

• called anion exchanger 1 and contributes up to 25% of RBC membrane proteins

• HCO₃⁻ OUT ↔ Cl⁻ IN (in tissues)

• HCO₃⁻ IN ↔ Cl⁻ OUT (in lungs)

Antiporter The Cl^- / HCO₃^- (AE1): Step 1

In respiratory tissues (where CO₂ is produced)

• waste CO₂ diffuses into RBCs and is converted to HCO₃^- by carbonic anhydrase

• HCO₃^- must leave the cell (prevent build up), otherwise reaction stops. It is more soluble in blood plasma than CO₂ (better carried to the lung)

• AE1 exports it and imports Cl^- (1:1 exchange)

  • this massively increases CO₂ transport efficiency

Antiporter The Cl^- / HCO₃^- (AE1): Respiratory Tissue Figure

Antiporter The Cl^- / HCO₃^- (AE1): Step 2

In lungs (where CO₂ is exhaled)

• HCO₃^- reenters RBC through AE1 and Cl^- leaves

• carbonic anhydrase converts HCO₃^- → CO₂ → exhaled

Antiporter The Cl^- / HCO₃^- (AE1): Lung Figure

Antiporter The Cl^- / HCO₃^- (AE1): Membrane Potential

• even though ions move, AE1 exchanges one negative ion for another

• no net charged moved

• membrane potential stays constant (electroneutral transport)

Antiporter The Cl^- / HCO₃^- (AE1): Obligatory Coupling

AE1 cannot move just HCO₃^- or just Cl^-

• if Cl^- is absent, HCO₃^- transport stops

Antiporter The Cl^- / HCO₃^- (AE1): Hemoglobin

• when Cl^- enters the RBC in tissues, it slightly lowers the cystolic pH (more acidic) and Hb released O₂ more readily at low pH

• when Hb reaches the lungs and O₂ binds, the structural changes makes certain His residues release H⁺ 

  • this H⁺ reacts with HCO₃^- to form CO₂ to be exhaled

Symporter vs. Antiporter

• both cotransporters in secondary active transport, but differ in direction

• symporter: move both substrates in the same direction

• antiporter: move substrates in opposite directions

Primary Active Transport

• directly uses cellular energy, primarily from ATP, to move substances (e.g Na⁺, K⁺) across membranes against their concentration gradient

• use transmembrane proteins (pumps) that change shape after binding ATP, thus pumping molecules out or in

• eg. Na⁺/K⁺ ATPase

Facilitated Diffusion

Type of passive transport where molecules move across a cell membrane down their conc. gradient, with the help of a specific carrier or channel protein

• doesn’t require ATP

• eg. Aquaporins, GLUT1

ABC Transporter Family

• ATP binding casette

• 48 types in humans; mostly exporters (e.g bile salts, lipids), many are linked to disease

• in bacteria, 5% of the genome encodes for ABC transporters, including importers of nutrients

• each transporter is specific for a class of substrates (e.g sugar, vitamins, a.a’s, proteins)

• share a conserved structure: nucleotide binding domain (NBDs) for ATP binding/hydrolysis, transmembrane domains (TMDs) that form the transport pathway

Structural variations found across ABC Transporters

General Mechanism for ABC Transporters: Steps

• substrate binding to TMDs on one side of membrane

• NBD dimerization: 2 ATP molecules bind and are sandwiched at the NBD dimer interface, causing it to dimerize

• ATP hydrolysis → Pi released first then ADP

• Resetting the transporter: NBDs dissociate, then transporter returns to original conformation

General Mechanism for ABC Transporters: Steps FIGURE

ABCB1 (MDR1)

Multidrug Resistance Protein

• ABCB1 is an ABC transporter that normally exports hydrophobic metabolites into bile or urine

• in cancer: tumor cells overexpress ABCB1, leading to resistance to multiple chemotherapic drugs

  • chemotherapy selects for cells with high ABCB1 expression → these cells survive and overgrow

ABCB2 TAP Transporter

• TAP is an ABC transporter composed of TAP1 and TAP2

• plays central role in adaptive (not innate) immunity

• foreign proteins enter the cell and are degraded in cytosol into short peptides

• these peptides are recognized and transported by TAP1-TAP2 into ER lumen → peptides are loaded onto MHC class 1 molecules

  • peptides are not taken up by Sec61 (don’t have signal peptide)

• Peptide-MHC 1 complexes traffic to the PM

• CD8⁺ cytotoxic T cells recognize the foreign antigen and kill the cell (immune surveillance)

ABCB2 TAP Transporter FIGURE

Orientation of MHC Molecules

• MHC 1 proteins are synthesized in ER membrane w/ a fixed orientation

  • luminal side of ER = extracellular side after secretion, cytosolic domains always face the cytosol

• during transport (ER → Golgi → PM), proteins move in vesicles

• vesicle fusion does NOT flip membranes (cytosolic leaflet remains cytosolic, luminal leaflet becomes extracellular)

  • in other words, the fusion of a membrane vesicle to the cell surface maintains the membrane polarity

ABCC7-CFTR

• CFTR is in the ABC transporter family, but functions an ion channel, not a pump

  • forms a Cl^- channel in the PM of epithelial cells

• ATP binding (not hydrolysis) regulates channel gating

  • ATP binding to NBD = channel open

  • ATP release/hydrolysis = channel closes

• Cl^- moves passively thru CFTR down its electrochemical gradient

• CFTR activity regulates ion and water balance in epithelial surfaces (e.g lungs)

• mutations in the CFTR protein cause cystic fibrosis

Overall Pathogenesis of Cystic Fibrosis: Normal Lung

• CFTR is located on apical surface of airway epithelial cells, allowing Cl^- secretion into the airway lumen

• Cl^- movement draws water osmotically, maintaining a hydrated mucus layer

• proper hydration allows cilia function and efficient clearance of particles

Overall Pathogenesis of Cystic Fibrosis: Cystic Fibrosis

• defective or absent CFTR → reduces Cl^- secretion

• altered ion balance disrupts osmotic water movement

• mucus becomes dehydrated and viscous

• thick mucus traps bacteria → persistent infection and inflammation

Over 300 mutations cause CF but ∆F508 in about 90% of patients

Precision Medicine

• precision medicine treats disease by targeting the molecular defect, not just the symptoms

• Trikafta is a combination of ivacaftor + tezcaftor + elexcaftor

  • these drugs act as CFTR correctors

• tezcaftor + elexcaftor → improve CFTR folding and trafficking to the membrane

• Ivacaftor → increases channel opening (gating) of CFTR already at the membrane

Typical Ion Concentrations in Mammalian Cells: Chemical & Electrical Gradients

• Na⁺/K⁺ ATPase (P-type ATPase) establishes and maintains ion gradients across the PM

• chemical gradient: created by unequal ion conc. (high K⁺ inside, high Na⁺ outside)

  • maintained by continuous ATP hydrolysis by the Na⁺/K⁺ pump

• electrical gradient: the pump is electrogenic (net +1 charge leaves the cell each cycle) → inside of cell is electrically negative relative to ouside

  • additionally, the cytosol contains many (-) charged proteins

How Na⁺/K⁺ ATPase Works

• maintains Na⁺ and K⁺ gradients across the PM

• binds 3 Na⁺ from cytosol → ATP hydrolysis → conformation change → Na⁺ is expelled to extracellular space → 2 K⁺ ions bind from outside → dephosphorylation → conformation change → K⁺ released into cytosol

• high K⁺ inside, high Na⁺ outside

• contributes to resting membrane potential

• the pump costs a lot of energy: constantly counteracting Na⁺ and K⁺ leakage

The Na⁺/K⁺ ATPase Drives Nutrient Transport

• ATP is not used directly to transport glucose, it is used to create a Na⁺ gradient which then powers glucose uptake

  • strong inward Na⁺ electrochemical gradient that is the energy source for nutrient uptake

• SGLT uses the Na⁺ gradient to import glucose (glucose is carried up its concentration gradient)

• GLUT2 passively releases glucose into the bloodstream (down glucose gradient)

  • prevents glucose buildup

• excess Na⁺ is removed (otherwise dissipates gradient) by Na⁺/K⁺ ATPase

The Na⁺/K⁺ ATPase Drives Nutrient Transport FIGURE

The Na+/K+ ATPase is ElectrogenicFIGURE

P-type ATPase

• P-type ATPases are phosphorylated by ATP

• are cation pumps; maintain ionic differences b/w cytosol and extracytosolic environment

• Na⁺/K⁺ ATPase is an antiporter for Na⁺ and K⁺

• H⁺/K⁺ is an antiporter for H⁺ and K⁺ (acidifying stomach)

• Ca²⁺ ATPase is an uniporter for Ca²⁺ (Ca²⁺ leads to muscular fiber contraction in myocytes).

P-type ATPase: SERCA (P-type ATPase)

• sarcoplasmic ER calcium ATPase; located in sarcoplasmic reticulum (SR) of muscle cells

• pumps Ca²⁺ from cytosol → SR lumen, using ATP (maintains low cytosolic Ca²⁺)

Muscle contraction cycle:

• small Ca²⁺ influx across PM opens Ryanodine receptors (Ca²⁺ release channels) → Ca²⁺ binds to troponin → muscle contraction

• relaxation: SERCA pumps Ca²⁺ back into SR, restoring low cytosolic Ca²⁺

V-type ATPase (V for vesicle/vacuolar)

• pumps H⁺ into organelles (e.g. lysosomes, endosomes, vacuoles) and synaptic vesicles to acidify them

  • allows for lysosomal enzyme activity, vesicle trafficking, receptor-mediated endocytosis

  • 1-2 V-type ATPases per vesicle

• ATP-driven electrogenic pump: uses ATP hydrolysis to move H⁺ against their gradient

• contains multi-subunit rotary: V₁ domain (ATP hydrolysis), V₀ domain (proton translocation)

• H⁺ gradient drives neurotransmitter uptake via H⁺/neurotransmitter antiporter

F-type ATPase

• found in mitochondria, chloroplasts and bacterial PMs

• primarily synthesizes ATP using the proton gradient (protons flow down their electrochemical gradient)

• reversible, can hydrolyze ATP to pump protons in reverse if needed

• contains multi-subunit rotary motor: F₀ domain (proton channel embedded in membrane), F₁ domain (catalytic ATP synthesis)

• similar structure to V-type ATPase

F-type ATPase in Mitochondria

• proton enters half channel 1, binds Asp-61 on C subunit (Asp becomes neutral and can enter bilayer)

• neutral Asp allows c-ring rotation, moving the next Asp61 into position

• Arg-210 interacts to guide proton down half channel 2 → proton exits

• C-ring rotates, repeating the cycle

  • rotating C subunit is driven by proton gradient

F-type ATPase in Mitochondria FIGURE