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CARRIER-MEDIATED TRANSPORT
Solute carrier superfamily
• All carriers that do not couple to ATP or electron transport belong to SLC superfamily
• SLC organised based on amino acid homology ~20-25% homology
o SLC1 – the high affinity glutamate and neutral amino acid transporter family
o SLC2 – the facilitative GLUT transporter family
o SLC3 – the heavy subunits of the heteromeric amino acid transporters
o SLC4 – the bicarbonate transporter family
o SLC5 – the sodium-glucose transporter family
o SLC6 – the sodium- and chloride-dependent neurotransmitter transporter
family
o SLC7 – the cationic amino acid transporter / glycoprotein-associated family
o SLC8 – the Na+/Ca2+ exchanger family
o SLC9 – the Na+/H+ exchanger family
o SLC10 – the sodium bile and cotransporter family
o SLC11 – the proton-coupled metal ion transporter family
o SLC12 – the electroneutral cation-Cl- cotransporter family
o SLC13 – the human Na+-sulphate/carboxylate cotransporter family
Facilitated diffusion
• Carriers have specific affinity for binding particular solutes
• Can only mediate passive (downhill) transport
• Limited number of carriers and limited maximal transport rate = saturation (Jmax)
• Kinetics similar to Michaelis-Menten - km

• Also called passive transport
• All channels and many transporters allow solutes to cross the membrane passively
• If the solute does not have a net charge, it will move down its concentration gradient
• If the solute has a net charge, it will move down its electrochemical gradient
Pore vs
...
carrier
• Pore (non-gated channel)
o Pores are conduits that are always open
• Channel (gated pore)
o Channels are conduits that are gated by a “door”
o Channel is closed or open

•

Carrier
o Carriers are conduits that are gated by two “doors” that are never open at
the same time
o The carrier is open to the outside
o X enters from the outside and binds at a binding site
o The outer gate closes and X becomes occluded, still attached to its binding
site
o The inner gate opens with X still bound
o X exits and enters the inside of the cell
o The outer gate closes, occluding an empty binding site – this cycle can also
flow in the reverse order

SLC2 family hexose transformers – GLUT isoforms
• GLUT1
o Red blood cell
o Brain
o Kidney
o Placenta
• GLUT2
o Liver
o Intestine
• GLUT3
o Astrocytes

o Neurones
• GLUT4
o Adipocytes
o Muscle – insulin responsive
• GLUT5
o Intestine
High degree of homology but different affinities for glucose and fructose
Structure of GLUT1

•

Amphipathic helices surround the putative conduit

•

GLUT protein family is part of the major facilitator superfamily – largest superfamily
of proteins involved in membrane transport and are ubiquitous
• Integral membrane proteins of all eukaryotic cells
• Isoforms differ in kinetic properties, sugar specificity, tissue localisation and
regulation
• Some transport other substrates such as galactose, water and painkiller
glycopeptides
• Entry of glucose through GLUT is often the rate-limiting step for the performance of
cells with high energy metabolism
• Abundant in epithelial cells lining the walls of small blood vessels, particularly on
blood-brain barrier – high metabolic demand for glucose utilisation
• GLUT1 expressed in endothelial cell membrane at its interfaces with the blood and
intercellular space, as well as astrocyte PM (important for function of blood-brain
barrier)
• GLUT1 transports glucose from blood into the endothelial cells and then into the
astrocytes – convert glucose into other energy sources that are transported into
neurons
Facilitated transport

Mechanism

GLUT1 deficiency syndrome or De Vivo disease
• Rare autosomal dominant disease due to mutations in GLUT1
• In children, the brain glucose demand is 3-4 times higher than in adults and
represents 80% of the glucose utilisation of the body
• Impaired glucose transport across the blood-brain barrier
• Characterised by seizures and developmental delay caused by impaired glucose
transport into the brain
• Symptoms
o Microcephaly (small head)
o Mental and motor developmental delays
o Infantile seizures refractory to anticonvulsants
o Ataxia

o Dystonia
o Spasticity
• Seizures start between 1 and 4 months in 90% of cases
• Treatment – α-ketogenic diet
GLUT4 is regulated by insulin
• In adipocytes and muscle cells, insulin stimulates translocation of GLUT4 to the
plasma membrane

•
•

GLUT4 mediates glucose uptake by muscle and adipose tissues
During and after a meal, one of the actions of insulin is to increase glucose uptake
into the cells of these tissues
• GLUT4 isoform – also called insulin-responsive transporter – undergoes regulated
transport to the cell surface
• GLUT4 located in intracellular vesicles that fuse with PM – delivers GLUT4 to PM to
increase glucose transport capacity
• Binding of insulin to its cell-surface receptor initiates intracellular signalling cascades
that result in the rapid fusion of these vesicles with the cell membrane – allows rapid
increase in transport of glucose through GLUT4 transporters
• In T2DM, uptake of glucose from blood plasma into muscle and adipose tissue is
impaired, apparently due to diminished targeting of GLUT4 to the plasma membrane
Active transport – your life depends on it!
• Primary active transport
• Secondary active transport
Primary active transport
• P-type ATPases / Na+-K+-ATPases

•

o Structurally and functionally related multi-pass transmembrane proteins
o Phosphorylate themselves during the pumping cycle
o Includes many of the ion pumps that are responsible for setting up and
maintaining gradients of Na+, K+, H+ and Ca2+ across cell membranes
V type (vaculor) H+-ATPase
o Structurally similar to F-type ATPases
o Normally pump H+ rather than synthesise ATP

o Pump H+ into organelles such as lysosomes, synaptic vesicles and plant
vacuoles to acidify the interior

•

F-type ATPase
o Turbine-like proteins, constructed from multiple different subunits
o Found in PM of bacteria, the inner membrane of mitochondria and the
thylakoid membrane of chloroplasts
o Often called ATP-synthases – normally work in reverse – instead of using ATP
hydrolysis to drive ATP transport, they use the H+ gradient across the
membrane to drive the synthesis of ATP from ADP and phosphate
o H+ gradient is generated either by electron-transport steps of oxidative
phosphorylation in aerobic bacteria and mitochondria, during photosynthesis
in chloroplasts or by the light-activated H+ pump bacteriorhodopsin

Ion pumps are enzymes
Hydrolysis of ATP provides energy to move ions against energetically unfavourable
gradients
...
The protein binds a molecule of the substance to be transported on
one side of the membrane, changes shape, and releases it on the other side
...

The protein pumps are also ATPase enzymes, since they catalyse the splitting of ATP g ADP +
phosphate (Pi), and use the energy released to change shape and pump the molecule
...

The Na+-K+ Pump
...

• ATP-driven antiporter, actively pumping Na+ out of the cell against its steep
electrochemical gradient and pumping K+ in
• K+ concentration is typically 10-30 times higher inside the cell than outside, whereas
the reverse is true of Na+
• Gradient produced drives the transport of most nutrients into animal cells and also
has a crucial role in regulating cytosolic pH
• A typical animal cell devotes a third of its energy to fueling this pump, and it
consumes even more energy in their dissociation
• Structural changes in cytosolic domains are thought to be transmitted to the
transmembrane segments, driving cycles of conformational changes and alternately
expose substrate-binding sites on one side of the membrane and then on the other
Categories
• P-type ATPases (E1E2-ATPases)
o Na+-K+-ATPase
o Ca2+-ATPase
o H+-K+-ATPase
o Metal ion pumps
o Flippases
• V-type ATPases
o “Vesicular” H+-ATPases
• F-type ATPases (F0F1-ATPases)
o Mitochondrial ATP synthase
• ABC transporters – ATP-binding cassette (ABC) domains
o P-glycoprotein
o MsbA multi-drug resistant proteins
+
+
Na -K -ATPase
• The “sodium” pump
• Hydrolysis of ATP provides energy for active transport
• It’s a carrier and an enzyme
• Maintains gradients for excitability and secondary active transport

Jens Skou – 1997 Nobel Prize for Chemistry “for the first discovery of an ion-transporting
enzyme”
Na+-K+-ATPase key facts
• Most important pump in animal cells
• Only primary active Na+ pump in animal cells
• Responsible for inward Na+ gradient that drives many secondary active processes

Structure

There is also a regulatory  subunit of FXYD family
...
g
...
NCX can move Ca2+ either into
or out of cells, depending on the net Na+, Ca2+, and K+ gradient across the membrane
...

NCX1 (rat 971 aa, human 970 aa, mouse 970 aa) is most prominently expressed in the heart
where it plays a major role in excitation-contraction coupling, but is also present in most
other tissues
...
E1
...
E1
...
3Na+
Three Na+ ions bind

3
...
(3Na+)
ATP hydrolysis phosphorylates α-subunit, causing conformational change (occluded)

4
...
3Na+
Conformation spontaneously changes to E2, Na+ free to leave

5
...
E2-P
...
E2
...
E1
...
2K+
ATP-binding changes conformation back to E1, K+ free to leave

9
...
ATP
Cycle starts again

P-type: sarco / endoplasmic reticulum Ca2+-ATPase (SERCA)

P-type: H+-K+-ATPase
• The gastric hydrogen-potassium ATPase or H+-K+-ATPase is the proton pump of the
stomach
• Important site of drug action for treatment of dyspepsia, peptic ulcer disease, and
gastro-oesophageal reflux disease
• Proton pump inhibitors (PPIs) like omeprazole (Prilosec)
• Ranitidine (Zantac) is a histamine H2-receptor antagonist that inhibits stomach acid
production

V-type ATPases
• V-type ATPases acidify a wide array of intracellular organelles (e
...
lysosomes) and
pump protons across the plasma membranes of numerous cell types
• Many physiological roles where acidification is required
• Multiple subunits identified to form a functional V-ATPase complex

Cortical collecting duct showing V-ATPase staining of intercalated cells
(yellow/green)
...
This is an
intriguing example of the same protein being targeted to different plasma membrane
domains
...

The importance of V-ATPase activity in renal proton secretion is highlighted by the
inherited disease distal renal tubular acidosis
...

F-type ATPases and ATP synthase

Comparison of pores, channels and carriers

Cortical collecting duct showing V-ATPase staining of intercalated cells
(yellow/green)
...
This is an
intriguing example of the same protein being targeted to different plasma membrane
domains
...

The importance of V-ATPase activity in renal proton secretion is highlighted by the
inherited disease distal renal tubular acidosis
...




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