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CARRIER MEDIATED TRANSPORT
SECONDARY AND TERTIARY ACTIVE TRANSPORT
SECONDARY ACTIVE TRANSPORT
General properties of secondary active transporters
• Can move solute uphill – against an electrochemical gradient
• Do not consume ATP, instead harness existing gradient: Na+, K+, H+
• Saturable and defined affinities
• Symport – driver and driven in the same direction
• Antiport – driver and driven in opposite directions

Cotransport (symport)
• Moves two or more solutes in the same direction
• Usually driven by Na+ or H+ gradient into the cell

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Harvest energy stored in an electrochemical gradient of one solute to transport the
other
Free energy released during the movement of an inorganic ion down an
electrochemical gradient is used as the driving force to pump other solutes uphill,
against their electrochemical gradient
In PM of animal cells, Na+ is the usual co-transported ion, the electrochemical
gradient of which provides a large driving force for the active transport of a second
molecule – pumped out later by ATP-driven Na+ pump in the PM – maintains the
gradient – indirectly drives transport

Na+-glucose cotransporter SGLT: SLC5

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The 664 amino acid protein contains 14 transmembrane α-helical domains
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Highlighted
in yellow is the architecture of common members of the solute symporter family (SSF) gene
family with the sodium:SSF signature sequence: [GS]-x (2)-[LIY]-x (3)-[LIVMFYWSTAG] (7)-x
(3)-[LIV]-[STAV]-x (2)-G-G-[LMF]-x-[SAP]
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The
glucose-binding and -translocation domain (C5) is located at the COOH-terminal end of the
protein, and the residues proposed to be involved in glucose binding at the extra- and
intracellular sides of the membrane are highlighted
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• GGM is characterised by neonatal onset of watery and acidic severe diarrhoea,
which is fatal within a few weeks unless lactose (glucose and galactose) is removed
from the diet
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Location of the 23GGM missense mutations in the secondary structure of SGLT1 – most
mutations result in either truncated SGLT1 protein or mistrafficking of the transporter in
the cell

Location of the 23 GGM missense mutations in the secondary structure of SGLT1
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Glycosylation
tree indicates the extracellular surface of the protein
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k
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NRAMP1 / DCT1

Summary of the molecular characteristics of Divalent Metal Transporter 1 (DMT1)
• 561 amino acid polypeptide – 60kDa
• Predicted to have 12 membrane-spanning domains
• Glycosylated extracellular loop
• N- and C-terminal in cytosol

Summary: functional characteristics of DMT1
• Km for Fe2+ - 6μm
• pH dependent
• Transports Mn2+, Cu2+, Co2+, Cd2+, Zn2+, Ni2+ and Pb2+
Two electrode voltage clamps as used to study DMT1

Divalent Metal Transporter 1 (DMT1)

Sequence antibody was raised to is common to isoforms containing both exon 1a and exon
1b because the epitope is in exon 2
Staining of apical duodenum membranes by affinity purified rat DMT1 antiserum

Iron responsive elements
• FPN1, Ferritin H- and L- subunits
• IREs are cis-acting nucleotide sequences
• They form a stem-loop structure that by association with trans(IRP) protein
molecules can modulate mRNA translation
Summary: molecular components of duodenal translocation

Iron responsive elements IREs
IREs are cis-acting nucleotide sequences
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Receptor is a disulphide linked transmembrane glycoprotein homodimer
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Cytoplasmic domain (N-terminal) is essential for receptor internalization and a
tetrapeptide sequence in the cytoplasmic tail acts as a signal sequence for high-efficiency
endocytosis
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Regulation of receptor synthesis is post-translational and involves modulation of
transferrin receptor mRNA stability
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Affinity of transferrin receptor for transferrin varies depending on the conditions
under which it is measured (10-7 to 10-9 mol/litre)
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• Control of iron levels in mammals by the proteins ferritin and transferrin –
translational regulation
o Iron is a toxic metal that can cause oxidative damage to cellular components
– important to keep excess iron stored safely as a complex with ferritin in the
blood stream until needed for synthesis of haemoglobin and other ironcontaining enzymes
o Transferrin helps iron enter cells through transferrin receptor-mediated
endocytosis
o When iron levels are low, it is advantageous to the cell to inhibit the levels of
ferritin so that any remaining free iron can be available for endocytic
transport by transferrin via the transferrin receptor
• To reduce the levels of ferritin and increase the levels of transferrin receptor, an IRP
binds to a site located on the ferritin and transferrin receptor mRNAs called IRE
• When IRE is bound by the IRP, the complex blocks the scanning 40S ribosome from
reaching the start codon

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In the case of the transferrin receptor, there are multiple IREs located in the 3’UTR –
binding of an IRP to the IRE in the transferrin receptor greatly stabilises the
receptor’s normally unstable mRNA
• When iron levels are low in the blood, the cells act to decrease the level of ferritin
translation initiation and to increase the stability of the transferrin receptor mRNA
o IRP has an iron-binding site; when it is filled with iron, the affinity of IRP for
the IRE is significantly reduced
• When iron levels are high in the blood, ferritin synthesis is increased to enhance iron
storage
o High iron levels also release the binding of IRPs to the transferrin receptor
IREs and thereby stimulate the degradation of IRP
Exchangers (antiporters)
• Move one or more “driving” solutes in one direction
• One or more “driven” solutes in the opposite direction
• Usually exchange cations for cations or anions for anions

The AE family (SLC4)

Band 3 proteins (AE1)
• Highly abundant in red cell membrane (>1 million copies cf
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g
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(Zhu et al
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Met66 (arrow) marks the start of kidney AE1
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The mutations associated with
hereditary spherocytic anemia and ovalocytosis are orange, and include missense,
nonsense, splicing and deletion mutations
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Mutations associated with dominant and recessive
distal renal tubular acidosis are green
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Upper left: scanning electron micrographs of wild-type erythrocytes and AE1–/– bovine
spherocytes (HS) (Inaba et al
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Upper right: consecutive semithin sections from rat
kidney cortex immunostained with antibodies recognizing vH+-ATPase (left) and kAE1
(right)
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, 1989)
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Scale bars 10 μm at top left; 7 μm, top right
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, 2007)
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Secondary active transporters (e
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system A/SNAT2) generate net movement of amino acids from the extracellular
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Secondary active transporters
(e
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, System A/SNAT2) generate net movement of AA from the extracellular to the
intracellular pool, whereas tertiary active transport through exchangers such as LAT1
(System L) allows for redistribution of individual AAs without affecting total pool sizes

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