Transporters in cell physiology

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Transporters in cell physiology
Hana Sychrová

• Department of Membrane Transport
• Institute of Physiology, AS CR, v.v.i.
• Videnska 1083, 142 20 Prague 4
• Tel [phone]
• E-mail sychrova_at_biomed.cas.cz

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Membranes

• Integrity of cells, organelles
• Flow
• material - transport of substances
• energy - radiation
• - oxidation
• - electrochemical potential
• information - physical signals -
  light
• - heat
• - electrical potential
• - magnetic field
• - mechanical pressure
• - chemical signals - attractants and
  repellents
• - sense signals

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Transport mechanisms
1. Nonspecific permeation (diffusion down the
concentration gradient) 2. Specific transport
(membrane protein) Transport systems Pore
- (oligomer) permitting nonspecific passage of
solutes of different size ranges Transporter -
relatively high specificity
- binding site opens alternately to the one and
to the other membrane side
- antiporter/symporter -
uniporter -
exporter/importer - old
names permease (symporter)
carrier (transporter)
Channel - one protein or
an oligomeric cluster -
when open, the specific site in the channel can
transiently bind solutes
from both sides of the membrane

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• Transporter
• membrane protein showing a relatively high
  specificity
• - binding site opens alternately to the one and
  to the other membrane side
• - antiporter/symporter (at least one ionic)
• - uniporter
• - exporter/importer

Facilitated (mediated) diffusion without input of
energy (glc in yeast) Active transport against
solutes chemical or electrochemical potential
gradient primary
driven by exergonic chemical and photochemical
reactions
(ATP hydrolysis, mainly ions)
- secondary driven by
electrochemical potential gradients
of H and
Na (exceptionally of K)
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• Channel
• membrane protein (or an oligomeric cluster)
• specific transport of ions or uncharged
  molecules
• down their chemical/electrochem. potential
  gradient
• two conformational states, open and closed
• opening by
• (a) spreading electric field
  (potential-gated)
• (b) binding a specific ligand (chemically
  gated)
• (c) mechanical stress (mechanically
  gated)
• when open, the specific site in the channel can
• transiently bind solutes from both sides of the
  membrane

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Transport mechanisms
1. Nonspecific permeation (diffusion down the
concentration gradient) 2. Specific transport
(membrane protein) Transport systems Pore
- (oligomer) permitting nonspecific passage of
solutes of different size ranges Transporter -
relatively high specificity
- binding site opens alternately to the one and
to the other membrane side
- antiporter/symporter -
uniporter -
exporter/importer - old
names permease (symporter)
carrier (transporter)
Channel - one protein or an oligomeric
cluster - when open, the
specific site in the channel can transiently bind
solutes from both sides
of the membrane

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• Nonspecific permeation
• diffusion down the concentration gradient
• 1.1 hydrophobic domains of membranes    
  (various small, medium-sized and large lipophilic
  molecules)
• 1.2 water-filled pores
• (polyene antibiotics)
• some opening only after a stimulus
  (connexons between adjacent cells -    
  all solutes up to a certain size)
• 1.3 true pores in the lipid bilayer
  transiently formed at higher
• temperature and with applied
  transmembrane electric potential    (all
  solutes, including macromolecules
  ("electroporation"))
• 1.4 via non-receptor endocytosis in membrane
  vesicles    (all solutes present in
  extracellular aqueous medium)

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• 2. Specific transport
• 2.1 Selective channels
• 2.2 Specific transporters
• 2.3 Group translocation
• Gram-positive and Gram-negative
  bacteria, brain tissue
• (mono- and disaccharides amino
  acids)
• 2.4 Receptor-mediated pinocytosis
• 2.4.1 endocytosis, mainly in animal
  cells    (ferritransferrin)
• 2.4.2 exocytosis, mainly in fungal and
  animal cells    (hormones)

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• 2. Specific transport
• 2.1 Selective channels
• 2.1.1 nongated (porins in bacterial, outer
  mitochondrial and chloroplast
• membranes,
  aquaporins)     (cations,
  anions, water also nonelectrolytes - glycerol)
• 2.1.2 electrically or potential-gated
  (nerve and muscle-cell)
• (generation and
  propagation of action potential, Na, K, Ca2)
• 2.1.3 chemically or ligand-gated (hormone
  receptor-channels, acetylcholine receptor)   
  (cations)
• 2.1.4 mechanically or stress-gated
  (channels in blood capillary walls, inner ear
• hair cells)   
  (cations, especially K)
• 2.2 Specific transporters
• 2.3 Group translocation
• 2.4 Receptor-mediated pinocytosis

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2. Specific transport 2.1 Selective
channels 2.2 Specific transporters
2.2.1 mediated (or facilitated)
diffusion    (monosaccharides in
animal, noninsulin-dependent tissues, yeast
cells) 2.2.2 primary active transport
2.2.3 secondary active transport
2.3 Group translocation 2.4 Receptor-mediated
pinocytosis
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2.2.2 primary active transport
2.2.2.1 driven by ATP or diphosphate hydrolysis
P-type, F-type and ABC-type
ATPases in all cells   
(cations, anions, amino acids, sugars,
xenobiotics) 2.2.2.2 driven by
oxidation reactions, in
bacterial, inner mitochondrial and chloroplast
membranes    (H, Na)
2.2.2.3 driven by light absorption,
in halobacteria (bacteriorhodopsin)  
 (H, Cl-)
2.2.2.4 driven by decarboxylation,
in alkalifilic bacteria   
(Na) 2.2.2.5 driven by
methyl transfer, some
methanobacteria    (Na)
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2.2.3. secondary active transport
2.2.3.1 symport type,
using H, Na and, exceptionally, K as driving
ion, bacterial, fungal,
plant and animal cells, net charge
transporting    (various
nonelectrolytes, mainly nutrients)
with H - lactose in Gram- bacteria
with Na - glucose in kidney
tubules with K - lysine in
insect intestines 2.2.3.2 antiport
type, electrically silent,
in a variety of cells   
(cations) 2.2.3.3 antiport type,
net charge transporting, in
outer mitochondrial membranes   
(ADP/ATP)
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Transport mechanisms
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http//www.tcdb.org/
Na or KH antiporter Nha1 2.A.36.4.1
2. Electrochemical-potential-driven
transporters 2.A. Transporters or carriers
(uniporters, symporters and antiporters)
2.A.36 Univalent cationH antiporter-1
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Nomenclature Committee of the International Union
of Biochemistry and Molecular Biology (NC-IUBMB)
Membrane Transport Proteins http//www.chem.qmul.
ac.uk/iubmb/mtp/
Introduction Glossary of terms used in membrane
transport work List of families and subfamilies
of the TC system 1. Pores and channels 1.A
a-Helical channels 1.B b-Strand porins 1.C
Pore-forming toxins 1.D Non-ribosomally
synthesized channels 1.E Holins 2.
Electrochemical-potential-driven transporters
2.A Transporters or carriers (uniporters,
symporters and antiporters) 2.B Non-ribosomally
synthesized transporters 3. Primary active
transporters
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Characterization of transport systems

• Kinetic parameters - KT , Jmax , ( sin/sout ),
  substrate specificity
• pH optimum, inhibitors, source of energy (active
  transport)
• Isolation of the gene and sequencing ? primary
  structure and properties of the protein
• Protein purification ? reconstitution,
  crystalization
• existence of similar systems (overlap of
  substrate specificity)
• small amount of protein (low expression, short
  half-life)
• denaturation of protein during membrane
  solubilization loss of characteristic
  properties
• ? expression of the gene in host organisms
  (yeast Saccharomyces cerevisiae)

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Transport mechanisms in S. cerevisiae

• 400 transport systems
• ? 23 families

Passive transport simple diffusion -
O2, CO2, glycerol facilitated diffusion -
channels metal cations, Cl-
transporters monosaccharides Active
transport primary 89
H-ATPasa (gen PMA1) secondary
201 symport with H - aa, K, Pi
antiport
with H - Na, K André B., Yeast 11, 1575-1611
(1995) Nelissen B., FEMS Microbiol. Rev. 21,
113-134 (1997)
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• sugars - facilitated diffusion (glc, gal), 17
  regulated systems
• - active symport with H
  (maltose)
• glycerol - simple diffusion
• - facilitated diffusion - channel (FPS1)
  only outward
• - active symport with H - inward

amino acids (nitrogen source) -
active symport with H -
accumulation 102 - 105 - 24
genes - 20 transporters (permeases)
characterized kinetically - substrate
specificity overlap - 16 mutants
isolated (resistance to toxic aa analogs)
- 14 gene isolated, products characterized
(homology 20 - 60 )
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Transport of K and Na in S. cerevisiae
Growth 1 ?M 2.5 M K 0 1.8 M
Na Intracellular concentration
200 - 300 mM K lt 100 mM Na

• 10 transport systems
• Channels (1 -2)
• ATPases (1)
• Symporters (2 4)
• Antiporters (5)

K influx Trk1,2p si/so 3 x 105
Sequestration in organellles antiporters Nhx1p
Kha1p Vnx1p, Mkh1p(?)
Alkali-metal-cation efflux (Na, Li, K,
Rb) Nha1p Na/H antiporter (acidic
pHout) Ena1/Pmr2p Na-ATPase (neutral pHout)
Tight regulation expression, biogenesis
(targeting, post-translational modif.),
degradation
(post-translational modif., delocalization)
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Transport of K and Na in S. cerevisiae
X
X
Growth 1 ?M 2.5 M K 0 1.8 M
Na Intracellular concentration
200 - 300 mM K lt 100 mM Na
X
X
Gene deletion Phenotype
growth on YNB (pH 5.0) salt mM
Strain NaCl LiCl
KCl