Transport of small molecules across membranes

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Transport of small molecules across membranes
Cell Structure and Function 2003 - 2004
Part I Basic Principles

• Svetlana Lutsenko
• MRB 625, phone [phone]
• lutsenko_at_ohsu.edu

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Learning Objectives

• 1. Know different modes of transport.
• 2. Know the two requirements for transport to
  occur.
• 3. Be able to distinguish between passive
  transport and active transport.
• 4. Be able to distinguish between simple
  diffusion and facilitated diffusion.
• 5. Know the factors that affect permeability.
• 6. Be able to describe a simple carrier model for
  membrane transport.
• 7. Know the mode of action of ionophores.
• 8. Be able to distinguish, with illustrative
  examples, between carriers and channels.

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Reading Assignment Alberts et al. "Essential
Cell Biology", pp. 372-385.
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Why transporters are important?
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Completion and analysis of various genomes
revealed that about 10 of all proteins function
in transport (in E.coli 427 transporters)
In eucaryotic cells, 2/3 of cellular energy at
rest is used to transport ions (H, K, Na,
Ca) About 200 families of transporters are
recognized The largest family ABC-transporters
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Wilsons disease protein (ATP7B) is a key
regulator of copper concentration in the liver
Normal liver
ATP7B -/- liver
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Membrane Transport and Human Disease

• Cystic Fibrosis and CFTR (the most common fatal
  childhood disease in Caucasian populations).
  Inadequate secretion of pancreatic enzymes
  leading to nutritional deficiencies, bacterial
  infections of the lung and respiratory failure,
  male infertility.
• Bile Salt Transport Disorders Several ABC
  transporters, specifically expressed in the
  liver, have a role in the secretion of components
  of the bile, and are responsible for several
  forms of progressive familial intrahepatic
  cholestasis, that leads to liver cirrhosis and
  failure.
• Retinal Degeneration The ABCA4 gene produts
  transports retinol (vitamin A) derivatives from
  the photoreceptor outer segment disks into the
  cytoplasm. A loss of ABCA4 function leads to
  retinitis pigmentosa and to macular dystrophy
  with the loss of central vision.
• Mitochondrial Iron Homeostasis ABCB7 has been
  implicated in mitochondrial iron homeostasis. Two
  distinct missense mutations in ABCB7 are
  associated with the X-linked sideroblastic anemia
  and ataxia
• Multidrug Resistance ABC genes have an important
  role in MDR and at least six different ABC
  transporters are associated with drug transport

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• Transporters vary enormously in their
  architecture and size from small organic
  molecules and peptides to multi-subunit complexes
  (the V-type and Fo-type ATPase may have more that
  10 subunit in a complex). The size of the
  individual molecules could be as large as 5000
  amino-acid residues (Ryanodine receptor)
• Some transporters are ubiquitous (aquaporins,
  glycerol facilitators, copper-transporting
  ATPases), others are kingdom specific (bacteria
  and yeast cells lack Na,K-ATPase, one of the
  most abundant transporter in higher eucaryots.
  At the same time, only bacterial cells have
  phosphoenolpyruvate-dependent phosphotranspherase
  system)
• Transporters often have dual function acting as
  enzymes or receptors in addition to transport
  function

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Despite their enormous variety, the transporters
utilize common rules to transport ions and
molecules across cell membranes
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The Modes of Membrane Transport
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Two major modes of membrane transport

• I. Simple (Passive)Diffusion
• no carriers is involved

• Molecules that are transported through the cell
  membrane via simple diffusion include organic
  molecules, such as benzene and small uncharged
  molecules, such as H2O, O2, N2, urea,
  glycerol,and CO2

• II. Mediated Diffusion
• is carried out by proteins, peptides, and small
  molecular weight carriers
• (ions, uncharged organic compounds, peptides, and
  even proteins can be transported)

There are two major modes of mediated diffusion
passive transport (or facilitated diffusion) and
active transport
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Passive transport (facilitated diffusion)
energy independent, down the concentration
gradient

• Mobile carriers -ionophores (valinomycin,
  nigericin, dinitrophenol, etc)
• Protein-translocators - (Band 3, porins,
  erythrocyte glucose transporter)
• Channels - channels-forming ionophores
  (gramicidin)- voltage-gated channels (Na-, K-
  and Ca2 -channels)- ligand-gated channels-
  mechanosensitive channels

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Active transport - energy-dependent, against
concentration gradient
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Primary Active Transport - utilizes energy of ATP
hydrolysis

• P-type ATPases (Na,K-ATPase, H,K-ATPases,
  Ca-ATPase, Zn2/Pb2transporting ATPase of
  bacteria)
• V-type ATPases and F1F0-ATPases (Na-ATPase and
  H-ATPase)
• ATPases that transport peptides and drugs
  (multidrug-resistance protein, P-glycoprotein,
  yeast a-factor transporter

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Secondary Active (Coupled) Transport - utilizes
ion-gradients generated by primary transporters
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Types of Secondary Transporters

• Symporters (two solutes move in same direction)
  Lac- permease, Na/glucose transporter)
• Antiporters (two solutes move in opposite
  directions
• Na/Ca2 exchanger)
• Uniporters (mitochondrial Ca2 uniporter and
  NH4-transporter in plants require H gradient)

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Thermodynamics of membrane transport
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Simple (passive) diffusionis a non-mediated and
non-saturable transport

• Molecules that are transported through the cell
  membrane via simple diffusion include small
  organic molecules, H2O, O2, N2, urea,
  glycerol,and CO2
• Applications of simple diffusion drugs delivery,
  analysis of membrane topology using
  membrane-permeable and impermeable reagents,
  regulation of osmotic pressure, etc.

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• Simple diffusion of molecules through the
  membrane thermodynamically resembles chemical
  equilibrium.
• A chemical potential is generated by the
  differences in concentration of the transported
  molecules.

If C2ltC1 then DG is negative and transport occurs
spontaneously down the concentration gradient If
C2gtC1 then DG is positive and then the energy
source, such as ATP, is required to transport the
molecules against concentration gradient
DG DG2- DG1RTln(C2/C1)
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For Uncharged Molecules

• Rate of flow or flux Jc can be expressed as
  follows
• Jc -P (C2-C1)
• where P is permeability coefficient and C1 and C2
  are concentrations of the transported molecule in
  two different compartments across the membrane.
• P reflects dependence of the rate of simple
  diffusion on charge, hydrophobicity, size of the
  molecules, as well as the effect of the membrane
  thickness and composition on the rate of flow
• PKD/X
• K - partition coefficient ( in general, it
  depends on properties of the solute, such as
  
  hydrophobicity and charge)
• D - diffusion coefficient (in general, it
  depends on the size of the transported
  molecule and the membrane viscosity)
• X- thickness of the membrane
• Therefore Jc can be expressed as Jc
  -KD(C2-C1)/X Ficks equation

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For Charged Moleculesthe rate of flow depends
not only on difference in the solute
concentration on the both sides of the membrane,
but also on charge difference across the membrane

• Consequently, DG for transfer of charged
  molecules across the membrane includes both
  chemical and electrical components
• chemical
  electrical
• DG RTln(C2/C1) ZFDY
• DG - electrochemical potential
• C2 and C1 concentrations of the molecule
• Z- ionic charge of the molecule
• T - absolute temperature
• R - gas constant
• F - Faraday constant
• DY - membrane potential

Y2-Y1DY lt 0 Z 1 ZF DY lt 0
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Facilitated diffusion - transport of molecules in
an energy-independent fashion down the
electrochemical gradient

• Protein or carrier-mediated
• Characterized by saturation kinetics
• much faster than simple diffusion
• Facilitators have chemical and stereochemical
  specificity for transported molecules (for
  example, glucose transporter would transport
  D-glucose, but not L-glucose, valinomycin
  transport K ions 20,000 times better than Na)
• susceptible to competitive inhibition

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How to distinguish experimentally facilitated
diffusion and passive diffusion?
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Passive-Mediated Glucose Transportfacilitates
glucose uptake about 50,000 fold

• Erythrocytes glucose transporter is a 55 kDa
  glycoprotein with 12 transmembrane segments
• The transporter is believed to function through
  alternating conformation mechanism
• Transport can occur in either direction and
  serves mainly to equilibrate glucose
  concentration

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Mobile carriers (ionophores) the non-protein
transporters and small organic molecules

• Ionophores serve as simple models for analysis
  of the mechanisms of membrane transport
• They transport ions down the concentration
  gradient to equilibrium, and often used as
  convenient tools to load cells with certain ion
  or to analyze property of more complex
  transporters by disrupting concentration
  gradients.
• number of antibiotics function as ionophores

Key feature have two forms with markedly
different hydrophobicity hydrophylic ion-free
form and lipid-soluble ion-bound form
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Valinomycin

• A potassium ionophore
• A dodecadepsipeptide (has both peptide and ester
  bonds). It is a cyclic structure composed of
  4-unit sequence repeated three times
• Exists in two forms with markedly different
  hydrophobicity
• Destroys K-gradient without affecting DpH
• Increases K-permeability up to 10,000 K-ions/sec
• Highly selective for K

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• High selectivity of valinomycin for K-ion is due
  to
• a) perfect fit into coordination sphere
• b) more favorable energetics
• (K ion radius is 0.133 A, radius of Na is
  smaller 0.095, but the free energy of hydration
  is significantly higher for Na (300 kJ/mol)
  than for K (230 kJ/mol), consequently it takes
  more energy to dehydrate Na )

These two major principles of ion selection are
also utilized by other more complex transporters
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Channels

• Greatly increase permeability for the transported
  molecules
• Have the highest rate of transport among all the
  transporters, 105 - 107 ions/sec
• valinomycin (carrier) transports up to 104
  K/sec
• gramicidin (channel) permeability is up to 107
  K/sec

• Transport through the channels is unaffected by
  temperature, while facilitated transport mediated
  by the mobile carrier is temperature- dependent

• The selectivity of channels towards transported
  molecules varies. As a rule, the b-barrel-based
  channels (pores and porins) and antibiotic-based
  channels are less selective and not as highly
  regulated as the channels that utilize the
  a-helix as their major structural element

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Simple channels

• Gramicidin A
• a 15-mer polypeptide composed of alternating L
  and D amino-acids
• forms a b-helix (6,7 amino acid residues per
  turn), which then dimerizes head-to head by
  hydrogen bonding association between their
  N-formyl ends to cross the membrane. The
  diameter of the pore is 4 A.
• greatly increases permeability for monovalent
  cations, but not divalent cations i.e. it is
  less selective than valinomycin, but much more
  permeable

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The hydrophobic side chains of gramicidin contact
the lipid bilayer, while the polar carbonyl
groups surround the aqueous pore and transiently
coordinate cation when it passes through the
channel very similar to the structure of more
complex channels