Chapter 7.1-7.

Transport of ions and small molecules across cell membranes

Yi-Fang Tsay, 蔡宜芳 Institute of Molecular Biology, Academia Sinica

A cell

B cell

• Different mixtures of low-molecular weight compounds • The plasmamembrane of each cell type contain a specific set of transport proteins that allow certain ions and molecules to cross • Different organells

What kinds of molecules are permeable to membrane?

Passive and active transport?
For uncharged molecules: • Passive: move from higher concentration to lower concentration • Active: need energy input in order to move from lower concentration to higher concentration

Passive v.s. active transport
Sugar: chemical gradient Ions: electrochemical gradient







Overview of membrane transport proteins
Active or passive? Energy source? Active or passive? Energy source?

•Facilitated diffusion •Gated or non-gated •Transport rate

Active or passive? Energy source?

= Carriers or permease

•Facilitated diffusion •Transport rate

Passive diffusion
diffusion rates: 1. Concentration gradients 2. Hydrophobicity • Partition coefficient K: equilibrium constant for its partition between oil and water, K=Cm/Caq • Diethylurea has a K of 0.01, urea 0.0002, which one move faster across membrane? • Fatty acids with longer hydrocarbon chain, fatty acids with shorter hydrocarbon chain, which one move faster?

facilitated diffusion
The differences between passive diffusion and facilitated diffusion: 1. Transporting rate 2. Certain Vmax: limited number of transporters presented on the membrane. 3. Specificity: Km

KM=1.5 mM
Blood glucose is 5 mM, 77% of the maximal rate

KM=20 mM
After a meal, blood glucose increase from 5 mM to 10 mM, GLUT1 or GLUT2 expressing cells double their glocose uptake?


GLUT glucose transporter can transport D-Glucose, DMannose, D-Galactose, but not L-Glucose




Km= 1.5 mM

20 mM

30 mM

Operating mechanism of uniporter GLUT1

• • •

Conformational change Can transport on either direction depending on the gradient Kinetics: like an enzyme, can be described by MichaelisKinetics Meton equation.


Vmax _________ 1+ Km/C

The human genome encodes 12 GLUT proteins, GLUT1-12
• 12 α-helix TMs, in the binding sites, side chain of the amino acid residures form hydrogen bonds with the hydroxyl groups of glucose • GLUT1, Km=1.5mM, erythrocytes • GLUT2, Km=20 mM, liver and insulin-secreting β cells of pancreas • GLUT4, fat and muscle cells, insulin induce PM targeting of GLUT4 to lower the blood glucose level • GLUT5, transport fructose

Two common experimental systems for studying the function of transporters 1. Using liposome reconstitution to study the functional properties of transport proteins

2. Xenopus oocyte expessing system


ATP-powered pumps

•Form phosphoprotein • β Subunit: regulatory

Vacuole pH 5.5 pH 7.2

Plant cell
pH 5.5

Muscle Ca2+ ATPase pumps Ca2+ ions from the cytosol into the Sarcoplasmic Reticulum (SR)
10-2 M

high-affinity binding

10-7 M (resting cells) 10-6 M (contracting cells)

~80% of integral protein in SR membrane

low-affinity binding

A domain P domain N domain

Nature 2004, 430:529-535

1. On its cytoplasmic side, there is a large empty space in which several water molecules were identified in the crystal structure (Fig.a) 2. site II Ca2t (blue) will be ready to dissociate, if the Glu 309 carboxyl detaches from it by thermal movement.



Na+/K+ ATPase (pump)
• 25% of the ATP produced by
> <

nerve and kidney cells is used for ion transport, and 50% of the ATP produced by human erythrocytes is used for ion transport. • Most of this ATP is used to powered the Na+/K+ pump. • What happen to a cell, if it is treated with poisons that inhibit the production of ATP?

> <

145mM 4mM


12mM 139mM
High affinity for Na+ (Km=0.6mM, [Na+]c=12 mM) Low affinity for K+

High affinity for K+ (Km=0.2mM, [K+]0=4 mM) Low affinity for Na+

Keep cell contain high concentration of potassium and low concentration of sodium. Potassium gradient is the main factor for generating membrane potential, and sodium gradient is the driving force for a lot of substrate to be transported into animal cells The β-subunit help α-subunit to fold properly

V-class pump
• Vacuolar membrane of plant, yeast and fungi, endosomal and lysosomal membrane of animal cells, plasamembrane of acid-secreting animal cells (osteoclasts: dissolve bone) • multiple subunits • No phophorylation and dephosphorylation • 2 domains, one (V1) is hydrophilic cytosolic-facing domain required for ATP binding and hydrolysis; the other (V0) is transmembrane domain include c and a (or K and I) subunit which form ion-conducting channel

Science 2005 308:654-659

Science 2005 308:659-662

10 subunits

11 subunits

4 TMs

2 TMs

A model for ion translocation by the V-ATPase

H+ ATPase (pump)
•In order to generate proton gradient, anions have to be transported in the same direction (lysosomes and plant vacuole), or cations have to be transported in the reverse direction (the lining of stomach using H+/K+ ATPase). Otherwise the electric potential generated will prevent further transportation of proton.

Plant cells use proton gradient and membrane potential to drive the transport of other substrate, animal cells use sodium gradient
pH=5.5 pH=7
Vm=-180 mV
Proton -ATPase
Primary transporter

[Na+]=145 mM [Na+]=12 mM

Na+, K+ -ATPase

H+ H+

Vm=-70 mV



Plant cells
Proton-coupled transporter
secondary transporter

Aninal cells
sodium-coupled transporter

ABC proteins
Gram-negative bacteria

5% of prokaryotic genome, transport amino acids, sugars, vitamins or peptides

Vitamin B12

1. 2. 3. 4.

10 TMs L1 and L2: interface between C and D Gate ATP binding site: interface between two D subunits, binding of ATP will bring two units together. Referred by comparing the nucleotide-free state of
BtuCD with Rad50: DNA repair enzyme in its dimeric ATP-bound form.

1. initiation of a productive transport cycle requires coupling of substrate binding to alterations in the nucleotide-binding site. 2. ATP hydrolysis is coupled to substrate translocation. Binding of ATP may induce a shortening of the distances between analogous regions of the B12 importer, resulting in a BtuD cassette interface similar to that observed in the Rad50 dimer.

ABC proteins
Prokaryotic Eukaryotic

~50 ABC pumps are known in Mammals
Multiple drug transport protein (MDR)
• Hydrolyze ATP to export large variety of drugs • Normal function: detoxification in liver, kidney and intestine, substrate include natural and metabolic toxins • Other human ABC transporter can transport sugars, amino acids, cholesterol, peptides, proteins, toxin, and xenobiotics.

X-linked adrenoleukodystrophy (ALD)
• ABCD1 is localized to the peroxisomal membranes which regulates import of very long chain fatty acids into peroxisomes, where they undergo oxidation. In its absence, these fatty acids accumulate in the cytosol.

Flippase model of transport

E coli lipid flippase

MDR2 has been shown to have flippase activity. Liver cell membrane that faces the bile duct. Generate an excess of phospholipids in the exoplasmic leaflet, then peel off into the bile duct

CFTR: cystic fibrosis transmembrane regulator • Affects 1/2000 people in Northern Europe and US, 1/20 are carrier • Genetic disease, defective in the Cl channel of epidermis in lung, intestinal tract, sweat glands, pancreas. Dehydrated mucus clogs ducts and then fosters lethal bacterial infection • In 1989, identified by positional cloning • Cl- channel for reabsorption of Cl- into cells

CFTR function as a channel gated by ATP


Required ATP, but not use ATP as energy to pump Cl-. The Cl- channel is gated by the binding of ATP

Only one of the bound ATPs will be hydrolysed in CFTR


ABC transporter serve as a regulator

7.3 Nongated ion channels and the resting membrane potential
pH=7 Vm=-70 mV for animal cell
How to keep this membrane potential and ion concentration? How the electro-chemical gradient affect in and out of other substrate? Plasma membrane 3.5 nm, therefore – 70 mV = 200,000 volts per cm High-voltage transmission lines: 200,000 volts per km

ENa=(RT/ZF)*ln([Na]l/[Na]r) (Nernst equation) R:gas constant, 1.987 cal/degree.mol T:absolute temperature F:Faraday constant, 23062 cal/mol.V At 20℃ Na+: ENa(V)=(0.059/Z)*log([Na]l/[Na]r) 15 mM 150 mM ENa(mV)=(59/Z)*log([Na]l/[Na]r) ENa(mV)=(59/1)*log(15/150) ENa(mV)= -59mV Vm=(59/Z)*log([Na]r/[Na]l)= 59 mV

The membrane potential in animal cells depends largely on resting K+ channels

Their opening and closing are not affected by the membrane potential or by small signaling molecules

Vm=(59/Z)*log([K]o/[K]i)=(59/1)*log([4]o/[139]i)= -90 mV

Membrane potential of animal cells is about –70 mV. This is determined by two factors: 1. Na+/K+ pump (Naout, K in) to generate K+ gradient 2. membranes contain many K+ channels but few Na+, Ca+2 channel. This type of K channel is called K leak channel or resting K channel.

• Selectivity (specificity) • gating

Whole cell two-electrode voltage clamping

Patch clamping

Potassium channel

Ion channels contain a selectivity filter formed from conserved transmembrane a helices and P segmemts

glycine, Gly-Tyr-Gly of each P segment

•Activation energy for passage of Na+ ions is 1000 times higher than that of K+. •Then, how Na channel selectively transport only Na?

• Selectivity (specificity) • gating

Ligands, voltage, temperature, or mechanical stress


Voltage-gated Na+ channels remain inactive for several milliseconds after opening

Symporter and antiporter
• Primary transporter: pump • Secondary transporter: symporter or antiporter
Na+ Na+ Glucose Na+



K+ Na-K-ATPase

Ca+2 symporter antiporter

Properties of transporters: (1) stoichiometry Vmax V = _________ 1+ Km/C

Free energy of Na+ entry
Two forces govern the movement of ions: voltage gradient and concentration gradient,

∆G= ∆Gc+ ∆Gm ∆Gc=RT*ln([X]in/[X]out) R:1.987 cal/degree.mol T:293K for 20℃ ∆Gm=FE F:23062 cal/mol.V E:membrane potential for –70 mV, ∆Gm=-1.61 Kcal/mol

Sodium-glucose transporter

∆G= ∆Gc+ ∆Gm ∆G=RT*ln([Glucose]in [Na]in2 /[Glucose]out [Na ]out2)+2FE Vm=-70mV, [Na]in=12 mM, [Na]out=145 mM, ∆G for Vm and Na is –6 Kcal/mol 6 Kcal/mol=RT*ln ([Glucose]in /[Glucose]out), [Glucose]in =30,000[Glucose]out ∆G= ∆Gc+ ∆Gm ∆G=RT*ln([Glucose]in [Na]in /[Glucose]out [Na ]out)+FE Vm=-70mV, [Na]in=12 mM, [Na]out=145 mM, ∆G for Vm and Na is –3 Kcal/mol 3 Kcal/mol=RT*ln ([Glucose]in /[Glucose]out), [Glucose]in =170[Glucose]out Higher transporting rate but lower gradient generated, at the end of tubing, change to 2Na+/Glucose

• In intestine, 2Na+/Glucose

• In kidney, Na+/Glucose

Properties of transporters:
• (2) binding
– Random binding – Ordered binding

• (3) transport mechanism
– Simultaneous – Sequential

Wright, E. W. at UCLA

JBC 2004, 279(29):30150-30157

E. Coli lactose permease
UCLA, Ronald H. Kaback

Monomer is the functional unit; One binding site alternatively accessible to each side of the membrane; two fold symmetry; a hydrophilic cavity is formed between TM I, II, IV, and V of N-terminal and TM VII, VIII, X and XI of the C-terminal

Substrate-binding site

At a similar distance from either side of the membrane; Arg144→Lys abolishes binding; both N-terminal and C-terminal are involved

Antiporter (exchanger)

Ca+2in/3Na+out exchanger in cardiac muscle cells
• Transport one Ca++ out by transporting three Na+ in • Cardiac muscle cell, high [Ca+2]in triggers contraction • [Ca]out=10000[Ca]in • In cardiac muscle cell, Ca+2/3Na+ exchanger plays the principal role in maintaining a low concentration of Ca++ in the cytosol • Drugs ouabain and digoxin used to treat congestive heart failure will increase heart muscle contraction by inhibiting Na/K ATPase

Several antiporters regulate cytosolic pH
Metabolic will make cytosol acidic, use Na+in/HCO3-in/Cl-out exchanger and Na+in/H+out increase cytosolic pH, and Band 3 to balance out their reaction

Carbonic anhydrase: HCO3- → CO2 + OH-

Numbers transport protein enable plant vacuoles to accumulate metabolites and ions

Salt Tolerance Conferred by Overexpression of a Vacuolar Na+/H+ Antiport in Arabidopsis
Maris P. Apse, Gilad S. Aharon, Wayne A. Snedden, and Eduardo Blumwald

Science 1999 August 20; 285: 1256-1258.

pH 5.5

pH 7.0 pH 5.5




200 mM 150 mM 100 mM 50 mM 0 mM

Water channel

Osmosis: water move across membrane from a solution of low solute concentration to a solution of high concentration

Osmotic pressure of 0.5 M NaCl =Osmotic pressure of 1 M glucose


Various mechanisms for controlling cell volume
Hypotonic solution (lower concentration): cell will swell. animal cell will export inorganic ions, require Na+/K+pump to generate sodium gradient for active transport of inorganic ions. Inhibit production of ATP, cell will ? Hypertonic solution (higher conc.): cell will shrink, pH will drop; pH drop will activate Na+/H+ antiporter (Na+in/H+out) and band3 (HCO3-out/Cl-in) to increase salt concentration of cytosol.

•Plant cells are surrounded by cell wall, therefore osmotic influx of water will increase intracellular pressure (turgor pressure) but not cell volume. Turgor pressure is important in supporting the plant and plant cell elongation. •Turgor pressure is also important in regulating stomata opening

To prevent osmotic lysis, the vacuole of Paramecium caudatum (a protozoan) is filled by radiating canals that collect fluid from the cytosol. When the vacuole is full, it fuses for a brief period with the plasma membrane and expels its contents

Full vacuole

Empty vacuole

Water channel

Xenopus oocyte expressing erythrocyte mRNA encoding aquaporin

0.28 nm in diameter, 2nm long

Aquaporin1: erythrocytes Aquaporin2: kidney epithelial cells disease: diabetes insipidus, large volume of dilute urine

7.6 transepithelial transport

Amino acids



Diarrhea:a toxin of bacteria will activates chloride secretion and lead to water lost. Rehydration of diarrhea patient can be done by giving a solution of glucose and salt to drink to increase cell salt concentration.

Parietal cells acidify the stomach contents while maintaining a neutral cytosolic pH

0.1 M HCl

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