Membrane Transport

1
Transport proteins control

• Ionic composition of intracellular fluid ?
  osmolarity

• Cell volume

• Intracellular pH

• Membrane potential

• Ions gradients

• Exchange of molecules

Ion In Out Potassium 140 mM 1 4.5
mM Sodium 5 15 mM 145 mM Magnesium 5 mM 1
2 mM Calcium gt 0.5 mM 2.5 5 mM Chloride
4 mM 110 mM
2
Passive flux down the gradient of chemical
potential
3
Something controls the membrane potential
4
Movement of molecules across cell membranes
1) Diffusion through bilayer
2) Difusion through a pore (107-108 ions/sec)
3) Facilitated diffusion (102-104 ions/sec)
4) Active transport (1-1000 ions/sec)
5
Two requirements of membrane transport

• Energy to move substances

1. Light - powers H pumping - bacteriorhodopsin
3. ATP - large class of ATP-driven ion
transporters.
6
Active transport
Direct coupling of metabolism to the transport
process.
Inhibited by metabolic inhibitors such as cyanide
and dinitrophenol.
7
Primery active transport (direct energy
utilizing active transport)

• Transport protein must be activated

• ATP activates the protein by giving up a
  phosphate

Na/K-ATPase Ca2-ATPase H-ATPase H/K-ATPase
8
Secondary Active Transport (Coupled Transport)
Uniport transport of a single solute driven
only by ??
9
Symport (cotransport) amino acids and sugers
10
Antiport (countertransport) restricted to ions
11
The consequence of the transfer of charged
malecules
12
Master pump !!!
13
The master pump concept

• Creates transmembrane gradient of a selected ion.

• Other ions and molecules are transported across
  the membrane by coupling their movement to the
  movement of the selected ion.

• The electrochemical potential energy is stored
  only across the membrane in which the pump is
  located.

• Ion gradients generally store smaller packets of
  energy than ATP - coupled transporters
  (increased efficiency).

• Coupling transport to a single master pump serve
  a control function.

14
Atributes of a master pump

• Low dissipation (leakage current) is the reason
  that pumps almost exclusively transport the
  relatively impermeant inorganic cations.

• High capacity the ion gradient involve
  concentrations that are relatively large compared
  to the concentrations of the compounds that are
  to be transported.

• High efficiency

15
Na,K-ATPase

• Abundance reflects importance
• Erythrocyte 20-30 copies
• Heart cell or neuron gt 100,000 copies

Pump Activity is Electrogenic
16
Na,K-ATPase Functions

• Maintenance of high intracellular K needed for
  optimal intracellular enzyme activity.

• Maintenece of osmotic stability and cell volume.

• Restoration of potentials in excitable cells.

• Generates anergy for transport in the form of
  Na gradient.

17
A transport system might not be coupled to the
master pump

• When the transport system has a high capacity
  itself, it may adversely affect the ion gradients
  established by the master pump.

• When the transported substrate serves a
  regulatory function, then it may be desirable to
  control its concentration separately.

18
Integration of a transport systems !!!
19
Accumulation of ions and sucrose in the plant
vacuole.

• Two types of proton pumps
• V-class H ATPase
• a pyrophosphate-hydrolyzing proton pump
• They generate a lowered luminal pH and an
  inside-positive electric potential the inward
  pumping of H ions.

The inside-positive potential powers the movement
of Cl- and NO3- from the cytosol through separate
channels.
Proton antiporters, powered by the H gradient,
accumulate Na, Ca2, and sucrose inside the
vacuole.
20
Na/glucose cotransporter

• Glucose transport requires Na gradient

21
Acidification of the stomach lumen The role of
H/K ATPase
The neutral pH and electroneutrality of the
cytosol is continuously maintained.
This is the largest concentration gradient across
a membrane in eukaryotic organisms!
22
Ion channels are enzymes that catalyze the flow
of ions across cell membranes causing picoamp
current.
The catalytic rate is on the order of 107 per
second.
23
Channels (gated pore) secondary active transport
24
Properties of Ion Channels
Membrane-spanning protein
25
Voltage Gated Sodium Channel
26
Ligand-gated channel acetyl choline gated
channel
Receives acetyl choline released from the
presynaptic cleft and reinitiates an action
potential by allowing Na and K ions to pass
through the channel
2. An allosteric protein (three conformations
open, closed, and inactive).
3. Acetylcholine binding promotes opening the
closed channel
4. Open channels allow Na but not Cl to pass.
27
Methods for Studying Ion Channels

• Biochemistry
• agonist, antagonist or drug binding
• isolation and purification
• reconstitution
• radioactive ion flux

• Molecular biology
• sequencing, cloning, mutagenesis

• Structural biology
• microscopy, crystallography, NMR, ...

28
(No Transcript)
29

• Each water molecule stabilizes the ion by
  approximately 24 kT.

Roux McKinnon, Science (1999)
30
Voltage-gated K channels mediate outward K
currents during nerve action potentials.
31
Selectivity filter
Zhou et al. Nature (2001)

• K ions encounter four layers of carbonyl oxygen
  atoms a layer of threonine hydroxyl oxygen
  atoms.

• Four K ion binding sites.

• K is surrounded by eight oxygen atoms from the
  protein
• - four above and four below.
• - very similar to water molecules around hydrated
  K.

32
(No Transcript)
33
Why does the ion coordination required for high
selectivity not cause the ions to bind too
tightly prevent rapid diffusion through the
pore?
Selectivity filter contains more than one ion
repulsion between closely spaced ions will helps
overcome the intrinsic binding site affnity.
34

• They form hydrogen bonds which acts as tight
  springs that will not allow the pore to collapse.

35
The conductive conformation of the filter
requires the two K.
Entry of the second K ion induces a
conformational change.
A simple thermodynamic consequence

• Some fraction of the ion binding energy is used
  to change the filters structure.

• Consequently ions bind less tightly than if a
  conformational change did not occur.

• Weak binding is a prerequisite for high
  conduction rates.

Zhou et al. Nature (2001)
Transfer is isoenergetic ? conductivity close to
diffusion limit.