Membrane Channels and Pumps
1. Mechanism and Regulation
2. Clinical and Physiological Relevance
Channels and pumps
Pumps:
Passive and active transports
Active: Uniport, symport and antiport
Energy sources for active transport: gradient, ATP and light
Mechanism for transport: 4 stages for P-type ATPase
Channels:
Gap junction and bystander effect.
Models of Pumps
P-type ATPase pumps:
Ca2+ pump
Na+-K+ pump
Co-transporters:
Na+-glucose pump
Na+- Ca2+ exchange
Free energy across a membrane
∆G = RT ln(c2/c1) + ZF∆V
=2.303RT log10 (c2/c1) + ZF∆V
Z: electrical charge of the passing molecule
F: faraday (23.1 kcal/V•mol )
R: gas constant (2 cal/mol•K)
T: absolute temperature (K)
∆V: membrane potential
Specific Channels
Special channels
Ligand (transmitter)-gated channels
Mechanism for activation and desensitization: acetylcholine
binds to the receptor and induces a conformational change.
Voltage-gated channels
Mechanism for operation: actin potential, depolarization and
repolarization.
Equilibrium potential (∆Veq)
Measurement of membrane conduct:
Patch-clamp techniques
Isolation of receptors using nature receptor-binding molecules.
Structures of the ion channels
Structure similarity among K+, Na+ and Ca2+ channels.
Mechanism for ion selection:
Energetic based selection (K+ channel)
Size exclusive selection (Na+ channel).
The role of amino acid residues of the selectivity filter.
Models for ion channels:
Two-site model
Ball and chain model.
Using trypsin and mutagenesis to study the mechanism of
ion channels.
Natural acetylcholine receptor binding
molecules
Nicotine
Cobratoxin
Zeng and Hawrot, J. Biol. Chem, 2002
Isolation of acetylcholine
receptors using nature
resources
Cell extract
from torpedo
Elute bound
proteins
Bungarotoxin
or
Corbratoxin
Selection of
snake toxin
binding molecule
Coordination of ion channels
Fugu (puffer fish): A fish to die for
Tetrodotoxin (from puffer fish) is
270 times more toxic than cyanide.
Clinical importance of membrane pumps and
channels
Cardiovascular disease:
Digitalis, a drug for congestive heart failure
Caner:
MDR - multidrug resistance protein
Gap junction and gene therapy for cancer
Cystic fibrosis (CF):
Cystic fibrosis transmembrane regulator (CFTR)
Electrical activation in the 3D heart model
Reproduced with permission of Physiome Sciences, Inc., 307 College
Road East, Princeton, New Jersey
MDR and Cancer
About 40% of cancer developed
multiple drug resistance due to
amplification of a part of the genome
contains Mdr1.
Gap junctions and bystander effect
Griffin GD, Oak Ridge National Laboratory
Subauste MC, et al, J. Biol. Chem., 2001
CFTR coordinates Cl-, Na+ and water transport
Sound induced signaling pathway
Touching
Pressure
Hot pepper
Temperature
Signal Transduction
1. 7TM, GH and RTK Signaling Pathways
2. Clinical and Physiological Relevance
CREB won the Nobel Prize
2000 Nobel Prize for Physiology or Medicine
was awarded to three prominent scientists:
Arvid Carlsson, Paul Greengard and Eric Kandel
Dr. Kandel, Columbia University, found
that for the long-term memory to occur,
certain genes needed to be “turned on” or
activated through the release of a protein
known as CREB 1.
Aplysia (boasts large neurons) - a
tool to study learning and memory
Short-term memories, which are stored for
minutes to hours, rely on the phosphorylation of
certain ion channels. This increases calcium
flows and thereby indirectly promotes the transfer
of neurotransmitters, thus reinforcing the
protective reflex.
Long-term memory can persist for weeks and
relies on more widespread changes affecting the
entire cell. Altered gene expression patterns mean
that new proteins are produced, which can
permanently affect the shape, size and sensitivity
of the synapse.
Dr. Kandel’s Lab
How molecular changes in a synapse may
produce short-term memory and longterm memory in the sea slug, Aplysia
GH signaling pathway
Mouse teeth and Nobel Prize
The Nobel Prize in Physiology or Medicine 1986
Dr. Stanley Cohen, Vanderbilt University School of
Medicine Nashville, TN, USA
Dr. Rita Levi-Montalcini, Institute of Cell Biology
of the C.N.R Rome, Italy
Regulation of MAP
kinase pathways
Major signaling pathways relevant to cancer in
human
Molecular biology methods for
studying signal transduction
Know ligand
binding domain
Sequencing
Dominant
negative mutant
database
search
Known
RTK
Unknown
RTK
Ligand binding domain
Affinity column
for ligand
purification
Signal transduction and diseases
Src
•
Structure: two SH domains and one kinase domain
with Tyr phosphorylation site on tail (Y527).
•
Mechanism for activation: three ways of activation.
•
V-Sar vs. c-Src: Tail or no tail, that is the problem.
Cholera
•
Lock GTP in Gαs and keep it active.
Whooping cough
•
Lock GDP in Gαi and keep it inactive.
Nobel price for studying Rous sarcoma virus
Peyton Rous, Rockefeller University, 1966 Nobel Laureate in
Medicine.
In 1910, Dr. Rous found that sarcomas in hens could be transmitted
to fowl of the same inbred stock not only by grafting tumor cells but
also by injecting a submicroscopic agent extractable from them; this
discovery gave rise to the virus theory of cancer causation.
v-Src
vs. cSrc
Tyr 527
V. Cholera in action
Whooping cough
Light induced
signaling
TRP-transient receptor
potential family: votagegated K+ or cNTP gated
channel
INAD complex
with 5 PDZ
(postsynaptic
density protein)
domains
Olfactory signaling
pathways
Bitter and sweet tastes induced signaling pathways
Bitter signaling
Sweet signaling
Development of GleevecTM (STI-571)
1. Identifying Target Gene
2. Developing GleevecTM
CML Diagnosis - Bone marrow
An abnormality of chromosomes:
The presence of the Philadelphia
chromosome (Ph), a shortened
chromosome number 22, in the
marrow cells.
95% of CML cases are associated
with a particular chromosomal
translocation [t(9;22)], which creates
a new gene - bcr-abl.
Philadelphia chromosome
FISHing the Philadelphia
Multi color FISH with three
different fluorochromes.
Chromosome painting
probes are labeled in three
fluorochromes which would
display seven different
colors.
The paints are hybridized to
chromosomes from a
chronic myeloic leukemia
(CML) cell line showing
various chromosome
rearrangements.
Brc and Abl genes
GleevecTM binds to the tyrosine kinase domain of the Abl
protein
Milestones for the discovery of GleevecTM
Cancer Res. 1996 Jan 1;56(1):100-4.
Inhibition of the Abl protein-tyrosine kinase in vitro and in vivo by a 2phenylaminopyrimidine derivative.
Buchdunger E, Zimmermann J, Mett H, Meyer T, Muller M, Druker BJ, Lydon NB.
Nat Med. 1996 May;2(5):561-6.
Effects of a selective inhibitor of the Abl tyrosine kinase on the growth of Bcr-Abl
positive cells.
Druker BJ, Tamura S, Buchdunger E, Ohno S, Segal GM, Fanning S, Zimmermann J, Lydon
NB.
N Engl J Med. 2001 Apr 5;344(14):1031-7.
Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine kinase in chronic
myeloid leukemia.
Druker BJ, Talpaz M, Resta DJ, Peng B, Buchdunger E, Ford JM, Lydon NB, Kantarjian H,
Capdeville R, Ohno-Jones S, Sawyers CL.
N Engl J Med. 2001 Apr 5;344(14):1038-42.
Activity of a specific inhibitor of the BCR-ABL tyrosine kinase in the blast crisis of
chronic myeloid leukemia and acute lymphoblastic leukemia with the Philadelphia
chromosome.
Druker BJ, Sawyers CL, Kantarjian H, Resta DJ, Reese SF, Ford JM, Capdeville R, Talpaz M.