now playing:

the Electric Slide

Lecture 10- membrane

transport 2

Alberts chapter 12

announcements
audio
grading

clicker lightning round!!

True (1)/false (2):
Everything that enters a cell
goes down a concentration
gradient.

clicker lightning round Q2!

By color code, choose the best labels for
this picture:

A) protein layer,
phospholipids,
phospholipids
transported molecules
B) plasma membrane,
protein-lipid complex,
complex
ion ion
C) phospholipid bilayer,
transmembrane protein,
protein
transported molecules

clicker lightning round Q3!

Potassium moves across
membranes via:

A) voltage gated ion channels

B) Na+/K+ pump
C) leak channels
D) A and B
E) A and B and C
F) because of charge, it cannot
move across a cell membrane

clicker lightning round Q4!

passive transport
H20

H2
0
second law of thermodynamics: entropy spontaneously increases

-G is energetically favorable molecules spontane

move down a gradient

that also means - you need to do work/expend energy to add ord

to a system or to move up a gradient

differences in ion concentrations

are established/maintained by
active transport
Na+

if everything
always moved
down its gradient,
you couldnt have this:

5-15mM

Na+

145m

H+ pH 7.2

K+
5
Mg++ 1-2
Ca++ 1-2
H+ pH 7.4

Cl-

Cl-

K+

140

Mg++ 0.5
Ca++ 10-4

5-15

110

extracellular
typical mammalian cell

how does a cell make gradients?

active transport

Cells USE
ENERGY in
different forms
to establish
gradients

and gate keepers

is this transport active

or passive??

for example, the K+ ion channel

the K+ channel is
1.a pore
2.voltage-gated
3.ligand-gated
4.heat-gated
5.a facilitated transporter
6.always open

a fly mutant for the K+ channel

http://www.pnas.org/content/suppl/2005/02/22/
0406164102.DC1/06164Movie1.mov
Episodic Ataxia Type-1 (EA-1) is considered the human equivalent
of the Shaker mutation in Drosophila. EA-1 is a rare autosomal
dominant neurological disorder that results in uncoordinated
movements (ataxia) that may last from several seconds to hours.
Genetic linkage studies have identified the gene responsible for
EA-1 as the Shaker-related Kv1.1 gene.

major differences in ion concentrations

in and outside the cell store energy
Na+

145mM

K+

Mg++
Ca++

1-2
1-2

H+

pH 7.4

Cl-

110

Na+

5-15mM

K+

140

Mg++ 0.5
Ca++

H+
Cl-

10-4

pH 7.2
5-15

(note that overall

the cell is neutral
with respect to
charge)

extracellular

typical mammalian cell

while were in the intestine.

consider: cholera toxin

intestine

What if several ion channels were

disrupted such that the salt
concentration in the lumen
(outside of the cells) was
HIGHER than that inside of the
cells in this tissue?
water?

many ATP-powered pumps

Na+/K+ pump is only in animal cells

H+/K+ pump in the stomach (pumps acid into the

stomach; pump translocates to plasma membrane
after eating)
H+ proton pump in plants and bacteria is
important for import of solutes and control of pH
ABC (ATP-binding cassette) superfamily of pumps
present in bacteria through mammals
pump ions, sugars, peptides, polysaccharides,
proteins!
Defects in pumps, transporters, or channels often

ACTIVE transport moves ions to against their

electochemical gradients
example: the Na+/K+ ATPase pump
3:2
extracellular

INTRAcellular

animation: http://highered.mcgraw-hill.com/olc/dl/120068/bio03.

critical elements of Na+/K+ pump

Pumps ions AGAINST the concentration gradient
Hydrolysis of ATP is required (P-type pump: hydrolysis of ATP
results in phosphorylation of the transport protein)
Transport protein must have a higher binding affinity for Na+
inside the cell and a lower binding affinity for Na+ outside of the
cell
Transport protein must have a higher binding affinity for K+
outside of the cell and a lower binding affinity for K+ inside the
cell
Different affinities are achieved by phosphorylating the
transport protein
Pump is necessary to maintain steep gradient required for
nerve and muscle cell activation.

Q5: with respect to the Na+/K+

ATPase,
which of the following is TRUE?
a) ions move more slowly through a pump than a
channel because the movement of 3 Na+ and 2 K+
molecules can only occur once per ATP hydrolysis
instead of continuously
b) ion movement via the Na+/K+ ATPase is an example
of passive transport
c) this pump stores energy in the form of
electrochemical gradients that is only required for
nerve cell activation.
d) the crystal structure of this molecule was determined

model of an ABC transporter

eption
c
x
e
n
a
CFTR is
ed but
P
s
i
t
i
t
in tha
se that
u
t
o
n
s
doe
ove Clm
o
t
y
g
ener

multi-drug resistance (MDR)

tumor cells become resistant to chemotherapy
MDR-1 protein is part of a pump (ABC
transporter) that pumps toxic materials out of cells
MDR-1 expressed in normal liver and kidney to
export toxic molecules (e.g. bile, urine)
MDR-1 transports many drugs/toxic materials
BUT in cancer cells: MDR-1 gene is amplified and
over-expressed
so chemotherapy drugs that diffuse through the
cancer cell membrane are pumped out

another pump: Ca++ ATPase

transports Ca++ to keep it at a
low in [ ] the cytosol
hydrolyses one ATP
for each Ca++ ion
transported
can export Ca++
(on plasma
membrane)or
pump it into the ER
(or sarcoplasmic
reticulum) for
storage when
needed

coupled transport
(coupled pumps)

the Na+/Ca++ exchanger

(NCX) works by antiport to
control Ca++ levels
out

3Na+

removes calcium from the cytosol

by using energy stored by the Na+
gradient (one Ca++ out for 3 Na+ in)
secondary active transport (coupled)

cytosol

has a low affinity for Ca++ but high

capacity (ie, fast at high
concentrations)
Ca++ is an important messenger
1 Ca++
within the cell and can be released
to send signals, for example for the
heart muscle to contract

ouabain
ouabain and digoxin have
been used to treat congestive heart
failure
Na+

these drugs bind to the Na+/K+ pump and

inhibit it, resulting in higher intracellular sodium.
this reduces the NCX function and yields higher
intracellular calcium, making heartbeats
Na+
stronger and more frequent.

1Ca++

Ca2+ is an important second

intracellular messenger
[Calcium] low in the cytosol
due to pumps and the
membrane is highly
impermeable to the ion.

intracellular
calcium release
within a neuron

Calcium channels can be

transiently opened by action
potential or calcium itself.
Calcium binds to calciumbinding proteins, which
can initiate downstream
signals.

diseases linked to ion channels

case study: Cystic Fibrosis (CF)

abnormal secretion of fluid by epithelial cells of
the airways
CF patients make thick, sticky mucous that stays
in airways
cilia cant move properly so bacteria remain
infections and inflammation
destroys lung function
lethal
1/25 people of Northern European origin are
genetic carriers; 1/2500 infants affected

Cystic fibrosis patients have a defect in an ABC

transporter (cystic fibrosis transmembrane conductance
regulator, CFTR)

CFTR functions:
forms a cAMP-regulated
Cl- channel (passive
transport)
transports bicarbonate
(HCO3-) ions
blocks Na+ channel
activates several
chloride/bicarbonate
transporters

why do the mutations lead to

disease?
CF patients have a higher-than-normal
salt (solute) concentration inside cells of
the lungs. according to our recent
lectures, more water will?
less water secreted by the cell results in thicker
mucus; causes airway obstruction

Cystic Fibrosis patients

commonly have a particular
amino acid change in the CFTR
protein, F506
CFTR is a ABC transporter that acts at the
plasma membrane by transporting Cl- and
suppresses Na+ ion channel function in
epithelial cells
to study this mutation, you clone both normal
and F506 mutant copies of the gene and add a
GFP tag. you then express the protein in cells
and look for the GFP expression. you see:

CFTR expression experiment

white light image

normal CFTR+GFP
CFTR+GFP
blue stain for nucleus

F506

fluorescent light images

clicker 6: based on this data, you conclude

A.the F506 CFTR mutant protein is no longer
expressed in the cell
B.the F506 CFTR mutant protein is no longer
expressed on the cell surface
C.the expression of F506 CFTR mutant protein disrupts
nuclear staining
D.the F506 CFTR mutant protein is expressed on the

CFTR expression experiment

2
normal CFTR+GFP
F506 CFTR+GFP
white light image
blue stain for nucleus
+ red marker for ER

fluorescent light images

note, for
clicker 7: based on this data, you conclude

LIGHT
GREEN AND
normal CFTR protein is expressed in the
REDER
MAKE
YELLOW
F506 CFTR mutant protein is no longer

A.the
B.the
expressed in the cell
C.the F506 CFTR mutant protein is localized to the
Golgi
D.the F506 CFTR mutant protein is localized to the ER

it is thermodynamically favored
to move down a concentration
gradient thus, movement down
a concentration gradient can
occur by passive transport
At room temperature ~ 25C

G = (1.4 Kcal/mole) log10 Ci/Co

in this example, Ci/Co will be less than 1 and G
will be negative and a net INFLUX of solute is
thermodynamically favored.

Co
Ci

Cell
if G is negative you do not need to put
energy in.

ENERGY IS STORED BY DIFFERENCES IN

CONCENTRATION

10

for an electrolyte to move across the

membrane,
2 gradients must be considered
1) chemical gradient determined by the
concentration difference of the substance on the
two
sides
of the membrane
2) the
electro-potential
gradient determined by the
difference in charge between the two sides of the
membrane
thermodynamically unfavorable for
an electrolyte to come into a
o
compartment having a charge of the
same sign.

thermodynamically favored for

an electrolyte to move to a
compartment of the opposite
charge.

Ci

10

free energy difference for movement

of an electrolyte across a membrane
into a cell:
G =RT ln Ci/Co + zFEm

z is the valence of the solute

F is the Faraday constant = 23.06 Kcal/volt equivalent (amount
of electrolyte having 1 mole of charge)
Em = potential difference in volts between compartments

Moving ions against a concentration OR electrical

gradient requires work (input of energy)

ENERGY IS STORED BY DIFFERENCES IN CHARGE (POT

the difference in charge across

the membrane is the
membrane potential
largely due to Na+ K+ ATPase and
some flow of K+ back via leak
+++++++
channels
other channels
essentially closed

+++++

----------- -70mV is a standard -potential,

but
----

varies according to cell type/organism

NERST EQUATION: V = 62
log Co/Ci

changes in membrane potential

regulate the K+ channel
the voltage required
to open/close the channel
is cell/protein type dependent

there are many

different genes encoding
K+ channels

depolarization
of membrane

spontaneous

reset

major differences in ion concentrations

in and outside the cell store energy
Na+

145mM

K+

Mg++
Ca++

1-2
1-2

H+

pH 7.4

Cl-

110

Na+

5-15mM

K+

140

Mg++ 0.5
Ca++

H+
Cl-

10-4

pH 7.2
5-15

extracellular

typical mammalian cell

how are these

differences
generated?

ACTIVE transport moves ions to against their

electochemical gradients
example: the Na+/K+ ATPase pump
3:2
extracellular

INTRAcellular

BUT
K+ also goes back out
through
LEAK CHANNELS

the difference in charge across

the membrane is the
membrane potential
largely due to Na+ K+
ATPase and some flow of K+
back via leak channels (rest
closed)
-70mV (depending on cell)
described by Nerst
Equation
in a nerve or muscle cell
resting potential
can be measured with
electrodes-

+++++++
+++++
----------------

the difference in charge across

the membrane is the
membrane potential
largely due
to Na+ K+
ATPase and
some flow
of K+ back
via leak
channels
(rest closed)

an action potential is the rapid

change in charge across the
membrane
+++++++

occurs in excitable cells, ie,

nerves, muscle, beta
pancreatic cells, endocrine
cells, some plant cells
takes place via the rapid
opening of voltage gated
ion channels (then closing)

+++++
----------------

can be measured with

electrodes- especially if
you are working with giant
squid nerve cell (1 mm
diameter!)

msec timeframe

nerve cell anatomy

d here
e
v
i
e
c
e
r
signals

sign
a

ls re
leas
ed h
ere

the myelin sheath acts as an

insulator
membrane!
Schwann cells or
oligodendrocytes

the disease
Multiple
sclerosis
(MS)
is caused
by
disruption
of the
myelin
sheath

action potential
stimulus depolarizes the
membrane
Na+ channel opens, Na+
influx +40mV (close to
equilibrium)
change in potential opens
K+ gates -80mV
(repolarization)

this uses stored energy ions flow down their electrochem

action potential
Na+
3) Na+
1) stimulus
voltage
2) depolarization gated
Na+
(past threshold) channels
open in

-70mV

K+

-50mV

+40mV

4) K+
voltage
gated
channels
open (Na+
channels
close)

K+
out

5) K+
channels
close
(although
leak channe
are open)

-80mV

(does slowly
http://www.youtube.com/watch?v=ifD1YG07fB8
leak out) also, animations at:
http://highered.mcgraw-hill.com/olc/dl/120107

be a sodium channel

action potential

stimulus depolarizes the

membrane
Na+ channel opens, Na+
influx +40mV (close to
equilibrium)
change in potential opens K+
this
usesstored
gates
-80mVenergy ions flow down their electrochem

action potentials are

propagated as current flow

myelinated cells propagate

impulses 20x faster by
restricting areas of Na+
influx to nodes 120 m/s

resting experiment

Q9: you expect to find:

a)no labeled Na+ or K+
b)labeled Na+ only
c)labeled K+ only
d)labeled Na+ and K+

action potential experiment

Q10: you expect to find:

a) no labeled Na+ or K+
b) mostly labeled Na+
c) mostly labeled K+
d) equal labeled Na+ and K+

action potential experiment

2

Q11: can you fire an

action potential?
a) yes
b) no

nerves talk to other cells

synaptic signaling

transmitter gated
ion channel

- many types of neurotransmitters and receptors:

acetylcholine, glutamate, serotonin, GABA (-aminobuteric acid), glycine
- excitatory -> Na+ channels; inhibitory -> Cl- channels;
- these molecules and receptors/ion channels are targeted by classes
of drugs used to treat depression and other mental health disorders

nerves talk to muscles at the

neuromuscular junction

for more on
all of these
issues, take
Biol 305
(animal physiology)

receptor for the neurotransmitter

acetylcholene is an ion channel
cryo-EM/
electron
crystallography
resolution is
not as good as
x-ray, but can
use protein+
membrane

how can
(membrane)
proteins be
studied?
combinations of
approaches are
always better!

reminder/clarifications on
specifications grading
final current (add - convert Hw grade to 6 pts)
A > 496 points 124
B > 448 points 112
C > 352 points 88
D > 304 points 76
F > 304 points

next time:
Dr. Whitworth will
lecture on
mitochondria;
see Blackboard
site for online
homework