now playing: go outside

the cults

Lecture 5- transport across

membranes

Alberts, Chapter 12

matters arising

membrane transport goals

by the end of this topic, you should
know why membrane transport is important to cell
biology

know what kinds of molecules enter a cell easily

and which must be actively moved in or out
know what proteins allow transport and what
aspects of their protein structure are important to
their function and selectivity
compare the properties of channels vs active
transporters and know how they relate to energy
be able to describe how disruptions in membrane
transporters contribute to diseases or mutant
animal behaviors
be able to describe the key transporters and ions required
for maintaining resting potential and generating an action
potential

transport across membranes - overview

Simple diffusion of uncharged molecules (down
a gradient)
Osmosis
Ion channels (regulated, but diffusion down a
gradient)
Facilitated diffusion
Pumps (active transport UP a gradient)
Energy storage in electrochemical gradients
Defective transport leads to disease
Membrane potentials and nerves

hydrophobicity
N-term 1

how can you tell the topology?

50

100AA C-term

(and you understand

that actual proteins
are
more complex than
my
line drawings)

N
N

rest of the cell

C
rest of the cell

a test for membrane

experiment 1 topology
add
purify
trypsin membranes

SDS
page

trypsin is an enzyme that breaks proteins into very small pieces,

too small (generally) to see in an SDS-PAGE gel
trypsin cannot get into a cell thus transmembrane and internal proteins
are protected

a test for membrane

experiment 1 topology
SDS
page

add
purify
trypsin membranes
experiment 2

purify
membranes
or permeabilize

add
trypsin

SDS
page

return to membrane
transport
already in progress

if you put a cell in water

what happens?
how do you interpret the result?

embranes selectively allow molecules acr

semi-permeable
some molecules penetrate
the lipid bilayer:
those that are soluble in oils
or very small molecules
These may also go through
PORES
Large uncharged polar
molecules like glucose,
sucrose, amino acids
or IONS
are not permeable and
must be transported by
proteins.

(in and out)

active vs. passive transport

Passive transport: from area of high to low

concentration
Active transport: from area of low to high

active vs. passive transport

H20

H2
0
second law of thermodynamics: entropy spontaneously increases

-G is energetically favorable things move down a gra

that also means - you need to do work/expend energy to add order
to a system or to move up a gradient

energetics of solute movement

The free energy change that occurs when a solute diffuses across a
membrane is dependent on the difference in concentration on the two
sides of the membrane.
For an uncharged substance - non-electrolyte moving into the
cell:

G = RT ln Ci/Co

or

G is the free energy

R is the gas constant
T is the absolute
temperature in degrees
Kelvin
Ci/Co is the ratio of [solute]
inside to [solute] outside the
membrane

G = 2.303 RT log10 Ci/C

H2O can diffuse quickly through

membranes

H2O moves more quickly through membranes than

dissolved ions or small solutes
Osmosis: movement of H2O from an area of low
solute concentration to an area of higher solute
concentration

ghost
can get
hypotonic
lysis

Plant cells maintain turgor pressure

H2O enters/exits cells through

specialized pores called aquaporins

simple diffusion doesnt account for all H2O movement across membran

H2O molecules
pass through one by
one

Channel wall is
positively charged
and binds to
negatively charged
O; thereby
disrupting H bonds
that link H2O
http://nobelprize.org/nobel_prizes/chemistry/laureates/2003/c
molecules
together
hemanim1.mpg

facilitated diffusion (transport) of glucose

through membranes via transporter
various polar solutes are
transported by this
mechanism, down a
[gradient]

Transport 102-105
molecules/sec

Most cells contain a glucose

transporter that facilitates the
diffusion of glucose from the blood
stream into the cell to be used for

facilitative
(passive)
transporter

glucose uptake is controlled in part by

regulation of its transporter on the cell
surface

Insulin hormone
produced by endocrine
cells of the pancreas - it
maintains blood sugar
levels.

1) at low insulin levels few

transporters are on the cell
surface.
2) Insulin stimulates the
exocytosis of the glucose
transporters

Some adult type 2 diabetes results from deficiency in the Glut 4

glucose transporter.

What if a cell already has high

glucose?

it is thermodynamically
favored to move down a
concentration
gradient
At room temperature
~ 25C
G = (1.4 Kcal/mole) log10 Ci/Co

(log Ci logC0)

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

if G is negative you do not need to put

energy in.
if G is positive, it represents the energy
i
required to pump the solute across the
Cell
membrane
For diffusion of a solute to the outside of the cell Co becomes the
numerator in the concentration ratio term where the solute is
going is the numerator.

10

ENERGY IS STORED BY DIFFERENCES IN CONCENTRAT

major differences in ion concentrations

in and outside the cell
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

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

it is thermodynamically favored to move

down a concentration gradient which
can allow something else to move up its
concentration gradient

transport of glucose through

membranes via active transporters
intestinal and
kidney cells take
up glucose from
the intestinal
lumen or kidney
tubules where the
glucose
concentration is
low and actively
transport it
across the
membrane using
energy from
coupled import of
Na+ ion --an example of
symport

about these ions - why doesnt

everything go to equilibrium? how
does the cell
make
gradients?
Na+
5-15mM

Na+

145mM

K+

Mg++
Ca++

1-2
1-2

H+

pH 7.4

Cl-

110

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

how does a cell make gradients?

hard work

Cells USE
ENERGY in
different forms
to establish
gradients

movement against a concentration

gradient occurs by active transport
Na+
150 mM

15 mM

Ca2+
10-3 M

10-7 M

Active transport
requires energy
(ATP hydrolysis)
pumps mediate active
transport drive a given
ion in only one direction

movement against a concentration

gradient occurs by active transport

Pumps are critical:

maintain low pH inside lysosomes
maintain low pH inside the

key example: Na+/K+ ATPase pump

we will return to this but first more ways ions move

many kinds of ion/tranport systems

bacteriorhodopsin is a light-activated
proton pump
a photon of light
changes the
structure of
retinal, starting a
relay that results
in proton release
on the
extracellular side
then the retinal
is re-protonated

charged ions move through

gated channels
small ions (K+, Na+, Ca2+, Cl-) cant diffuse through
lipid bilayers
necessary for nerve impulses, muscle contraction,
etc.
most ion channels are selective for specific ions
when the gate is
open,
ions flow down
their concentration
gradient
-no other energy
what would supplied
happen if there was
no gate?
(fast! 10 million

who/what are the gate keepers?

voltage-gated channels: open based on differences in
membrane potential
ligand-gated channels: open based on a ligand binding to
the channel (conformational change)
mechanosensory gated channels: open in response to force
or other stimuli

conformational changes close the

channel
this is due to changes
in voltage more on
that next time

K+ channel of bacteria (KcsA

channel)
hydrophobic alpha helices
span membrane (integral
membrane protein)
tetramer
selectivity filter only
permits K+ ions to bind
K+ is normally hydrated (O);
Filter provides Os
(electronegative); Only K+
ions fit into the selectivity
filter
mammalian channels are
very very
similar

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.

a cool, hot channel

TRPV is
Transient Receptor Potential Vanilloid

gated by
ligands or by
heat (& pain)
they allow
cations such
as Ca++, Mg+
+

through

en.wikipedia.org/wiki/TRPV

TRP family
members
have diverse
activators
and initiate
signal

you discovered a new

channel!
you name your new channel Ch 303 - HD
your observation: it multimerizes and folds
in such a way that there is a very small
pore lined with amino acids with a positive
or partially positive charge

you try a patch clamp

experiment
measures ion flow by
measuring electrical current

the Na+/K+ ATPase pump

(1)
Na+ binds to the
transport protein on
the inside of the cell
with high affinity
ATP is hydrolyzed
and the released P
binds to the
transport protein
binding of P changes the
transporters configuration and
affinity for Na+ ; Na+ is released
to the outside of the
membrane

this is a P pump

flashback: for reversible chemical reactions, forward

and reverse reaction rates are equal at equilibrium

for the reaction

at equilibrium,

krev[C]

kfor
krev
and,

kfo

A +k B
r

rev

kfor[A]

k is the rate
constant,
and the forward
rate is determined
by kfor [A][B]
where [ ] denotes
Molar
concentrations

[B] =

for a given concentration

of reactants/products,
you can calculate Keq.
if Keq > 1, the forward
reaction is favored
but this does not describeeq
reaction ENER

[C]
[A][B]
=

=K

an aside on affinity
most of the time proteins are
associated with something else in the
cell in a complex (C) via non-covalent
bonds
in protein (P) -ligand (L) interactions,
affinity is described
by aofdissociation
when amount
protein
constant Kd: (alone) = the amount of
K
= [P] [L]
(ind moles)
[C]

protein in a complex (ie, HALF

of the total protein is in a
complex with the ligand),
Kd=[L]
if the protein and ligand have

the Na /K ATPase pump

(2)
+

K+ binds to the pump

P dissociates causing the pump resumes its original

conformation
this lowers K+ binding affinity
K+ diffuses into the cytoplasm

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

long-awaited crystal structures of

the Na+ K+ Pump
Ogawa H, Shinoda T, Cornelius F,
Toyoshima CCrystal
structure of the sodium-potassium pum
p (Na+,K+-ATPase) with bound potassium
and
ouabain
Proc
. Natl. Acad. Sci. U. S. A. v106, p.1374213747
Shinoda T, Ogawa H, Cornelius F,
Toyoshima CCrystal
structure of the sodium-potassium pump
at 2.4 A
resolution
Nature v459, p.446-450
Morth JP, Pedersen BP, Toustrup-Jensen
MS, Sorensen TL, Petersen J, Andersen JP,
Vilsen B, Nissen PCrystal
structure of the sodium-potassium pump
Nature v450, p.1043-1049
review: Structural
biology:Ion pumps made crystal
clear
David C. Gadsby
Nature
450, 957-959(13 December 2007)doi
:10.1038/450957a

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

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 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
lead to disease

model of an ABC transporter

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

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
autosomal recessive

Cystic fibrosis patients have a defect in an ABC transporter ( cystic fibrosis

transmembrane
conductance regulator, CFTR) with several functions

forms a cAMP-regulated Cl- channel

transports bicarbonate (HCO3-) ions
blocks Na+ channel
activates several chloride/bicarbonate
transporters

why do the mutations lead to

disease?

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:

next time:
membrane potentials,
using the energy you
have stored,
neurotransmitters,
diseases related to
membrane transport