Epigenetics and Membrane Transport

8/11/2019 Epigenetics and Membrane Transport

http://slidepdf.com/reader/full/epigenetics-and-membrane-transport 1/82

Cell and Membrane Trafficking

• Kid #3: Hey, mister. Ain't you got a car?

Eddie Valiant: Who needs a car in L.A.? Wehave the best public transportation system

in the world.

•Judge Doom: A few weeks ago I had the

good providence to stumble upon a plan of

the city council. A construction plan of epic

proportions. We're calling it a freeway.

Eddie Valiant: Freeway? What the hell's a

freeway?Judge Doom: Eight lanes of shimmering

cement running from here to Pasadena.

Smooth, safe, fast. Traffic jams will be a thing

of the past.

From “Who Framed Roger Rabbit?”

http://www.imdb.com/name/nm0802648/












Announcements

• Next exam is Tuesday, Nov. 12, at 8 PM.• This is the last exam before the final.

• Quiz 7 is now available and is due Thursday, Nov

7th, at 11 pm.

• Keep working on CELLLS Part 2.



Epigenetics

• Barbara Stefanska, PhD

– [email protected] – MS, Public Health at Medical University of Lodz, Poland in 2003

– PhD, Biomedical Sciences - Nutritional Epigenetics at Medical University ofLodz, Poland in 2007

– Post-Doc, Cancer Epigenetics at McGill University, Montreal, Canada in2013

– “Epigenetics refers to the molecular events controlling gene expression thatare independent of changes in the underlying DNA sequence. These eventsinclude DNA methylation, covalent histone modifications, and non-codingRNA-related mechanisms. Epigenetic modifications of DNA have beenshown to contribute to the etiology of chronic diseases like cancer.However, epigenetic changes are dynamic and serve as an adaptivemechanism to a wide variety of environmental factors including diet.”

mailto:[email protected]

mailto:[email protected]



Clicker Question

Who is interested in

potentially helping with

research?

A. Sorry – too busy.

B. Maybe – can you tell

me more?

C. Love to – where can I

sign up?



5

Purdue UniversityNovember 5, 2013, West Lafayette, IN

Nutriepigenomics:

New Horizons in CancerPrevention and Therapy

Dr. Barbara Stefanska

Assistant Professor

Department of Nutrition Science

Purdue University, West Lafayette, IN, [email protected]



6

Outline

1. Gene-diet interaction

The role of epigenetic modifications in regulation of gene

transcription

Reversibility of epigenetic alterations upon dietary exposures:

• Effects of vitamins and polyphenols on tumor suppressor genes

• Genome-wide remodeling of DNA methylation patterns by nutrients

involved in one carbon metabolism

2. Research projects in Dr. Stefanska’s Lab

• Epigenetic mechanisms of polyphenols in cancer prevention and

therapy

• Epigenetic biomarkers for solid tumors



7

A twin approach to unravelling epigenetics

Identical twins who were given up for adoption to different families

at birth and only discovered this in their mid-30s.

Stefania Moretti, CBC News

• Identical twins start with the same

genetic make-up but over time in

phenotype they drift apart

• Epigenetic variation as a dynamic

quantitative trait

• High monozygotic twin discordance

rates for common diseases



8

Components of the epigenome

Ac Me Ac Ac

D I E T

A

B U S I V E B E H A V I O

U R

Non-coding RNA



9

Role of DNA methylation in regulation of gene transcription

Portela and Esteller, Nat Biotechnol 2010

Hypermethylation

Hypomethylation

Silencing

Activation



DNA methylation alterations in cancer

Fernandez AF et al., Trends in Genetics, 2012 (modified)

Tumor suppressors Oncogenes

Hypomethylated and

Activated

Hypermethylated and

Silenced

Healthy tissue

Early/Late Cancer stage Early/Late Cancer stage

Healthy tissue

Repetitive

sequences/Transposons

Healthy tissue

Chromosomal

rearrangements/Genome

instability

OFF



11

ATRA

0

0.5

1

1.5

2

PTEN APC RARbeta2

F o

l d c

h a n g

e

( A T R A / C t r l )

Methylation Expression

Vitamin D3

0.0

0.5

1.0

1.5

2.0

PTEN APC RARbeta2

F o

l d c

h a n g

e

( V i t . D 3 / C t r l )


Resveratrol

0.0

0.5

1.0

1.5

2.0

PTEN APC RARbeta2

F o

l d c h

a n g e

( R E S / C t r l )


Activation of tumor suppressor genes in breast cancer

**

**

* * ** *

*

** *

* P <0.05 vs. control

*



12

Sensitizing breast cancer cells to anticancer agents

ATRA Vit.D3 RES

+ 2CdA

0

20

4060

80

ATRA 2CdA 2CdA +

ATRA

+ F-ara-A

0

20

40

60

80

ATRA F-ara-A F-ara-A

+ ATRA

+ 5-aza-dCyd

0

20

40

60

80

ATRA 5-aza 5-aza +

ATRA

+ 2CdA

0

20

4060

80

Vit.D3 2CdA 2CdA +

Vit.D3

+ F-ara-A

0

20

40

60

80

Vit.D3 F-ara-A F-ara-A

+ Vit.D3

+ 5-aza-dCyd

0

20

40

60

80

Vit.D3 5-aza 5-aza +

Vit.D3

+ 2CdA

0

20

4060

80

RES 2CdA 2CdA

+ RES

+ F-ara-A

0

20

40

60

80

RES F-ara-A F-ara-A

+ RES

+ 5-aza-dCyd

0

20

40

60

80

RES 5-aza 5-aza +

RES

C e l l v i a b i l i t

y [ ( v i a b l e t r e a t e d / v i a b l e u n t r e a t e d ) x 1 0 0 %

] **

** ** ** ** ****##^^ **##^^

**##

** ** ****

** ****##^ **#^^ **##^^

** **** ** **

****##^^

**#^^ **##^^

*P <0.05, ** P <0.01 vs. control, #P <0.05, ##P <0.05 vs. natural, ^ P <0.05, ^^ P <0.05 vs. analog



DNA methylation alterations in cancer

Fernandez AF et al., Trends in Genetics, 2012 (modified)

Tumor suppressors Oncogenes

Hypomethylated and

Activated

Hypermethylated and

Silenced

Healthy tissue

Early/Late Cancer stage Early/Late Cancer stage

Healthy tissue

Repetitive

sequences/Transposons

Healthy tissue

Chromosomal

rearrangements/Genome

instability

OFF



14

N o r m a l # 1 5

N o r m a l # 1 4

N o r m a l # 5

N o r m a l # 6

N o r m a l # 4

N o r m a l # 1

N o r m a l # 1 1

N o r m a l # 9

N o r m a l # 1 0

N o r m a l # 1 2

N o r m a l # 8

C a n c e r # 8

C a n c e r # 1 2

C a n c e r # 9

C a n c e r # 1 0

C a n c e r # 1 1

N o r H e p

H e p G 2

C a n c e r # 1 4

C a n c e r # 6

C a n c e r # 5

C a n c e r # 1 5

C a n c e r # 4

C a n c e r # 1

Blue – Normal Red - CancerColor key

and histogram g en e s

-2 2

Hypermethylated

1,572

Suppressed

3,182322

Hypomethylated

1,752

Induced

2,667230

Landscape of DNA methylation in liver cancer patients



15

Pathways associated with differentially methylated genes

Pathways repressed in liver cancer, associated

with hypermethylated genes

0 2 4 6 8 10 12

Cell adhesion molecules (CAMs)

Focal adhesion

Protein export

ABC transporters - General

Taste transduction

Apoptosis

Metabolism of xenobiotics by P450

Folate biosynthesis

Calcium signaling pathway

Cytokine-cytokine receptor interaction

p53 signaling pathway

Toll-like receptor signaling pathway

% genes

Pathways induced in liver cancer,

associated with hypomethylated genes

0 2 4 6 8 10

Metabolic pathways

Basal transcription factors

Cytokine-cytokine

interaction

Chemokine signaling

MAPK signaling

GnRH signaling

WNT signaling

PPAR signalingmTOR signaling

JAK-STAT signaling

VEGF signaling

Hedgehog signaling

TGF-beta signaling

Glycolysis/gluconeogenesis

% of genes



16

SNORA/D, SLC,

KIAA families

BCAS, CDH families

ZNF family KLK, SIGLEC, CEACAM families

FAM family KRT family SLC family

CCL family

SULT1A family CDH family

KRTAP family

GAGE family

MAGE family

CECR, DGCR, GSTT families

Chromosome 17

Chromosome 16

Chromosome 19

Chromosome 20

Chromosome 21

Chromosome 22

Chromosome X

Hypomethylated genes clustered across the genome



17

Prostate cancerLiver cancer 14122030 8139

Hypomethylated genes

Non-invasive LNCaP

prostate cancer cell line andinvasive PC3 prostate

cancer cell line

6 days

Genes hypomethylated in liver and prostate cancer

Illumina 450K

DNA isolation



18

S-adenosyl-Lmethionine (SAM) as hypermethylating agent

Folate

THF

5,10-MTHF

serine

glycine SHMT

B6

5-CH3-THFB2

Methionine

Homocysteine

MSB12 BHMT

betaine

dimethyl

glycine

choline

SAHH

DNMTs

MTs

HMTs

Methylation

reactionsglutathione

CBS B6

SAH

SAM

MAT

GNMT

MTHFR

DHFR

One carbon metabolism



19

Hypomethylated in

prostate cancer

SAM

hypermethylated11149 9440

Hypomethylated genes targeted by SAM

Genes targeted by SAM in prostate cancer

6 days

Illumina 450K

DNA isolation

PC3 prostate cancerSAM treatment

(250 µM) every 2 days



20

Cell Growth

0

100

200

300

400

500

0 2 4 6 8

C e

l l n u m

b e r

( t h o u s a n

d )

Ctrl SAM 250µM

Invasion assay

0

50

100

150

N u m

b e r o

f c e

l l s

i n v a

d e

d

Ctrl SAM 250µM

Soft agar assay

0

10

20

30

40

N u m

b e r o

f c o

l o n i e s

Ctrl SAM 250µM

Ctrl SAM 250µM

Functional role of DNA methylation changes upon SAM treatment

**

*

*

*P <0.05 vs. control



21

PC3 prostate cancer

SAM treatment(250 µM) every 2 days

6 days

Functional role of DNA methylation changes upon SAM treatment

Ctrl

SAM



22

Conclusions

1. Environmental factors including diet trigger epigenetic changes, in particular

alterations in DNA methylation, and modulate the disease states.

2. Resveratrol, vitamins A and D3:

• decrease DNA methylation and activate tumor suppressor genes in breast

cancer (DNMT1, p21),

• inhibit cancer cell growth and sensitize the cells to anti-cancer agents.

3. Hypomethylation in cancer is as persistent as hypermethylation and associated

with activation of cancer driving genes.

4. SAM, the ubiquitous methyl donor:

• hypermethylates (silences) genes involved in functions that are essential

for cancer progression and metastasis,

• suppresses cell growth, anchorage independent growth and invasiveness

in vitro, and inhibits skeletal metastasis

in vivo.



23

Research Projects in Dr. Stefanska’s Lab

1. Epigenetic mechanisms mediating the beneficial effects of polyphenols

in cancer prevention and therapy

2. Epigenetic biomarkers in cancer



24

Resveratrol

Pterostilbene

EGCG

50% down-regulation of DNMT1 in MCF-7 cells

Activation of methylation-silenced tumor suppressor genes

such as PTEN and RARbeta

Binding to the catalytic domain of DNMT1 and inhibiting its activity

Activation of methylation-silenced tumor suppressor genes

such as p16, RARbeta, MGMT, hMLH1 in cancer cells

Similar to resveratrol anti-cancer, anti-oxidant effects,

lowering blood lipids and cholesterol, lowering bloodglucose level (diabetes)

Project 1 - Polyphenols



25

Fisher-344 Rats

Methyl donor deficient diet

+defined amino acid diet

1, 4, 24, 56 weeksLiver cancer

Resveratrol

Pterostilbene

EGCG

Resveratrol

Pterostilbene

EGCG

Prevention Treatment

Project 1 – Animal studies



26

Liver cancer: HepG2,

SkHep1, NorHep

High density promoter

Microarray(MeDIP-seq)Methylated DNAimmunoprecipitation

(MeDIP)

RNA pol II CTD phospho-

Ser5 chromatin

immunoprecipitation (ChIP)

Methylation

Transcription onset

(expression)

Histone activating mark

H3K4methylation

(ChIP)


microarray

(ChIP-seq)


microarray

(ChIP-seq)

Resveratrol

Pterostilbene

EGCG

Project 1 – Genome-wide studies



27

European Prospective Investigation into Cancer and Nutrition (EPIC)

Cancer Prevention Study (CPS) of American Cancer Society

Diet (methyl-donors and polyphenols)

and liver/ovarian cancer risk

Epigenetic biomarkers for liver/ovarian cancer

EPIC cohort and CPS cohort

Project 2 – Biomarkers in prospective cohorts



28

Risk development biomarkers Prognostic biomarkers

Blood samples prior

and after diagnosis

Liver cancer/Ovarian cancer

Genome-wide DNA methylation analysis

(Illumina 450K microarray)

DNA isolation and

bisulfite conversionMethylation

Biomarkers(validation by pyrosequencing)

Project 2 – Epigenetic biomarkers

Diagnostic biomarkers



29

Research Projects in Dr. Stefanska’s Lab

1. Epigenetic mechanisms of polyphenols in cancer prevention and therapy

• prevention and attenuation/reversal of liver cancer induced by methyldonor deficient diet;

• genome wide changes in DNA methylation, transcription onset and histone

modifications upon treatment with polyphenols (identifying targets,

pathways, networks, possible mechanisms);

• importance of epigenetic mechanisms for gene regulation in cancerprevention and therapy.

2. Epigenetic biomarkers in cancer

• early diagnosis, prognosis, therapy outcome;• epigenetic alterations analysed in blood instead of tissue biopsies have

predictive potential;

• nutritional aspects (polyphenol intake and cancer risk).

[email protected]



TRANSPORT ACROSS THE CELL

MEMBRANE

• Cell membrane mediatescommunication between

cell and external

environment.

• Movement of nutrients,ions, waste products etc.

through cell membrane.



Passive and Active Transport



• Diffusion in a liquid or within the

membrane is the motion from a high

to low concentration.

• The change in concentration along

a spatial dimension is the

concentration gradient.

• Diffusion can be passive, in which

molecules move from high to low

concentrations along the

concentration gradient

• Or facilitated, in which molecules

are chaperoned across the

membrane along the gradient

• Transport can be active, in which

molecules move from low to high

concentrations against the

concentration gradient. Active

transport requires energy.

F i l h b



Four non-vesicular ways to cross the membrane

a) Passive diffusion across membrane

b) Passive diffusion through aqueous channel

c) Facilitated diffusion through channel

d) Active transport through channel

e) In A-C, diffusion is from high concentration to low along a gradient

while it is against the gradient in D.

Examples of each type of movement across



Examples of each type of movement across

the membrane

• O2 and nonpolar substances can passively diffuse across (slowly)

• When a channel is opened, ions flow along their electrochemical gradient

• Glucose entry is facilitated by a glucose transporter

• Na-K ATPase is an exchange mechanism that uses energy to transport both ionsagainst their concentration gradient, from [Low] to [High].



Cystic Fibrosis

• Hydrating mucous layer used to move bacteriaout of airway

– Chronic infections often follow



Clicker Question

Cystic Fibrosis is caused by a mutation to what

type of channel?

A. Passive Na+ and Cl- ion channel

B. Active Na+ and Cl- ion channel

C. Passive H20 channel

D. Active H20 channel



Our respiratory and circulatory systems use very large

areas, steep concentration gradients, and relatively short

paths to transport oxygen via diffusion

Our lung alveoli together form a surface area the size of a tennis court!



For a membrane, permeability is determined by:

• The ability to pass through the membrane directly

• The ability to pass through an aqueous pore in the membrane

• Can estimate permeability through the membrane by measuring

the partition coefficient

• Partition coefficient is the ratio of solubility in nonpolar versus polar

solvent (typically water)



Drug permeability is quite correlated with lipid solubility



TRANSPORT OF WATER: OSMOSIS

• Water flows from a region of LOW SOLUTECONCENTRATION (High water potential) to

HIGH SOLUTE CONCENTRATION (Low water

potential) across a SEMI PERMEABLE

MEMBRANE.



Solute Concentration

?



HOW DOES WATER CROSS THE CELL MEMBRANE?

• Water, as expected has a very low oil/water partition coefficient

• Its permeability is far above what would be expected based on this.

WHY?

Lik t f h l A i



Like water for channels: Aquaporins

• Aquaporins are a family of water permeable channels discovered by Peter

Agre’s group in the early 1990’s (Nobel Prize 2003)

• Very high flux rate, up to 1 billion waters/second

• Left, one subunit, with multiple transmembrane portions

• Tetramers each with a specific, water permeable channel

King et al. 2004



Aquaporin



Proposed mechanism of

aquaporin action

• Hydrophobic side chains intomembrane and protein backbone linespore

• H atoms of water form hydrogen bonds

with carbonyl O of backbone• In center of pore, two + charged amines

from backbone align O of watermolecule and interrupt H-bond network



Practical importance of aquaporin

• Aquaporins rapidly regulate osmotic balance

• Aquaporins are expressed throughout the body, but are particularly

important for water balance in the kidneys.

• ADH, or vasopressin, is released from the posterior pituitary and

stimulates insertion of AQP2 channel in collecting ducts of kidney

• This allows water to be absorbed as it passes through the kidneys

• Alcohol suppresses ADH (vasopressin) release, so water that would

normally be absorbed passes through to the bladder instead, with obvious

consequences.

The free energy of solute movement



The free energy of solute movement

DG= 1.4 log10[Cin/Cout] for movement of a solute into a cell.

• So if Cout is greater than Cin, DG is negative, because log10[Cin/Cout]

will be negative.

FACILITATED DIFFUSION



FACILITATED DIFFUSION

• Solute binds to transporter

molecule• No energy required

• Bidirectional, follows concentration

gradient

• In this example, glucose binds to thetransporter

• Glucose then dissociates and the

transporter reforms in its original

conformation• Like enzymes, transporters are

highly specific, including

stereospecific



RATE OF FACILITATED DIFFUSION

• Much faster than passive

diffusion

• Slower than channels, only up

to about 102 to 104

molecules/s for facilitated

diffusion vs 107 or 108 /s for

channels

• Saturation kinetics

determined by number of

transporters

Glucose transport during insulin stimulation



Glucose transport during insulin stimulation

• During basal conditions, few GLUT4 glucose transporters in fat cells

(adipocytes), endocytosis>exocytosis

• Upon activation of insulin receptors, GLUT4 are recruited to membrane in

order to remove excess glucose from the blood, which it can then convert

to adipose tissue (fat).

• Humans have at least 5 transporters, GLUT1-5, and there are at least two

others found in other species

Mueckler 1994

GLUT4: INSULIN

DEPENDENTGLUCOSE

TRANSPORTER



Active transport

• It takes lots of energy to maintain the highly non-equilibrium conditions that

drive cell processes

• In active transport, the endergonic movement against a concentration

gradient is coupled with an exergonic process, such as ATP hydrolysis, electron

transport, electrical potentials, or light absorbance



Channel Rhodopsin

• Light-driven proton pump• Responsible for the perceptionof light

• Absorption of a photon of light

causes change in electronicstructure

• Important for Optogenetics

– Control and monitor theactivities of individual neurons inliving tissue

– http://www.youtube.com/watch?v=v7uRFVR9BPU

Active transport: Na+/K+ ATPase example

http://www.youtube.com/watch?v=v7uRFVR9BPU







Active transport: Na+/K+-ATPase example

• Na+out=150 mM, Na+

in=10 mM

• K+out=5 mM, K+

in=100-150 mM

• These large concentration imbalances are maintained by the Na/K pump

• Na/K ATPase: Hydrolizes ATP & transport of Na and K.

• 3 Na+ are pumped out for each 2 K+ that come in, so the pump is

electrogenic and will also create a charge difference across the

membrane

http://www.youtube.com/watch?v=awz6lIss3hQ&feature=related

Na+ binds on intracellular side and ATP is hydrolyzed





Na+ binds on intracellular side and ATP is hydrolyzed

to start the exchange

• 3 Na+ ions bind within the pump, which is in the resting (E1) conformation

• ATP hydrolysis phosphorylates an aspartate on pump

• Phosphorylation alters the pump protein conformation

Phosphorylation alters the pump conformation to E2 and



Phosphorylation alters the pump conformation to E2 and

changes the binding affinity for Na+

• Lowering the affinity and closing the path back to the cell release Na+ ionsinto the extracellular environment

Following Na+ release, K+ ions are able to bind while pump is



g , p p

in E2 conformation

• 2 high affinity binding sites for K+

• As K+ binds, the pump protein dephosphorylates

The dephosphorylated pump protein changes back to E1



conformation and releases K+ into the cell

• Regulation of this pump is critical, because its responsible for 1/3 of energy use innon-neuronal cells, and 2/3 in neurons

• Not present in plant cells• Orientation in membrane is important. In resealed RBC ghosts, K+ had to be

outside, Na+ and ATP inside & pump in membrane

• Blocking Na/K pump can actually be beneficial in heart disease and is a commondrug treatment

H+/K+ P-type pumpV i l



H+/K+ P type pump

• Stomach lining H+/K+-ATPase - P-type pump. Secretes acid solution (up to0.16 N HCl) into stomach.

• With food, hormones cause pump-containing membranes to move to apicalsurface and secrete acid. Ridges in cell surface called canaliculi.

• Overactivity of this pump can lead to heartburn.

• Prilosec directly blocks this pump. Zantac and Tagamet block the hormone

receptors that enable pump insertion into membranes.

Vesicles

containing pumps

in membrane

GOING MY WAY? ACTIVE TRANSPORT BY



COUPLING WITH CONCENTRATION GRADIENTS

favorable electrochemical gradient + an unfavorable one

secondary active transport

• Sodium ions cotransport of glucoseintestinal epithelium

• Na+ moves glucose against its gradient asNa+ flows down its gradient across apicalmembrane

• Na+/glucose cotransporterprotein binds 2 Na+ ions & 1glucose molecule

• When Na+ ions release on inside of cell,

protein changes conformation and losesaffinity for glucose. Glucose is released aswell

• Glucose diffuses through cell & movesacross basal membrane by facilitateddiffusion

Na+out=150 mM,

Na+in=15 mM

Sodium ->along

glucose-> against

cotransport

FREE ENERGY OF CO-TRANSPORT:



FREE ENERGY OF CO-TRANSPORT:

CLICKER QUESTION

This coupling of a favorable electrochemical gradient with an unfavorable

one for active transport is called secondary active transport

ENERGY GAINED FROM Na+

For 1 Na+ ion moved ALONG ,

D

G=-3.1 kcal/mol

For 2 Na+ ions moved ALONG ,

DG=-6.2 kcal/mol

DG= 1.4 log10[Cin/Cout]

Roughly what Cin/Cout glucose ratio couldbe achieved when Na+ ions are

cotransported with glucose across the

intestinal epithelium?

A. 0.5:1

B. 100:1C. 8,000:1

D. 27,000:1

E. 100,000:1



Clicker Question

Roughly what Cin/Cout glucose ratio could be

achieved when Na+ ions are cotransported with

glucose across the intestinal epithelium?

A. 0.5:1

B. 100:1

C. 8,000:1

D. 27,000:1

E. 100,000:1

Cl ifi ti f t t



Classification of transporters

• Uniport system - transport one solute at atime, such as facilitated diffusion of glucose

• Cotransport systems - >1 solute movedacross membrane at same time by a single

transport molecule • 2 transported solutes move in same

direction (symport) such as Na+/glucosetransport

• Transported solutes move in oppositedirections (antiport or exchanger), such asNa+/K+ pump or Na+/H+ pump

ION CHANNELS



ION CHANNELS

Integral membrane proteins forion transport

GATING: Channel

opening/closing is

regulated.

Selectivity: Only

specific ion (Na+, K+,

Ca++, Cl-) can passthrough the channel.

KcsA Channel: Bacterial K+ ion channel



KcsA Channel: Bacterial K+ ion channel

http://www.youtube.c

om/watch#!v=UqxzS

rjzJ70&feature=relat

ed

Zagotta, 2006

What determines the selectivity of KcsA channels?

http://www.youtube.com/watch










y

• Conserved residue sequence GYGVT of P segment

lines selectivity filter• The double-bonded O in pore are just large

enough to admit K+ and replace hydration shell of

K+ (3.0 A diameter vs 2.7 A diameter for K+)

• Na+ ions do not interact with the pore O atoms

well and cannot easily desolvate. It is moreenergetically favorable to remain hydrated.

• Each K+ stabilized by 8 O atoms, 4 above and 4

below.

• 4 potential binding sites, but only two occupied at

any given time

Hint: Not

these guys



GATING MECHANISMS

• Voltage-gated is when the membrane potential determineswhether the channel is open or closed (e.g. Na+ channel inneurons)

• Ligand-gated channel is when binding of a ligand to a receptoropens the channels (e.g. glutamate, acetylcholine)

• Mechano-gated is when mechanical force opens channels (e.g.hair cells in inner ear)

Mechanisms of KcsA gating and opening



Mechanisms of KcsA gating and opening

• Gated by low pH < 4.0.

• Previous figure was closed with K+ ions in pore

•M2 helices form a helix bundle in closed conformation in a related channel

• In low pH conditions, a glycine allows M2 to bend like a hinge and open



K+ channel inactivation terminates K+ flux



• Inactivation is a critical

mechanism to limit ion flux in

many different channels

• Cytoplasmic inactivation peptide

swings into open channel

• Channel inactivated until voltage

returns when membrane

potential returns to negative

resting potential, called de-

inactivation



Patch Clamping



ION TRANSPORT: BIOENERGETICS



ION TRANSPORT: BIOENERGETICS

• Now must consider the concentration gradient and the voltage

gradient, or the electric potential produced by an unequal balance

of charges

• Together this is the electrochemical gradient.

DG= 1.4 log10[Cin/Cout] + zF DE

AtDE =

Eion (equilibrium pot.), DG = 0

1.4 log10[Cin/Cout] + zF Eion = 0

zF Eion = 1.4 log10[Cin/Cout]

• In millivolts, Eion=-60/z *log10[Cin/Cout] - EQBM POTENTIAL OF

ION

This is the Nernst equation.

Z: Valency of ion

F: Faraday constantDE: Potential Diff across

membrane



NERNST EQUATION EXAMPLES

• In millivolts, Eion=-(60/z )* log10[Cin/Cout]

• Na+out=150 mM, Na+

in=15 mM : ENa=+60 mV

• K+

out=5 mM, K+

in=100 mM : EK= -78 mV

BYOC (Build your own channel)



BYOC (Build your own channel)

Biological or non-biological

(chemical sensor, electrical component, etc.)

• What is it permeable to?

• What triggers it?

• What, if anything, inactivates and then de-inactivates it?

• Anything fancy or special about it?

Very interesting “Inner Life of a Cell” video



Very interesting Inner Life of a Cell video

• http://www.studiodaily.com/main/technique/tprojects/6850.html (video)

• http://www.sciencechatforum.com/bulletin/viewtopic.php?p=24688 (explanation of video)

SOLUTE PROPERTIES

http://www.studiodaily.com/main/technique/tprojects/6850.html


http://www.sciencechatforum.com/bulletin/viewtopic.php?p=24688








SOLUTE PROPERTIES

DETERMINING DIFFUSION RATE

• Partition coefficient:Solubility in

lipid/solubility in water

- Higher partition

coefficient, greater

membrane permeability.

• Size: Smaller molecule-

better permeability.

h l



The Glucose Transporter

E l f i



Examples of osmosis

• Cycle of secretion and osmotic reabsorption

in digestive tract

• Animal cells usually isotonic

• (a) Plant cells usually hypotonic. They are

swollen and have an internal osmotic

pressure called turgor.

• Turgor provides support for plants

• (b) If placed in a hypertonic solution (e.g.

seawater), the cell will shrink away from the

cell wall, or wilt, aka plasmolysis.

General principles of biological ion channels



General principles of biological ion channels

• Selective permeability

• Triggers can change channel conformation between closed

and open states

• Channel open time is often limited by inactivation



Good luck studying for exam 3!

Epigenetics and Membrane Transport

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