This lecture: This lecture:

• Essential Cell Biology, 3rd ed.: chapters 11-12• Essential Cell Biology, 3rd ed.: chapters 11-12

• ion channels, the pumps and mechanisms relevant to Ca, osmo- and pH regulation, also included in this Ca, osmo- and pH regulation, also included in this chapter of the book, will be discussed in otherlectureslectures

• + lecture notes (web pages)• + lecture notes (web pages)

Cell membrane, transport

~50 membrane proteins

mutation

altered morphology

Figure 4. Red cell morphology. Hereditary spherocytosis (HS; top panel); nonhemolytichereditary elliptocytosis (HE; middle panel); hereditary elliptocytosis (HE; middle panel); elliptocytes, poikilocytes, and fragmented red cells in hemolytic HE (bottom panel). BLOOD, 2008, 112(10)

Membrane transportMembrane transport

→ Energetics

→ Membrane– artificial → Energetics

– passive

– active→ Transported

substance

– artificial

– real

– active substance– hydrophylic

– hydrophobic→ Number of transported

– hydrophobic→ Number of transported

substances/direction of transporttransport– uniport

– symport, antiport– symport, antiport

Real membranes: passive and active transport

see Active transportprevious

slide

Active transport

Carrier-mediated - passive - transportCarrier-mediated - passive - transport

uniport ionophore

Valinomycin (ionophore)

Main categories of active transportMain categories of active transport

Secondary active

P V F ABCNa/K Pgp,etc

Secondary active transporters, carriers

symport antiportvacuolar

mitochondrial

Intestinal glucose Intestinal glucose transport

IntestinalglucosetransportIntestinalglucosetransport

http://academic.brooklyn.cuny.edu/biology/bio4fv/page/sympo.htm

Categories of transport according to solubility ofsolubility of

„S”:

A. hydrophylic substratesA. hydrophylic substrates

B. hydrophobic substratesB. hydrophobic substrates

1C

Caq

m

>=RCaq

Transport of hydrophobic molecules

• Passive• Passive

– R (lipid / waterpartitioncoeff.)– R (lipid / waterpartitioncoeff.)– i.c. partition - Henderson-Hasselbach eq.

pH = pK + log(M/M+)• Active

pH = pK + log(M/M+)

– ABC (ATP binding casette) transporters• pump• pump• channel• regulator

– Other transporters– Other transporters

•

Passive diffusionis governed by the lipid-water partition coefficient and its pH

dependence (expressed by the Henderson-Hasselbach relationship):

concentrationreached in reached in membrane/efficiency

http://molinterv.aspetjournals.org/cgi/content/full/1/5/258

hydrophylic anaesthetichydrophylic anaesthetic

Partition coefficient may be pH-dependent -consequencesconsequences

R-NH3+

R-NH2

lysosome

R-NH2

lysosomepH ≈ 5

cytoplasmpH ≈ 7R-NH + pH ≈ 7R-NH3

+

Membrane transportMembrane transport

→ Energetics– Passive (simple

→ Membrane– Passive (simple

diffusion, also through channels, or facilitated,

→ Membrane– artificial

uniport-mediated diff.)

– Active (coupled or pumped)

– real

pumped)

→ Transported → Number of

→ Transported substance– hydrophylic

→ Number of transported substances– uniporter

– hydrophylic

– hydrophobic

– uniporter

– symporter, antiporter

General structure of ABC transporters

TMcell membrane

TM

– 2 transmembrane domains (TM)NBD

TM– 2 nucleotide binding domains

(= NBD = ATP binding domain, the „engine” that energizes 3 kinds

TM

the „engine” that energizes 3 kinds of activities, see a,b,c on next slide)

ABC: ATP-Binding Cassette

NBD

ABC: ATP-Binding Cassette

Most important ABC transporters

• Drug transporters in primitive cells (a)

• MDR 1 Pgp (a)• MDR 1 Pgp (a)

• PC“flippase” (a)• PC“flippase” (a)

• CFTR (b,c)

• TAP (a)• TAP (a)

• SUR (c)(a) pump

(b) channel• SUR (c)(b) channel

(c) regulator

Drug transporters in primitive cellsDrug transporters in primitive cells

• Plasmodium falciparum (malaria): chloroquin resistence

• Drug transporters in thebacterial (inner, cytoplasmic)membrane:

– Specific for antibiotic produced by the same cell– Multidrug transporters

"He that will not apply new remedies, must expect new evils." Francis Bacon (1561-1626); English philosopher, essayist, statesman.Francis Bacon (1561-1626); English philosopher, essayist, statesman.

Drug transporters in prokaryotes

Alberts, Fig. See also Lodish, Fig. 15-15

Alberts, Fig. 11-17

15-15

Drug transporters in human Drug transporters in human cells: e.g. mdr1 gene coded P-glycoprotein (also called

Pgp or ABCB1)Pgp or ABCB1)

P-glycoprotein coded by the mdr1 genecoded by the mdr1 gene

-Structure:-Structure:2x6 transmembrane domain,2 ABC motif („engine”)2 ABC motif („engine”)

-Expression:adrenalglands, intestines,pancreas,BBB,adrenalglands, intestines,pancreas,BBB,

cancer cell surfaces

-Substrates:endogeneous: cholesterol ?exogeneous: xenobiotics, drugs, “reversing agents”

-Knock-out: BBB insufficiency

-Mechanism: membrane→ aqeousphase-Mechanism: membrane→ aqeousphase

-Significance: cancer chemotherapy

Phosphatidyl choline (PC) flippase (MDR2, also

called ABCB4)

-Expression: hepatocytes, apical -Expression: hepatocytes, apical membrane

-Substrates: endogeneous: PC-Substrates: endogeneous: PC-Mechanism:

PC flippase, exports PC to the outer leafletleafletto be extracted from there by the bile acids acids

-Knock-out, genetic disease:PC- depletion in bile → cholangitis, death, progressive familial intrahepatic death, progressive familial intrahepatic cholestasis (PFIC) type 3

In normal bile the inherent toxicity of bile acids is quenched by PC. In the bile of PFIC 3 patients bile acid monomers are toxic.

ABC pumps of intestines

grapefruit juice(flavonoids)(flavonoids)

Pgp

ABCG2MRP1

ABCG2

Multi-drug transporters in the blood-brain barrier (BBB)

BBBBBB ABC-pumps of the liver ABC-pumps of the liver

ABCG5/G8cholesterol

CFTR:(„cystic fibrosis(„cystic fibrosis

transmembrane

conductanceregulator”):conductanceregulator”):

(ATP-dependent) chloride(ATP-dependent) chloridechannel, as well as

(ATP-dependent) regulator(ATP-dependent) regulatorof certain accompanyingchannels;channels;

In its absence(geneticdefect):In its absence(geneticdefect):

viscous mucus in bronchioli

http://en.wikipedia.org/wiki/Cystic_fibhttp://en.wikipedia.org/wiki/Cystic_fibrosis_transmembrane_conductance_regulator

Nature Cell Biology 8, Nature Cell Biology 8, 908 - 909 (2006)

TAP (oligopeptid TAP (oligopeptid transzporter):

Viral and cellular proteins decomposed to harmless oligopeptides decomposed to harmless oligopeptides by proteasomes in the cytosol are pumped into the lumen of the endoplasmatic reticulum by TAP I/II. endoplasmatic reticulum by TAP I/II. The peptides, „signatures” of the proteins they are derived from, become complexed with MHC I become complexed with MHC I proteins and appear on the cell surface.They are exhibited to the immune system as a „moving exhibition”, system as a „moving exhibition”, eliciting specific immune response by T cells.

Alberts, Figure 24-58

.

http://www.bio.davidson.edu/courses/Immunology/Students/spring2000/buxton/MFIP.htm

http://en.wikipedia.org/wiki/Sulfonylurea_receptor

Hetero-octamer

K+

ulfonylurea_receptor

octamer

SURSURGlucose ↑ →ATP↑ → closed state is stabilized → depol. → insulin secr.

ATP↓ → hydrolysis → opening → hyperpol.

Vertical heterogeneity

Asymmetric lipid composition

PS exposure on the surface of dying PS exposure on the surface of dying cells: eat-me signal for the phagocytes

dynamic equilibrium between flippases-

phagocytes

dynamic equilibrium between flippases-floppases (vectorial lipid transporters) and scramblases

The following slides are included either as reminders, or as illustrations / reference reminders, or as illustrations / reference

material rso

ghostRBC

hypotonic normotonic iso

Model of secondary-active transport

Arteficial membranes

5-8 nm

Vesicles and MBLs

MW SDS gelelectrophoretogramm(SDS-PAGE)(SDS-PAGE)(RBC membrane proteins)

Lateral pressure profileLateral pressure profile

„surfactants”

“Scanning calorimetry”

QQ

T

T<TmT<Tm

T>Tm

See also:See also:Z. Cournia,http://zarbi.chem.yale.edu/~zoe/

„mdr reversion” = „modulation”

ATP-depletedPgp+ cells

„mdr reversion” = „modulation”

ATP-depletedPgp+ cellsor Pgp- cellsdrug

cc..icc..i

ATP-depletedPgp+ cells

tt

ATP-depletedPgp cells+ATPATP

tt

Human NK cells readily form membrane nanotubes.

Chauveau A et al. PNAS 2010;107:5545-5550Chauveau A et al. PNAS 2010;107:5545-5550

©2010 by National Academy of Sciences