D103 Membrane Proteins (ALS2, Videos 3 and 4)

a lipid bilayer is permeable to

• small hydrophobic molecules

• small uncharged polar molecules

a lipid bilayer is impermeable to

large polar molecules

ions

purpose of transport proteins

make the lipid bilayer selectively permeable

3 different transport proteins 

transporters (ex: GLUT1)

ATP-powered pumps (ex: Na+/K+ pump) 

ion channels

common features of transport proteins

• transmembrane proteins with multipleTMDs

• protein-lined pathway across lipid bilayer, so that water-soluble molecules can pass without contacting hydrophobic core of membrane

• conformational changes (most important!!)

protein conformations

determined by

• AA sequence of the protein

• interactions between individual amino acids (hydroen bonds, salt bridges, disulfide bonds, hydrophobic interactions)

conformational changes

are induced by 

• binding of a ligand 

• posttranslational mods of the protein 

• change in the pH, voltage, or temperature 

hexokinase

ex of ligand-induced conformational change

• glucose binding brings 2 domains closer together so that they are then surrounding glucose

EF-Tu

ex of ligand-induced conformational changes 

• GTP binding induces EF-Tu (a cofactor in protein synthesis) to become more compact 

GLUT1 deficiency syndrome (GDS)

genetic disorder that impairs brain metabolism

• brain is not supplied with sufficient glucose

• brings glucose from blood to nerve

• symptoms: seizures, developmental delay, motor disorders

• treatment: ketogenic (high fat) diet

how does GLUT1 transport glucose

ex of uniporter

• one or more specific binding sites for a solute (= substrate)

• binding of solute induces a reversible conformational change in the transporter

• direction of transport is inversed if the direction of the concentration gradient is changed (down conc gradient)

transporter-mediated transport and vmax 

reaches a Vmax 

vmax

each is working at its maximal rate

• all binding sites are filled (saturable transport)

Km

concentration at ½ Vmax

• it is a measure of the transporter’s affinity for its solute

• low Km = high affinity (inverse relationship)

how can we test experimentally how a mutation affects GLUT1 function

approach: isolate wt and mutant GLUT1 aend examine ability to transport glucose into a liposome 

problem: GLUT1 is a transmembrane protein

1. extract protein from membrane with detergent molecules

2. reconstitute into liposomes 

3. perform functional activity assays 

passive transport

no ATP and down conc gradient

• channel-mediated and transporter-mediated

active transport

transport against an electrochemical gradient with energy

energy input from

• ATP driven pump; direct input of energy

• electrochemicla gradient for one molecules drives the transport of the other; indirect input of energy

Na+/glucose symporter 

2 Na+ goes with conc gradient, providing energy for the 1 glucose going against conc gradient 

• cooperative binding 

• binding stimulates conformational change of transporter 

• Na+ and glucose disassociate from transporter 

• disassociation allows symporter to revert to its original conformation 

Na+/K+ pump

establish Na+ electrochemical gradient for glucose; both move against their gradients

• 3 Na+ out; 2 K+ in

what membrane protein has the highest transport rate

ion channels (always passive)

types of gating in ion channels 

voltage, ligand-gated extracellular, ligand-gated intracellular, mechanically gated 

• ligands are not transported; just for opening 

selectivity of ion channels 

selective for a specific ion (size and charge) 

• selectivity filter arranges molecules to pass in a single file 

• no actual ion binding site as seen in carriers 

bacterial K+ channels

ion channel; homotetramer that experiences conformational change to allow the transport of K+ ions

dravet syndrome (myoclonic epilepsy of infancy)

initially has normal milestones, but has fever-induced (febrile) seizures in the first year

• after that, seizures are triggered by slight changes in temp

• developmental delays and features of austism

• mutations in a Na+ channels involving gene SCN1A

mutations encoded by gene SCN1A 

> 1250 mutations in the sodium channel Na v1.1 have been identified 

• 26 TMDs in total 

why are flies used as a model system to study epilepsy

Nav1.1 (in mammals): 9 isoforms that mediate Na+ transport

Para (in flies): the only voltage-gated sodium channel (1 vs 9 channels)

• domains organization is conserved

• regions in para that are critical for function are conserved

forward genetic screen

induce random mutations and study those that cause paralysis as well as temperature-induced seizures (novel mutation)

knock-in studies

express mutated human gene in drosphila → temp induced seizures 

• mutate and see how they repeat 

what is an experimental model system

• simple system to study a biological question

• extrapolation of results to higher organisms

• availability of genetics (good for larger organisms)

• best to use the simplest possible system that allows a scientist to study a process

3 types of experimental model systems

1. prokaryotic cell (ex: e coli)

2. single cell eukaryotes (ex: yeast)

3. multicellular organisms (ex: mouse, fish, etc)

yeast model system

most simple (least number of genes)

haploid: esily generate mutants; good for genetics and screens

• basic cell biological and genetics questions

fruit fly model system

diploid: genetics, screens for components

• development from single cell to multicellular organism; cell biology

worm model system 

diploid: genetics, screens for components 

• precise timing of development from single cell to adult with 959 cells; cell bio 

mouse model system

most complex

diploid: gene deletion mutants and transgenic animals can be generated; genetics are possible

expensive

• dvelopment, immunology, mammalian genetics and cell bio

which hydrophobicity plot most likely describes the protein with 3 TMDs

1. look at y axis

• water → apolar = -△G for TMDs

2. look at x axis and range

TMDs from apolar → water

hydrophobic domains (△G > 0) 

TMDs from water → apolar

hydrophobic domains (△G < 0)

length of potential TMD 

20 AAs = 3 nm 

how many TMDs are in the protein

4

apolar → water = + G

which of the following is most likely to induce conformational change in Na+ transporter that is necessary for Na+ transport

binding of Na+

• in transporters, what is being transported causes the change; no separate ligand

in glucose/ Na+ symporter, what is driving 

na+ is the driving force in the apical membrane

epithelial cells contain 2 types of glucose transporter because

the glucose concentration in the intestine varies during the day (ex: before and after a meal)

• need a symporter and uniporter for this reason

which of these graphs describes the transport of Ca2+ across a biological membrane

depends if a channel or if it is a transporter