Key points
categories of transport
enegetics: electrochemical gradients
permeability of bilayer to solutes
facilitated diffusion: mech. and example re glucose transporter
active transport
mech and examples Na/K exchange
ABC class
Lecture Outline
- Intro to topic:
- dual function of PM:
- retention of cytosol
- exchange of materials
- future perspective from Lodish
- Categories of transport
- diffusion: through bilayer
- ion channel: diffusion driven 10e7-10e8 ions/sec
- facilitated transport: uniporters, symporters, antiporters 10e2-10e4/sec
- active transport: pumps 10e0-10e3 ions/sec
- Energetics:
- delta S (entropy drives diffusion) proportional to conc. gradient
- delta G (free energy change) can be calculated for influx of non charged substances
- dG=RT ln Ci/Co
- delta G for influx of charged molecules
- dG=RT ln Ci/Co +zF Em
- => electrochemical gradient determines driving force on solutes
- Simple diffusion through bilayer:
- first step is movement from aqueous to hydrophobic interior of bilayer
- hydrophobicity of substance measured by partition coeff. K= Cm/Caq
- next step diffusion across bilayer: this is rate limiting because its >100X H20 viscosity
- next step movement into aqueous environment
- rate can be calculated using modified Ficks Law
- dn/dt= A (KD)/x (Ci-Co)
- diffusion proport. to part. coef (K). & diffusion constant (D)
& inverse of mem thickness (x)
- =>partition coef. is main determinant=> hydrophobicity dictates
- freely diffusible:
- gases
- small hydrophobic (uncharged molecules) eg ETOH
- partially diffusible (membrane is semipermeable)
- H20: discussion of osmotic force, its importance & reg.
- urea
- non diffusible
- charged polar molecules including ions & phosphorylated molecules
- large uncharged molecules
- Uniporters: facilitate movement down gradient
- already favored, same delta G w or w/out the uniporter but...
- rate is far higher
- transport is specific to single molecule or group of closely related molecules
- limited # of transport sites=>Vmax reached at high conc.
- glucose transport is example for mech of action (GLUT1-5)
- glucose used by all cells, obtained from blood
- transporter has 2 conformations
- binding site faces outside
- binding site faces inside
- glucose binding to outside triggers the conformational change
- after glucose release inside the conformation changes back
- experimental support
- glucose uptake rate is saturable
- kinetics fit curve predicted by Km of glucose binding to transporter
- L-isomer of glucose, D-mannose, etc very poorly transported & high Kms
- cytoplasmic glucose is rapidly P04=> conc. gradient of glucose maintained
- 12 TMD domain protein w/ internal H-bonding side chains that are glu binding sites
- activity regulated by insulin
- high glucose-> insulin secetion
- insulin -> fusion of GLUT4 vesicles-> increased glucose uptake
- lowered glucose -> endocytosis of GLUT4-> decreased glu uptake
- P-class ATP powered pumps (P for phosph. intermediate)
- phosphorylated during transport
- alpha sub containing ATP binding site, regulatory beta sub, often tetrameric
- conserved family evolved ligand specificity and other unique features
- Ca ATPases (example muscle calcium pump)
- maintain low (0.1µM) cytoplasmic Ca, by pumping against gradient
- this is important for signaling, muscle contraction
- key features!
- 4 Ca++ binding sites: 2 high affinity cyto sites, 2 low affinity exo sites
- ATP binding site and hyrolysis with phosphorylation intermediate
(aspartyl phosphate)
- 2 conformations: change driven by difference in free energy of P hydrolysis
- defined order in reaction
- structural considerations:
- catalytic alpha subunit has 10 TMDs that form transport pore
hydrophilic residues important for Ca binding in TMD
also has cytoplasmic domain that forms stalk for energy transduction
also has cytoplasmic domain for ATP binding
also has cytoplasmic domain w/ site for aspartyl-phosphate formation
- and ATP
- NaK ATPase pump
- again tetramer with catalytic alpha subunits containing stalk, ATP binding etc.
- key features
- low & high affinity binding sites allow movement of Na out and K in
- 2 conformations
- H+ ATPase of stomach
- F & V class: these are structurally related, we will discuss the V for vesicle pump
- V-type acidifies vesicles eg lysosome to 4.5-5.0
- pumps H+, counter ion (Cl-) moves in same direction through channel
- very complex structure
- Vo=TM portion= a1 b1 c9-12 multispanning subunits
- V1=cyto portion=alpha3 beta3 gamma delta epsilon peripheral subunits
- no phosphorylation intermediate
- ABC superfamily
- >100 types each has specific ligand or group of ligands
- characterstic structure: 2 TM regions (6 TMDs each) & 2 cyto domains with ATP binding
- flipase model for transport mechanism