Where would you find active transport?

•interface with the environment….

•maintain cell volume

•control internal environment

•signaling….Ca++ gradient

Lecture 16

Membrane TransportActive transport

Characteristics of a Transporter

•Saturability…characterized by KM and Vmax

•Stereospecificity..or specificity unrelared to biophysical characteristics

•Higher rate than expected from oil/water partition coef.

GLUT = sugar transporters

GLUT1-GLUT12

Michaelis-Menten equation for enzyme/transport reactions is very similar to

the Langmuir isotherm

][

][max sK

sVV

m

[s], mM

0 10 20 30 40 500

0.2

0.4

0.6

0.8

1

Km = 1 mM

Km = 10 mM

Vmax

A “simple explanation” says that the rate of reaction should be proportional to the occupancy of the binding site as long as Vmax is constant.

Bacterial Lac permease (lacY): Lactose-proton co-transporter

from Abramson et al. 2003

from Abramson et al. 2003

The Lac permease functional cycle, an example of coupled transport

Note: the proton is always taken up first, but is released at last, which ensures strict coupling of transport without H+ leakage

FzS

SRT Ss )

][

][ln(

2

1energy in gradient:

Example:

Na+-glucose symport: stoichiometry of 2:1

at equilibrium: Δμglu= -2ΔμNa

)][

][ln(2)

][

][ln(2

out

in

in

out

glu

gluRTF

Na

NaRT

)][

][log(

3.2

2)

][

][log(2

out

in

in

out

glu

glu

RT

F

Na

Na

Aspartate Transporter:

Na+ - dependent transport of aspartate

(from Boudker et al., Nature 2007)

apical

basolateral

Tight junction

K +

N a+

N a+

N a+ G LU

G LUK +N a+

G LU

K +

N a+

Na-K ATPase = the primary active transport, generates concentration gradients of Na+ and K+

utilizing ATP Na-Glucose co-transporter, utilizes Na+ gradient as a secondary energy source

Glucose diffusion facilitator (no energy consumed, passive transport)

H2O

G LUGLUT

ATPases that couple splitting of ATP with ion motion across the membrane

pump only protons

ATP synthase(works in reverse)

During contraction of the striated and cardiac muscle, Ca2+ is released into the cytoplasm, but during the relaxation phase it is actively pumped back into SR. Ca2+ ATPae (SERCA) constitutes >80% of total integral protein in SR.

High-affinity stateopen inside

Low-affinity stateopen outside

Muscle Ca2+ pump (SERCA)

The activity of SERCA, especially in the heart is regulated by Phospholamban, a small (single-pass) transmembrane protein. Phosphorylation of phospholamban by PkA removes its inhibitory action and increases the activity of SERCA by an order of magnitude.

The activity of plasma membrane Ca2+ pump (p-class) is regulated by calmodulin, which acts as a sensor of Ca concentration. Elevated Ca2+ binds to calmodulin, which in turn causes allosteric activation of the Ca2+ pump.

Post-Alberts Cycle for the Na+/K+ ATPase

FpHRTFH

HRT

H)

][

][ln(

2

1

HATP n at equilibrium:

Vacuilar or Lysosomal V-type ATPases work in conjunction with Cl- channels

BtuCD ATPase pumps vitamin B12 (ABC transporter)

Many ABC transporters work as flppases or pump lipid-soluble substances (MDR)

MDR1

flippase