polycystic renal disease 1 in 500 autopsies 1 in 3000 hospital admissions accounts for ≈10% of...

POLYCYSTIC RENAL DISEASE

1 in 500 autopsies

1 in 3000 hospital admissions

Accounts for ≈10% of end-stage renal failure

Autosomal dominant inheritance

CYSTIC FIBROSIS

1/2000 births in white Americans

Median age for survival late 30s

Autosomal recessive inheritance

Intracellular Extracellular Concentration ConcentrationComponent (mM) (mM)

Cations Na 5-15 145 K 140 5 Mg 0.5 1-2 Ca 10-4 1-2 H 8 x 10-5 (pH 7.1) 4 x 10-5 (pH 7.4)

Anions Cl 5-15 110 Because the cell is electrically neutral the large deficit in intracellular anions reflects the fact that most cellular constituents are negatively charged. The concentrations for Mg and Ca are given for free ions.

COMPARISON OF ION CONCENTRATIONS INSIDE AND OUTSIDE A TYPICAL MAMMALIAN CELL

A Cross between Human Beings and Plants . . .

SCIENTISTS ON VERGE OF CREATING PLANT PEOPLE . . .

Bizarre Creatures Could do Anything You Want

Tuesday, July 1, 1980

Bra

in w

ate

r (g

/10

0 g

dry

wt)

Simple DiffusionF

lux

[S]o

• Flux is proportional to external concentration

• Flux never saturates

PROTEIN MEDIATED MEMBRANE TRANSPORT

• PRIMARY ACTIVE

• SECONDARY ACTIVE TRANSPORT

• FACILITATED DIFFUSION

• ENDOCYTOSIS/TRANSCYTOSIS

Membrane Flux (moles of solute/sec)

• Simple Diffusion

• Carrier Mediated Transport• Facilitated Diffusion• Primary Active Transport• Secondary Active Transport

• Ion Channels

TRANSPORT OF MOLECULES THROUGH MEMBRANES

CARRIER MEDIATED TRANSPORT

Membrane Potential Review• The lipid bilayer is impermeable to ions and acts like an

electrical capacitor.

• Cells express ion channels, as well as pumps and exchangers, to equalize internal and external osmolarity.

• Cells are permeable to K and Cl but nearly impermeable to Na.

• Ions that are permeable will flow toward electrochemical equilibrium as given by the Nernst Equation.

Eion = (60 mV / z) * log ([ion]out / [ion]in) @ 30°C

• The Goldman-Hodgkin-Katz equation is used to calculate the steady-state resting potential in cells with significant relative permeability to sodium.

outClinNainK

inCloutNaoutKm [Cl]P[Na]P[K]P

[Cl]P[Na]P[K]Plog60mVV

• Higher flux than predicted by solute permeability

• Flux saturates• Binding is selective

(D- versus L-forms)• Competition• Kinetics:

[S]o << KmM [S]

[S]o = Km M = Mmax / 2

[S]o >> Km M = Mmax

Carrier-Mediated Transport

[S]oKm

Mmax

0.5Flu

x

MEMBRANE ION TRANSPORT PROTEINS

dSCo/dt = k+ [S]o [C]o – k- [SC]o = 0 at equilibrium

k+ [S]o [C]o = k- [SC]o

k- / k+ = ([S]o [C]o)/[SC]o = Km [SC]o = ([S]o [C]o)/Km

Fractional Rate = M / Mmax = [SC]o / ([C]o + [SC]o)

M = Mmax / (1 + [C]o/[SC]o) = Mmax / (1 + Km/[S]o)

Transport Kinetics

So + Co SCo Si S = Solute C = Carrierk +

k -

Reversible Transport

Co Ci

So Si

SCo SCi

Mnet = Min – Mout =

Mmax 1 11 + Km / [S]o 1 + Km / [S]i

-( )

• Uses bidirectional, symmetric carrier proteins

• Flux is always in the directions you expect for simple diffusion

• Binding is equivalent on each side of the membrane

Facilitated Diffusion

Facilitated Diffusion: Band 3/AE1

Facilitated Diffusion: Band 3/AE1

Cytoskeletal/AE1 Interactions

Primary Active Transport: Driven by ATP

• Class P – all have a phosphorylated intermediate• Na,K-ATPase• Ca-ATPase• H,K-ATPase• Cu-ATPase

• Class V • H+ transport for intracellular organelles

• Class F• Synthesize ATP in mitochondria

Primary Active Transport: Na,K-ATPase

• 3 Na outward / 2 K inward / 1 ATP• Km values: Nain = 20 mM Kout = 2 mM• Inhibited by digitalis and ouabain• Palytoxin “opens” ion channel• 2 subunits, beta and alpha (the pump)• Two major conformations E1 & E2• Turnover = 300 Na+ / sec / pump site @ 37

°C

3 Na

2 K

ATP

ADP + Pi

Na,K-ATPase Reaction Scheme

Membrane Transport and Cellular Functions that Depend on the Na,K-ATPase

Amino Acid Homology Among the Na,K-ATPase Subunit Isoforms

The Na,K-ATPase As a Receptor For Signal Transduction

Association of Src With the Na,K-ATPase

SR Ca-ATPase

FoF1 ATPase

Experimental Evidence for Rotation

Secondary Active Transport

• Energy stored in the Na+ gradient is used to power the transport of a variety of solutes

glucose, amino acids and other molecules are pumped in (cotransport)

Ca2+ or H+ are pumped out 2 or 3 Na+ / 1 Ca2+ ; 1 Na+ / 1 H+

(countertransport)

• These transport proteins do not hydrolyze ATP directly; but they work at the expense of the Na+ gradient which must be maintained by the Na,K-ATPase

Energy available from ATP H2OATP ADP + Pi

G = Gproducts – G reactants

Chemical Energy (G) = RT ln [C]

G = G° + 2.3 RT (log ([ADP] [Pi]) – log [ATP])

2.3 RT = 5.6 kiloJoules / mole @ 20° C

G° = -30 kiloJoules /mole @ 20°C, pH 7.0 and 1M [reactants] and [products] “Standard Conditions”

Energy Depends on Substrate Concentrations

G = -30 – 5.6 log [ATP] kJ / mole [ADP] [Pi]

The energy available per molecule of ATP depends on: [ATP] 4mM, [ADP] 400 µM, [Pi] 2 mM

per mole of ATP hydrolyzed:

G = -30 kJ – 5.6 kJ * log 4 x 10-3 2 x 10-3 * 4 x 10-4

= -30 kJ - 21 kJ = -51 kiloJoules per mole of ATP

Converting to approximately -530 meV/molecule of ATP

Energy in the Sodium Gradient

Consider Na+ movement from outside to inside:

G = Gproducts – Greactants = Ginside – Goutside

Gtotal = Gelectrical + Gchemical

Conditions for our sample calculation: Vm = -60 mV [Na+]out = 140 mM [Na+]in = 14 mM

and 2.3 RT = 60 meV / molecule

Energy in the Na Gradient: Electrical Term

Gelectrical = e * mVin – e * mVout

= +1e * -60 mV – (+1e) * 0 mV

= -60 meV

• negative sign means energy is released moving from outside to inside

• 60 meV is the energy required to move a charged ion (z=1) up a voltage gradient of 60 mV (assuming zero concentration gradient)

Energy in the Na Gradient: Chemical Term

Gchemical = 2.3 RT (log [Na+]in – log [Na+]out)

= 60 meV * (-1)

= -60 meV

• negative sign means energy is released moving from outside to inside

• 60 meV is the energy required to move a molecule up a 10 fold concentration gradient (true for an uncharged molecule or for a charged molecule when there is no voltage gradient)

Energy in the Na Gradient: Total

Gtotal = Gelectrical + Gchemical = -120 meV

• 120 milli-electron-Volts of energy would be required to pump a single Na+ ion out of the cell up a 10 fold concentration gradient and a 60 mV voltage gradient.

• Hydrolysis of a single ATP molecule can provide at least 500 meV of energy – enough to pump 4 Na+ ions.

• A single Na+ ion moving from outside to inside would be able to provide 120 meV of energy, which could be used to pump some other molecule, such as glucose, an amino acid, Ca2+ or H+ up a concentration gradient

Example: Na+/Ca2+ exchange

Compare the internal [Ca2+] for exchange ratios of

2 Na+ : 1 Ca2+ vs. 3 Na+ : 1 Ca2+

Vm = -60 mV, [Ca2+]out = 1.5 mM [Ca2+]in = ?

Ca2+ moves from inside to outside

G = Gproducts – Greactants = Goutside – Ginside

Gelectrical = (+2e) * (0 mV) – (+2e) * (-60 mV)

= +120 meV

Gchemical = 60 meV (log 1.5 – log ?)

Na+/Ca2+ exchange

2 Na+ 240 meV 240 = 120 + 60 log (1.5 / ?)120 / 60 = log (1.5 / ?) 102 = 1.5 / ? ? = 15 µM

3 Na+ 360 meV 360 = 120 + 60 log (1.5 / ?)240 / 60 = log (1.5 / ?) 104 = 1.5 / ? ? = 0.15 µM

Gtotal = GE + GC = 120 meV + 60 meV log (1.5 / ?)

Internal [Ca2+]can be reduced 100 fold lowerfor 3 Na : 1 Cavs 2 Na : 1 Ca

Structure of the Na/Ca Exchanger

Summary: Energetics

Transport Energetics • A molecule of ATP donates about 500

meV• It takes 60 meV to transport up a 60

mV electrical gradient• It takes 60 meV to transport up a 10

fold concentration gradient• A single sodium ion donates

approximately 120 meV

Summary: Membrane Flux (moles of solute/sec)

Simple Diffusion• Flux is directly proportional to external concentration• Flux never saturates

Carrier-Mediated Transport • Higher flux than predicted by solute permeability• Flux saturates• Binding is selective D- versus L-forms• Competition• Kinetics

Facilitated Diffusion• Uses bidirectional, symmetric carrier proteins• Flux is in the direction expected for simple diffusion• Binding is equivalent on each side of the membrane

Primary Active Transport – driven by ATP hydrolysis Secondary Active Transport – driven by ion gradients

III. Ion Channels

Transporters Regulated by Signaling Cascades

Na/H Exchangers

Na/Phosphate Cotransporter

Na/K/2Cl Cotransporter

Na/Cl Cotransporter

K/Cl Cotransporter

Na/Ca Exchanger

Na Channels

K Channels

Na,K-ATPase

H,K-ATPase

Unidirectional Transport Assays

Cells growingin multi-well plates

1. Cells washed in isotonic buffered solution

2. Required transport inhibitor(s) added

3. Flux medium containing radioactive isotope added

4. At required times flux medium rapidly removed and cells washed (3-4 x) in ice-cold isotonic saline

5. Final wash removed, cells lysed and radioactivity and protein content of samples determined

Calculations:

Specific Activity of medium:

Measure radioactivity in known volume of flux medium.

For example: For unidirectional uptake of Na into cells in medium containing:

50 mM Na100 mM choline Cl25 mM K-Hepes, pH 7.422Na (≈ 1 µCu/ml)

Measure radioactivity in 5 µl flux medium

Measure radioactivity and protein content in sample.

Determine Na influx using specific activity of mediumDetermine transport rate/protein content (Na uptake nmoles/µg protein/min)

Unidirectional Transport Assays

cpm (22Na) 1 L 1 mole cpm (22Na)

5 x 10-6 L 0.050 moles Na 109 nmoles nmoles NaX X =

THICK ASCENDING LIMB CELL

GASTRIC PARIETAL CELL

SMALL INTESTINAL CELL