Membranes

CH339K

This has nothing to do with anything, but you all seemed appalled by the picture of the New Zealand weta, I figured a really big arthropod was worth a slide.

Theraphosa blondi – the Goliath Bird-Eater of South America can reach a legspan of 11 inches and a weight of 6-7 ounces. Fangs up to 2 cm.

Membrane Proteins

Transmembrane Helices

• Thickness of hydrophobic layer ~30Å

• Rise of helix 1.5 Å/aa• ~20 aa helix to span

membrane• Example glycophorin

Transmembrane Helices (cont.)

• Several characteristic motifs for helical membrane proteins

Bacteriorhodopsin

Purple membrane of halobacteriaLight-powered proton pumpTrimer – each monomer (above) contains 7 helices + retinal

Action of retinal (for anyone who cares)

Inside Outsidehν

H+

Hydrophobicity Plots

Beta Barrels

OmpG Protein of Gram-negative Bacteria• Adjacent b-sheets can also form membrane-spanning proteins• Common in bacteria, mitochondria, and chloroplasts• “Omp” = Outer membrane protein• 14-stranded -barrel• 34 kDaltons• Allows mono-, di-, and trisaccharides through outer membrane

Glycosylphosphatidylinisotol (GPI) anchored proteins

functionality ranges from enzymatic to antigenic and adhesion

What HAS to cross the membrane

(1) Water*, gases (O2, CO2), inorganic ions (Na+ , K+ , Cl- , Mg2+ , PO4

-3 , Ca++ , HCO3-,

NH3 / NH4+)

(2) small molecular weight organic molecules (sugars, amino acids, small lipids and lipid precursors, urea)

(3) large organic molecules and cell fragments

What DOES cross?

1. Impermeable to charged (inorganic ions, charged organic molecules etc); interior of membrane is very hydrophobic and resists penetration by charged atoms and molecules

2. Permeable to lipid-soluble (non-polar) molecules; permeability decreases as size of molecule increases (here we mean molecular volume).

3. Generally permeable to small slightly polar but uncharged molecules like water and dissolved gases (O2, CO2, CH3CH2OH); these molecules are literally small enough to pass beween phospholipid molecules.

4. Impermeable to proteins, carbohydrates, nucleotides, any “molecules of size.”

For the more visually oriented

Rate of movement depends on 2 factors

Partition coefficient Diffusion coefficient

Rates and Permeability Constants

where:P = permeability coefficient (cm-sec-1)K = partition coefficient (dimensionless)D = diffusion coefficient (cm2-sec-1)r = membrane thickness (cm)

The actual rate of transfer across the membrane then becomes:

J = -P*A*C

where:J = rate of passive diffusionP = permeability coefficient for the substance in questionA = surface area of the membraneC = difference in substance concentrations, [destination] - [source]

(usually in mol-cm-3.)

r

KD P

(Fick’s Law)

Correlation of K and P

Permeability varies with

temperature

D is temperature dependent, and thus so is P

Plot is the change in P for glycerol with T for two kinds of mouse sperm.

From Biology of Reproduction October 1, 1999 vol. 61 no. 4 1031-1041

Permeability versus Diffusion Coefficient

Correlation between permeability and diffusion coefficients of selected aliphatic and aromatic constituents of JP-8 jet fuel.From: Singh, S. and Singh, J. (2003) Percutaneous absorption, biophysical, and macroscopic barrier properties of porcine skin exposed to major components of JP-8 jet fuel, Env, Tox. Pharm. 14: 77-85.

P for some molecules of interest

Should be 1(you get pictures from the internet, you take your chances)

Sugar permeabilities

Membrane Preparation D-Glucose D-Mannitol

Synthetic Lipid Bilayer 2.4 x 10-10 4.4 x 10-11

Calculated Passive Diffusion 4 x 10-9 3 x 10-9

Intact Human Erythrocyte (red blood cell)

2.0 x 10-4 5 x 10-9

Anomalous Point!!! – Something must be letting glucose through the membrane!

Facilitated DiffusionIonophores– Valinomycin (K+ carrier) from Streptomyces

Facilitated Diffusion - Channels and Transporters (Permeases)

Facilitated diffusion is saturable

Kinetics of Facilitated Diffusion

Channels

Gramicidin from Bacillus brevis• 15 amino acids• Passage for monovalent cations

Gramicidin channel formation.

Lundbæk J A et al. J. R. Soc. Interface 2010;7:373-395

©2010 by The Royal Society

Channels

•-latrotoxin from Latrodectes mactans • channel for cations through presynaptic membrane• triggers neurotransmitter release• 130 kDal, forms tetramers

Pore Electron Micrographs

From: Zh. I. Andreeva-Kovalevskaya, A. S. Solonin, E. V. Sineva, and V. I. Ternovsky (2008) Pore-Forming Proteins and Adaptation of Living Organisms to Environmental Conditions, Biochemistry (Moscow)73: 1473-1492.

Channels

Streptococcus Hemolysin A (heptamer): inserts into susceptible cell membranes, causing cell death by leakage (note -barrel)

A Uniporter

Erythrocyte Glucose Transporter (A permease)

Two states:T1 = open to the outsideT2 = open to the intside

Glucose binding lowers G‡ for T1⇌T2 conversion

An Antiporter – ADP/ATP Exchanger(inner mitochondrial membrane)

Active Transport

Na+-K+ Transporter

Pumps 3 Na+ outPumps 2 K+ inBurns 1 ATP per cycle

Symport (and Secondary or Indirect Active Transport)

Secondary Active Transport- Galactoside Permease of bacteria- Uses extracellular H+ concentration to drive import of lactose

Membrane potentials

Membrane potentials

ZFC

CRTG

1

2ln

What happens when there’s a charge across the membrane?

• R = Gas constant• T = Absolute temperature• Keq = Equilibrium constant• Z = Charge on the ion• F = Faraday’s constant• = charge across the membrane

By convention:• If inside is positive with respect to outside, then > 0• If outside is positive with respect to inside, then < 0

Relationship of and Concentration

2

1lnor

1

2ln

:for Solving1

2ln

:so 0,G m,equilibriuAt 1

2ln

C

C

ZF

RT

C

C

ZF

RT

C

CRTZF

ZFC

CRTG

Nernst Equation

out

in

C

C

zF

RTln

Where:ΔΨ = Membrane Potential (V)R = Gas Constant (Jmol-1K-1)T = Temperature (K)z = Charge on the ionF = Faraday’s Constant (JV-1mol-1)

Goldman Equation

OutOutInIn

InInOutOut

CPCP

CPCP

F

RTln

Where:

ΔΨ = Membrane Potential (V)R = Gas Constant (Jmol-1K-1)T = Temperature (K)F = Faraday’s Constant (JV-1mol-1)P = Permeability (cm-sec-1)

Example – Giant nerve cells of Aplysia

Ion Cin (mM) Cout (mM) P (relative)

K+ 280 10 1

Na+ 61 485 0.12

Cl-1 51 485 0.44

ΔΨ ≈ -45 mV

)485)(.44(.)061)(.12(.)280)(.1(

)051)(.44(.)485)(.12(.)01)(.1(ln

F

RT

Nerve Cell TransmissionVoltage Gated Channels Pore

Voltage Sensors

Voltage sensors change conformation, open and close pore

Nerve Cell TransmissionVoltage Gated Channels

ActionPotential

1) Initial depolarization causes the Na channels to open

2) Na becomes controlling ion3) Potential goes positive4) Na channels close as K channels

open5) K becomes controlling6) Potential goes negative and

overshoots7) K channels close8) Potential returns to normal9) Na/K exchanger restores

concentrations

Action Potential

Scorpion -toxins

Bind to voltage-gated sodium channelsInhibit closure of the channelProlonged action potential disrupts nervous system

Leiurus quinquestriatus – death stalker (North Africa to Western India)

Impact of Scorpion Toxin on Sodium Channel Closure

Tohru Gonoi, Bertil Hille, and William A. Catterall (1984) Voltage clamp analysis of sodium channels in normal and scorpion toxin-resistant neuroblastoma cells, J. Neurosci. 4, 2836 - 2842

Cardiac Glycosides

Foxglove (Digitalis sp.)

The Bad:

Irregular and slow pulse, tremors, various cerebral disturbances, unusual color vision with objects appearing yellowish to green, and blue halos around lights, convulsions, and deadly disturbances of the heart.

The Good:

Positive inotropic* effect; also used in reentrant cardiac arrhythmias and to slow the ventricular rate during atrial fibrillation.

And it’s pretty.

* inotropic: altering the energy of muscle contraction

Cardiac glycoside action

1. Digitalis blocks the Na+/K+ exchanger

2. Intracellular Na+ rises

3. Na+/Ca++ exchanger goes into reverse

4. Intracellular Ca++ rises, stimulating heart muscle (positive inotropic effect)

Cystic FibrosisMost common genetic disease of Europeans.1 in 25 carry the alleleMembrane protein – chloride ion permease.

Cystic FibrosisHigh intracellular ion concentration pulls water from outside the cell.

Thick, sticky mucus; frequent chest infections, shortness of breath, poor nutrient absorption, male infertility, and death (avg age 37 in 2008)

Molecular Formula C129H223N3O54

LD50 <100 ng/kg

Source Palythoa toxica (soft coral)Limu make o hana (Seaweed of Death from Hana)

Palytoxin

Deeds JR, Handy SM, White KD, Reimer JD (2011) Palytoxin Found in Palythoa sp. Zoanthids (Anthozoa, Hexacorallia) Sold in the Home Aquarium Trade. PLoS ONE 6(4): e18235. doi:10.1371/journal.pone.0018235

Maitotoxin (new winner of most toxic molecule)

Molecular Formula C164H256O68S2Na2

LD50 ~50 ng/kg

Source Gambierdiscus toxicus (dinoflagellate)

Effect Activates Ca+2 channels, leading to cell death through complex pathways