chapter_10 | Transmembrane Protein | Ion Channel

Biochemistry 2/e - Garrett & Grisham

Chapter 10
Membrane Transport
to accompany Biochemistry, 2/e by Reginald Garrett and Charles Grisham
All rights reserved. Requests for permission to make copies of any part of the work should be mailed to: Permissions Department, Harcourt Brace & Company, 6277 Sea Harbor Drive, Orlando, Florida 32887-6777 Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham

Outline
‡ ‡ ‡ ‡ ‡ ‡ 10.1 Passive Diffusion 10.2 Facilitated Diffusion 10.3 Active Transport 10.4 - 10.6 Transport Driven by ATP, light, etc. 10.7 Group Translocation 10.8 Specialized Membrane Pores

‡ 10.9 Ionophore Antibiotics

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Biochemistry 2/e - Garrett & Grisham

Passive Diffusion
No special proteins needed ‡ Transported species simply moves down its concentration gradient - from high [c] to low [c] ‡ Be able to use Eq. 10.1 and 10.2 ‡ High permeability coefficients usually mean that passive diffusion is not the whole story
Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e .Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .

Biochemistry 2/e .Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .

3 ‡ Two important distinguising features: ± solute flows only in the favored direction ± transport displays saturation kinetics Copyright © 1999 by Harcourt Brace & Company .Garrett & Grisham Facilitated Diffusion (G negative.Biochemistry 2/e . but proteins assist ‡ Solutes only move in the thermodynamically favored direction ‡ But proteins may "facilitate" transport. increasing the rates of transport ‡ Understand plots in Figure 10.

Biochemistry 2/e .Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .

Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .Biochemistry 2/e .

Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .Biochemistry 2/e .

Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .Biochemistry 2/e .

Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .Biochemistry 2/e .

light or a concentration gradient ‡ ‡ ‡ Copyright © 1999 by Harcourt Brace & Company .Garrett & Grisham Active Transport Systems ‡ Energy input drives transport Some transport must occur such that solutes flow against thermodynamic potential Energy input drives transport Energy source and transport machinery are "coupled" Energy source may be ATP.Biochemistry 2/e .

Garrett & Grisham The Sodium Pump ‡ ‡ ‡ ‡ ‡ aka Na.120 kD and 35 kD subunits Maintains intracellular Na low and K high Crucial for all organs. but especially for neural tissue and the brain ATP hydrolysis drives Na out and K in Alpha subunit has ten transmembrane helices with large cytoplasmic domain Copyright © 1999 by Harcourt Brace & Company .Biochemistry 2/e .K-ATPase Large protein .

Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .Biochemistry 2/e .

Biochemistry 2/e .Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .

Garrett & Grisham Na.K Transport ‡ ATP hydrolysis occurs via an E-P intermediate ‡ Mechanism involves two enzyme conformations known as E1 and E2 ‡ Cardiac glycosides inhibit by binding to outside Copyright © 1999 by Harcourt Brace & Company .Biochemistry 2/e .

Biochemistry 2/e .Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .

Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .Biochemistry 2/e .

Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .Biochemistry 2/e .

Ca accumulation ‡ Studies show this inhibitor to be ouabain! Copyright © 1999 by Harcourt Brace & Company .Garrett & Grisham Na.Biochemistry 2/e .K Transport ‡ Hypertension involves apparent inhibition of sodium pump. Inhibition in cells lining blood ‡ Vessel walls results in Na.

K-ATPase ‡ Aspartyl phosphate E-P intermediate is at Asp351 and Ca-pump also fits the E1-E2 model Copyright © 1999 by Harcourt Brace & Company .Garrett & Grisham Calcium Transport in Muscle A process akin to Na.K transport ‡ Calcium levels in resting muscle cytoplasm are maintained low by Ca-ATPase .Biochemistry 2/e .a Ca pump ‡ Calcium is pumped into the sarcoplasmic reticulum (SR) by a 110 kD protein that is very similar to the alpha subunit of Na.

Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .Biochemistry 2/e .

Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .Biochemistry 2/e .

Biochemistry 2/e .Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .

Biochemistry 2/e .K-ATPase pumps protons from these cells into the stomach to maintain a pH difference across a single plasma membrane of 6.4 ‡ H.8 ‡ The parietal cells of the gastric mucosa (lining of the stomach) have an internal pH of 7.K-ATPase The enzyme that keeps the stomach at pH 0.6! Copyright © 1999 by Harcourt Brace & Company .Garrett & Grisham The Gastric H.

Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .Biochemistry 2/e .

Biochemistry 2/e .K-ATPase is similar in many respects to Na.K-ATPase ‡ This is the largest concentration gradient across a membrane in eukaryotic organisms! ‡ H.K-ATPase and Ca-ATPase Copyright © 1999 by Harcourt Brace & Company .Garrett & Grisham The Gastric H.

acid dissolves the Caphosphate matrix of the bone ‡ An ATP-driven proton pump in the membrane does this! ‡ Osteoclast Proton Pumps Copyright © 1999 by Harcourt Brace & Company .Garrett & Grisham How your body takes your bones apart! ‡ Bone material undergoes ongoing remodeling ± osteoclasts tear down bone tissue ± osteoblasts build it back up ‡ Osteoclasts function by secreting acid into the space between the osteoclast membrane and the bone surface .Biochemistry 2/e .

Biochemistry 2/e .Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .

Biochemistry 2/e .Garrett & Grisham The MDR ATPase aka the P-glycoprotein ‡ Animal cells have a transport system that is designed to recognize foreign organic molecules ‡ This organic molecule pump recognizes a broad variety of molecules and transports them out of the cell using the hydrolytic energy of ATP Copyright © 1999 by Harcourt Brace & Company .

Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .Biochemistry 2/e .

Biochemistry 2/e .Garrett & Grisham The MDR ATPase ‡ MDR ATPase is a member of a "superfamily" of genes/proteins that appear to have arisen as a "tandem repeat" ‡ MDR ATPase defeats efforts of chemotherapy Copyright © 1999 by Harcourt Brace & Company .

Biochemistry 2/e .Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .

halobium are 75% bR and 25% lipid .Garrett & Grisham Light-Driven H Transport The Bacteriorhodopsin story ‡ Halobacterium halobium.Biochemistry 2/e . it can survive by using bacteriorhodopsin and halorhodopsin to capture light energy ‡ Purple patches of H.a "2D crystal" of bR . carries out normal respiration if O2 and substrates are plentiful ‡ But when substrates are lacking. the salt-loving bacterium.ideal for structural studies Copyright © 1999 by Harcourt Brace & Company + .

Biochemistry 2/e .Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .

Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .Biochemistry 2/e .

Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .Biochemistry 2/e .

Garrett & Grisham Bacteriorhodopsin Protein opsin and retinal chromophore ‡ Retinal is bound to opsin via a Schiff base link ‡ The Schiff base (at Lys-216) can be protonated. and this site is one of the sites that participate in H+ transport Copyright © 1999 by Harcourt Brace & Company .Biochemistry 2/e .

Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .Biochemistry 2/e .

see Figure 10. and retinal lies mostly parallel to the membrane and between the helices ‡ Light absorption converts all-trans retinal to 13-cis configuration .Garrett & Grisham Bacteriorhodopsin ‡ Lys-216 is buried in the middle of the 7TMS structure of bR.22 Copyright © 1999 by Harcourt Brace & Company .Biochemistry 2/e .

.Garrett & Grisham Bacteriorhodopsin The protons visit the aspartates...Biochemistry 2/e . ‡ Asp-85 and Asp-96 lie on opposite sides of a membrane-spanning helix ‡ These remarkable aspartates have pKa values around 11! (Why?) ‡ Protons are driven from Asp-96 to the Schiff base at Lys-216 to Asp-85 and out of the cell Copyright © 1999 by Harcourt Brace & Company .

instead of H + ‡ Halorhodopsin has Lys-242 Schiff base but no aspartates and no deprotonation of Schiff base during the transport cycle Copyright © 1999 by Harcourt Brace & Company .Biochemistry 2/e .Garrett & Grisham Halorhodopsin ‡ Halorhodopsin transports Cl .

Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .Biochemistry 2/e .

Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .Biochemistry 2/e .

Garrett & Grisham Secondary Active Transport Transport processes driven by ion gradients ‡ Many amino acids and sugars are accumulated by cells in transport processes driven by ion gradients Copyright © 1999 by Harcourt Brace & Company .Biochemistry 2/e .

ion and the amino acid or sugar are transported in the same direction across the membrane ‡ Antiport .Garrett & Grisham Secondary Active Transport ‡ Symport .ion and transported species move in opposite directions ‡ Several examples are described in Table 10.Biochemistry 2/e .2 Copyright © 1999 by Harcourt Brace & Company .

HPr. EII. coli cells ‡ Four proteins required: EI.Garrett & Grisham Group Translocation The phosphotransferase system (PTS) ‡ Discovered by Saul Roseman in 1964 ‡ Sugars are phosphorylated from PEP during transport into E.Biochemistry 2/e . and EIII Copyright © 1999 by Harcourt Brace & Company .

Biochemistry 2/e .Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .

Garrett & Grisham Group Translocation ‡ EI and HPr are universal and work for all sugars ‡ EII and EIII are specific for each sugar ‡ Mechanism involves transfer of P from PEP to EI and then to HPr and then to 2 sites on EIII and then finally phosphorylation of sugar Copyright © 1999 by Harcourt Brace & Company .Biochemistry 2/e .

Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .Biochemistry 2/e .

Biochemistry 2/e .Garrett & Grisham Porins ‡ ‡ ‡ ‡ ‡ Found both in Gram-negative bacteria and in mitochondrial outer membrane Porins are pore-forming proteins .30-50 kD General or specific .exclusion limits 600-6000 Most arrange in membrane as trimers High homology between various porins Porin from Rhodobacter capsulatus has 16stranded beta barrel that traverses the membrane to form the pore (with eyelet!) Copyright © 1999 by Harcourt Brace & Company .

Biochemistry 2/e .Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .

Biochemistry 2/e .Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .

Biochemistry 2/e .Garrett & Grisham Why Beta Sheets? ‡ ‡ ‡ ‡ for membrane proteins?? Genetic economy Alpha helix requires 21-25 residues per transmembrane strand Beta-strand requires only 9-11 residues per transmembrane strand Thus. with beta strands . a given amount of genetic material can make a larger number of trans-membrane segments Copyright © 1999 by Harcourt Brace & Company .

Biochemistry 2/e .Garrett & Grisham The Pore-Forming Toxins ‡ Lethal molecules produced by many organisms ‡ They insert themselves into the host cell plasma membrane ‡ They kill by collapsing ion gradients. facilitating entry by toxic agents. or introducing a harmful catalytic activity Copyright © 1999 by Harcourt Brace & Company .

coli) ‡ Single colicin molecule can kill a host! ‡ Three domains: translocation (T).Biochemistry 2/e . coli ‡ Inhibit growth of other bacteria (even other strains of E.Garrett & Grisham Colicins ‡ Produced by E. receptor-binding (R). and channelforming (C) Copyright © 1999 by Harcourt Brace & Company .

Biochemistry 2/e .Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .

with others splayed on the membrane surface ‡ A transmembrane potential causes the amphipathic helices to insert! Copyright © 1999 by Harcourt Brace & Company . with H8 and H9 forming a hydrophobic hairpin ‡ Other helices amphipathic (Fig.30) ‡ H8 and H9 insert.Biochemistry 2/e . 10.Garrett & Grisham Clues to Channel Formation ‡ C-domain: 10-helix bundle.

Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .Biochemistry 2/e .

Biochemistry 2/e .Garrett & Grisham Other Pore-Forming Toxins ‡ Delta endotoxin also possesses a helixbundle and may work the same way ‡ There are other mechanisms at work in other toxins ‡ Hemolysin from Staphylococcus aureus forms a symmetrical pore ‡ Aerolysin may form a heptameric pore with each monomer providing 3 beta strands to a membrane-spanning barrel Copyright © 1999 by Harcourt Brace & Company .

Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .Biochemistry 2/e .

Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .Biochemistry 2/e .

Garrett & Grisham Amphiphilic Helices form Transmembrane Ion Channels ‡ Many natural peptides form oligomeric transmembrane channels ‡ The peptides form amphiphilic E-helices ‡ Aggregates of these helices form channels that have a hydrophobic surface and a polar center ‡ Melittin (bee venom). magainins (frogs) and cecropin (from cecropia moths) are examples Copyright © 1999 by Harcourt Brace & Company .Biochemistry 2/e .

26 residues ‡ Cecropin A .35 to appreciate helical wheel presentation of the amphipathic helix Copyright © 1999 by Harcourt Brace & Company .bee venom toxin .Biochemistry 2/e .37 residues ‡ Magainin 2 amide .cecropia moths .frogs .Garrett & Grisham Amphipathic Helices ‡ Melittin .23 residues ‡ See Figure 10.

Biochemistry 2/e - Garrett & Grisham

The Magainin Peptides
‡ Discovered by Michael Zasloff ‡ He noticed that incisions on Xenopus laevis (African clawed frog) healed without infection, even in bacteria-filled aquarium water ‡ He deduced that the frogs produced a substance that protected them from infection!
Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham

Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham

The Cecropins
‡ Produced by Hyalophora cecropia (the cecropia moth - see Figure 10.36) ‡ Induced when the moth is challenged by bacterial infections ‡ These peptides are thought to form Ehelical aggregates in membranes, creating an ion channel in the center of the aggregate
Copyright © 1999 by Harcourt Brace & Company

Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .Biochemistry 2/e .

Biochemistry 2/e .Garrett & Grisham Gap Junctions ‡ ‡ ‡ ‡ Vital connections for animal cells Provide metabolic connections Provide a means of chemical transfer Provide a means of communication Permit large number of cells to act in synchrony Copyright © 1999 by Harcourt Brace & Company .

as response to stress Copyright © 1999 by Harcourt Brace & Company .g. e.Biochemistry 2/e .Garrett & Grisham Gap Junctions ‡ Hexameric arrays of a single 32 kD protein ‡ Subunits are tilted with respect to central axis ‡ Pore in center can be opened or closed by the tilting of the subunits.

Biochemistry 2/e .Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .

Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .Biochemistry 2/e .

so transport rates are highly sensitive to temperature.Biochemistry 2/e . so transport rates are approximately constant over large temperature ranges ‡ Carriers depend on the fluidity of the membrane. especially near the phase transition of the membrane lipids Copyright © 1999 by Harcourt Brace & Company .Garrett & Grisham Ionophore Antibiotics Mobile carrier or pore (channel) ‡ How to distinguish? Temperature! ‡ Pores will not be greatly affected by temperature.

Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .Biochemistry 2/e .

Biochemistry 2/e .a molecule with both peptide and ester bonds Valinomycin is a dodecadepsipeptide The structure places several carbonyl oxygens in the center of the ring structure Potassium and other ions coordinate the oxygens Valinomycin-potassium complex diffuses freely and rapid across membranes Copyright © 1999 by Harcourt Brace & Company .Garrett & Grisham Valinomycin ‡ ‡ ‡ ‡ ‡ A classic mobile carrier A depsipeptide .

Biochemistry 2/e .Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .

but affinities for Na + and Li + are about a thousand-fold lower Radius of the ions is one consideration Hydration is another .Biochemistry 2/e .see page 324 for solvation energies It "costs more" energetically to desolvate Na+ and Li+ than K+ ‡ ‡ ‡ Copyright © 1999 by Harcourt Brace & Company .Garrett & Grisham Selectivity of Valinomycin ‡ Why? K + and Rb + bind tightly.

Garrett & Grisham Gramicidin ‡ ‡ ‡ ‡ A classic channel ionophore Linear 15-residue peptide .4 nm or 4 A diameter ‡ Ions migrate through the central pore Copyright © 1999 by Harcourt Brace & Company .6.alternating D & L Structure in organic solvents is double helical Structure in water is end-to-end helical dimer Unusual helix .Biochemistry 2/e .3 residues per turn with a central hole .0.

Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .Biochemistry 2/e .

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