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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Outline 10.1 Passive Diffusion 10.2 Facilitated Diffusion 10.3 Active Transport Transport Driven by ATP, light, etc Group Translocation 10.8 Specialized Membrane Pores 10.9 Ionophore Antibiotics

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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 and 10.2 High permeability coefficients usually mean that passive diffusion is not the whole story

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 Facilitated Diffusion  G negative, 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 10.3 Two important distinguising features: –solute flows only in the favored direction –transport displays saturation kinetics

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 - 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 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, light or a concentration gradient

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company The Sodium Pump aka Na,K-ATPase Large protein 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

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 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

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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Calcium Transport in Muscle A process akin to Na,K transport Calcium levels in resting muscle cytoplasm are maintained low by Ca-ATPase - 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,K-ATPase Aspartyl phosphate E-P intermediate is at Asp- 351 and Ca-pump also fits the E1-E2 model

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 - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company The Gastric H,K-ATPase The enzyme that keeps the stomach at pH 0.8 The parietal cells of the gastric mucosa (lining of the stomach) have an internal pH of 7.4 H,K-ATPase pumps protons from these cells into the stomach to maintain a pH difference across a single plasma membrane of 6.6!

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

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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Osteoclast Proton Pumps 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 - acid dissolves the Ca- phosphate matrix of the bone An ATP-driven proton pump in the membrane does this!

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

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

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

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

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

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

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 - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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

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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Bacteriorhodopsin Lys-216 is buried in the middle of the 7- TMS structure of bR, and retinal lies mostly parallel to the membrane and between the helices Light absorption converts all-trans retinal to 13-cis configuration - see Figure 10.22

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Bacteriorhodopsin The protons visit the aspartates.... 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

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

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 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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Secondary Active Transport Symport - ion and the amino acid or sugar are transported in the same direction across the membrane Antiport - ion and transported species move in opposite directions Several examples are described in Table 10.2

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

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

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

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

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

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 Why Beta Sheets? for membrane proteins?? Genetic economy Alpha helix requires 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

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

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

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

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

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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Other Pore-Forming Toxins Delta endotoxin also possesses a helix- bundle 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

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 Amphiphilic Helices form Transmembrane Ion Channels Many natural peptides form oligomeric transmembrane channels The peptides form amphiphilic  -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

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

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

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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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  - helical aggregates in membranes, creating an ion channel in the center of the aggregate

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

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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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, e.g. as response to stress

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 Ionophore Antibiotics Mobile carrier or pore (channel) How to distinguish? Temperature! Pores will not be greatly affected by temperature, so transport rates are approximately constant over large temperature ranges Carriers depend on the fluidity of the membrane, so transport rates are highly sensitive to temperature, especially near the phase transition of the membrane lipids

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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Valinomycin A classic mobile carrier A depsipeptide - 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

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

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

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

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