Transport of Ions and Small Molecules Across Cell Membranes

1. Transport of Ions and Small Molecules Across Cell Membranes • Objectives: • Review how membrane transport is accomplished with the help of proteins – facilitated diffusion, active transport (uniport, antiport, symport) • Understand the characteristics, locations, and functions of the major classes of ATP-powered pump proteins • Appreciate how non-gated channel proteins influence membrane potential, and how membrane potential and channel functions are investigated experimentally • Understand the process of cotransport – characteristics, examples of where it is used and why • Review how cells regulate water movement across the plasma membrane • Understand how and why transepithelial transport works in certain epithelial cells. What is necessary for this to occur? • Appreciate how gated channels are used in neural function and how this is examined experimentally

2. What do you know? • Order the molecules on the following list according to their ability to diffuse through a lipid bilayer, from most to least permeant. Explain your order. Ca2+, CO2, ethanol, glucose, RNA, H2O • If a frog egg and a red blood cell are placed in pure water, the RBC will swell and burst, but the frog egg will remain intact. Although a frog egg is about one million times larger than a red cell, they both have nearly identical internal concentrations of ions so that the same osmotic forces are at work in each. Why do you suppose RBCs burst in water, while frog eggs do not? • True/False. The plasma membrane is highly impermeable to all charged molecules. Explain your answer. • How is it possible for some molecules to be at equilibrium across a biological membrane and yet not be at the same concentration on both sides? • True/False. A symporter would function as an antiporter if its orientation in the membrane were reversed (that is, if the portion of the protein normally exposed to the cytosol faced the outside of the cell instead). Explain your answer. • Name three ways in which an ion channel can be gated. • True/False. Carrier proteins saturate at high concentration of the transported molecule when all their binding sites are occupied; channel proteins, on the other hand, do not bind the ions they transport and thus the flux of ions through a channel does not saturate.

3. Channels • Carriers • Pumps

6. Four Major Classes of Active Transporters • One or more binding sites for ATP on the cytosolic face • ATP is hydrolyzed only when a molecule is being transported

7. Animation Na/K pump

11. Therapeutic targets Cardiac glycosides Acid blockers Antifungals

12. Acid Blockers Proton-pump inhibitors protonix prevacid

13. Function: acidify the lumen of their associated organelle (vacuole, lysosome, etc.) *Do not have a phosphorylated intermediate

18. ABC = ATP Binding Cassette

22. ABC transporters involved in drug resistance. Gene Substrates Inhibitors ABCB1 Colchicine, doxorubicin, VP16,Verapamil, PSC833, GG918, V-104, Adriamycin, vinblastine, digoxin, Pluronic L61 saquinivir, paclitaxel ABCC1 Doxorubicin, daunorubicin, vincristine, Cyclosporin A, V-104 VP16, colchicines, VP16, rhodamine ABCC2 Vinblastine, sulfinpyrazone ABCC3 Methotrexate, VP16 ABCC4 Nucleoside monophosphates ABCC5 Nucleoside monophosphates ABCG2 Mitoxantrone, topotecan, doxorubicin, Fumitremorgin C, GF120918 daunorubicin, CPT-11, rhodamine a VP16, etoposide.

23. hepatomas, lung or colon carcinomas, breast cancers, malaria, HIV The ATP-binding cassette (ABC) superfamily, the major facilitator superfamily (MFS), the multidrug and toxic-compound extrusion (MATE) family, the small multidrug resistance (SMR) family and the resistance nodulation division (RND) family. Piddock Nature Reviews Microbiology4, 629–636 (August 2006) | doi:10.1038/nrmicro1464

24. Two forces govern the movement of ions across selectively permeable membranes: • the membrane electric potential and • the ion concentration gradient, • These forces may act in the same or opposite directions.

26. If a membrane is permeable only to Na+ ions, then the measured electric potential across the membrane equals the sodium equilibrium potential in volts, ENa. The magnitude of ENa is given by the Nernst equation, which is derived from basic principles of physical chemistry: ENa = RTln[Nal] ZF [Nar] where R (the gas constant) = 1.987 cal/(degree · mol), or 8.28 joules/(degree · mol); T (the absolute temperature) = 293 K at 20 °C, Z (the valency) = +1, F (the Faraday constant) = 23,062 cal/(mol · V), or 96,000 coulombs/(mol · V), and [Nal] and [Nar] are the Na+ concentrations on the left and right sides, respectively, at equilibrium.

27. Assume a temperature of 20oC • RT/ZF = 0.059 • If the ratio in Na+ concentration between the inside and outside is 0.1 and the membrane is permeable only to Na+, • Then you can predict the mV difference across the cell membrane using the Nernst equation. • -59 mV

28. What will the equilibrium membrane potential be if the membrane is permeable only to K+?

29. More open K+ channels • Few open Na+ or Ca2+ channels Thus resting potential is close to that of the K+ equilibrium potential

30. Voltage-gated ion channels • Open in response to changes in the membrane potential (voltage gating); • Over time following opening, will close and become inactive; and • Have specificity for those ions that will permeate and those that will not. Voltage gated K+ channel

34. Aquaporins

36. Patch Clamping

38. (a) Na+ movement (b) K+ movement in response to different clamping voltages

39. Transporters in Disease CFTR