1. Transmembrane and Intracellular Transport Tissue Engineering & Drug Delivery BBI 4203 LECTURE #13

2. Transport across cell membrane http://www.people.virginia.edu/ ~rjh9u/cellmemb.html How do molecules and nanoparticles enter cells? How to determine the rate of transmembrane transport?

3. I. Mechanisms of transmembrane transport They depend on physical and chemical properties of drugs Transport of small molecules across a membrane can be either passive or active transport.

4. Passive transport (i.e., energy-independent) Diffusion Facilitated transport: mediated by channels (e.g., ion channels) or passive carriers (e.g., glucose carrier)

5. Passive diffusion across the membrane

6. Facilitated transport Types of carriers: Uniporter – transport one molecule at a time Symporter (or cotransporter) – transport several molecules simultaneously in the same direction Antiporter (or exchanger) – transport several molecules simultaneously in different directions 1. Carriers Drug 2 H+H+ Drug 3 HCO 3 - Drug 1

7. Glucose transport through a carrier proteins (or transporters) bindingconformational change conformation recovery http://personal.tmlp.com/Jimr57/textb ook/chapter3/cmf4a.htm An example of uniporter

8. Aquaporins http://virtuallaboratory.mmawe b.net/Biofundamentals/lectureN otes/Topic2E_Membranes.htm 2. Channels A typical ion channel http://personal.tmlp.com /Jimr57/textbook/chapter 3/cmf1a1.htm

9. Regulation of ion channels

10. Active transport (i.e., is driven by thermodynamic potential ) In general, the secondary active transport is coupled with the primary active transport in order to maintain the intracellular concentration of ions. Primary active transport: consumes “energy” directly. Secondary active transport: consumes “energy” indirectly through coupling with the transport of a second molecule in an energetically favorable direction.

11. Primary active transport (e.g., Na + /K + ATPase) (The process consumes energy directly) Outside cell Inside cell http://personal.tmlp.com/Jimr57 /textbook/chapter3/cmf4a.htm Na + /K + ATPase, an active antiporter

12. Secondary active transport is mediated through exchangers/co-transporter (e.g., Na + /glucose co-transporter) (Although no metabolic energy is used in the process, maintaining the intracellular concentration of Na + requires Na + /K + ATPase)

13. Molecules and particles can be engulfed into the cell through at least three distinct, multimolecular processes. They are the mechanisms for cellular uptake of macromolecules and particles that are too large to diffuse through the membrane or fit into channels or binding pockets in transporters. http://personal.tmlp.com/Jimr57/ textbook/chapter3/cmf4a.htm Nature, 422:37, 2003 PhagocytosisMacropinocytosisEndocytosis

14. Doherty and McMahon, Annu. Rev. Biochem. 2009. 78:857–902

15. (Phagocytosis) (Clathrin-dependent endocytosis)

16. Molecular transport of across nuclear envelope Transport of macromolecules is selective. It requires specific amino acid sequences called nuclear localization sequences (or signals) (NLS). NLS can bind to a cytoplasmic receptor (importin), which facilitates the transport across the nuclear pore complex in an energy-dependent manner. www.scripps.edu/~stoffler/proj/NPC/npc.html Diffusion for small molecules and facilitated transport for macromolecules (e.g. histones, DNA and RNA polymerases, transcription factors, nucleic acids, and some therapeutic agents)

17. Translocation ATP GTP ADP GDP Pore (9 nm dia) Importin mediated transport across nuclear pore complexes

18. for small and uncharged molecules (e.g., O 2, CO 2, glycerol, and ethanol). governed by a phenomenological equation. II. Quantitative analysis of transmembrane transport Where J S – rate of solute transport in mass/time (g/s) S – vessel surface area (cm 2 ) P – membrane permeability coefficient in length/time (cm/s)  C – concentration difference (g/cm 3 ) (1) Diffusion of small uncharged molecules Units of mass/time (e.g. g/s)

20. Permeability coefficient is related to diffusion coefficient P = D  / h where D - diffusion coefficient of solutes in the membrane  - partition coefficient of solutes h - thickness of the membrane For K +,  = 1.04  10 -39 Need facilitated transport Impedance too high for ion to diffuse across membrane

21. Nernst Potential Across Semi- Permeable Membrane Start with equimolar concentrations of X+ and Y- on the left and right with [XY]i>[XY]o. X+ can pass through semi- permeable membrane but Y- is deflected back. Over time X+ follows the concentration gradient from left to right making left side negative relative to the right side. This establishes an electrical potential across the membrane

22. The transport of ions is mediated by ion pumps, channels, and transporters, which are specific to a subset of ionic species, e.g., potassium ions, chloride ions, and bicarbonate ions. Phospholipids, membrane proteins, and carbohydrate chains contain charged groups on the surface, which attract counter ions from the surrounding media. Different concentration gradients of ions across the membrane Genesis of transmembrane potential (2) Facilitated transport of ions via channels

23. Generalized Nernst – Planck equation convection diffusion due to  C iontophoresis due to  Where:N x - flux of electrolytes across the membrane C – concentration of electrolytes V - convective velocity of electrolytes D - diffusion coefficient of electrolytes in the membrane z - valence of electrolytes F - Faraday’s constant R - gas law constant T – temperature (K)  /  x – the potential field difference across membrane

24. At equilibrium (i.e., Donnan equilibrium), N x = 0 where, C is the concentration of ions. It can be either M +, X -, or A -. Rearrangement of Eq.1 gives Const = RTD/zF

25. The potential across the cell membrane is governed essentially by the Nernst Equation Potential across cell membrane

26. Electric potential difference across a membrane due to selective permeability of ions, that establishes concentration differences of electrolytes. Donnan potential On left K+ and Pr- inside cell. On right K+ and Cl- outside cell. Lack of Cl- on the left pulls Cl- into the cell along a concentration gradient. Since Pr- cannot cross membrane it pulls K+ into the cell along an electrical gradient. This makes [Cl-] and [K+] higher inside cell than outside. https://www.youtube.com/watch?v=MhSfQio8mp0

27. Carrier proteins move specific substrates across the cell membrane through conformation changes. (3) Facilitated transport via carrier proteins Facilitated transport by carriers can be modeled, mathematically, as enzymatic reactions.

28. Example: facilitated diffusion of glucose across the cell membrane. bindingconformational change conformation recovery Kinetic Analysis of Uniport http://personal.tmlp.com/Jimr57/textb ook/chapter3/cmf4a.htm

29. where (compatibility condition) Truskey, Yuan, Katz, 2009

30. (Ping-Pong model) In this model, the antiporter or exchanger has only one binding site for both A and S. Kinetic Analysis of Antiport

31. (4) Active transport across membrane Kinetic analysis of active transport is molecule-dependent. The concentration ratio between intracellular and extracellular compartments, [S i ]/[S e ], can be estimated through Gibbs free energy analysis. Example: Primary active transport of Na + /K + across the cell membrane is coupled with ATP hydrolysis. The transport can be modeled by the following reaction. Outside cell Inside cell 3 Na + 2 K +

32. Thermodynamic analysis of Na + /K + ATPase ATP + H 2 O + 3Na + i + 2K + e  ADP + P i + 3Na + e + 2K + i The change in free energy, due to solute transport through the pump, is related to the concentration ratio across the membrane. where is Standard free energy difference of solute between intracellular and extracellular compartment is Standard free energy change due to ATP hydrolysis. R is gas law constant; and T is temperature (K).

33. Modeling clathrin-dependent endocytosis Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y receptor recycling or exocytosis coated pit (driven by coated protein, eg. clatherin) ligand coated vesicle uncoating process early endosome late endosome lysosome from the nucleus receptor synthesis pinocytosis Truskey, Yuan, Katz, 2009