1. Gas Channels Workshop September 7, 2012 Cleveland, Ohio Mathematical Modeling of Gas Movements in an Oocyte Department of Physiology & Biophysics Case Western Reserve University School of Medicine 10900 Euclid Avenue Cleveland, OH 44106-4906 Rossana Occhipinti, Ph.D.

2. CO 2 HCO 3 – H+H+ H2OH2O CO 2 H2OH2O HCO 3 – H+H+ pH S [CO 2 ] S Bulk Extracellular Fluid (BECF) 2 min pH 7.5 7.7 7.3 7.0 1.5% CO 2 / 10 mM HCO 3 – pH S pH i (data kindly provided by Dr. Musa-Aziz)  [HCO 3 – ] Xenopus oocyte: pH Changes Caused by CO 2 Influx

3. A spherical cell Transport of CO 2 across the plasma membrane Reactions of a multitude of extra- and intracellular buffers Diffusion of solutes through the extra- and intracellular spaces Temporal and spatial variations of solute concentrations Carbonic anhydrase (CA) activity at specific loci An appropriate mathematical model should include…

4. The Mathematical Model Somersalo, Occhipinti, Boron, Calvetti, J Theor Biol, 2012

5. The Key Components of the Model Bulk extracellular fluid (BECF) Infinite reservoir where convection could occur but not reaction or diffusion Extracellular unconvected fluid (EUF) Thin layer adjacent to the surface of the oocyte where no convection occurs, but reactions and diffusion do occur Plasma membrane Infinitely thin and permeable only to CO 2 In both EUF and intracellular fluid (ICF) Slow equilibration of the CO 2 hydration/dehydration reactions Competing equilibria among the CO 2 /HCO 3 – and a multitude of non-CO 2 /HCO 3 – buffers

7. Assuming spherical symmetry, we write a reaction-diffusion equation for each species j, with r distance from the center of the oocyte Diffusion term (Fick’s second law) Reaction term (law of mass action) R R∞R∞ Oocyte EUF BECF

8. R R∞R∞ R r 0 = 0 R r t r1r1 r2r2 rjrj R ∞ = r N Method of Lines r3r3 Intracellular fluid (ICF) Extracellular Unconvected Fluid (EUF) Center of Cell Somersalo, Occhipinti, Boron, Calvetti, J Theor Biol, 2012

9. Numerical Experiments The BECF, EUF, ICF and plasma membrane have same properties as water The EUF has thickness d = 100 µm Small CA-like activity uniformly distributed inside the oocyte and on the surface of the plasma membrane The BECF and EUF - contain 1.5% CO 2 /9.9 mM HCO 3 – / pH 7.50 - have a single mobile non-CO 2 /HCO 3 – buffer with pK = 7.5 (e.g., HEPES) and [TA] = 5mM The ICF - has initial pH i = 7.20 - [CO 2 ] = [H 2 CO 3 ] = [HCO 3 – ] = 0 mM - has a single mobile non-CO 2 /HCO 3 – buffer with pK = 7.10 and [TA] ≈ 27.31mM Assumptions

10. Results Extracellular concentration-time profiles for solutes (A) (B)(C) (F)(D)(E)

11. (F) (D) (E) (A) (B)(C) Intracellular concentration-time profiles for solutes

12. 020040060080010001200 7.500 7.502 7.504 7.506 7.508 Time (sec) pH S (A) 020040060080010001200 7.00 7.05 7.10 7.15 7.20 Time (sec) pH i (C) 10 -4 10 -2 10 0 2 (  pH S ) max P M,CO 2 (cm/sec) x 10 -3-3 (B) (D) 0 x 10 -3 10 -4 10 -2 10 0 2 1 2 3 -( dpH i /dt ) max P M,CO 2 (cm/sec) Effects of Decreasing CO 2 Membrane Permeability

13. Implications The background permeability of the membrane (i.e., in the absence of gas channels) must be very low Given a sufficiently small P M,CO2, gas channels could contribute to CO 2 permeability even in the presence of a large d (in our numerical experiments d = 100µm) With additional refinements to the model, we ought to be able to estimate absolute permeabilities

14. ULs are thin, diffuse layers of fluid, always present near the surface of solid bodies immersed in a fluid, where molecules move predominantly via diffusion (Dainty and House, J Physiol, 1966; Korjamo et al, J Pharm Sci, 2009) The EUF is a generalization of the concept of unstirred layer (UL) R R∞R∞ EUF BECF d Oocyte For a particular solute, the width of the UL ( ) is defined as where D is the diffusion constant and P is the empirically measured permeability Effects of Changing the Width of the EUF The width of the UL: 1.A steady-state concept 2.Solute-dependent 3.Ignores the effects of chemical reactions It is because our system is dynamic, involves multiples solutes, and solutes can react in the “UL”, that we decided to define the EUF

15. (A) 020040060080010001200 Time (sec) 7.500 7.505 7.510 7.515 pH S d = 150m  d = 100m  d = 50m  d = 25m  d = 10m  d = 5m  d = 1m  050100150 d (  m) 0 0.005 0.010 0.015 (  pH S ) max 020040060080010001200 Time (sec) 7.00 7.05 7.10 7.15 7.20 pH i 050100150 3 4 5 6 7 8 x 10 -3-3 d (  m) -( dpH i /dt ) max (B) (D) (C)

16. pH S H+H+ CO 2 H2OH2O – HCO 3 diffusion pH electrode Implications There is competition between diffusion and reaction in replenishing the lost CO 2 near the outer surface of the oocyte DRR rises as the width d of the EUF decreases We quantify this competition by introducing the diffusion reaction ratio (DRR)

17. The Vitelline Membrane: pH S Spike Additional diffusion barrier to the movement of solutes Implemented by reducing the mobility D of each solute near the outer surface of the oocyte by the same factor γ, i.e., D * = D/γ

18. As we increase γ, the maximal height of the pH S spike, (ΔpH S ) max, increases Implementation of the vitelline membrane reduces the contribution of diffusion and enhances the contribution of reaction at the surface 1/  = 0.03 1/  = 0.06 1/  = 0.12 1/  = 0.25 1/  = 0.50 No Vit Membrane 0200400600 7.50 7.52 800 7.54 7.56 Time (sec) 00.51 0 0.02 0.04 0.06 1/   = 1/32 1/  = 1/16 1/  = 1/8 1/  = 1/4 1/  = 1/2 No Vit Memb pH S (  ) max

19. Implications Implementation of the vitelline membrane – which reduces the contribution of diffusion and enhances the contribution of the reaction – can explain the height of the pH S spike Because the pH S electrode creates a special environment with restricted diffusion, our implementation of the vitelline membrane somehow mimics this environment diffusion H+H+ CO 2 H2OH2O HCO 3 - CO 2 pH S diffusion pH S electrode

20. Conclusions The model can reproduce the pH transients observed experimentally The simulations predict that: 1.The background permeability of the oocyte membrane must be very low 2.Given a sufficiently small P M,CO2, gas channels could contribute to CO 2 permeability even with a large EUF The model provides new insights into the competition between diffusion and reaction processes near the outer surface of the plasma membrane

21. Future Directions Apply the model to investigate the movements of ammonia and ammonium across the plasma membrane Model the pH S electrode’s touching on the oocyte surface to explore the special environment underneath the pH S electrode

22. Acknowledgments Principal Investigator Walter F. Boron, M.D., Ph.D. Collaborators Erkki Somersalo, Ph. D. (CWRU) Daniela Calvetti, Ph. D. (CWRU) Raif Musa-Aziz, Ph.D. (University of Sao Paulo)