1. Experimental Biology 2004 Cellular Homeostasis Refresher Course Walter F. Boron Dept. of Cellular & Molecular Physiology Yale University Regulation of Intracellular pH

2. Outline A helpful model: Temperature-regulation in your house The real thing: Overview of pH i homeostasis Conclusions

3. Outline A helpful model: Temperature-regulation in your house –Heat capacity –Perturbations: Acute & chronic heat/cold loads –Regulation: Furnaces & air conditioners –Changes in steady-state temperature –Sensors for snow and sunlight The real thing: Overview of pH i homeostasis Conclusions

4. Temperature Homeostasis in a House Target: 22 ºC

5. Temperature Homeostasis in a House Heat capacity … 1 Mass equivalent to 50,000 kg H 2 O 1000 W  2.4 d = 50  10 6 cal 22 ºC  23 ºC C = 50  10 6 cal 1 ºC = 50  10 6 cal/ºC

6. Target: 22 ºC Add a 50-m 3 indoor swimming pool Temperature Homeostasis in a House Heat capacity … 2

7. Temperature Homeostasis in a House Heat capacity … 3 Mass equivalent to 100,000 kg H 2 O 1000 W  2.4 d = 50  10 6 cal 22 ºC  22.5 ºC C = 50  10 6 cal 0.5 ºC = 100  10 6 cal/ºC

8. Temperature Homeostasis in a House Heat capacity … 4 The heat capacity represents the ability to buffer loads of heat or cold. Increasing the heat capacity increases the ability to resist temperature fluctuations … … but does not eliminate temperature fluctuations … and does not return temperature to “normal.”

9. Outline A helpful model: Temperature-regulation in your house –Heat capacity –Perturbations: Acute & chronic heat/cold loads –Regulation: Furnaces & air conditioners –Changes in steady-state temperature –Sensors for snow and sunlight The real thing: Overview of pH i homeostasis Conclusions

10. Temperature Homeostasis in a House Perturbations: Acute heat loads Target: 22 ºC

11. Temperature Homeostasis in a House Perturbations: Acute cold loads (heat sinks) Target: 22 ºC

12. Heat loss to the environment (0 ºC) Temperature Homeostasis in a House Perturbations: Chronic cold loads (heat sinks) Heat loss to the environment (0 ºC)

13. Outline A helpful model: Temperature-regulation in your house –Heat capacity –Perturbations: Acute & chronic heat/cold loads –Regulation: Furnaces & air conditioners –Changes in steady-state temperature –Sensors for snow and sunlight The real thing: Overview of pH i homeostasis Conclusions

14. Furnace-like appliances Air-conditioner– like appliances Target: 22 ºC Temperature Homeostasis in a House Regulation

15. . Temperature Homeostasis in a House Regulation: Dumb controller Temperature (ºC) 2025303540101505 Heat flow (cal) FurnacesAir conditioners

16. . Temperature Homeostasis in a House Regulation: Smart controller … 1 Temperature (ºC) 2025303540101505 Heat flow (cal) FurnacesAir conditioners Steady-state temperature

17. . Temperature Homeostasis in a House Regulation: Smart controller … 2 Temperature (ºC) 2025303540101505 Heat flow (cal) FurnacesAir conditioners 2. Heating exceeds cooling, so that temperature rises, rapidly at first. 3a & b. Temperature keeps climbing until the rising rate of cooling comes into balance with the falling rate of heating. 1. The blast of cold air lowers the temperature. 3a 3b

18. Temperature Homeostasis in a House Regulation: Smart controller … 3 Time Temperature ( º C) 0 20 30 40 10 50 Blast of cold air Heat capacity limits initial transient Normal heat capacity C = 50  10 6 cal/ º C High heat capacity C = 100  10 6 cal/ º C Furnaces speed up … Air conditioners slow down

19. Temperature Homeostasis in a House Regulation: Smart controller … 4 Time Temperature ( º C) 0 20 30 40 10 50 Blast of hot air Heat capacity limits initial transient Normal heat capacity C = 50  10 6 cal/ º C High heat capacity C = 100  10 6 cal/ º C Furnaces slow down … Air conditioners speed up

20. Outline A helpful model: Temperature-regulation in your house –Heat capacity –Perturbations: Acute & chronic heat/cold loads –Regulation: Furnaces & air conditioners –Changes in steady-state temperature –Sensors for snow and sunlight The real thing: Overview of pH i homeostasis Conclusions

21. . Temperature Homeostasis in a House Changes in steady-state temperature … 1a Temperature (ºC) 2025303540101505 Heat flow (cal) Wimpy furnaces Steady-state temperature falls Air conditioners Furnaces

22. . Temperature Homeostasis in a House Changes in steady-state temperature … 1b Temperature (ºC) 2025303540101505 Heat flow (cal) 2. Cooling exceeds heating, so that temperature falls, rapidly at first. 2 2 3a & b. Temperature keeps declining until the falling rate of cooling comes into balance with the rising rate of heating. 3a 3b 3a 3b 1. Heating rate abruptly falls. Furnaces Air conditioners

23. Temperature Homeostasis in a House Changes in steady-state temperature … 1c Time Temperature ( º C) 0 20 30 40 10 50 Wimpify the furnaces  Heat capacity slows attainment of new steady state … … but does not affect the steady state. Normal heat capacity C = 50  10 6 cal/ º C High heat capacity C = 100  10 6 cal/ º C

24. . Temperature Homeostasis in a House Changes in steady-state temperature … 2a Temperature (ºC) 2025303540101505 Heat flow (cal) Furnaces Lower outside temperature … stable A/C Steady-state temperature falls Air conditioners

25. Temperature Homeostasis in a House Changes in steady-state temperature … 2b Time Temperature ( º C) 0 20 30 40 10 50 Lower outside temperature  Heat capacity slows attainment of new steady state … … but does not affect the steady state. Normal heat capacity C = 50  10 6 cal/ º C High heat capacity C = 100  10 6 cal/ º C

26. . Temperature Homeostasis in a House Changes in steady-state temperature … 3a Temperature (ºC) 2025303540101505 Heat flow (cal) 1a. Deliver a blast of cold air … 1b … and lower outside temperature Furnaces Air conditioners

27. . Temperature Homeostasis in a House Changes in steady-state temperature … 3a Temperature (ºC) 2025303540101505 Heat flow (cal) 1b … and lower outside temperature 1a. Deliver a blast of cold air … 3a 3b 3a 3a & b. Temperature keeps climbing until the rising rate of stimulated cooling comes into balance with the falling rate of heating at a lower-than-normal temperature. 2. Heating exceeds cooling … temperature rises.

28. Temperature Homeostasis in a House Changes in steady-state temperature … 2b Time Temperature ( º C) 0 20 30 40 10 50 Supercharge the air conditioners Blast of cold air Temperature “regulates” back to a lower-than-normal value.

29. Outline A helpful model: Temperature-regulation in your house –Heat capacity –Perturbations: Acute & chronic heat/cold loads –Regulation: Furnaces & air conditioners –Changes in steady-state temperature –Sensors for snow and sunlight The real thing: Overview of pH i homeostasis Conclusions

30. Temperature Homeostasis in a House Special sensors … 1 Modulate sensitivities of furnaces & air conditioners Snow detector Sun detector

31. . Temperature Homeostasis in a House Special sensors … 2 Temperature (ºC) 2025303540101505 Heat flow (cal) FurnacesAir conditioners By themselves, these modulations would raise steady-state temperature … … but by anticipating an increased rate of heat loss, they would tend to stabilize steady-state temperature …

32. . Temperature Homeostasis in a House Special sensors… 3 Temperature (ºC) 2025303540101505 Heat flow (cal) FurnacesAir conditioners By themselves, these modulations would lower steady-state temperature … … but by anticipating increased passive heating, they would tend to stabilize steady-state temperature …

33. Time Out Although we have not yet mentioned the word “pH,” don’t despair. If you understand the concepts that we have already discussed, you really understand all you really need to understand to be a mid-level expert in the field of pH i regulation. Now … by merely changing the names of the players (e.g., from temperature to pH i ), you will be able to talk the talk and walk the walk at least as well as half of the people who consider themselves experts in the field of pH i regulation!

34. Outline A helpful model: Temperature-regulation in your house The real thing: Overview of pH i homeostasis –Buffering (Heat capacity) –Perturbations: acute & chronic acid loads (Heat/cold loads) –Regulation: acid extruders & loaders (Furnaces & A/C) –Changes in steady-state pH i (Temperature) –Sensors for CO 2 & HCO 3 – (Snow & sun) Conclusions

35. pH i Homeostasis Buffering … 1 … pH pH  –log 10 [H + ] 1  10 – 6 M 6.00 1  10 – 7 M 7.00 1  10 – 8 M 8.00 2  10 – 8 M 7.70 8  10 – 8 M 7.10 4  10 – 8 M 7.40  =1.00  =10   =0.30 =2=2 =2=2 =2=2

36. pH i Homeostasis Buffering … 2 B (n)   Weak Acid Conjugate Weak Base Conjugate Weak Acid NH 4 +   NH 3 + H + H 2 CO 3 HCO 3 – + H + HB (n+1) + H +   H 2 PO 4 –   + H + HPO 4 = pH litermoles AcidStrong pH litermoles BaseStrong       / ][ / ][ 

37. pH i Homeostasis Buffering … 3a … closed systems See Figure 27-7, parts 1, 2a, and 2b In: Boron & Boulpaep, Medical Physiology, 2003. Philadelphia, PA: Elsevier Science.

38. pH i Homeostasis Buffering … 3b … closed system ([H + ] + K) 2 [H + ] K ][3.2TB closed =  [TB] = [HB (n+1) ] + [B (n) ] … Total Buffer See Figure 27-2 A and B In: Boron & Boulpaep, Medical Physiology, 2003. Philadelphia, PA: Elsevier Science.

39. pH i Homeostasis Buffering … 4a … open system See Figure 27-3 In: Boron & Boulpaep, Medical Physiology, 2003. Philadelphia, PA: Elsevier Science.

40. pH i Homeostasis Buffering … 4b … open system 3.2[HCO 3 – ] = open  See Figure 27-4 In: Boron & Boulpaep, Medical Physiology, 2003. Philadelphia, PA: Elsevier Science.

41. Outline A helpful model: Temperature-regulation in your house The real thing: Overview of pH i homeostasis –Buffering (Heat capacity) –Perturbations: Acute & chronic acid loads (Heat/cold loads) –Regulation: Acid extruders & loaders (Furnaces & A/C) –Changes in steady-state pH i (Temperature) –Sensors for CO 2 & HCO 3 – (Snow & sun) Conclusions

42. pH i Homeostasis Perturbations: Acute acid & alkali loads … 1 HClH+H+ B (n) HB (n+1) pH i 7.2 Alkaline Acid Time pH i HCl KOH 7.2 Alkaline Acid Time pH i KOH OH – B (n) HB (n+1) H+H+ H2OH2O pH i

43. pH i Homeostasis Perturbations: Acute acid & alkali loads … 2 H+H+ B (n) HB (n+1) 7.2 Alkaline Acid Time pH i CO 2 H2OH2OH 2 CO 3 HCO 3 – pH i CO 2 CO 2 / HCO 3 – 7.2 Alkaline Acid Time pH i B (n) HB (n+1) H+H+ pH i NH 3 NH 3 / NH 4 + NH 4 +

44. pH i Homeostasis Perturbations: Chronic acid loads … 1 H+H+ OH – HCO 3 – Because V m is more negative than E H, H + tends to leak into the cell. OH – and HCO 3 – tends to leak out of the cell. Therefore, all leaks tend to acidify the cell … relentlessly (i.e., chronically).

45. Outline A helpful model: Temperature-regulation in your house The real thing: Overview of pH i homeostasis –Buffering (Heat capacity) –Perturbations: Acute & chronic acid loads (Heat/cold loads) –Regulation: Acid extruders & loaders (Furnaces & A/C) –Changes in steady-state pH i (Temperature) –Sensors for CO 2 & HCO 3 – (Snow & sun) Conclusions

46. pH i Homeostasis Regulation: Acid extruders & loaders … 1 H+H+ V-type H + pump H+H+ Na + H+H+ Na-H Exchanger Cl – HCO 3 – 2 Na + Na-Driven Cl-HCO 3 Exchanger Na/HCO 3 Cotransporter (1:2 stoichiometry) HCO 3 – 2 Na + HCO 3 – 3 Na + Na/HCO 3 Cotransporter (1:3 stoichiometry) HCO 3 – Cl – Cl-HCO 3 Exchanger Acid ExtrudersAcid Loaders pH i OH – HCO 3 – Metabolism H+H+

47. pH i Homeostasis Regulation: Isolating an acid extruder … 1 Na + H+H+ H+H+ H+H+ EIPA Metabolism H+H+ Acid ExtrudersAcid Loaders pH i 8.0 7.6 7.2 6.8 6.4 6.0 2 min NH 3 / NH 4 + EIPA pH i Acute acid load Recovery from the acid load Unmasking of EIPA-insensitive acid loading Cultured Renal Mesangial Cell Graph Adapted from Figure 2A in Boyarsky et al, PNAS 87:5921–5924, 1990 Free access at http://www.pnas.org/cgi/reprint/87/15/5921

48. pH i Homeostasis Regulation: Isolating an acid extruder … 2 dpH i dt &  dpH i dt &  From Boyarsky et al, PNAS 87:5921–5924, 1990 Free access at http://www.pnas.org/cgi/reprint/87/15/5921 See Fig. 1A See Fig. 1C See Fig. 1B See Fig. 1D

49. Outline A helpful model: Temperature-regulation in your house The real thing: Overview of pH i homeostasis –Buffering (Heat capacity) –Perturbations: Acute & chronic acid loads (Heat/cold loads) –Regulation: Acid extruders & loaders (Furnaces & A/C) –Changes in steady-state pH i (Temperature) –Sensors for CO 2 & HCO 3 – (Snow & sun) Conclusions

50. pH i Homeostasis Changes in steady-state pH i … 1 Cultured 3T3 Fibroblasts: Effect of ras See Figure 12 from Kaplan & Boron, Journal of Biological Chemistry 269:4116-4124, 1994. Freely accessible at http://www.jbc.org/cgi/reprint/269/6/4116 http://www.jbc.org/cgi/reprint/269/6/4116

51. pH i Homeostasis Changes in steady-state pH i … 2 Cultured Rat Hippocampal Neurons: Effect of Metabolic Acidosis See Figure 4 A&B from Bouyer et al., Journal of Physiology 559: 85-101, 2004. Freely accessible at http://jp.physoc.org/cgi/content/full/559/1/85 http://jp.physoc.org/cgi/content/full/559/1/85

52. Outline A helpful model: Temperature-regulation in your house The real thing: Overview of pH i homeostasis –Buffering (Heat capacity) –Perturbations: Acute & chronic acid loads (Heat/cold loads) –Regulation: Acid extruders & loaders (Furnaces & A/C) –Changes in steady-state pH i (Temperature) –Sensors for CO 2 & HCO 3 – (Snow & sun) Conclusions

53. pH i Homeostasis Sensors for CO 2 … 1 See Zhao et al, Nature 374:636-639, 1995 Under access control Out-of-Equilibrium (OOE) Solutions A.Diagram of 2 solutions being mixed of “pure” CO 2 : Syringe 1 with solution of pH 5.40 and 10% CO 2 Syringe 2 with solution of pH 7.55 and 0 CO 2 /HCO 3 - B. Diagram of 2 solutions being mixed of “pure” HCO 3 - : Syringe 1 with solution of pH 6.99 and 0 CO 2 /HCO 3 - Syringe 2 with solution of pH 9.40 and 44 mM HCO 3 -

54. pH i Homeostasis Sensors for CO 2 … 2 See Figure 3 in Zhou et al, PNAS 102:3875–3880, 2005 Freely accessible at http://www.pnas.org/cgi/content/full/102/10/3875 http://www.pnas.org/cgi/content/full/102/10/3875

55. pH i Homeostasis Sensors for CO 2 … 3 See Figure 1 (top row) in Zhou et al, PNAS 102:3875–3880, 2005 Freely accessible at http://www.pnas.org/cgi/content/full/102/10/3875 http://www.pnas.org/cgi/content/full/102/10/3875

56. pH i Homeostasis Sensors for HCO 3 – … 1 (a reprise) Cultured Hippocampal Neurons: Effect of Metabolic Acidosis See Figure 4 A&B from Bouyer et al., Journal of Physiology 559: 85-101, 2004. Freely accessible at http://jp.physoc.org/cgi/content/full/559/1/85 http://jp.physoc.org/cgi/content/full/559/1/85

57. pH i Homeostasis Sensors for HCO 3 – … 2 Model: Neuron that acidifies in response to Metabolic Acidosis HCO 3 – Na + H+H+ Cl – HCO 3 – Cl – Na + H+H+  pH i Acid Extruders Acid Loader pH i Metabolic Acidosis pH i [HCO 3 ] –

58. pH i Homeostasis Sensors for HCO 3 – … 3 Model: Neuron that resists Metabolic Acidosis Na + HCO 3 – H+H+ Cl – HCO 3 – Cl – Na + H+H+ HCO 3 – Na + HCO 3 – H+H+ Cl – HCO 3 – Cl – Na + H+H+ HCO 3 –  pH i Normal [HCO 3 – ] o Low [HCO 3 – ] o

59. Outline A helpful model: Temperature-regulation in your house The real thing: Overview of pH i homeostasis –Buffering (Heat capacity) –Perturbations: Acute & chronic acid loads (Heat/cold loads) –Regulation: Acid extruders & loaders (Furnaces & A/C) –Changes in steady-state pH i (Temperature) –Sensors for CO 2 & HCO 3 – (Snow & sun) Conclusions