9 th PBL in Calcium and Phospholipid Signaling Department of Clinical Science and Education;

1. 9 th PBL in Calcium and Phospholipid Signaling Department of Clinical Science and Education; Department of Physiology and Pharmacology. May 4-15, 2009 Md. Shahidul Islam, M.D., Ph.D. Associate Professor Department of Clinical Research and Education Karolinska Institutet Forskningscentrum, Södersjukhuset 118 83 Stockholm, Sweden Shaisl@ki.se

2. [L.Ca] Ka = [L].[Ca2+] Preparation of multiple ligand-ion solutions Association constant or binding constant L + Ca2+ L.Ca

3. [L].[Ca2+] Kd = [L.Ca] At half saturation of the ligand: [L]=[L.Ca] And Kd = [Ca2+] Dissociation constant Kd=1/Ka

4. There are many Ca2+ buffers to choose from • EGTA • BAPTA • Dibromo BAPTA • Quin 2 • Fura-2 • EDTA

5. Affinity of the ligand for Ca2+ depends on • pH • EGTA more than BAPTA • Temperature • Ionic strength =0.5SCi.׀Zi׀

7. How do I know which buffer to use? • Kd should be close to the desired [Ca2+] • Affinity for Ca2+ must be much higher than Mg2+ • pH-sensitivity of affinity for Ca2+ • Costs etc

8. Ca2+ buffers exist in multiple states of protonation [Lt]-[CaL]=[L]+[HL]+[H2L]+[H3L]+[H4L] At pH 6 to just over 7, 99% of EGTA is in the form of H2EGTA Only two forms of EGTA, EGTA4- and EGTA3- bind Ca2+

9. Some important properties of Ca2+ buffers Ca2+ buffer Kd KCa/KMg EGTA 67 nM 6.2 72.2 KCa(pH 7.4) (pH 7.4) BAPTA 192 nM 1.14 158.24 KCa(pH 7.0) DiBromo BAPTA 1.83 mM 1.02 63.00

10. EGTA is the ”devil we know” • Kd near intracellular [Ca2+]i • 100,000 times higher affinity for Ca2+ over Mg2+ • Marked pH-dependence of Ca2+-affinity • Variable purity of EGTA • 30 times cheaper than BAPTA • EGTA itself may have non-specific effetcs • on processes studied

11. A. Multiple Ligand Approach When solutions contain more than one ligand Use computer programs Two methods of making Ca2+ buffers B. Two solutions approach Mix two solutions in various proportions Single ligand Ca2+ calibration solutions

12. Making of Ca2+ buffers • Use High Purity Water • Use high purity EGTA, BAPTA • Use Plastic Wares • Use Ca2+ standard solutions • And not CaCl2.2H2O • Accuracy in measurement of pH • Check by Ca2+ eletrodes

13. Lowering Extracellular [Ca2+] A. Reducing to <1 mM Stock solution of 1 M Na2H2EGTA (pH 7.4) Add Na2H2EGTA three times the concentration Of Ca2+ B. Reducing Ca2+ to 0 Nominally Ca2+-free medium Add Na2H2EGTA, 1 mM C. Precisely known concentration of Ca2+ Use computer programs Check with Ca2+ electrodes Or use two solutions approach