Effects of endogenous and exogenous Ca++ buffers in shaping Ca++ signals

1. Effects of endogenous and exogenous Ca++ buffers in shaping Ca++ signals Md. Shahidul Islam, M.D., Ph.D. Department of Clinical Sciences and Education, Södersjukhuset, Karolinska Institutet Forskningscentrum, Södersjukhuset 118 83 Stockholm, Sweden shaisl@ki.se

2. Ca++ signals and Ca++ buffers • Buffer means reversible reaction between Ca++ and a chelator – and not sequestration of Ca++ into organelles • Ca++ -indicator dyes are buffers and influence the Ca++ signal, particularly diffusion gradients • Effects of buffers on Ca++ signals are often ignored by many investigators

3. Endogenous Ca++ buffers Exogenous Ca++ buffers

4. Endogenous Ca2++ buffers • Slow mobility • Has low affinity • Does not saturate at Ca++ levels as high as 1 mM • Shapes the Ca2++ signal • Normally binds most of the Ca++ entering the cell (only 1/90 of Ca++ entering the cell stay as free Ca++ • Addition of exogenous buffers change the Ca++ signal

5. Endogenous buffers • Anexins (low affinity) • Parvalbumins (Ca++ affinities in the submicromolar range) • Calmodulin • Others

6. Exogenous ca++ buffer • Ca++ indicators • The time course of decay become slower in the presence of Ca++ indicators • At high Ca++ concentration all the Ca++ is bound by the indicator and fluorescence signal is proportional to the amount of Ca2++ entry

7. Neher and Augustine 1992, J. Physiol, 450:273-301

8. 400 mM K-Fura-2 200 ms 50 ms Neher and Augustine 1992, J. Physiol, 450:273-301

9. 50 mM K-Fura-2 50 ms Neher and Augustine 1992, J. Physiol, 450:273-301

10. Estimation of cytoplasmic Ca++ binding capacity • Analysis of rate of slow decline as a function of added buffer • Added buffer method. Changes in fluorescence signal due to addition of exogenous buffer E. Neher. The use of fura-2 for estimating Ca buffers and Ca fluxes. Neuropharmacology 34 :1423-1442, 1995.

11. The simplest case:The buffering reaction in a homogeneous single compartmentCa++ + L2- = CaL (Low concentration limit) • In the low-concentration limit bound Ca++ is proportional to free Ca++ • Addition of a buffer reduces the amplitude of a Ca++ -transient, induced by a shortinflux-pulse • It lengthens the time constant of decay of the transient by the same factor, such that the area under the transient (or its integral) is unchanged • Consequently any effector, which is linear in Ca++ , is not affected by the addition of a buffer • The buffer-effect depends on the non-linearities - either of those of the buffer or of the effector‘s E. Neher. Cell Calcium (1998) 24, 345-357