>
> Martin:
>
> I'm afraid you're mixing apples and oranges here.  The concern is not
> really how much total calcium is "lost" into calcium-indicator complexes
> (e.g. "1000% buffering"), but rather how much the indicator alters the
> normal calcium homeostasis of the cell.  That is to say, how does the
> indicator alter the amplitude and kinetics of calcium transients.  And
> that, to over simplify slightly, is determined by the concentrations and
> affinities of the other buffering systems already in the cells.  If the
> [indicator] is small relative to the [total buffers], then the indicator
> will have only a small effect on the transient. Cells have an enormous
> capacity for buffering calcium (important for survival!).  Most of this
> buffering capacity is slow and with low affinity.
>
> Unfortunately, most studies to date have concluded that even relatively
low
> indicator levels (30-50 uM) can dominate the endogenous buffering system
in
> cells, especially for brief transients.  Therefore, the general
conclusion
> is probably correct.  We do need to be careful about indicator
> concentrations in interpreting results, especially if conclusions are
going
> to be based on quantitative analysis of the magnituded or kinetics of
rapid
> calcium transients.
>
> Peter Guthrie
> Department of Neurobiology & Anatomy
> University of Utah School of Medicine
> 50 N Medical Drive
> Salt Lake City, UT  84132
> (801) 581-8336       (801) 581-4233 fax
> [log in to unmask]

Peter,

I agree with you entirely!  My point is that fura2 and other Ca indicators
generally work well in practice BECAUSE there is so much extra buffering
present, and NOT because their intrinsic buffering is low.  The situation
is indeed likely to be less favorable under transient conditions, where one
also needs to take into account the rate constants for Ca binding by the
indicator and by all the competing buffer systems.  There is a possibility
that things could get quite interesting here – if anyone has any data on
this  (I don't), perhaps they would like to comment?


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