Biophys J, December 2000, p. 2783-2784, Vol. 79, No. 6

NEW AND NOTABLE
Calcium Buffers in Flash-Light

Department of Membrane Biophysics, Max Planck Institute for Biophysical Chemistry, D-37077 Goettingen, Germany

	ARTICLE

Ca²⁺-binding proteins are dear to neuroanatomists because antibodies against them selectively stain specific subpopulations of neurons in the brain (Andressen et al., 1993). Thus, one might conclude that they have very specific functions, conferring distinct properties on the cell populations in which they are expressed. Unfortunately, it must be concluded, however, that we hardly understand what the staining pattern tells us, as the functional significance of Ca²⁺ binding proteins is only slowly emerging. Part of the reason for this ignorance is the fact that there is a not so trivial interplay between Ca²⁺ binding proteins, other cellular Ca²⁺ buffers, Ca²⁺ release, and Ca²⁺ extrusion mechanisms, not to mention the Ca²⁺ indicator dyes that are used to measure Ca²⁺ signals. To understand some aspects of this interaction we not only need to know the amounts and affinities of the Ca²⁺ chelators, but also their kinetic properties. The paper by Naegerl et al. (2000) in this issue represents a big step toward providing such basic information by measuring rate constants for Ca²⁺ binding and dissociation of one of the most abundant Ca²⁺-binding proteins, calbindin-D28k. This is achieved by analyzing the time course of binding of Ca²⁺ to the buffer after uncaging Ca²⁺ with a UV flash. The method should be readily applicable to other Ca²⁺-binding proteins and should be portable to in vivo experiments.

The action of fast Ca²⁺ buffers (the term Ca²⁺ buffer will be restricted here to proteins and other ligands binding Ca²⁺ and will not be used for other Ca²⁺ sequestration mechanisms) seems to be quite trivialto bind Ca²⁺ rapidly and thereby lower its concentration. This, however, is true only transiently; for instance, after a short episode of Ca²⁺ influx. As time goes on, Ca²⁺ pumps will remove Ca²⁺ from the cytosol and from the buffers, establishing a steady state that is determined by a balance between Ca²⁺ fluxes across the boundaries surrounding the space occupied by cytosol. The presence of buffers inside will not influence that balance and, therefore, not change steady-state Ca²⁺ levels. Buffers will, however, lengthen the time required for the steady state to be achieved. In simple cases this lengthening will be by exactly the same factor by which the amplitude of the transient is reduced, such that the area under the transient (or the product of amplitude and time constant) will not be influenced by the presence of the buffer. The same will be true for the cumulative effect on any process that is linearly dependent on Ca²⁺ (Neher, 1998). This simple consideration gives a hint as to why it is so difficult to pinpoint the action of fast Ca²⁺ buffers: it resides mainly in deviations from linearity of the Ca²⁺-effector systems. In order to appreciate this, accurate numbers on affinities are required.

Another issue is kinetics. While a fast buffer will simply diminish the amplitude and slow down a Ca²⁺ transient (unless it becomes saturated), a slow buffer will affect the time course of the Ca²⁺ signal in a more complicated manner: a rapid decay phase, which represents binding of Ca²⁺ to the buffer, will be followed by a return to baseline, which is slower than what would occur in the absence of buffer (Markram et al., 1998). Complex time courses can result when Ca²⁺ binding to buffers and sequestration by pumps occur on the same time scale (Lee et al., 2000). Furthermore, saturation of Ca²⁺-binding proteins can lead to nonlinear summation of Ca²⁺ signals and to changes in the range of action of Ca²⁺ signals (Allbritton et al., 1992; Maeda et al., 1999). For all these reasons accurate numbers are required for simulating and interpreting the observed properties of the Ca²⁺ signal. Given the fact that any cell may contain several Ca²⁺ buffer species (including the indicator dyes) and multiple extrusion mechanisms, the number of model parameters in a simulation is large. Therefore, being able to fix any one of them by an independent measurement is a blessing.

Naegerl et al. work with droplets of "flashing solution," which is a mixture of salts, a caged Ca²⁺ compound (DM-Nitrophen), and a low-affinity Ca²⁺ indicator dye. They measure free Ca²⁺ in the focal spot of a confocal microscope (with the resting beam) and elicit transients in free Ca²⁺ by photolysing DM-Nitrophen with a flash of UV light. They analyze the decay of [Ca²⁺] in the presence of Ca²⁺ buffers (first EGTA to validate the method, then calbindin-D28k) to determine kinetic rates of Ca²⁺ binding. The method should be applicable to intracellular measurements in any cell type that can be loaded with Ca²⁺ indicator dyes and caged Ca²⁺. This should complement other methods in which intracellular buffers are titrated by continuous release of Ca²⁺ from caged Ca²⁺ or by multiple flashes (Xu et al., 1997; Maeda et al., 1999).

Naegerl et al. find that calbindin-D28k Ca²⁺-binding kinetics are best described by assuming two types of Ca²⁺-binding sites that differ in affinity and rate constants by factors of 2 to 6. The binding rate constants (~1 and 8 · 10⁷ M¹ s¹ for the two sites, respectively) are surprisingly slow, the first one being comparable to that of EGTA (a typical "slow" buffer) and the second one being only 6-8 times faster. However, the value for EGTA obtained with this method (1.05 · 10⁷ M¹ s¹) is somewhat fast compared to recent T-jump experiments under very similar conditions (Naraghi, 1997). Based on the comparison with EGTA, calbindin-D28k might be considered to be situated between fast and slow Ca²⁺ buffers. Its exact role in shaping the Ca²⁺ signal within a living cell will depend on its concentration and on influences exerted by other components of the intracellular milieu.

In cerebellar Purkinje cells, known to contain large amounts of calbindin-D28k, dendritic Ca²⁺ transients evoked by subthreshold parallel fiber stimulation were significantly affected when experiments were performed on calbindin-D28k-deficient (CB /) mice (Airaksinen et al., 1997). Peak amplitudes were clearly enhanced, demonstrating that binding of Ca²⁺ to calbindin-D28k was sufficiently fast to affect [Ca²⁺]_i within the first 100 ms after the influx of Ca²⁺. Caged Ca²⁺ experiments on Purkinje cells have revealed a high concentration of a high-affinity Ca²⁺-binding protein with an affinity similar to the numbers reported here (Maeda et al., 1999). It was tentatively assigned to calbindin-D28k, but was reported to have cooperative Ca²⁺ binding, with an estimated Hill coefficient between 2 and 4. A complication in the analysis of these results arises from the fact that Purkinje cells not only express high levels of calbindin-D28k, but also equally high concentrations of the "slow" mobile buffer parvalbumin. It will be interesting to apply the method of Naegerl et al. to such cells and to see how calbindin-D28k injected into different types of neurones of CB/mice will affect Ca²⁺ transients.

	ACKNOWLEDGMENTS

I thank B. Schwaller, Fribourg, for helpful suggestions regarding the manuscript.

	FOOTNOTES

Received for publication 13 October 2000 and in final form 13 October 2000.

	REFERENCES

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Biophys J, December 2000, p. 2783-2784, Vol. 79, No. 6
© 2000 by the Biophysical Society   0006-3495/00/12/2783/02  $2.00

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