Ca²⁺ signals play a crucial role in all eukaryotic cells in the regulation of many cellular processes including gene expression, cytoskeletal alterations, cell cycle, cell death, neurotransmission, and signal transduction. To achieve its role as a messenger, the intracellular Ca²⁺ concentration ([Ca²⁺]) has to be very tightly regulated in time, space and magnitude. In neurons at rest [Ca²⁺] is maintained at relatively low levels, around 100 nM. During neuronal activation, [Ca²⁺] can rapidly rise up to the millimolar range via the influx through voltage or ligand gated channels or via its release from intracellular stores such as the endoplasmatic reticulum. The [Ca²⁺] returns to its resting levels through the action of extrusion pumps, Ca²⁺-exchangers, calcium binding proteins (CBPs) and other endogenous buffers.  Apart from playing an essential role in modulating the shape of Ca²⁺ transients, CPBs are essential as detectors of Ca²⁺ signals.  The Ca²⁺ signal is shaped by buffering CBPs, but some CBPs also ‘translate’ the Ca²⁺ ‘message’ into changes in cellular function. When the signaling CBPs bind Ca²⁺ conformational changes result in a signal by ‘activating’ other proteins. Calmodulin, the BK-channel, synaptotagmin and troponin C are but a few examples of signaling CBPs.
How a Ca²⁺ signal is ‘interpreted’ by a cell depends on the timing and duration of the Ca²⁺ binding to signaling CBPs.  This is the critical event in the regulation of physiologically relevant processes such as opening of ion channels or activation of gene transcription. Accordingly, Ca²⁺ signaling strongly depends on the competition for Ca²⁺ ions between buffering CBPs and signaling CBPs. Three key features of CBPs determine the spatio-temporal characteristics of Ca²⁺ signals and their transduction: binding kinetics, Ca²⁺ affinity and localization within the cell.  The kinetics of Ca²⁺ binding is probably the most critical component of cellular Ca²⁺ signaling. In the CNS, the distribution of CBPs is highly specific. This has resulted in their use as specific anatomical markers, e.g. parvalbumin (PV) has been considered as an excellent marker for certain types of interneuron. Such distinct distribution supports the idea that selective physiological properties of CBPs contribute to the specific physiological function of various neurons. The presence of various CBPs in certain types of neuron has advanced our anatomical view of the mammalian brain. Several features have been determined for CBPs of the CNS: their biochemical or crystal structure, and their specific neuronal distribution. Yet, with the single exception of calmodulin (CaM), their specific function in nerve cells remains a mystery. It is my goal to quantify the physiological properties of the most important CBPs and to determine how their particular properties play a role in neuronal function. I have, in collaboration with Drs. Mody and Vergara at UCLA, developed an in-vitro technique using Ca²⁺ fluorescent dyes capable of measuring ultra-fast (<20 ms) kinetics of Ca²⁺-binding to CBPs following flash photolysis of caged Ca²⁺. In combination with compartmental kinetic modelling using a new model for cooperative binding kinetics we were able to resolve for the first time the kinetics of a cooperative Ca²⁺ binding process.  By determining the non-equilibrium association and dissociation rates of Ca²⁺ to the most commonly present buffering CBPs and to the signaling CBP CaM, one can make realistic models and predictions about their roles in a wide variety of physiological processes including short- and long-term synaptic plasticity, the regulation of cellular Ca²⁺ microdomains, or determining the susceptibility of neurons to various insults. The first accomplishment was to determine the kinetic properties of calretinin (CR) one of major buffering CBPs of the CNS. The cooperative binding of Ca²⁺ to CR renders the buffering speed of CR to be dependent on ambient Ca²⁺ levels. This is an entirely novel insight, revealing that the Ca²⁺ signal filtering properties of a CBP can be frequency tuned by ambient Ca²⁺ levels making these CBP far more versatile compared to what has been assumed so far. Presently, I am working on measurements to evaluate the properties of calbindin, parvalbumin, and calmodulin. My goal is to expand this research to CBPs essential for synaptic transmission including synaptotagmin and to ion channels affected by direct Ca²⁺ binding (e.g. the BK-channels) independent of calmodulin. 
Written By:          Dr. Guido Faas, UCLA David Geffen School of Medicine
Colloborator(s): Dr. Beat Schwaller, Fribourg University, Fribourg Switzerland