Calcium ion has been widely known as the secondary messenger, which is associated with calmodulin, calbindin, calcineurin and other calcium binding proteins in a variety of organs and tissues, to mediate muscle contraction, heart beating, neurotransmitter release, and gene ex- pression (Figure 1.1) (4, 5, 138-140). As the first messenger, via the calcium sensing receptor or group 1 metabotropic glutamate receptors, it controls calcium homeostasis in organs such as par- athyroid gland, liver, kidney, blood vessels, as well as axon and dendrite outgrowth in nervous system (141-145). Calcium signaling is determined by the amplitude and kinetics of calcium transients, which is contributed by the activation and kinetics of calcium pumps, channels and the buffer proteins. Those calcium binding proteins including calcium channels sophistically control the spatial-temporal calcium gradient and transient, resulting in different physiological signal transduction.
Calcium (Ca2+) transients originate from the calcium concentration gradients across bio- logical membranes and are determined by the calcium binding affinity and kinetics of calcium channels/pumps as well as intracellular calcium binding proteins. The spatial-temporal calcium concentration change results in different physiological signal transduction, including muscle con- traction, heartbeat, neurotransmitter release, gene expression, etc. (4, 5, 138, 139, 141). The time scale of calcium signaling varies from milliseconds to minutes. The fast calcium signaling, espe- cially associated with action potentials usually occurs with a rapid local calcium rise (millisec- onds) due to the calcium influx via the membrane voltage gated calcium channel and the calcium release from the internal stores, for example, the excitation-contraction coupling (EC coupling)
in muscle cells and the neuron-transmitter release in neuron cells (21, 139, 146-155). On other hand, the slower calcium signaling usually happens in cellular events such as immune response, which can last minutes and even hours (7). In those slow calcium signaling pathways, the calci- um transient is controlled by several factors and second messengers like DAG, IP3 and ATP, in- volving more complicated regulation mechanisms (156).
To accurately monitor the calcium transients in terms of the kinetics, amplitude, and du- ration, calcium indicators/sensors are required to have several key properties. It is necessary to match the dissociation equilibrium constant (Kd) of calcium indicators to the resting calcium
concentration of the sub-cellular compartment. On the other hand, to detect fast action potential related calcium release from internal calcium pools such as the endoplasmic reticu-
lum/sarcoplasmic reticulum (ER/SR) in skeletal muscle and neuronal cells, calcium binding af- finity in the range of 0.1-1.0 mM and a calcium dissociation rate greater than 200 s-1 is necessary (considered the time constant range of action potential is 1-5 ms).
Understanding calcium signaling was advanced by the development of calcium dyes and genetically encoded calcium indicators (GECIs) (50, 67-72). High-affinity organic calcium dyes are often used to monitor calcium transients in the cytosol because the Kd is in the magnitude of
hundreds of nanomollar. However, the detection of Δ[Ca2+] induced by a train of high frequency, especially the rapid release from the ER store, is largely limited by the availability of calcium indicators. It has been reported that it is not suitable for the calcium dye Oregon Green BAPTA 1 to detect the calcium transients upon action potential stimulation of more than 50 Hz because the slow decay (koff=2.6 s-1) results in the accumulation of signals (67, 73-75).
The low affinity calcium dyes, with their low affinity and rapid off rate, such as Fluo-5N (76-78), Mag-Fluo-4 (79), Calcium-green-5N (80, 81) were used to monitor calcium change in
internal calcium stores including the mitochondria and the endoplasmic reticulum (82-84). How- ever, the reported kinetics and the amount of calcium concentration change during EC coupling in skeletal and cardiac muscle cells as well as neurons were largely varied. For example, it was reported a broad range from 3.3 to 8.0 ms of the calcium release presented by the full duration at half-maximum (FDHM) at room temperature from the mouse fast-twitch fibers upon the action potential (85-88). Such variations are likely a result of limitations associated with calcium dyes, including the spatial localization of dyes, binding affinity, and kinetics (kon and koff) (71, 89).
The development of genetically encoded indicators (GECI), such as GCaMP, GECO, TN and Cameleon series, enables to probe spatial-temporal cellular events and cell signaling in real time (19, 54, 60, 90-97). GECI are composed with a fluorescent protein moiety and native cyto- solic calcium trigger proteins (CBPs) such as calmodulin (CaM) or troponin C (TnC) that are used to sense calcium concentration change and calcium-induced global conformational rear- rangement. Each CaM or TnC binds four calcium ions cooperatively with a strong calcium bind- ing affinity (Kd=10-7 M) and calcium binding on rates in the magnitude of 107 M-1s-1 that enable
them to sense the immediate [Ca2+] rise in the cytosol (98-101). Meanwhile, these GECIs have slow dissociation rate around 0.1-10 s-1 likely due to the cooperativity associated with multiple calcium binding sites and multiple layers of conformational change (102, 103). Their slow kinet- ics of signal decay hampers their applications in probing physiological fast calcium transient es- pecially in the neuron and skeletal muscle cells (56, 91, 104). Further efforts to reduce the calci- um binding affinities in Cameleon D1ER results in a multiple Kds around 0.8 and 60 μM and the
off rate around 256 s-1 (38). Such sensor, however, was not able to capture the calcium release from SR upon the stimulation in the mouse FDB fibers (105, 106).
The statistical analysis of calcium binding geometry and charged residue preference has been study by our group (66, 157, 158). The classic calcium binding pocket is consisted with 6-7 oxygen atoms, and forms a bipyramidal shape, resulting in the high calcium binding affinity and selectivity. To obtain different calcium binding capability, alternation of the classic calcium binding rule was allowed. For example, to lower the calcium binding affinity, the geometry would be modified to form 3/4 or 1/2 shell of the classical bipyramidal; the negatively charged residues could be replaced by the neutral glutamine or asparagine and the non-polar amino acids; the glutamate and aspartic acid could be substituted with each other to change the side chain length.
To fulfill the unmet need of a fast calcium indicator required for monitoring calcium re- leases in high concentrations such as the ER/SR, we reported a calcium sensor called CatchER without using the native calcium binding domain (66). CatchER was created by designing a cal- cium binding site into a single enhanced green fluorescent protein (EGFP). The binding stoichi- ometry is 1:1 and the Kd is 0.18 mM in vitro and 0.8 mM calibrated in situ allowing measure-
ment of basal calcium in different cell lines and their changes in response to different drugs. Compared to Cameleon D1ER, CatchER exhibits faster kinetics, allowing it to catch the SR cal- cium change in the skeletal muscle cells (107). To date, however, there is no sensors available for measuring calcium responses at the red wavelength region with an advantage of better tissue penetration to complement the green sensors developed by us and others (66). In this chapter, we report the achievement of designing calcium binding protein RapidER using a red florescent pro- tein mCherry by integrating key factors for calcium binding kinetics and metal induced optical property change. We show that the designed red calcium binding sensor RapidER, on the sur- face of mCherry, is able to report calcium concentration by fluorescence in the range of 10-4-10-3
M with an unprecedented dissociation rate in the magnitude of 103 s-1. Our finding reveals essen- tial factors needed for future design of fast calcium binding proteins, fulfilling a pressing need for monitoring calcium dynamics in fast biological processes.