Cellular and Molecular Pharmacology

Unfolding cellular mechanisms by Ca²⁺ signaling-based research

Ca²⁺ signaling is one of the most important signaling mechanisms regulating myriads of cellular functions (Fig. 1). We have identified basic mechanisms of the diverse spatiotemporal dynamics of Intracellular Ca²⁺ signals, which underlie the versatility of Ca²⁺ in cell signaling. Based on these key findings, we are aiming at deciphering physiological and pathophysiological functions of Ca²⁺ at all levels spanning from the molecules to whole body.

1. BASIC MECHNISMS OF Ca²⁺ RELEASE

While Ca²⁺ signal can be generated by an influx of Ca²⁺ from the extracellular space, Ca²⁺release from the intracellular store also plays important roles in generation of Ca²⁺ signals. The endoplasmic reticulum functions as the major intracellular Ca²⁺ store, and Ca²⁺ can be released via two types of Ca²⁺ release channel; ryanodine receptors and inositol 1,4,5-trisphosphate (IP₃) receptors (Fig. 2).

Our laboratory has clarified basic mechanisms of the Ca²⁺ release and Ca²⁺ signaling mechanisms as summarized below.

Ca²⁺ signals show very dynamic spatiotemporal changes. This property allows the Ca²⁺ signal to be an extremely versatile cellular switch regulating diverse cell functions (Fig. 1). One of the most notable spatiotemporal patterns of Ca²⁺ signals is a wave-like increase in intracellular cytoplasmic Ca²⁺ concentration ([Ca²⁺]_Cyt), or Ca²⁺ wave. Another remarkable spatiotemporal pattern is an oscillatory change in [Ca²⁺]_Cyt, or Ca²⁺ oscillation. Indeed, many cellular functions are regulated by the frequency of Ca²⁺ oscillation. However, fundamental questions remain. How and what for does [Ca²⁺]_Cyt oscillate? We addressed these important questions.

First, we studied IP₃-induced Ca²⁺ release mechanism, which is one of the most important Ca²⁺ mobilizing mechanisms in many types of cell. We showed that activation of the IP₃ receptor (IP₃R) requires not only IP₃ but also Ca²⁺ (J. Gen. Physiol., 1990; Nature, 1992). Therefore, Ca²⁺release via the IP₃R is under the positive feedback control of Ca²⁺ that is released through the IP₃R. The positive feedback loop makes Ca²⁺ release via IP₃R regenerative. This property nicely explains the reason for the generation of Ca²⁺ wave (Fig. 3).

There are three subtypes of IP₃R, which have different sensitivities to IP₃ and Ca²⁺ concentrations. We showed that these subtypes generate different patterns of Ca²⁺ oscillation (EMBO J. 1999). We then identified the Ca²⁺ sensor region of the IP₃R, and experimentally showed that the positive feedback regulation of IP₃R via the Ca²⁺ sensor of IP₃R indeed plays an essential role in regulating Ca²⁺ oscillations (EMBO J., 2001). Thus, our results clarified that the regeneratively of IP₃-induced Ca²⁺ release is fundamental to the generation of Ca²⁺ oscillation.

What for, then, does [Ca²⁺]_Cyt oscillate? We addressed this question looking at transcription by the nuclear factor of activated T cells (NFAT), which is one of the important cellular functions that are regulated by the Ca²⁺ oscillation frequency. NFAT is dephosphorylated by Ca²⁺-dependent phosphatase, calcineurin. Upon dephosphorylation, NFAT is translocated from the cytoplasm to the nucleus to initiate transcription. We analyzed the kinetics of the dephosphorylation and translocation of NFAT, and found that the dephosphorylated form of NFAT functions as a working memory of transient increases in [Ca²⁺]_Cyt (EMBO J. 2003). With increasing frequency of Ca²⁺ oscillation, dephosphorylated NFAT accumulates in the cytoplasm to enhance its nuclear translocation. This is the molecular basis of the mechanism that decodes the Ca²⁺ oscillation frequency. We further showed that Ca²⁺oscillation is more cost-effective in regulating cell functions than a continuous increase in Ca²⁺ (EMBO J. 2003). These studies provide us with an insight into the reason why [Ca²⁺]_Cyt has to oscillate.

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2. IMAGING STUDIES

Our study on Ca²⁺ signaling made us realize the importance of visualization of signaling molecules within living cells. Thus, our laboratory has been involved in the generation of new indicators of signaling molecules upstream and downstream Ca²⁺ signals.

IP₃ imaging

We generated an IP₃ indicator based on the PH domain of PLC{delta}1, and have succeeded in imaging IP₃ signaling in various cells including intact neurons within cerebellar slice preparations. Our study showed that IP₃ signaling in cerebellar Purkinje cells are cooperatively regulated by both metabotropic and ionotropic glutamate receptors (Fig. 5).

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Nitric oxide (NO) imaging

We generated a nitric oxide (NO) indicator, HBR-GFP, based on the heme-binding region of soluble guanylyl cyclase (the physiological target of NO). This indicator was successfully used in cerebellar slice preparations to image NO signals in response to parallel fiber (PF) stimulation. We found that the NO signal intensity decreases steeply with distance from the activated synapse, and NO generates synapse-specific long-term potentiation (LTP) of PF-Purkinje cell synapses. We also showed that the NO signal intensity depends biphasically on the frequency of PF stimulation. (This means that there is an optimal firing frequency for NO generation.) Importantly, the LTP depends similarly on the frequency of PF stimulation (Fig. 5). Thus, our NO indicator provided us with valuable information regarding the role of NO signals in the central nervous system.

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Glutamate imaging

Glutamate is the most important excitatory neurotransmitter in the brain. We generated a new glutamate indicator (EOS) based on the glutamate binding site of the ionotropic glutamate receptor (GluR2). We have successfully used this indicator to image glutamate dynamics in acute brain slices and in cerebral cortex in vivo (Fig. 5).

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Intraorganellar Ca²⁺ imaging

As described above, Ca²⁺ is released from the ER. Therefore, Ca²⁺ concentration within the ER ([Ca²⁺]_ER) is an important information. However, [Ca²⁺]_ER) is several thousand-fold higher than that of [Ca²⁺]_Cyt, and It has been difficult to measure [Ca²⁺]_ER with a high signal-to-noise ratio. Based on a genetically encoded Ca²⁺ indicator, we successfully generated intraorganellar Ca²⁺ indicators (CEPIAs) that are optimized for measuring [Ca²⁺]_ER. We also generated variants of CEPIA that are suitable for measuring Ca²⁺ in mitochondria ([Ca²⁺]_Mit). Since we generated CEPIAs with three different fluorescence colors, we can now measure [Ca²⁺]_Cyt, [Ca²⁺]_ER, and [Ca²⁺]_Mit, simultaneously. Using CEPIA we have succeeded in imaging ER Ca²⁺ dynamics in postsynaptic neurons in the cerebellum. We observed synaptic input-dependent Ca²⁺ release from the ER. Furthermore, we observed activity-dependent accumulation of Ca²⁺ in the ER, so that ER functions as a memory of nervous activities.

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3. SEARCH FOR NEW ROLES OF Ca²⁺ AND RELATED SIGNALING MOLECULES

Our laboratory has been trying to identify hitherto unrecognized functions of Ca²⁺ signals especially in the brain in both health and disease. We have a very powerful tool to study the role of Ca²⁺ signals, i.e., the IP₃ hydrolyzing enzyme, IP₃ 5-phosphatase, which effectively shuts down IP₃ signaling. Indeed, in many cell types expression of the enzyme virtually abolishes agonist-induced Ca²⁺ release.

Physiological functions

Using the method to inhibit IP₃-induced Ca²⁺ release, we have so far identified an activity-dependent synaptic maintenance mechanism in the cerebellar cortex (Fig. 5) and a neuron-astrocyte interaction in terms of the promotion of neurite growth.

We also studied the role of NO signaling in the brain. We showed that NO induces Ca²⁺ release from the intracellular sore in cerebellar Purkinje cells. Our in-depth analyses showed that NO S-nitrosylates the type 1 ryanodine receptor (RyR1) at a specific cysteine residue (Cys3636 in mouse, Cys3635 in human) to induce activation of the Ca²⁺ release channel. This NO-induced Ca²⁺ release (NICR) leads to long-term potentiation (LTP) of the parallel fiber-Purkinje cell synapse (Fig. 5).

Pathophysiological functions

We then generated a knock-in mouse line, in which the important cysteine residue (Cys3636) is replaced with alanine so that NICR is deficient in these animals. Interestingly, we found that NICR also has a pathophysiological role in neuronal injury during hyperactivity of neurons (excitotoxicity). In NICR-deficient mice, seizure-induced neuronal injury was significantly ameliorated. Furthermore, we found that a pharmacological agent, dantrolene, which inhibits NICR has a neuroprotective effect. Thus, NICR is involved in excitotoxic neuron injury (Fig. 6), and inhibition of NICR may have a therapeutic value for excitotoxicity.

Our in vivo Ca²⁺ imaging in astrocytes showed that these cells generate robust Ca²⁺ oscillations after brain injury. These Ca²⁺ oscillations are absent in IP₃R2-KO mice. By comparing the effect of brain injury between WT and IP₃R2-KO mice, we found that astrocytic Ca²⁺ oscillations are required for reactive astrogliosis and neuroprotection (Fig. 7).

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4. IN VIVO Ca²⁺ IMAGING

Our goal of Ca²⁺ imaging is to image Ca²⁺ signals (and other signaling molecules) within individual cells of a living animal. We have succeeded in imaging Ca²⁺ signals and other signaling molecules such as glutamate in the brain of living mice (Fig. 8).

We are now trying to expand the list of organs for in vivo imaging.

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5. FUTURE DIRECTIONS

Combining new imaging tools and the specific method to inhibit Ca²⁺ signals, we wish to contribute to the advancement of our knowledge in cell signaling especially in conjunction with brain functions.

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