"Ian Parker is distinguished for his elegant and innovative contributions to calcium signalling. Our understanding of how intracellular calcium waves are generated encompasses a remarkable and complex series of events: they are initiated by stochastic release from clusters of IP3 receptors, leading to a hierarchical recruitment of such clusters, which culminates in a 'catastrophic' generation of a wave via a self-amplified calcium-induced release. This understanding has been derived almost entirely from Parker's work, which includes path breaking exploitation of fluorescent microscopy using equipment of his own design and construction. Amongst more recent innovations is to image calcium influx through many individual plasma membrane channels at once by high-speed TIRF microscopy, thus taking the quantitative analysis of such channels into new dimensions."

[Citation for election to the Royal Society, 2008]

The entry of Ca 2+ ions into the cytosol through ligand- and voltage-gated channels in the plasma and endoplasmic reticulum (ER) membranes regulates numerous functions in virtually all types of cells. The specificity of Ca 2+ signals arises from their spatio-temporal patterning, which is organized hierarchically involving openings of single channels (‘fundamental’ events), concerted openings of clustered channels (‘elementary’ events, such as Ca 2+ puffs), and global calcium waves mediated by Ca 2+ diffusion and Ca 2+-induced Ca 2+ release (CICR) between clusters of intracellular release channels such as inositol trisphosphate (IP 3) receptors. Fundamental and elementary events serve localized signaling functions in their own right, as well as forming the building blocks underlying the complex spatio-temporal patterns of cellular Ca 2+ signals. Advanced imaging techniques now offer a unique opportunity to analyze, in intact cells, the hierarchical organization of Ca 2+ signaling from single channels to global responses.

Our overall goals are to elucidate the generation, interaction and functional consequences of fundamental, elementary and global Ca 2+ signals, so as to better understand the physiological and pathological functioning of the ubiquitous Ca 2+ messenger pathway.

1. Development of techniques for imaging and analyzing single-channel Ca 2+ signals. We recently demonstrated, for the first time, imaging of ‘fundamental’ Ca 2+ signals with resolution sufficient to measure the kinetics of single Ca 2+-permeable channels and map their locations with sub- m m precision. These optical techniques hold promise as a complement to patch-clamping for single-channel analysis, and we propose to build on our achievements by;

• Developing improved techniques of TIRF microscopy for imaging voltage-and ligand-gated plasmalemmal channels with sub-ms kinetics, and extend these techniques to mammalian cell lines and cultured neurons.
• Developing a novel method of ‘multi-focal’ microscopy for fast 4-D imaging of fundamental and elementary intracellular Ca 2+ signals.
• Generating computer routines for massively-parallel analysis of single-channel imaging data.

2. Massively-parallel and spatially-correlated studies of channel function. Imaging of single-channel function has some major advantages over existing electrophysiological techniques, enabling simultaneous recording from >500 spatially-mapped channels. We propose to capitalize on these advantages to explore several fundamental aspects of channel properties that would otherwise be inaccessible to experimental study. Specifically, we will;

• Characterize differences in gating properties among nominally-identical channels and explore possible spatially-defined modulation of function.
• Analyze Ca 2+-mediated interactions between individual Ca 2+-permeable channels.
• Utilize TIRFM for imaging gating of IP 3 receptor/channels in nuclear and B cell plasma membrane to measure activation kinetics and study functional modulation by Ca 2+ flux through individual channels.

3. Mechanisms underlying elementary Ca 2+ events. Elementary events involve stochastic opening of a single release channel followed by concerted, transient opening of neighboring channels clustered at functional release sites. The mechanisms underlying these events remain unclear, and we will apply multi-focal microscopy to address the following questions in the context of IP 3-evoked Ca 2+ puffs:

• How are events initially triggered, and what distinguishes single-channel IP 3R signals that evoke puffs from those that do not?
• How are IP 3Rs distributed in a cluster, and how is their recruitment orchestrated?
• How does luminal [Ca 2+] in the ER change during puffs, and may this contribute to puff termination?

4. Elementary and global Ca 2+ signals in neuronal physiology and pathophysiology

Our previous work utilizing Xenopus oocytes as a model system helped elucidate many of the basic mechanisms of IP 3/Ca 2+ signaling. We have now begun to explore how the hierarchy of Ca 2+ signals are coordinated and utilized by neuronal cells to serve their more complex signaling functions. Our aims are to:

• Determine the properties and spatial localization of elementary Ca 2+ events in cultured neurons, and their coordination to generate Ca 2+ waves.
• Explore the interactions of intracellular IP 3-mediated signals with Ca 2+ entry through voltage-and ligand-gated channels.
• Examine the role of disruptions of Ca 2+ signaling in the pathogenesis of Parkinson’s disease (PD) by imaging elementary and global Ca 2+ signals in neuroblastoma cells overexpressing PD-linked mutations associated with defects in mitochondrial function.