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Structure and function of calcium signalling pathways

Our research is concerned with understanding how receptors responding to diverse extracellular stimuli evoke release of Ca²⁺ from intracellular stores and how that then regulates cellular activities. How can the same simple cationic element, Ca²⁺, be used by so many different receptors to control such diverse cellular activities without wires getting crossed? One recurrent theme is the importance of spatial organization: a Ca²⁺ signal in one part of a cell can evoke a very different response to a Ca²⁺ signal in another part of the same cell. A second theme is reliability. How do Ca²⁺ signals that involve small numbers of proteins in individual cells, and which thereby differ between cells and include considerable randomness, reliably regulate cellular activity? To tackle these issues, we address IP₃-evoked Ca²⁺ signalling at levels that range from atomic structures, through single-molecule functional analyses and organelle dynamics to cellular behaviours. Our work is supported by national and international collaborations with experts in other disciplines that include X-ray crystallography, chemistry and mathematics.

The wasp stings the elephant. How does IP₃ gate IP₃ receptors?

Opening of all IP₃ receptors is initiated by binding of IP₃, a molecule that is some 5000x smaller than the IP₃ receptor. How does binding of IP₃ cause such a large protein to change its behaviour? IP₃ binds to the IP₃-binding core located within the N-terminal region of the IP₃ receptor. A combination of structure-activity and structural studies demonstrates that all ligands that activate IP₃ receptors interact with each side of the clam-like binding site causing it to close. We suggest that this clam closure disrupts interactions between the four IP₃ receptor subunits leading to opening of the pore. Ryanodine receptors are the second major family of intracellular Ca²⁺ channel, and their N-terminal structures and gating mechanism seem remarkably similar to those of IP₃ receptors.

But IP₃ alone is not capable of gating IP₃ receptors: they must also bind Ca²⁺. This dual regulation of IP₃ receptors by IP₃ and Ca²⁺ is important because it allows IP₃ receptors to propagate Ca²⁺ signals across the cell as regenerative Ca²⁺ waves. The frequency with which these Ca²⁺ waves initiate increases as the extracellular stimulus intensity increases. This allows changes in the frequency of the resulting Ca²⁺ spikes to encode changes in stimulus intensity. But we know very little about the structural basis of Ca²⁺ binding to IP₃ receptors. Another unresolved issue relates to the peculiar pattern of IP₃-evoked Ca²⁺ release from intracellular stores. Stimulation of permeabilized cells with a submaximal concentration of IP₃ causes very rapid release of a fraction of the Ca²⁺ stores, after which release terminates without preventing responses to a subsequent increase in IP₃ concentration. This quantal pattern of Ca²⁺ release is likely to be important in cells responding to physiological stimuli, but its mechanisms are unresolved.

We are presently concerned with answering the following questions:

• What is the structural basis of the interaction between IP₃ and Ca²⁺ binding?
• How are IP₃-evoked structural changes within the N-terminal of the IP₃ receptor communicated to the distant pore to cause its opening?
• What mechanisms underlie quantal IP₃-evoked Ca²⁺ release.

Whispers and shouts. How are intracellular messengers delivered to IP₃ receptors?

From work examining how parathyroid hormone (PTH) regulates Ca²⁺ signals, we suggest that cAMP binds directly to IP₃ receptors to increase their sensitivity to IP₃. In this way, PTH potentiates the Ca²⁺ signals evoked by receptors that stimulate IP₃ formation. Surprisingly, however, even substantial inhibition of cAMP formation has no affect on the ability of PTH to potentiate IP₃-evoked Ca²⁺ signals. This led us to propose that cAMP is delivered directly to IP₃ receptors within junctions where the local concentration of cAMP is more than sufficient to maximally sensitize associated IP₃ receptors. We further propose that the concentration-dependent effects of PTH come from recruitment of these all-or-nothing hyperactive junctions, rather than from graded increases in activity within individual junctions.

We speculate that similar signalling junctions may allow delivery of IP₃ to IP₃ receptors. This has important implications. Firstly, it suggests mechanisms by which different receptors may use IP₃ to activate different IP₃ receptors and thereby evoke different Ca²⁺ signals. Secondly, receptors in the plasma membrane may either shout or whisper to IP₃ receptors. IP₃ receptors within signalling junctions will be robustly stimulated by receptor activation, while more distant IP₃ receptors will be only weakly stimulated and so perhaps respond only with help from additional messengers like cAMP. Finally, competitive antagonists of IP₃ will differentially affect signalling from the plasma membrane to junctional and extra-junctional IP₃ receptors.

We are addressing the following questions:

• How is IP₃ delivered from different receptors in the plasma membrane to IP₃ receptors?
• How are intracellular Ca²⁺ stores organized to allow different IP₃ receptors to release Ca²⁺ independently?
• How does cAMP regulate IP₃ receptors?

Intimate liaisons. How do dynamic proteins and organelles shape Ca²⁺ signals?

Most IP₃ receptors are expressed within membranes of the ER. But the ER is unique amongst intracellular organelles in the extent to which it forms close associations with the plasma membrane and with the membranes of most other intracellular organelles. These interactions have important consequences for both generating and decoding of Ca²⁺ signals. Furthermore, the ER and the organelles with which it interacts are dynamic, and so too are the proteins, like IP₃ receptors, that reside in ER membranes. How are these dynamic proteins and organelles regulated and how do the interactions between them shape cytosolic Ca²⁺ signals?

Fluorescence recovery after photobleaching (FRAP) analyses of IP₃ receptor mobility and patch-clamp recording of nuclear IP₃ receptors suggest that IP₃ receptors are mobile within ER membranes and that IP₃ causes IP₃ receptors to assemble into small clusters. We suggest that this clustering retunes the regulation of IP₃ receptors by IP₃ and Ca²⁺ better to allow them to mediate regenerative recruitment of Ca²⁺ signals. But other evidence has suggested that IP₃-evoked Ca²⁺ signals are initiated by immobile IP₃ receptors.

Recent work suggests that mobile lysosomes selectively sequester Ca²⁺ released by IP₃ receptors while ignoring that entering the cell via store-operated Ca²⁺ entry pathways. Lysosomes that are dynamic but closely associated with ER thereby shape the Ca²⁺ signals evoked by receptors that stimulate IP₃ formation. Neither the means by which lysosomes accumulate Ca²⁺ nor how they maintain such close contact with ER is understood.

Our work with signalling junctions also suggests that signalling pathways in the plasma membrane come into very close contact with IP₃ receptors in the ER. Are these interactions dynamic and regulated?

We are presently addressing the following questions:

• What is the relationship between the distribution of IP₃ receptors and the Ca²⁺ signals they evoke?
• Does IP₃ regulate IP₃ receptor distribution?
• How do lysosomes accumulate Ca²⁺?
• How do dynamic lysosomes maintain their associations with dynamic ER?
• Are signalling junctions between the plasma membrane and ER dynamically regulated?

Getting the message. How do cells decode Ca²⁺ signals?

How do Ca²⁺ signals selectively regulate different cellular activities? Ca²⁺ signals are initiated by random events involving tiny numbers of active IP₃ receptors, and individual cells differ in their sensitivities to extracellular stimuli. With so much variable and unpredictable behaviour, how do cells reliable encode extracellular stimuli as Ca²⁺ signals, and how do they then decode the Ca²⁺ signals?

Recent work suggests a role for Ca²⁺ release via IP₃ receptors in controlling cell migration, and we suggest that this may require delivery of IP₃ receptors to the leading edge of migrating cells.

Analysis of receptor-evoked Ca²⁺ spikes in a variety of cells suggests that cells respond to increases in stimulus intensity with fold-increases in the frequency of the Ca²⁺ spikes. This may allow cells reliably to encode changes in stimulus intensity despite considerable randomness and variability.

We are presently addressing the following questions:

• What is the role of IP₃ receptor movement in cell migration?
• How is information encoded and then decoded by Ca²⁺ spikes?