The use of isothermal titration calorimetry to unravel chemotactic signalling mechanisms

Chemotaxis is based on the action of chemosensory pathways and is typically initiated by the recognition of chemoeffectors at chemoreceptor ligand-binding domains (LBD). Chemosensory signalling is highly complex; aspect that is not only reflected in the intricate interaction between many signalling proteins but also in the fact that bacteria frequently possess multiple chemosensory pathways and often a large number of chemoreceptors, which are mostly of unknown function. We review here the usefulness of isothermal titration calorimetry (ITC) to study this complexity. ITC is the gold standard for studying binding processes due to its precision and sensitivity, as well as its capability to determine simultaneously the association equilibrium constant, enthalpy change and stoichiometry of binding. There is now evidence that members of all major LBD families can be produced as individual recombinant proteins that maintain their ligand-binding properties. High-throughput screening of these proteins using thermal shift assays offer interesting initial information on chemoreceptor ligands, providing the basis for microcalorimetric analyses and microbiological experimentation. ITC has permitted the identification and characterization of many chemoreceptors with novel specificities. This ITC-based approach can also be used to identify signal molecules that stimulate members of other families of sensor proteins.     

Cyclic ADP-ribose and inositol 1,4,5-trisphosphate mobilizes Ca from distinct intracellular pools in permeabilized lacrimal acinar cells

In permeabilized lacrimal acinar cells, cyclic ADP-ribose (cADP-ribose) and inositol 1,4,5-trisphosphate (Ins(1,4,5)P₃) release Ca²⁺ in a dose dependent manner from distinct thapsigargin-sensitive Ca²⁺ pools. Ryanodine specifically blocks the Ca²⁺ response to cADP-ribose, whereas heparin strongly reduces the response to Ins(1,4,5)P₃ application. GTP causes a rapid Ca²⁺ release by a ryanodine- and heparin-insensitive mechanism and potentiates Ins(1,4,5)P₃ but not cADP-ribose evoked Ca²⁺ release. It is estimated that cADP-ribose can release 16 μmol Ca²⁺/I cells, whereas Ins(1,4,5)P₃ can mobilize 55 μmol Ca²⁺/I cells. The results suggest that cADP-ribose and Ins(1,4,5)P₃ release Ca²⁺ from distinct internal stores and that a third Ca²⁺ pool exists which can selectively interact with the Ins(1,4,5)P₃-sensitive Ca²⁺ store by a GTP-mediated process.