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Question 1: Discuss the mechanisms by which cell-specific calcium signals with
characteristic spatial and temporal properties can be generated in cells and how these contribute to specificity in calcium signaling pathways.
Question 2: discuss the generation of stimulus -specific calcium signature and their role in encoding specificity in calcium signalling pathway
Use Figure 2 to comment on the gating of the channel (10%) and its likely function in nerve cell plasma membrane
Figure 1: The effect of eugenol and o-eugenol on survival (A & C) and cytosolic calcium (B & D, 3.2 mM eugenol/o-eugenol) in fibroblasts in culture media containing 10 μM CaCl2. Values are means +/- SEM from 4 independent experiments (B & D, SEM values = shaded areas).
a) Calculate the approximate concentration of eugenol and o-eugenol that decrease survival by 50% (i.e. the IC50 value) in culture media containing 10 μM CaCl2. (10%)
b) What can you conclude about the likely role of an increase in cytosolic calcium in eugenol toxicity from Figure 1 and your calculated IC50 values? Give brief details of how you arrived at your conclusion. (20%)
Dilution assays of eugenol concentration versus survival were subsequently performed for wild-type and mutant rat fibroblast cells in which a single gene had been knocked out (Mutant X) in culture media supplemented with 100 μM CaCl2 or containing the calcium chelator BAPTA (Figure 2)
Figure 2: The effect of external calcium on cell survival in response to varying concentrations of eugenol. (A) Wild-type fibroblasts: squares/dashed line, 100 μMCaCl2; circles/solid line, 50 μM BAPTA; diamonds/dotted line, 200 μM BAPTA. (B)Mutant X: squares/dotted line, 100 μM CaCl2; circles/solid line, 50 μM BAPTA. Valuesare means +/- SEM (n = 4).
c) Using the IC50 values for eugenol for wild-type fibroblasts in growth media supplemented with 100 μM CaCl2, 50 μM BAPTA and 200 μM BAPTA, explain how the efficacy of eugenol in the survival assay is effected by the concentration of external calcium. (10%)
d) What can you conclude about the role of an increase in cytosolic calcium in the tolerance of fibroblasts to eugenol? Give brief details of how you arrived at your conclusion. (20%)
e) Using your knowledge of the “calcium signalling toolkit” name ONE candidate for the protein encoded by the gene that has been knocked out in Mutant X. Discuss how your named protein might function in eugenol-calcium signalling including details of structure-function relationships for the protein. (40%)