Calcium dependence of aequorin bioluminescence dissected by random mutagenesis

Abstract

Aequorin bioluminescence is emitted as a rapidly decaying flash upon calcium binding. Random mutagenesis and functional screening were used to isolate aequorin mutants showing slow decay rate of luminescence. Calcium sensitivity curves were shifted in all mutants, and an intrinsic link between calcium sensitivity and decay rate was suggested by the position of all mutations in or near EF-hand calcium-binding sites. From these results, a low calcium affinity was assigned to the N-terminal EF hand and a high affinity to the C-terminal EF-hand pair. In WT aequorin, the increase of the decay rate with calcium occurred at constant total photon yield and thus determined a corresponding increase of light intensity. Increase of the decay rate was underlain by variations of a fast and a slow component and required the contribution of all three EF hands. Conversely, analyses of double EF-hand mutants suggested that single EF hands are sufficient to trigger luminescence at a slow rate. Finally, a model postulating that proportions of a fast and a slow light-emitting state depend on calcium concentration adequately described the calcium dependence of aequorin bioluminescence. Our results suggest that variations of luminescence kinetics, which depend on three EF hands endowed with different calcium affinities, critically determine the amplitude of aequorin responses to biological calcium signals.EF hand ͉ kinetics ͉ luminescence ͉ photoprotein ͉ transduction T he photoprotein aequorin is a stable luciferase intermediate formed from the reaction of the protein apoaequorin (luciferase) and the substrate coelenterazine (luciferin), which emits light upon Ca 2ϩ binding (1-5). Aequorin contains three EF-hand Ca 2ϩ -binding sites (6-8) located close to its N (EF1) or C terminus (EF2,3 pair). Aequorin mutagenesis and crystal structure suggest that these three EF hands indeed bind Ca 2ϩ , but their individual contribution to luminescence is still a matter of debate (9-13).The steep increase of luminescence intensity with [Ca 2ϩ ] makes aequorin a useful reporter of intracellular calcium signals (14). The formation of aequorin is a slow process (2), whereas the luminescence reaction is very fast and proceeds to completion in the continuous presence of Ca 2ϩ . The aequorin response thus occurs as a flash that decays exponentially and whose onset rate does not depend on [Ca 2ϩ ] (15). It has been observed early that the decay rate of this response increases with [Ca 2ϩ ], whereas the total light emitted (light integral) remains relatively constant (15). This suggests that the increase of luminescence intensity with [Ca 2ϩ ] is determined by variations of the decay rate but not of the light integral. In other words, the shorter the duration of the flash (i.e., the faster the decay), the larger the amplitude of the response (i.e., light intensity). However, the relationships among the intensity, the decay rate, and the integral of bioluminescence and their links to EF-hand occupancy have not been clearly established.To analyze the co...

Calcium imaging in single neurons from brain slices using bioluminescent reporters

“…Indeed, onset and decay time constants of 10 msec and 833 msec, respectively, were obtained for aequorin (Hastings et al, 1969), whereas these values were 2.5-4 msec and 139-294 msec, respectively, for obelin depending on the Obelia species (Morin and Hastings, 1971a;Stephenson and Sutherland, 1981;Illarionov et al, 2000;Stepanyuk et al, 2005). Similarly, the aequorin N26D mutant reportedly exhibits slower decay kinetics than wild-type aequorin (Tricoire et al, 2006). The present results further suggest that fusion of GFP at the N-terminal of the photoproteins resulted in only minor modifications of their kinetic properties.…”

“…It should be noted, however, that differences in GFP-photoprotein kinetics may fade at and below micromolar Ca 21 concentrations, as reported for aequorin and aequorin N26D (Tricoire et al, 2006). Indeed, although onset kinetics of photoproteins are reportedly independent of Ca 21 concentration (Hastings et al, 1969), their light emission rate markedly decreases with decreasing Ca 21 concentration (Hastings et al, 1969;Illarionov et al, 2000;Markova et al, 2002;Tricoire et al, 2006). Nonetheless, GO responses may be more intense than those of GA under physiological conditions in which excitatory synaptic transmission, or the presence of neuromodulators, combines with activities of voltage-gated channels to generate larger Ca 21 signals.…”

“…The apoaequorin gene was fully codon optimized for better expression in mammalian cells. The apoaequorin N26D mutation (Tsuzuki et al, 2005;Tricoire et al, 2006) was introduced in the humanized apoaequorin sequence by site-directed mutagenesis using the Quick Change Site-Directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer's instructions. The apo-obelin gene was purchased from LUX Biotechnology (Humanized obelin from Obelia longissima; LUX Biotechnology, Edinburgh, United Kingdom) and was modified by suppressing the codons between the last proline codon (which is the last amino acid of wild-type apo-obelin) and the stop codon.…”

“…The subthreshold light signal of GA N26D was similar to that of GA, suggesting that the sensitivity to low calcium levels is not enhanced in this aequorin mutant. Indeed, the N26D mutation increases the Ca 21 affinity of the first, lower affinity EF-hand but not that of the two high-affinity EF-hands of aequorin (Tricoire et al, 2006).…”

“…GFP-photoprotein fusions comprised the linker peptide described by Baubet et al (2000) for GA, where the Ca 21 sensitivity curve of the photoprotein is unchanged. In addition to GA and GO, a third sensor contained the aequorin N26D mutant (Tsuzuki et al, 2005), which exhibits a higher Ca 21 sensitivity (i.e., reaches maximal response at lower Ca 21 concentration) and slower kinetics than wild-type aequorin (Tricoire et al, 2006). The kinetics of their responses to Ca 21 and the inhibitory effect of physiological Mg 21 concentrations (Zhang et al, 1997) were first studied in vitro by using a fast-mixing apparatus.…”