Mechanistic insights in light-induced cAMP production by photoactivated adenylyl cyclase alpha (PACα)

Abstract

The flagellate Euglena gracilis contains as photoreceptor complex a heterotetrameric light-sensitive adenylyl cyclase (AC), consisting of the flavoproteins PACalpha and PACbeta. Previously, we demonstrated the functional expression of PACalpha and PACbeta in oocytes from Xenopus laevis and of PACalpha in different animal cell types. Both yielded a blue light-induced increase of cellular [cAMP]. Here, we report that the action spectrum of PACalpha is flavoprotein-typical, with maxima at approximately 380 and approximately 470 nm. Mutational analysis of PACalpha yields a model for its structure and function. PACalpha shows a basal AC activity in the dark which is unaffected by mutating the conserved tyrosines in the two flavin-binding domains (F1, F2), Y60 in F1 and Y472 in F2. Y60 in F1 is, however, essential for photoactivation as light-stimulation of cyclase activity is completely lost in the F1 mutant Y60F. This effect does not occur in the respective mutation in F2 (Y472F). Mutating the two cyclase domains (C1, C2) indicated that C1 and C2 form a heterodimeric catalytic center as in mammalian class III cyclases. Interaction of C1 with C2 in the same molecule could be excluded as coexpression of non-functional C1 and C2 mutants restored light-induced cyclase activity. Our results strongly suggest an intermolecular dimerization of C1 and C2 domains on PACalpha for a functional enzyme.

Light Modulation of Cellular cAMP by a Small Bacterial Photoactivated Adenylyl Cyclase, bPAC, of the Soil Bacterium Beggiatoa

“…the enzymatic active state lifetime will be more straightforward. (iii) It is likely that bPAC is active as a homodimer, like most prokaryotic Type III cyclases, resulting in a complex three or six times smaller than the tetrameric euPAC (4,33). (iv) Light stimulation of the purified euPAC complex resulted in an 80-fold cyclase activity (4), whereas we determined a 300-fold increase in activity for bPAC.…”

PACα- an optogenetic tool for in vivo manipulation of cellular cAMP levels, neurotransmitter release, and behavior in Caenorhabditis elegans

“…PACα and PACβ are independent in their catalytic function, and their ability to form cAMP can strongly and repeatedly be increased by blue light (Yoshikawa et al. 2005; Looser et al. 2009).…”

Optogenetic experimentation on astrocytes

“…As the field of optogenetics matures, more tools transpire (Airan et al 2009;Looser et al 2009). These constructs are similar to ChR2 in that they are activated by light but, unlike ChR2, they impinge on the intracellular signalling rather than acting via opening of ion channels in the plasma membrane.…”