Cholecystokinin 1 receptor (CCK1R) is activated photodynamically. For this to happen in situ, genetically-encoded protein photosensitizers (GEPP) may be tagged to natively-expressed CCK1R, but how to best tag GEPP has not been examined. Therefore GEPP (miniSOG or KillerRed) was tagged to CCK1R and light-driven photodynamic CCK1R activation was monitored by Fura-2 fluorescent calcium imaging, to screen for optimized tagging patterns. Blue LED irradiation of CHO-K1 cells expressing miniSOG fused to N- or C-terminus of CCK1R was found to both trigger persistent calcium oscillations - a hallmark of permanent photodynamic CCK1R activation. Photodynamic CCK1R activation was accomplished also with miniSOG fused to N-t... More
Cholecystokinin 1 receptor (CCK1R) is activated photodynamically. For this to happen in situ, genetically-encoded protein photosensitizers (GEPP) may be tagged to natively-expressed CCK1R, but how to best tag GEPP has not been examined. Therefore GEPP (miniSOG or KillerRed) was tagged to CCK1R and light-driven photodynamic CCK1R activation was monitored by Fura-2 fluorescent calcium imaging, to screen for optimized tagging patterns. Blue LED irradiation of CHO-K1 cells expressing miniSOG fused to N- or C-terminus of CCK1R was found to both trigger persistent calcium oscillations - a hallmark of permanent photodynamic CCK1R activation. Photodynamic CCK1R activation was accomplished also with miniSOG fused to N-terminus of CCK1R via linker (GlySerGly) , but not linker (GSG) or an internal ribosomal entry site (IRES) insert. KillerRed fused to N- or C-terminus of CCK1R after white light irradiation resulted in similar activation of in-frame CCK1R. Photodynamic CCK1R activation in miniSOG-CCK1R-CHO-K1 cells was blocked by singlet oxygen ( O ) quencher uric acid or Trolox C, corroborating a role of O as the reactive intermediate. It is concluded that photodynamic CCK1R activation can be achieved either with direct GEPP fusion to CCK1R or fusion via a short linker, fusion via long linkers might serve as internal control.
