Protein kinase C and calcium binding proteins in Balanus photoreceptors

Phototransduction in visual receptors consists of the conversion of light energy to an intracellular chemical signal which culminates in generation of a receptor potential. The intracellular mechanisms that mediate between these input-output events remain to be clearly elucidated. The purpose of the present investigation was to determine, largely with immunocytochemical techniques, whether certain putative second messengers of visual transduction could be identified and localized in giant Balanus eburneus photoreceptors. Based on previous eletrophysiological experiments, protein kinase C (PKC), phospholipase C (PLC) and certain Ca2+ binding proteins were of particular interest. Several different methods were employed to establish the existence and locale of the putative messengers: 1) light microscopy of thick frozen sections, 2) light and electron microscopy of thin and ultrathin plastic-embedded sections, and 3) SDS polyacrylamide gel electrophorsis and Western blotting of separated cell proteins. These methods were used in conjunction with antibodies to label different isoforms of PKC and the Ca2+ binding proteins Calbindin D28k (Cal D), Calmodulin and Calsequestrin. Results from three different anti-PKC antibodies used on cryosections collectively indicated the presence of PKC in the photoreceptors. SDS polyacrylamide gels of phtoreceptors revealed a positive reaction product when stained with anti-PKC (381-394); the band corresponded to the molecular weight of PKC (80,000). Experimental results from plastic sections proved to be less consistent than experiments with cryosections. Antiserum to Phospholipase C (PLC) showed no positve reaction product in the photoreceptors. A polyclonal antibody to Calbindin D28k proved reactive in cryosections of the photoreceptors. A calcium indicator, Stainsall," prouced several strong bands on protein gels suggesting the present of several Ca2+ binding proteins in the photoreceptor. Antibody reactions to calsequestrin and calmodulin proved negetive. These results provide evidence that one member of the phophoinositide cascade, PKC, is demonstrable in Balanus photoreceptors. It was not possible in these studies to demonstrate any qualitative differences in the amount or locale of this kinase in light an dark adapted specimens. However, the apparent location of PKC in the rhabdomes and PLC in a region adjacent to them is suggestive of a role for them in transduction. The significance of the finding that Cal D resides in the photoreceptors is unknown, but its identity and the tentative identifcation of other Ca2+ binding proteins indicate potential intracellular stores of this important ion.