Real-Time Modulation of Zebrafish Cone Phototransduction by Whole-Cell Delivery of zGCAP3 and of its Monoclonal Antibody

Abstract

Regulation of excitation and adaptation in photoreceptors of the vertebrate retina strongly depends on the cytoplasmic Ca2+ concentration and its interplay with Ca2+ sensor proteins like recoverin, calmodulin and the activating proteins (GCAPs) of guanylate cyclase (GC) (Scholten and Koch, 2011). Of the four GCAP isoforms exclusively transcribed in cones (zGCAP3, 4, 5 and 7), we investigated the physiological function of zGCAP3 in green-sensitive cones of zebrafish, by recording the effect on the photoresponse waveform by cytosol injection of exogenous zGCAP3 (to simulate “real time” protein over-expression), and its monoclonal antibody (to simulate protein knock-down). To identify a suitable antibody we screened several hybridoma fluids with respect to specificity and affinity towards zGCAP3, using immunoblotting and surface plasmon resonance (SPR) spectroscopy. The global fitting of an overlay of SPR sensorgrams obtained with increasing antibody concentrations gave a Ca2+-independent KD of 12 nM for the interaction of zGCAP3 with the antibody. Exogenous proteins were incorporated with a precise timing in the zebrafish cone cytosol by an internal perfusion system coupled to a pressure-polished patch pipette (Benedusi et al. 2011). Typical whole-cell recordings lasting even more than 20 min did not show any significant change in light sensitivity, dark current amplitude, response kinetics and light adaptation, proving also that the enzymatic cascade was not perturbed by the recording protocol. Injection of anti-zGCAP3 caused the complete shutdown of the dark current, indicating that zGCAP3 plays a major role in regulating GC. Injection of purified zGCAP3 did not alter the photoresponse, indicating that the target GC was already saturated with endogenous zGCAP3.Benedusi M, Aquila M, Milani A and Rispoli G (2011). Eur Biophys J 40: 1215-23.Scholten A, Koch KW. (2011). PLoS One 6(8):e23117.