Incorporating phototransduction proteins in zebrafish green cone with pressure-polished patch pipettes

Abstract

The neuronal Ca²⁺-sensor guanylate cyclase-activating protein 3 (zGCAP3) is a major regulator of guanylate cyclase (GC) activity expressed in zebrafish cone cells. Here, the zGCAP3, or a monoclonal antibody directed against zGCAP3, was injected in the cone cytoplasm by employing the pressure-polished pipette technique. This technique allows to perform "real time" zGCAP3 (or of any other phototransduction protein) over-expression or knock-down, respectively, via the patch pipette. Photoresponses were not affected by purified zGCAP3, indicating that GC was already saturated with endogenous zGCAP3. The cytosolic injection of anti-zGCAP3 produced the slowing down kinetics of the flash response recovery, as theoretically expected by a minimal phototransduction model considering the antibody acting exclusively on the maximal GC activation by low Ca²⁺. However, the antibody produced a progressive current decay toward the zero level, as if the antibody affected also the basal GC activity in the dark.

Keywords: Calcium signalling; Guanylate cyclase; Phototransduction; Retinal photoreceptors; Zebrafish retina; cGMP.