Abstract
Purpose: :
Biochemical, structural and functional characterization of Ca2+–modulated cyclic GMP signaling pathway in the inner retinal neurons, the ganglion cells.
Methods: :
Immunohistochemical analyses, Western blotting, RT–PCR, Ca2+–imaging, patch clamp recordings and in vitro reconstitution experiments in conjunction with enzyme activity assays were employed.
Results: :
Ca2+–dependent membrane guanylate cyclase transduction machinery is expressed in RGC–5 cells. Through immunological and functional analyses ROS–GC1 has been identified as the enzyme component of the system. Two Ca2+ sensor proteins, neurocalcin delta and S100B, co–exist with ROS–GC1 and regulate its activity. Staining of RGC–5 cells with specific antibodies against neurocalcin d and ROS–GC1 shows positive reaction in the processes of the cells. Overexpression of ROS–GC1 promotes Ca2+ entry into RGC5 cells as evidenced by the markedly reduced time–to–peak ratio and increased amplitude of the response. Transfection of neurocalcin d into the cells results in membrane depolarization.
Conclusions: :
Two Ca2+–modulated ROS–GC1 signaling pathways operate in isolated retinal ganglion cells. These pathways employ two Ca2+ sensors, neurocalcin delta and S100B, which via their specific sites on ROS–GC1 regulate Ca2+ traffic in and out of the cell and the membrane polarization. Thus, they may be linked with the visual transduction machinery of the retina.
Keywords: retina: proximal (bipolar, amacrine, and ganglion cells) • calcium • signal transduction: pharmacology/physiology