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Experimental Approaches for Defining the Role of the Ca2+-Modulated ROS-GC System in Retinal Rods of Mouse

  • Clint L. Makino
  • Teresa Duda
  • Alexandre Pertzev
  • Rameshwar K. Sharma
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1753)

Abstract

Our ability to see is based on the activity of retinal rod and cone photoreceptors. Rods function when there is very little light, while cones operate at higher light levels. Photon absorption by rhodopsin activates a biochemical cascade that converts photic energy into a change in the membrane potential of the cell by decreasing the levels of a second messenger, cGMP, that control the gating of cation channels. But just as important as the activation of the cascade are the shut-off and recovery processes. The timing of shutoff and recovery ultimately affects sensitivity, temporal resolution and even the capacity for counting single photons. An important part of the recovery is restoration of cGMP through the action of rod outer segment membrane guanylate cyclases (ROS-GCs) and guanylate cyclase-activating proteins (GCAPs). In darkness, ROS-GCs catalyze the conversion of GTP to cGMP at a low rate, due to inhibition of cyclase activity by GCAPs. In the light, GCAP enhances ROS-GC activity. Mutations in the ROS-GC system can cause problems in vision, and even result in blindness due to photoreceptor death. The mouse has emerged as a particularly useful subject to study the role of ROS-GC because the technology for the manipulation of their genetics is advanced, making production of mice with targeted mutations much easier. Here we describe some experimental procedures for studying the retinal rods of wild-type and genetically engineered mice: biochemical assays of ROS-GC activity, immunohistochemistry, and single cell recording.

Key words

Membrane guanylate cyclase GCAP Cyclic GMP Photoreceptor Visual transduction Knockout mouse Mouse genetics Confocal microscopy Single cell recording 

Notes

Acknowledgments

We thank Dr. Rikard Frederiksen for comments on the manuscript. Funded in part by NEI EY023980. The contents of this chapter are the sole responsibility of the authors and are not meant to convey the official views of the National Eye Institute.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Clint L. Makino
    • 1
  • Teresa Duda
    • 2
  • Alexandre Pertzev
    • 2
  • Rameshwar K. Sharma
    • 2
  1. 1.Department of Physiology and BiophysicsBoston University School of MedicineBostonUSA
  2. 2.Unit of Regulatory and Molecular Biology, Research Divisions of Biochemistry and Molecular BiologySalus UniversityElkins ParkUSA

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