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
Part of the Methods in Molecular Biology book series (MIMB, volume 1753)


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 



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.


  1. 1.
    Lamb TD (2013) Evolution of phototransduction, vertebrate photoreceptors and retina. Prog Retin Eye Res 36:52–119CrossRefPubMedGoogle Scholar
  2. 2.
    Koch KW, Dell’Orco D (2015) Protein and signaling networks in vertebrate photoreceptor cells. Front Mol Neurosci 8:67. CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Ingram NT, Sampath AP, Fain GL (2016) Why are rods more sensitive than cones? J Physiol Lond 594:5415–5426CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Wen X-H, Dizhoor AM, Makino CL (2014) Membrane guanylyl cyclase complexes shape the photoresponses of retinal rods and cones. Front Mol Neurosci 7:45. CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Retinal Information Network (1996) The University of Texas Health Science Center, Houston, TX. Accessed 30 Apr 2017
  6. 6.
    Hwang JY, Lange C, Helten A et al (2003) Regulatory modes of rod outer segment membrane guanylate cyclase differ in catalytic efficiency and Ca(2+)-sensitivity. Eur J Biochem 270:3814–3821CrossRefPubMedGoogle Scholar
  7. 7.
    Peshenko IV, Olshevskaya EV, Savchenko AB et al (2011) Enzymatic properties and regulation of the native isozymes of retinal membrane guanylyl cyclase (RetGC) from mouse photoreceptors. Biochemistry 50:5590–5600CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Brooks MJ, Rajasimha HK, Rogers JE et al (2011) Next-generation sequencing facilitates quantitative analysis of wild-type and Nrl(−/−) retinal transcriptomes. Mol Vis 17:3034–3054PubMedPubMedCentralGoogle Scholar
  9. 9.
    Margulis A, Goraczniak RM, Duda T et al (1993) Structural and biochemical identity of retinal rod outer segment membrane guanylate cyclase. Biochem Biophys Res Commun 194:855–861CrossRefPubMedGoogle Scholar
  10. 10.
    Hayashi F, Yamazaki A (1991) Polymorphism in purified guanylate cyclase from vertebrate rod photoreceptors. Proc Natl Acad Sci U S A 88:4746–4750CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Goraczniak RM, Duda T, Sitaramayya A et al (1994) Structural and functional characterization of the rod outer segment membrane guanylate cyclase. Biochem J 302:455–461CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Sambrook J, Fritsch EF, Maniatis T (1989) Expression of cloned genes in cultured mammalian cells. In: Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (eds) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor, NYGoogle Scholar
  13. 13.
    Duda T, Venkataraman V, Goraczniak R et al (1999) Functional consequences of a rod outer segment membrane guanylate cyclase (ROS-GC1) gene mutation linked with Leber’s congenital amaurosis. Biochemistry 38:509–515CrossRefPubMedGoogle Scholar
  14. 14.
    Duda T, Venkataraman V, Jankowska A et al (2000) Impairment of the rod outer segment membrane guanylate cyclase dimerization in a cone-rod dystrophy results in defective calcium signaling. Biochemistry 39:12522–12533CrossRefPubMedGoogle Scholar
  15. 15.
    Makino CL, Kraft TW, Mathies RA et al (1990) Effects of modified chromophores on the spectral sensitivity of salamander, squirrel and macaque cones. J Physiol Lond 424:545–560CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Mendez A, Burns ME, Sokal I et al (2001) Role of guanylate cyclase-activating proteins (GCAPs) in setting the flash sensitivity of rod photoreceptors. Proc Natl Acad Sci U S A 98:9948–9953CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Makino CL, Peshenko IV, Wen X-H et al (2008) A role for GCAP2 in regulating the photoresponse: guanylyl cyclase activation and rod electrophysiology in GUCA1B knock-out mice. J Biol Chem 283:29135–29143CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Makino CL, Wen X-H, Olshevskaya EV et al (2012) Enzymatic relay mechanism stimulates cyclic GMP synthesis in rod photoresponse: biochemical and physiological study in guanylyl cyclase activating protein 1 knockout mice. PLoS One 7:e47637. CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Olshevskaya EV, Calvert PD, Woodruff ML et al (2004) Y99C GCAP1 increases intracellular Ca2+ and causes photoreceptor degeneration in transgenic mice. J Neurosci 24:6078–6085CrossRefPubMedGoogle Scholar
  20. 20.
    Dizhoor AM, Olshevskaya EV, Peshenko IV (2016) The R838S mutation in retinyl guanylyl cyclase 1 (RetGC1) alters calcium sensitivity of cGMP synthesis in the retina and causes blindness in transgenic mice. J Biol Chem 291:24504–24516CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Howes KA, Pennesi ME, Sokal I et al (2002) GCAP1 rescues rod photoreceptor response in GCAP1/GCAP2 knockout mice. EMBO J 21:1545–1554CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Yang R-B, Robinson SW, Xiong W-H et al (1999) Disruption of a retinal guanylyl cyclase gene leads to cone-specific dystrophy and paradoxical rod behavior. J Neurosci 19:5889–5897PubMedGoogle Scholar
  23. 23.
    Baehr W, Karan S, Maeda T et al (2007) The function of guanylate cyclase 1 and guanylate cyclase 2 in rod and cone photoreceptors. J Biol Chem 282:8837–8847CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Baylor DA, Lamb TD, Yau KW (1979) Responses of retinal rods to single photons. J Physiol Lond 288:613–634PubMedPubMedCentralGoogle Scholar
  25. 25.
    Cooper N, Liu L, Yoshida A et al (1995) The bovine rod outer segment guanylate cyclase, ROS-GC, is present in both outer segment and synaptic layers of the retina. J Mol Neurosci 6:211–222CrossRefPubMedGoogle Scholar
  26. 26.
    Duda T, Koch KW, Venkataraman V et al (2002) Ca(2+) sensor S100beta-modulated sites of membrane guanylate cyclase in the photoreceptor-bipolar synapse. EMBO J 21:2547–2256CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Venkataraman V, Duda T, Vardi N et al (2003) Calcium-modulated guanylate cyclase transduction machinery in the photoreceptor—bipolar synaptic region. Biochemistry 42:5640–5648CrossRefPubMedGoogle Scholar
  28. 28.
    Krishnan A, Venkataraman V, Fik-Rymarkiewicz E et al (2004) Structural, biochemical, and functional characterization of the calcium sensor neurocalcin delta in the inner retinal neurons and its linkage with the rod outer segment membrane guanylate cyclase transduction system. Biochemistry 43:2708–2723CrossRefPubMedGoogle Scholar
  29. 29.
    Papermaster DS, Dreyer WJ (1974) Rhodopsin content in the outer segment membranes of bovine and frog retinal rods. Biochemistry 13:2438–2444CrossRefPubMedGoogle Scholar
  30. 30.
    Nickell S, Park PSH, Baumeister W et al (2007) Three-dimensional architecture of murine rod outer segments determined by cryoelectron tomography. J Cell Biol 177:917–925CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Calvert PD, Krasnoperova NV, Lyubarsky AL et al (2000) Phototransduction in transgenic mice after targeted deletion of the rod transducin alpha-subunit. Proc Natl Acad Sci U S A 97:13913–13918CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Mears AJ, Kondo M, Swain PK et al (2001) Nrl is required for rod photoreceptor development. Nat Genet 29:447–452CrossRefPubMedGoogle Scholar
  33. 33.
    Daniele LL, Lillo C, Lyubarsky AL et al (2005) Cone-like morphological, molecular, and electrophysiological features of the photoreceptors of the Nrl knockout mouse. Invest Ophthalmol Vis Sci 46:2156–2167CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    LaVail MM (1973) Kinetics of rod outer segment renewal in the developing mouse retina. J Cell Biol 58:650–661CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Carter-Dawson L, Alvarez RA, Fong SL et al (1986) Rhodopsin, 11-cis vitamin A, and interstitial retinol-binding protein (IRBP) during development in normal and rd mutant mice. Dev Biol 116:431–438CrossRefPubMedGoogle Scholar
  36. 36.
    Schalken JJ, Janssen JJ, Sanyal S et al (1990) Development and degeneration of retina in rds mutant mice: immunoassay of the rod visual pigment rhodopsin. Biochim Biophys Acta 1033:103–109CrossRefPubMedGoogle Scholar
  37. 37.
    Dorrell MI, Aguilar E, Weber C et al (2004) Global gene expression analysis of the developing postnatal mouse retina. Invest Ophthalmol Vis Sci 45:1009–1019CrossRefPubMedGoogle Scholar
  38. 38.
    Luo DG, Yau KW (2005) Rod sensitivity of neonatal mouse and rat. J Gen Physiol 126:263–269CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Lem J, Krasnoperova NV, Calvert PD et al (1999) Morphological, physiological, and biochemical changes in rhodopsin knockout mice. Proc Natl Acad Sci U S A 96:736–741CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Wenzel A, Remé CE, Williams TP et al (2001) The Rpe65 Leu450Met variation increases retinal resistance against light-induced degeneration by slowing rhodopsin regeneration. J Neurosci 21:53–58PubMedGoogle Scholar
  41. 41.
    Xue Y, Shen SQ, Corbo JC et al (2015) Circadian and light-driven regulation of rod dark adaptation. Sci Rep 5:17616. CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Mattapallil MJ, Wawrousek EF, Chan C-C et al (2012) The Rd8 mutation of the Crb1 gene is present in vendor lines of C57BL/6N mice and embryonic stem cells, and confounds ocular induced mutant phenotypes. Invest Ophthalmol Vis Sci 53:2921–2927CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Kueng-Hitz N, Grimm C, Lansel N et al (2000) The retina of c-fos −/− mice: electrophysiologic, morphologic and biochemical aspects. Invest Ophthalmol Vis Sci 41:909–916PubMedGoogle Scholar
  44. 44.
    Patton C (2014) Ca-Mg-ATP-EGTA calculator v1.0 using constants from NIST database #46 v8. Accessed 1 May 2017
  45. 45.
    Nambi P, Aiyar NV, Roberts AN et al (1982) Relationship of calcium and membrane guanylate cyclase in adrenocorticotropin-induced steroidogenesis. Endocrinology 111:196–200CrossRefPubMedGoogle Scholar
  46. 46.
    Winkler BS (1972) The electroretinogram of the isolated rat retina. Vis Res 12:1183–1198CrossRefPubMedGoogle Scholar
  47. 47.
    Raport CJ, Lem J, Makino C et al (1994) Down-regulation of cGMP phosphodiesterase induced by expression of a GTPase-deficient cone transducin in mouse rod photoreceptors. Invest Ophthalmol Vis Sci 35:2932–2947PubMedGoogle Scholar
  48. 48.
    Sung CH, Makino C, Baylor D et al (1994) A rhodopsin gene mutation responsible for autosomal dominant retinitis pigmentosa results in a protein that is defective in localization to the photoreceptor outer segment. J Neurosci 14:5818–5833PubMedGoogle Scholar
  49. 49.
    Azevedo AW, Rieke F (2011) Experimental protocols alter phototransduction: the implications for retinal processing at visual threshold. J Neurosci 31:3670–3682CrossRefPubMedPubMedCentralGoogle Scholar
  50. 50.
    Baylor DA, Matthews G, Yau KW (1983) Temperature effects on the membrane current of retinal rods of the toad. J Physiol Lond 337:723–734CrossRefPubMedPubMedCentralGoogle Scholar
  51. 51.
    Robinson DW, Ratto GM, Lagnado L et al (1993) Temperature dependence of the light response in rat rods. J Physiol Lond 462:465–481CrossRefPubMedPubMedCentralGoogle Scholar
  52. 52.
    Duda T, Wen X-H, Isayama T et al (2015) Bicarbonate modulates photoreceptor guanylate cyclase (ROS-GC) catalytic activity. J Biol Chem 290:11052–11060CrossRefPubMedPubMedCentralGoogle Scholar
  53. 53.
    Mazia D, Schatten G, Sale W (1975) Adhesion of cells to surfaces coated with polylysine. Application to electron microscopy. J Cell Biol 66:198–200CrossRefPubMedGoogle Scholar
  54. 54.
    Baylor DA, Lamb TD, Yau KW (1979) The membrane current of single rod outer segments. J Physiol Lond 288:59–611Google Scholar
  55. 55.
    Schnapf JL (1983) Dependence of the single photon response on longitudinal position of absorption in toad rod outer segments. J Physiol 343:147–159CrossRefPubMedPubMedCentralGoogle Scholar
  56. 56.
    Lamb TD, McNaughton PA, Yau KW (1981) Spatial spread of activation and background desensitization in toad rod outer segments. J Physiol Lond 319:463–496CrossRefPubMedPubMedCentralGoogle Scholar

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

Personalised recommendations