title: Characterization of guanylate cyclase activity in single retinal rod outer segments.
abstract: cGMP mediates vertebrate phototransduction by directly gating cationic channels on the plasma membrane of the photoreceptor outer segment. This second messenger is produced by a guanylate cyclase and hydrolyzed by a light-activated cGMP-phosphodiesterase. Both of these enzyme activities are Ca2+ sensitive, the guanylate cyclase activity being inhibited and the light-activated phosphodiesterase being enhanced by Ca2+. Changes in these activities due to a light-induced decrease in intracellular Ca2+ are involved in the adaptation of photoreceptors to background light. We describe here experiments to characterize the guanylate cyclase activity and its modulation by Ca2+ using a truncated rod outer segment preparation, in order to evaluate the enzyme's role in light adaptation. The outer segment of a tiger salamander rod was drawn into a suction pipette to allow recording of membrane current, and the remainder of the cell was sheared off with a probe to allow internal dialysis. The cGMP-gated channels on the surface membrane were used to monitor conversion of GTP, supplied from the bath, into cGMP by the guanylate cyclase in the outer segment. At nominal 0 Ca2+, the cyclase activity had a Km of 250 microM MgGTP and a Vmax of 25 microM cGMP s-1 in the presence of 1.6 mM free Mg2+; in the presence of 0.5 mM free Mg2+, the Km was 310 microM MgGTP and the Vmax was 17 microM cGMP s-1. The stimulation by Mg2+ had an EC50 of 0.2 mM Mg2+ for MgGTP at 0.5 mM. Ca2+ inhibited the cyclase activity. In a K+ intracellular solution, with 0.5 mM free Mg2+ and 2.0 mM GTP, the cyclase activity was 13 microM cGMP s-1 at nominal 0 Ca2+; Ca2+ decreased this activity with a IC50 of approximately 90 nM and a Hill coefficient of approximately 2.0.
authors.affiliation: Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Koutalos
authors.forename: K
authors.initials: K
authors.lastname: Nakatani
authors.forename: T
authors.initials: T
authors.lastname: Tamura
authors.forename: K W
authors.initials: KW
authors.lastname: Yau
mesh_terms: D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002462:Cell Membrane / Q000201:enzymology; D006152:Cyclic GMP / Q000032:analysis / Q000502:physiology; D004594:Electrophysiology; D006162:Guanylate Cyclase / Q000187:drug effects / Q000378:metabolism*; D015640:Ion Channel Gating / Q000502:physiology; D008274:Magnesium / Q000494:pharmacology; D008433:Mathematics; D011188:Potassium / Q000494:pharmacology; D017948:Retinal Rod Photoreceptor Cells / Q000737:chemistry / Q000201:enzymology* / Q000648:ultrastructure; D014562:Caudata; D014785:Vision, Ocular / Q000502:physiology
chemical_list: D006162:Guanylate Cyclase; D006152:Cyclic GMP; D008274:Magnesium; D011188:Potassium; D002118:Calcium