Characterization of guanylate cyclase activity in single retinal rod outer segments.
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.
Koutalos
Y
Y
Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Nakatani
K
K
Tamura
T
T
Yau
K W
KW
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
D006162:Guanylate Cyclase; D006152:Cyclic GMP; D008274:Magnesium; D011188:Potassium; D002118:Calcium