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Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21205.

1. In darkness, a single rod outer segment isolated from the toad retina was sucked partially, tip first, into a tight-fitting, Ringer solution-filled glass pipette for recording membrane current. The basal end of the outer segment outside the pipette was sheared off with a probe to allow internal dialysis. The potential between the inside and the outside of the pipette was held at 0 mV. 2. With cyclic GMP and IBMX (isobutylmethylxanthine) in the dialysis solution, a large inward current appeared across the plasma membrane of the outer segment; this current saturated at around 1 mM-cyclic GMP. IBMX by itself was ineffective. 3. The saturated cyclic GMP-induced current recorded varied in size with the length of outer segment (L) within the suction pipette. For L less than 25 micron, the relation was linear, with a current density of 4-20 pA micron-1. 4. At short L (less than 25 micron), the dose-response relation between current magnitude and cyclic GMP concentration was sigmoidal, with a Hill coefficient (n) of 1.8-3.1 and a half-saturating cyclic GMP concentration (K1/2) of 30-85 microM. 5. In the presence of IBMX and the absence of GTP, the dose-response relation was the same in continuous bleaching light as in darkness. This indicates that both the characteristics of cyclic GMP binding and the intrinsic conduction properties of the open conductance are not affected by light. 6. Removing IBMX from the dialysing solution had little effect on the saturated current, but substantially reduced the current induced at low concentrations of cyclic GMP. When the analogue 8-bromo cyclic GMP was used instead, however, the presence of IBMX was relatively unimportant even at low agonist concentrations. These observations indicated that significant phosphodiesterase activity was present within the truncated outer segment. 7. In the absence of IBMX and the presence of GTP, the cyclic GMP-induced current could be suppressed by light. When ATP was also present in the dialysing solution, the effect of light was significantly reduced and the suppression also became more transient. 8. We conclude from the above results that the cyclic GMP-gated conductance is indeed present in the plasma membrane of the rod outer segment, and that this conductance and the light-sensitive conductance are one and the same entity. 9. From the results, we estimate that only about 1% of the conductance is normally open in darkness. This fraction of open conductance corresponds to a free cyclic GMP concentration of a few micromolar.

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