[ABSTRACT]
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.