GATING, SELECTIVITY AND BLOCKAGE OF SINGLE CHANNELS ACTIVATED BY CYCLIC-GMP IN RETINAL RODS OF THE TIGER SALAMANDER

1. Patches in the inside-out configuration were excised from the membr ane of outer and inner segments of the larval tiger salamander, Ambyst oma tigrinum. The current flowing through single channels opened by cy clic GMP was studied with the voltage clamp technique. 2. Amplitude hi stograms of current recordings from patches containing only one flicke ring channel, excised from the inner segment and in the presence of 10 0 mu M cyclic GMP, could be fitted by a theoretical scheme in which th e single channel conductance was at least 55 pS at +40 mV and at least 45 pS at -40 mV. The mean open time was no longer than the time const ant of our recording system, about 35 mu s. Similar results were obtai ned by analysis of the amplitude histograms of patches from the outer segment containing many channels, and in the presence of 1-5 mu M cycl ic GMP. 3. In membrane patches excised from the outer segment, reducin g the temperature from 24 to 8 degrees C did not reduce the flickering , but changed the amplitude histograms of current fluctuations activat ed by 1 mu M cyclic GMP in a way consistent with a decrease of 50 % in the single channel conductance and a decrease of 50 % in the open pro bability. 4. In the presence of 1 mu M cyclic GMP at +60 mV, when Nawas replaced by NH4+ or K+, brief outward current transients flowing t hrough single channels were observed. When Na+ was replaced with Li+, Rb+ or Cs+, current transients were very small. 5. The shape of the po wer spectrum of current fluctuations induced by 1 mu M cyclic GMP at 60 mV did not change when the permeating ion was Na+, K+ or NH4+. Anal ysis of the amplitude histogram did not show any effect of the tested monovalent cations on the open probability or on channel gating. At +6 0 mV, the estimated single channel currents were at least 4, 2.8 and 2 pA for NH4+, Na+ and K+ respectively. 6. The addition of 0.5 or mM Ca 2+ to the medium bathing the cytoplasmic side of the membrane greatly reduced the frequency of openings, but single channel activity could s till be observed. The blocking effect of 1 mM Ca2+ on the channel acti vity induced by 2 mu M cyclic G;MP could be counterbalanced by increas ing the cyclic GMP concentration. The addition of 0.5 or 1 mM Ca2+ did not change the shape of power spectra obtained at membrane voltages b etween -100 and +100 mV. 7. The addition of 0.5 mM Mg2+, the medium ba thing the cytoplasmic side of the membrane did not affect the single c hannel activity observed at membrane voltages more negative than -40 m V. At positive membrane voltages, Mg2+ had a strong blocking effect on the single channel activity and caused a slight change in the shape o f power spectra of current fluctuations. 8. The present analysis of si ngle channel properties of the cyclic GMP-activated channel indicates that: (i) the gating of the channel is characterized by an intrinsic f lickering which is not abolished by lowering the temperature from 24 t o 8 degrees C; (ii) the gating of the channel is not effected by repla cing Na+ with NH4+ or K+ (iii) Ca2+ and Mg2+ block the channel by diff erent mechanisms.