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 membrane of outer and inner segments of the larval tiger salamander, Ambystoma tigrinum. The current flowing through single channels opened by cyclic GMP was studied with the voltage clamp technique. 2. Amplitude histograms of current recordings from patches containing only one flickering channel, excised from the inner segment and in the presence of 100 microM cyclic GMP, could be fitted by a theoretical scheme in which the 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 constant of our recording system, about 35 microseconds. Similar results were obtained by analysis of the amplitude histograms of patches from the outer segment containing many channels, and in the presence of 1-5 microM cyclic GMP. 3. In membrane patches excised from the outer segment, reducing the temperature from 24 to 8 degrees C did not reduce the flickering, but changed the amplitude histograms of current fluctuations activated by 1 microM cyclic GMP in a way consistent with a decrease of 50% in the single channel conductance and a decrease of 50% in the open probability. 4. In the presence of 1 microM cyclic GMP at +60 mV, when Na+ was replaced by NH4+ or K+, brief outward current transients flowing through single channels were observed. When Na+ was replaced with Li+, Rb+ or Cs+, current transients were very small. 5. The shape of the power spectrum of current fluctuations induced by 1 microM cyclic GMP at +60 mV did not change when the permeating ion was Na+, K+ or NH4+. Analysis of the amplitude histogram did not show any effect of the tested monovalent cations on the open probability or on channel gating. At +60 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 1 mM Ca2+ to the medium bathing the cytoplasmic side of the membrane greatly reduced the frequency of openings, but single channel activity could still be observed. The blocking effect of 1 mM Ca2+ on the channel activity induced by 2 microM cyclic GMP could be counterbalanced by increasing 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 between -100 and +100 mV.(ABSTRACT TRUNCATED AT 400 WORDS)
Sesti
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Dipartimento di Fisica, Università di Genova, Italy.
Straforini
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M
Lamb
T D
TD
Torre
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V
D000557:Ambystoma; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002412:Cations / Q000494:pharmacology; D002413:Cations, Divalent / Q000494:pharmacology; D000242:Cyclic AMP / Q000502:physiology*; D066298:In Vitro Techniques; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology*; D007473:Ion Channels / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D017948:Retinal Rod Photoreceptor Cells / Q000187:drug effects / Q000502:physiology*; D013696:Temperature
D002412:Cations; D002413:Cations, Divalent; D007473:Ion Channels; D000242:Cyclic AMP; D002118:Calcium