Conductance and kinetics of single cGMP‐activated channels in salamander rod outer segments.

Abstract

1. The conductance and kinetics of single 3',5'‐cyclic guanosine monophosphate (cGMP)‐activated channels of retinal rod outer segments were studied in inside‐out membrane patches. The size of the single channel currents was increased by using low concentrations of divalent cations. 2. At saturating cGMP concentration, the current flickered at high frequency. Occasionally, the current was interrupted by closures lasting tens or hundreds of milliseconds. At +50 mV the maximum current during an opening was slightly more than 1 pA, but the open channel level was poorly resolved due to the speed of the gating transitions. 3. Amplitude histograms confirmed the presence of a sublevel of current, roughly a quarter the size of the peak current, at low cGMP concentrations. The fraction of time in the sublevel decreased with increasing cGMP concentration, suggesting that the sublevel may be due to opening by the partially liganded channel. 4. Consistent with previous macroscopic current recordings, single channel activation by cGMP had an apparent dissociation constant of 8.6 microM, and a Hill coefficient of 2.8. 5. At saturating cGMP concentrations, the channel was modelled as a two‐state system with the following parameters. The open channel conductance was 25 pS. The opening rate constant, beta, was 1.5 x 10(4) s‐1 at 0 mV, and had a voltage sensitivity equivalent to the movement of 0.23 electronic charges outward through the membrane electric field. The closing rate constant, alpha, was 2.1 x 10(4) s‐1 and was voltage insensitive. Assuming that the open‐state chord conductance was voltage independent, the inferred voltage dependence of beta largely accounted for the outward rectification in the steady‐state macroscopic current‐voltage relation of multichannel patches, at saturating cGMP concentration.

Membrane current noise in dark‐adapted and light‐adapted isolated retinal rods of the larval tiger salamander

“…Adaptation of the second component of the dark noise is somewhat different in that the power spectral density shows an increase in power with a marked increase in half-power cut-off frequency. In truncated toad rods, this component scales with the light-sensitive current, and so has been attributed to slow gating processes of the light-sensitive channels (Rieke & Baylor, 1996), and long-lasting closures of about the right time scale have indeed been observed in isolated patches from salamander rods (Taylor & Baylor, 1995). This suggests the intriguing possibility that adaptation of the channels may occur by modulation of these long closures, perhaps, for example, via interaction with calcium and calmodulin (Gordon et al 1995).…”

Cyclic nucleotide-gated channels: shedding light on the opening of a channel pore

“…New evidence indicates that the selectivity filter of CNG channels undergoes a conformational change in association with gating. CNG channels that are partially activated have permeation properties that differ from those of fully activated channels [50][51][52] . In addition, intracellular tetracaine -a local anaesthetic -gives a statedependent block of CNG channels [53][54][55] .…”

Cyclic Nucleotide-Gated Ion Channels

“…Purified channel preparations do only contain these two polypeptides, and coexpression of the A1 and B1a subunits produces channels that recapitulate most, if not all, of the key properties of the native channel from rods (70,71,214). Notably, the rapid flickery gating that is so characteristic of the native channel (156,392,399,441) and the high sensitivity to blockage by L-cis-diltiazem (203,379) require the coexpression of the A1 and B1a subunits (71,214).…”