cGMP-dependent channel protein from photoreceptor membranes: single-channel activity of the purified and reconstituted protein.

Abstract

The cGMP-dependent channel protein has been purified from bovine rod photoreceptor membranes and incorporated into planar lipid membranes. At low divalent cation concentrations, cGMP stimulated single-channel current fluctuations. The probability P. of the channel being open strongly depended on the cGMP concentration (EC'50 = 31 ,IM;Hill coefficient, n = 2.3); whereas the single-channel conductance (A = 26 pS) was independent of the agonist concentration. The agonist-stimulated increase in the probability of an open channel was largely due to shorter closed times and, to a lesser extent, due to the channel staying open for a longer time. The current-voltage relationship of the single open channel deviated from ohmic behavior, and the open probability decreased at more negative membrane potentials. The rectification of the macroscopic cGMP-induced current in artificial bilayers that contained many channel copies can be accounted for by the voltage dependence of channel gating together with the nonlinearity of the current-voltage curve of an open channel. Current fluctuations exhibited a variety of sublevels, indicating that the channel may exist in more than one conductive state.Vertebrate photoreceptors hyperpolarize in response to light due to the closure of cation channels in the plasma membrane. It has been suggested that cGMP directly opens the light-regulated channel in the rod outer segment plasma membrane in a cooperative fashion (refs. 1-6; for reviews see refs. 7-10). A similar ion conductance also exists in the disk membrane (11-15).The cGMP-dependent channel protein from rod photoreceptor membranes has been solubilized, purified, and functionally reconstituted into liposomes and planar lipid bilayers (16)(17)(18) Purification of the cGMP-Dependent Cation Channel Protein. The channel protein was purified from bovine rod outer segments as described (16,17). Electrophoresis of the purified extract on 0.1% NaDodSO4/7.5% polyacrylamide gel revealed two polypeptides with molecular masses of63 kDa and 240 kDa (16). The 240-kDa component probably represents a spectrinlike (18) polypeptide that was absent in some of our preparations. The purified channel protein was incorporated into asolectin liposomes (16, 19). Approximately 5-10o of the liposomes contained at least one channel copy (19).Formation of Planar Lipid Bilayers and Channel Insertion. The purified channel protein was incorporated into planar bilayers by "spontaneous fusion" of channel-containing liposomes with a phospholipid bilayer formed from purified (19) asolectin (Sigma, Type IV-S) on the orifice of a small Teflon septum (diameter, 150 ttm; septum thickness, 5.6 ttm).The compartment into which the liposomes were injected was defined as the cis side of the recording chamber. The fusion events were monitored by continuously measuring the transmembrane current at a constant membrane potential. After the fusion of liposomes with the planar bilayer, cGMP was added to both compartments. The channel orientation was determined from the volta...

Self-assembling subnanometer pores with unusual mass-transport properties

“…An analysis 35 of the open/close probability under different concentrations of 1a in 4 M KCl leads to a fitted Hill coefficient (Fig. 4b) of 9 ± 3, consistent with the self-assembly of at least nine molecules of 1a to form a functional transmembrane channel.…”

Multiple conductance states of the light-activated channel of Limulus ventral photoreceptors. Alteration of conductance state during light.

“…In other channel types, the channel activity is more evenly distributed among its conductance states. This is the case for the glutamate-activated channel (CullCandy and Usowicz, 1987;Jahr and Stevens, 1987) and the light-dependent channel from vertebrate photoreceptors (Hanke et al, 1988;Haynes and Yau, 1990). For the Limulus light-activated channel, both states are expressed with roughly equal frequency in some cells, although not in all cells.…”

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

“…In two other studies of the salamander rod channel, the smaller current level was not observed. Matthews & Watanabe (1988) (Hanke et al 1988;Bennett & Clerc, 1989;Ildefonse & Bennett, 1991), but these channels appear to have slower activation kinetics and a lower sensitivity to cGMP than the channel of salamander rods. The value of 25 pS estimated here for the open-state conductance of the channel at +50 mV is in good agreement with two previous values for the amphibian rod channel (Haynes et al 1986, -25 pS;Zimmerman & Baylor 1986, -24 pS).…”

“…The cGMP-activated channels from mammalian rod cells have been incorporated into planar lipid bilayers and expressed in frog oocytes (Hanke, Cook & Kaupp, 1988;Kaupp et al 1989;Ildefonse & Bennett, 1991), but these channels appear to have different gating kinetics from those of excised patches from amphibian rod outer segments, which show much more rapid flicker. Some channels in inner segment patches from amphibia also flicker very little (Torre et at.…”