NELLY BENNETT , MICHtLE ILDEFONSE , SERGE CROUZY , YVES CHAPRON , AND ARMEL CLERC

The cationic conductances of purified bovine retinal rod membranes were studied by incorporation of vesicles into planar lipid bilayers. When the membranes were stripped of all peripheral proteins [guanine nucleotide-binding protein (G protein) and cGMP phosphodiesterase (3',5'-cyclicGMP 5'-nucleotidohydrolase), EC 3.1.4.35], sodium and calcium fluxes were almost only observed in the presence of cGMP. Reconstitution experiments in which purified cGMP phosphodiesterase alone or with G protein were reassociated to the vesicles in proportions similar to those found in the native rod provide evidence for a direct interaction between the cGMP-dependent channel protein and the phosphodiesterase. (i) In its inhibited state, phosphodiesterase markedly stimulates the activity of the channels in the presence of cGMP (situation in the dark-adapted rod) but is not capable of activating the channels in the absence of cGMP. (ii) In the absence ofcGMP, activation of the phosphodiesterase by G protein with GTP bound (equivalent to photoexcitation) induces the opening of cation channels that have the same conductance for sodium ions as cGMP-activated channels (20-22 pS, with two sublevels of about 7 pS and 13 pS). Photoexcitation induces hyperpolarization of the plasma membrane of retinal rods. The light-sensitive sodium conductance is regulated by direct binding of cGMP (1-3). Light-induced activation of the cGMP phosphodiesterase (PDE; 3',5'-cyclic-GMP 5'-nucleotidohydrolase, EC 3.1.4.35) via the transduction cascade [light -* excitation of rhodopsin activation ofguanine nucleotide-binding protein (G protein) with formation of the PDE activator GGTP (a subunit of G protein with GTP bound)] results in a reduction of the concentration of cGMP, which leads to the closing of sodium channels in the plasma membrane (reviewed in ref. 4). Several reports show that cGMP-activated channels are also present in the membrane of the discs (5-11), although this has been recently questioned (12). The characteristics of the channels have been described from patch-clamp (13, 14) and reconstitution experiments (15, 16). The cGMP-activated channels are studied here by incorporation of vesicles from bovine retinal rods into lipid bilayers. The results reveal the existence of a second regulation of the cGMP-activated channels by direct interaction with the phosphodiesterase.