Cyclic nucleotide-gated (CNG) channels are directly activated by the b
inding of several ligands(1-6). For these channels as well as for othe
r allosteric proteins, the functional effects of each ligand-binding e
vent have been difficult to assess because ligands continuously bind a
nd unbind at each site. Furthermore, in retinal rod photoreceptors the
low cytoplasmic concentration of cyclic GMP(7) means that channels ex
ist primarily in partially liganded states, so it is important to dete
rmine how such channels behave. Previous studies of single channels ha
ve suggested that they occasionally open to subconducting states at lo
w cGMP(2,3,8-10), but the significance of these states and how they ar
ise is poorly understood. Here we combine the high resolution of singl
e-channel recording with the use of a photoaffinity analogue of cGMP(1
1,12) that tethers cGMP moieties covalently to their binding sites to
show single retinal CNG channels can be effectively locked in four dis
tinct ligand-bound states. Our results indicate that channels open mor
e than they would spontaneously when two ligands are bound (similar to
1% of the maximum current), significantly more with three ligands bou
nd (similar to 33%), and open maximally with four ligands bound. In ea
ch ligand-bound state, channels opened to two or three different condu
ctance states, These findings place strong constraints on the activati
on mechanism of CNG channels.