We investigated the modulation of cGMP-gated ion channels in single cone photoreceptors isolated from a fish retina. A new method allowed us to record currents from an intact outer segment while controlling its cytoplasmic composition by superfusion of the electropermeabilized inner segment. The sensitivity of the channels to agonists in the intact outer segment differs from that measured in membrane patches detached from the same cell. This sensitivity, measured as the ligand concentration necessary to activate half-maximal currents, K 1/2, also increases as Ca2+ concentration decreases. In electropermeabilized cones, K 1/2 for cGMP is 335.5 ± 64.4 μM in the presence of 20 μM Ca2+, and 84.3 ± 12.6 μM in its absence. For 8Br-cGMP, K 1/2 is 72.7 ± 11.3 μM in the presence of 20 μM Ca2+ and 15.3 ± 4.5 μM in its absence. The Ca2+-dependent change in agonist sensitivity is larger in extent than that measured in rods. In electropermeabilized tiger salamander rods, K 1/2 for 8Br-cGMP is 17.9 ± 3.8 μM in the presence of 20 μM Ca2+ and 7.2 ± 1.2 μM in its absence. The Ca2+-dependent modulation is reversible in intact cone outer segments, but is progressively lost in the absence of divalent cations, suggesting that it is mediated by a diffusible factor. Comparison of data in intact cells and detached membrane fragments from cones indicates that this factor is not calmodulin. At 40 μM 8Br-cGMP, the Ca2+-dependent change in sensitivity in cones is half-maximal at K Ca = 286 ± 66 nM Ca2+. In rods, by contrast, K Ca is ∼50 nM Ca2+. The difference in magnitude and Ca2+ dependence of channel modulation between photoreceptor types suggests that this modulation may play a more significant role in the regulation of photocurrent gain in cones than in rods.

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We determined the Ca2+ dependence and time course of the modulation of ligand sensitivity in cGMP-gated currents of intact cone photoreceptors. In electro-permeabilized single cones isolated from striped bass, we measured outer segment current amplitude as a function of cGMP or 8Br-cGMP concentrations in the presence of various Ca2+ levels. The dependence of current amplitude on nucleotide concentration is well described by the Hill function with values of K 1/2, the ligand concentration that half-saturates current, that, in turn, depend on Ca2+. K 1/2 increases as Ca2+ rises, and this dependence is well described by a modified Michaelis-Menten function, indicating that modulation arises from the interaction of Ca2+ with a single site without apparent cooperativity. Ca K m, the Michaelis-Menten constant for Ca2+ concentration is 857 ± 68 nM for cGMP and 863 ± 51 for 8Br-cGMP. In single cones under whole-cell voltage clamp, we simultaneously measured changes in membrane current and outer segment free Ca2+ caused by sudden Ca2+ sequestration attained by uncaging diazo-2. In the presence of constant 8Br-cGMP, 15 μΜ, Ca2+ concentration decrease was complete within 50 ms and membrane conductance was enhanced 2.33 ± 0.95-fold with a mean time to peak of 1.25 ± 0.23 s. We developed a model that assumes channel modulation is a pseudo–first-order process kinetically limited by free Ca2+. Based on the experimentally measured changes in Ca2+ concentration, model simulations match experimental data well by assigning the pseudo-first-order time constant a mean value of 0.40 ± 0.14 s. Thus, Ca2+-dependent ligand modulation occurs over the concentration range of the normal, dark-adapted cone. Its time course suggests that its functional effects are important in the recovery of the cone photoresponse to a flash of light and during the response to steps of light, when cones adapt.

The ligand sensitivity of cGMP-gated (CNG) ion channels in cone photoreceptors is modulated by CNG-modulin, a Ca 2ϩ -binding protein. We investigated the functional role of CNG-modulin in phototransduction in vivo in morpholino-mediated gene knockdown zebrafish. Through comparative genomic analysis, we identified the orthologue gene of CNG-modulin in zebrafish, eml1, an ancient gene present in the genome of all vertebrates sequenced to date. We compare the photoresponses of wild-type cones with those of cones that do not express the EML1 protein. In the absence of EML1, dark-adapted cones are ϳ5.3-fold more light sensitive than wild-type cones. Previous qualitative studies in several nonmammalian species have shown that immediately after the onset of continuous illumination, cones are less light sensitive than in darkness, but sensitivity then recovers over the following 15-20 s. We characterize light sensitivity recovery in continuously illuminated wild-type zebrafish cones and demonstrate that sensitivity recovery does not occur in the absence of EML1.