Time Course and Ca²⁺ Dependence of Sensitivity Modulation in Cyclic Gmp-Gated Currents of Intact Cone Photoreceptors

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. ^CaK_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.