Gating of cyclic nucleotide-gated (CNGA1) channels by cGMP jumps and depolarizing voltage steps

Abstract

We expressed rod-type homotetrameric cyclic nucleotide-gated (CNGA1) channels in Xenopus oocytes and studied activation by photolysis-induced jumps of the 3',5'-cyclic guanosine monophosphate (cGMP) concentration and by voltage steps. cGMP jumps to increasing concentrations up to the EC50 value of 46.5 microM decelerate the activation gating, indicative that even at concentrations of cGMP << EC50 binding is not rate limiting. Above the EC50 value, activation by cGMP jumps is again accelerated to the higher concentrations. At the same cGMP concentration, the speed of the activation gating by depolarizing voltage steps is roughly similar to that by cGMP jumps. Permeating ions passing the pore more slowly (Rb+ > K+ > Na+) slow down the activation time course. At the single-channel level, cGMP jumps to high concentrations cause openings directly to the main open level without passing sublevels. From these results it is concluded that at both low and high cGMP the gating of homotetrameric CNGA1 channels is not rate-limited by the cGMP binding but by conformational changes of the channel which are voltage dependent and include movements in the pore region.

FIGURE 1

Concentration-response relationship for CNGA1 channels with free cGMP. The steady-state current at the indicated concentrations was normalized with respect to the steady-state current at 700 μM cGMP and the data points were fitted by 1/{(1 + (EC₅₀/[cGMP])^H} yielding the indicated values for EC₅₀ and H. To determine the free cGMP concentration in flash photolysis experiments, the actual steady-state current (I) was normalized with respect to the actual steady-state current (I_max) at 700 μM cGMP. The free cGMP concentration [cGMP] was obtained with Eq. 1 by using the values for EC₅₀ and H.

FIGURE 2

Activation of CNGA1 currents by jumps of the cGMP concentration. (A) Current traces at 3 cGMP jumps from zero to the indicated concentrations by flash photolysis. The transmembrane voltage was +100 mV. At the intermediate concentration of 21.5 μM, the activation time course was slower than at both higher and lower cGMP. The traces were fitted with the sum of two exponentials yielding the indicated time constants τ_j,fast and τ_j,slow. The dotted curves show the respective two components. (B) Plot of activation time constants as function of the cGMP concentration. Each data point was obtained from 5 to 11 individual measurements. The arrow indicates the EC₅₀ value for steady-state conditions. (C) Relative contribution of the fast and slow exponential, A_j,fast and A_j,slow, to the activation time course.

FIGURE 3

Voltage-dependent activation of CNGA1 channels. (A) Activating component of the current traces when stepping from −100 to +100 mV at 3 cGMP concentrations. The time courses were fitted with the sum of two exponentials yielding the indicated time constants τ_v,fast and τ_v,slow. The dotted curves show the respective two components. (B) Plot of τ_v,fast and τ_v,slow as function of the cGMP concentration. Each data point was obtained from 3 to 14 individual measurements. τ_j,fast and τ_j,slow from Fig. 2 B are shown for comparison. The arrow indicates the EC₅₀ value for steady-state conditions. (C) Contribution of the fast and slow exponential of voltage-dependent activation, A_v,fast and A_v,slow, respectively.

FIGURE 4

Mean time constants for activation by cGMP jumps and depolarizing voltage steps as a function of the cGMP concentration. The time constants were calculated according to Eq. 2. The vertical arrow indicates the EC₅₀ value for steady-state conditions.

Figure

FIGURE 5

Effect of voltage on the activation by cGMP jumps. The voltage was either −100, −50, +50, or +100 mV. (A) Current traces at 21.5 μM cGMP. The traces were recorded from the same patch. The traces were fitted with the sum of two exponentials and the mean activation time constants, τ_j,mean, were calculated by Eq. 2. (B) Current traces at 71.8 μM cGMP. The traces were recorded from the same patch. Same analysis as in panel A. (C) Plot of τ_j,mean as function of voltage. Each data point was obtained from 5 to 8 individual measurements.

FIGURE 6

Modulation of voltage-dependent activation of CNGA1 channels by ions. Patches were depolarized by pulses from −100 to +100 mV. The activation time courses were quantified by τ_v,mean. (A) Representative current traces illustrating the effect of intracellular, permeating ions. The traces were normalized with respect to the amplitude of the late current. The pipette contained Na⁺ ions, the bath either Na⁺, K⁺, or Rb⁺ ions. (B) Plot of τ_v,mean as function of the cGMP concentration for the three ionic conditions in A.

FIGURE 7

Representative consecutive single-channel recordings of CNGA1 channels activated by jumps of the cGMP concentration from zero to ∼81.2 μM cGMP. The cyclic nucleotide was liberated from 200 μM BCMACMcGMP. The channel gated directly from the closed to the main open level. The ensemble averaged current (bottom) was formed from 37 traces from 4 patches.