Figure 2. Proton block and current rectification in WT CNGA1 channel.

A and B, macroscopic current recordings in the inactivated state obtained 2 min after the addition of 1 mm cGMP at 0 mV elicited by voltage steps from −200 to +200 mV (ΔV = 20 mV) at pH_o 5 (A) and 7.4 (B). Leak and capacitative components were removed by subtracting from the cGMP-activated current those records obtained in response to the same voltage protocol but without cGMP. C, single-channel i–V (black circles) and macroscopic I–V relationships (red circles) obtained from noise analysis (D) and from the recording in A, respectively. The I–V relationship was scaled to the i flowing at +180 mV. D, stationary fluctuation analysis at the indicated positive (upper panels) and negative (lower panels) voltages. A linear regression fit throughout each dataset provided the indicated unitary current i. Currents were evoked by 30 μm intracellular cGMP and leak currents and variance observed in the absence of cGMP subtracted. Each point indicates the mean current (I) and the variance of the corresponding mean current (σ²) measured from one independent excised membrane patch. Recordings for each patch were of duration between 0.5 and 2 s. E, single-channel current recordings at +100 mV at the indicated pH_o. Amplitude histogram from recordings at pH_o 5 is shown at the right (grey area). Dashed brown and blue lines represent a two-component Gaussian fit to histograms obtained at pH_o 5 and 7.4, respectively. Black dashed lines indicate the 0 current level. C, O_b and O refer to the closed, blocked and open states respectively; i indicates the unitary current. F, as in E but at −100 mV. G, macroscopic current recording in the inactivated state elicited by a voltage prepulse held at +200 mV followed by test potentials ranging from −200 to +200 mV (ΔV = 20 mV). No tail currents were observed at negative membrane potentials, suggesting a fast mechanism of proton block. Black dashed line indicates the 0 current level. H, a sketch of the energy landscape for a symmetrical two-barrier Woodhull model of block. U_±i, k(0)_±i and δ indicate the heights of energy barriers, rate constants at 0 mV and the electrical distance, respectively. I, dependence of the fraction of unblocked channels (see Methods) on V at different pH_o (filled circles, open circles, filled triangles, open triangles, open squares and filled squares refer to pH_o 9, 8, 7, 6, 5 and 4, respectively). The red lines represent simultaneous fits of all the data with the model shown in F with k(0)₁, k(0)_–1, k(0)₂, k(0)_–2 and δ equal to 7.3 × 10⁷ m⁻¹ s⁻¹, 40.8 s⁻¹, 8.9 s⁻¹, 1.6 × 10⁷ m⁻¹s⁻¹ and 0.41, respectively.