Figure 3.

Effects of gramicidin on thylakoid membrane ionic conductance and on ΔpH. (A) Gramicidin channels increase the decay rate of the flash-induced electrochromic absorbance changes. Thylakoid vesicles in the presence of the indicated number of gramicidin channels per thylakoid were analyzed spectrophotometrically after dual-flash excitation under the same conditions used in Fig. 2. Monophasic exponential fits of the decay traces were used to determine half-times of decay: control, 0.37 s; 100 channels, 0.30 s; 500 channels, 0.18 s). Difference curves (control minus + gramicidin) accentuate changes in decay rates induced by increased ionic conductance through gramicidin channels. (B) Increased ion conductance observed with these low concentrations of gramicidin does not alter the transmembrane ΔpH formed under steady-state illumination. The fluorescent amine 9-aminoacridine reports the extent of acidification of the lumen after the onset of illumination (upward open arrow). The mean ΔpH of control thylakoids in the presence of ethanol alone (solid line) was 3.60 (SD = 0.16, n = 5); in the presence of 500 pores per thylakoid (dotted line), ΔpH = 3.61 (SD = 0.23, n = 5). Increased conductance provided by 500 nM gramicidin (∼7.5 × 10⁵ pores per thylakoid, dashed line) resulted in the inability of thylakoids to maintain a substantial pH gradient.