Ion channels in the thylakoid membrane were investigated by direct patch clamping on swollen thylakoids. A preparation method has been developed in order to release osmotically swollen intact thylakoids from pea protoplasts derived from cotyledons of young Pisum sativum plants. The swollen thylakoids with typical diameters between 10 μm and 20 μm formed reproducibly high‐resistance seals with patch pipettes. We observed a potassium channel with a main conductant state of @KL ≈ 40 pS and a conductance of @KL ≈ 90 pS (in asymmetric 20/100 mM KCl) for the fully open channel. Surprisingly, the thylakoid membranes also contained a high‐conductance channel with a main conductant state of @KL ≈ 620 pS (in asymmetric 20/100 mM KCl), revealing also higher and lower conductant states. With a different experimental approach we showed that thylakoids are able to accumulate transiently the membrane impermeant fluorescent dye Lucifer Yellow which likewise suggests the presence of a pore‐like channel with a diameter large enough to allow permeation of Lucifer Yellow.
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