Patch-clamp study on a Ca²⁺-regulated K⁺ channel in the tonoplast of the brackish characeae lamprothamnium succinctum

Cytoplasmic drops were prepared from internodal cells of the brackish Characeae Lamprothamnium succinctum. Applying the patch-clamp technique to single drops covered with tonoplast, we demonstrated the presence of Ca²⁺-regulated K⁺ channels in the tonoplast. In a cell-attached mode, the selectivity of such channels for K⁺ was about 50 times that for Na⁺. This channel showed a tendency to rectify in an outward direction. In the negative region of the pipette voltage, the conductance of this channel was 50 pS, while it was 100 pS in the positive voltage region. When the pipette voltage was increased above 50 mV, two conductance levels were found in the cell-attached mode as well as in the excised patch (cytoplasmic-side-out patch), which was obtained by pulling the patch pipette from the cytoplasmic drop under conditions of low levels of Ca²⁺. Using the excised patch, we controlled the level of Ca²⁺ on the cytoplasmic side of the channels. At a low level of Ca²⁺ (pCa=8) on the cytoplasmic side, the open frequency was very low and the opening time was short. An increase in Ca²⁺ on the cytoplasmic side (pCa = 5) increased both the frequency and the duration of opening. However, the conductance of the channels did not change. This regulation by Ca²⁺ of the K⁺ channels was reversible, that is, addition of EGTA on the cytoplasmic side inactivated the channels. The present study demonstrates a direct action of Ca²⁺ on the K⁺ channels. The physiological role of the K⁺ channel in the regulation of turgor in Lamprothamnium is discussed.