Figure 3

Functional expression and subcellular localisation of AKT1 and KAT1 channels in tobacco mesophyll protoplasts. (A) and (C) Typical recordings of the whole-cell inward and outward K⁺ currents in patch-clamped tobacco mesophyll protoplasts respectively transformed with AKT1-carrying and KAT1-carrying pFunct+Tag vectors. The voltage steps ranged from -200 mV to +100 mV in 20 mV increments. The holding potential was 0 mV and -40 mV respectively for AKT1 and KAT1 expressing protoplasts. The symbol above the records in a and c indicates the time of "steady-state" current sampling. (B) and (D) Current-voltage relationships at steady state in control tobacco mesophyll protoplasts (closed circles in both B and D) and in AKT1-expressing (open squares in B) and KAT1-expressing (open circles in d) ones (means ± SE; n = 16 for AKT1, n = 13 for KAT1). (E, F) Confocal microscopy sections of protoplasts transformed with AKT1-carrying (E) and KAT1-carrying (F) pLoc vectors. The left panels display protoplast sections analysed for the GFP fluorescence, the middle panels the same sections analysed for chloroplast auto-fluorescence and FM4-64 fluorescence and the right panels the overlay of the two former panels with the transmission light image from the same protoplast section. FM4-64 was 50 μM in both (E) and (F) and was incubated for 10 min on ice in (E) and for 40 min at room temperature in (F). Some places where GFP and FM4-64 fluorescence co-localises are marked by white arrows in (F). Scale bar = 20 μm.