Figure 3.

Eliminating the SYP121 membrane anchor recovers a subset only of KAT1 gating transitions. (a) Steady‐state current–voltage curves and representative current traces (insets) for KAT1 alone (filled circles) and co‐expressed with the cytosolic SNARE domain, SYP121^ΔC (triangles; KAT1: SYP121^ΔC ratios: 1:2, grey; 1:6, open). Voltage stepped from −20 mV to voltages between +20 and −180 mV. Solid curves are the result of the joint, least squares fitting to the Boltzmann function of Equation (3). Data are means ±SE (n ≥ 6). Insets: Representative current traces cross‐referenced by symbol. Scale: 2 μA (vertical), 1 s (horizontal). Corresponding Immunoblot analysis shown in Figure S1 and relative conductance curves shown in Figure S3. (b) Representative single‐channel currents recorded at −120 mV from one inside–out patch with KAT1 alone (above) and after adding SYP121^ΔC to the bath (below). Fitted amplitude histograms for the open (solid lines, grey fill) and close state (dashed lines) taken from the entire 8‐min recording. Open‐state curves are scaled (multiplied) by 4 relative to the closed state for clarity. (c) Frequency histograms of KAT1 open and close durations for KAT1 alone (above) and after adding SYP121^ΔC to the bath (below) plotted on logarithmic scales. Solid lines are fittings to Equation (2) to extract the dwell time constants. Bins below 1 ms (Nyquist limit) are shown but were not included in fittings. Open and closed lifetimes were fitted satisfactorily with sums of two and three exponentials, respectively. Mean dwell time constants ±SE (n = 6) for open and closed lifetimes are summarized by the bar charts (KAT1, open bars; KAT1+ SYP121^ΔC, filled bars). In the absence of the SNARE membrane anchor, a significant difference ± SYP121^ΔC was observed only for the longest closed dwell time constant, τ_C3 (see Table 1) [Colour figure can be viewed at http://wileyonlinelibrary.com]