Effects of ionic strength on gating and permeation of TREK-2 K2P channels

Figures

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  Fig 1. TREKΔm3 IV-curves in high internal ionic strength. a) Average normalized IV-curves in varying ionic strength. Shaded area represents SEM, n = 7 and 5 for KCL and NMDG respectively. Note that the IV curves are offset to each other on the x-axis, this is because the voltage-step protocols for each test-solution where modified in order to sample a ± 100 mV driving force range for K+. b) Absolute current values at 70 mV for the baseline condition (120 mM KCl) for each patch. c) Sequence alignment of the N-termini of ATI variants of TREK2 utilized in this study. d): Normalized currents in varying ionic strength, corrected for driving force. There was considerable variability in the response between different constructs and between replicates. n > 3. Faint lines and filled circles represent responses of individual patches.

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  Fig 2. High ionic strength, not osmotic gradients inhibit TREK2. a) Mean IV-curves in excised patches. Shaded areas represent ± SEM. One test solution was applied to every patch, then it was discarded (this yields separate control curves for every condition, n >8 paired recordings). b) Currents at +95 mV outward driving force. c) Relative current change. The mannitol test solution has no appreciable effect on current. Multiple comparisons here were tested with ANOVA and Tukey’s post-hoc test. d) Time course data of repeated application of 250 mM KCl solution to an inside out patch. Typical example shown from 3 independent measurements. Voltage ramps from -100 to +100 mV were continually applied to an inside-out patch. Full solution exchange was usually achieved in the course of 1 ramp (1.5 s) as shown by the expected change in reversal potential. Channel activity decreased spontaneously over the course of the measurement and then stabilized (rundown). The current size and relative reduction with these test solutions were variable, however every patch tested showed reduced current in response to application of more concentrated salt solutions to the intracellular side (see Fig 2b). By…

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  Fig 3. Single-channel behavior in high internal ionic strength. a) Example time-courses of low Po TREK2ΔM3 currents in solutions of varying K+ concentration and ionic strength. Outward (top row) and inward (bottom row) single channel currents at a net driving force of ± 70 mV. Different subconductances and channel flickers are apparent. Fitted amplitudes for the closed (grey) and open state (blue) are shown as horizontal lines, passive leak current was subtracted. b) Amplitudes were extracted from short stretches of recordings 0.95 s at voltages ranging from 100 mV either side of the calculated reversal potential in the given K+ gradient. Data collected at high ionic strength (500 mM) represent opening events recorded from residual activity of patches conducting macroscopic currents in the baseline condition (large amount of channels). Lines represent a third order polynomial fit. Number of patches for each reconstructed curve is at least 4 whereas the number of replicates at a given voltage can be less, if the amplitude could not be determined due to lack of openings. c) Estimates of γ plotted against the internal potassium concentration. Amplitudes were divided by the driving force…

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  Fig 4. BL1249 increases Po but not γ in high ionic strength. a) Single channel traces of TREK ΔM3 in 1M intracellular KCl at -104 mV (-50 mV driving force). a’ Enlarged view of individual openings in each trace (75 ms). b) All point amplitude histogram of the recordings shown (partially) in panel a, total length of the recording was 3 min each. c) Pair-wise comparison of the single-channel amplitudes plotted. The column label denotes the net driving force in mV. A paired t-test was used to test for a difference in amplitude. There was no statistically significant difference. d) Single-channel IV curves of TREK2ΔM3 in 1 M internal KCl concentration, with and without 3μM BL1249. e) Single-channel IV curves of TREK2ΔM3 in symmetrical 120 mM K+). No increase in γ was observed.

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  Fig 5. Combined action of BL1249 and high K+ relieves open-state instability. Comparison of open-state peaks of TREK2ΔM3 in different conditions. The open-state peaks of microscopic TREK2 recordings are often asymmetric or skewed (green trace, left). However, this is substantially reduced in the presence of BL1249 (red trace, left) and also in high ionic strength (purple trace, right). Data in the left panel was taken from a previously published dataset [17]. To compare the shape of the histograms the x-axis was aligned to the peak of the open-channel current, and the y-axis normalized to the peak. See insert for the raw amplitude histogram for the data plotted in the right panel. Membrane voltage was -100 mV except for the trace in 1000 mM K+ (-60 mV). At this voltage the open-channel current was closest in amplitude to the matching recording in 120 K+.