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Graphical Abstract
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Fig. 1. Localization of F256 and N257 and of the Ca2 + binding site on the homology model of CLC-Ka. (A) Surface representation of the CLC-Ka model [27]. The two subunits viewed from the extracellular side are indicated by two tones of gray. The residues involved in the response to NFA (F256 and N257) and those that coordinate Ca2 + (E259, E261, D278, E281) [14,27] are colored differently: green F256, yellow N257, red E259, blue E261, pink D278, and black E281. (B) Zoom of the region containing the residues of interest. Residues are depicted as sticks and colored as in A. (C) Sequence alignment around the I–J loop of CLC-Ka, -Kb, -K1, CLC-0, and EcCLC.
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Fig. 2. F256A CLC-Ka is hugely activated by NFA. (A–C) Effect of NFA on F256A and WT CLC-Ka in Xenopus oocytes. Typical currents of F256A (A) and WT CLC-Ka (B) in response to the IV-protocol (top) in control (left) and at 200 μM NFA (right). The inset (A) shows F256A currents at high magnification (C) Dose–response relationship of NFA modulation of WT and F256A CLC-Ka. The currents acquired at 60 mV are normalized to values measured in standard solution (i.e. 0 NFA) and plotted versus [NFA]ext (used concentrations: 1, 2, 5, 10, 50, 200, 500, 1000 and 2000 μM) (n ≥ 3).
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Fig. 3. Non-stationary noise analysis of F256A CLC-Ka. (A) Typical current traces recorded by patch clamp in configuration inside-out from different oocytes without NFA (left) and with 200 μM NFAext (right) evoked by the stimulation protocol (bottom). (B–C) Examples of non-stationary noise analysis of F256A CLC-Ka in standard conditions (B) and in the presence of 200 μM NFAext (C) at 60 mV. (left). Mean currents (upper) and variance (lower) are shown as a function of time. Right: Variance (symbols) is plotted versus the mean current and fitted with a parabola (red line) as described in the Materials and methods section. (D) Bars represent the absolute value of the single channel mean current in control solution and at 200 μM NFAext at two different potentials (− 100 mV and 60 mV) (n ≥ 4), p > 0.2 (unpaired Student's t-test) (background variance and leak currents were subtracted).
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Fig. 4. NFA increases the open probability of F256A CLC-Ka. Example of single channel recordings and analysis from a single patch (similar experiments allowing the estimation of the number of channels: n = 3; 2 additional patches with larger currents were allowed to estimate the degree of potentiation but not the number of channels). (A) Continuous recording from F256A CLC-Ka at 60 mV. Different colors represent the external solutions applied during the experiment (black: control, red: 200 μM NFA). (B) Portions of the recording in (A) shown at higher time resolution. (C) The amplitude histogram of the recording at 60 mV in control solution (black trace) and in the presence of 200 μM NFA (red trace). Fit curves are superimposed as dashed lines. (D) Mean current from the recording in (A) measured at 60 mV for 40 s in control solution and in 200 μM NFA. (E–F) Probabilities of the conductance states in 200 μM NFA and in control solution. The measured state probabilities are shown as bars, whereas the symbols are the expected values from the binomials fits (see the Materials and methods section) resulting in N = 5 channels, p = 20% in NFA and p = 0.3% in control (for the fit in control, the number of channels was fixed to 5, as obtained in the presence of NFA).
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Fig. 5. WT CLC-K1 is blocked by NFA both with and without barttin. Effect of 200 μM NFA on WT CLC-K1/barttin (A) and WT CLC-K1 (B). Currents are plotted as function of the time. Colors and symbols correspond to the different solutions applied during the experiment. (C) Dose response of NFA effect on WT CLC-K1 with and without barttin. Currents normalized to those recorded in standard solution are plotted versus [NFA]ext (concentrations tested on CLC-K1/barttin: 10, 50, 200, 2000 μM, n ≥ 3; concentrations tested on CLC-K1: 10, 200 μM, n ≥ 3).
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Fig. 6. Barttin involvement in the potentiation of F256A CLC-K1. Voltage-clamp traces of F256A CLC-K1/barttin (A) and F256A CLC-K1 (B) evoked by the IV-protocol in standard solution. (C–E) NFA effect on F256A CLC-K1 expressed with barttin or by itself in Xenopus oocytes. Current of F256A CLC-K1/barttin (C) and F256A CLC-K1 (D) at 60 mV is shown as function of time. Colors and symbols correspond to the solutions applied. Vertical arrows indicate the initial current (I0), the maximal NFA potentiation extrapolated by a single-exponential function (I1), and the steady state current (I2). (E) Dose–response relationship of NFA potentiation of F256A/barttin CLC-K1 and NFA inhibition of F256A CLC-K1/barttin and F256A CLC-K1. I1/I0 and I2/I0 are plotted versus [NFA]ext (used concentrations: 10, 50, 200, and 2000 μM, n ≥ 3). The lines represent the fit curves obtained from the equation I2 / I0 = 1 / (1 + (c / KD)) for NFA block of F256A/barttin (KD ~ 142 μM) and F256A (KD ~ 33 μM).
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Fig. 7. NFA potentiation and block of F256A CLC-Ka in HEK293 cells. (A) Representative current recordings of F256A CLC-Ka/barttin expressed in HEK293 cells in control solution (top right), at 20 μM NFA (bottom left) and at 500 μM NFA (bottom right). (B and C) Effect of [NFA]ext on F256A CLC-Ka. (B) Mean current at 60 mV is plotted vs. time. Colors and symbols correspond to the [NFA]ext applied (0, 20, and 500 μM). (C) Normalized currents acquired at different [NFA]ext are plotted vs. [NFA]ext (used concentrations: 20, 50, 200, and 500 μM) (n ≥ 4 for 20, 50, and 500 μM; n = 17 for 200 μM).
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Fig. 8. Dramatic inhibition of N257A CLC-Ka by NFA. (A) Typical currents of N257A CLC-Ka expressed in Xenopus oocytes in control solution (left) and at 5 μM NFA (right). (B) Dose–response relationship of NFA effect on N257A CLC-Ka. Normalized currents are plotted vs. [NFA]ext (used concentrations: 2, 5, 10, 50, 200, 500, and 1000 μM) (n ≥ 4 except n = 3 for 1000 μM). The red line represents the fit curve obtained from the equation I / I0 = 1 / (1 + (c / KD)) with KD ~ 1 μM. (C) Insensitivity to washing of N257A CLC-Ka inhibition. Mean currents plotted as function of time after a short NFA perfusion (left) (similar experiments n = 4) or a longer NFA perfusion (right) (similar experiments n = 10). Colors and symbols represent the solutions applied.
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Fig. 9. Non-stationary noise analysis of N257A CLC-Ka. (A) Typical patch clamp inside-out traces of N257A CLC-Ka measured from different oocytes in control conditions (left), at 4 μM NFAext (middle), at 4 μM NFAext and 100 μM CPAint (right). (B and C) Examples of non-stationary noise analysis of N257A CLC-Ka in the presence of 100 μM CPAint, in control external solution (B) and at 4 μM NFAext (C). Analysis was performed both for the deactivating currents at − 100 mV as well as for the unblocking relaxations at 60 mV. The traces shown are an example at 60 mV (left). Mean currents (upper) and variance (lower) are shown as a function of time (right). Variance (symbols) is plotted versus the mean current and fitted with a parabola (red line) as described in the Materials and methods section. (D) Bars represent the absolute value of the single channel mean current in the presence of 100 μM CPAint in external control solution and at 4 μM NFAext at two different potentials (− 100 mV and 60 mV) (n ≥ 3), p > 0.3 (unpaired Student's t-test) (background variance and leak currents were subtracted).
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Fig. 10. Single channel recordings from N257A CLC-Ka. (A) Representative traces from a single patch at − 100 mV in internal control solution and in 50 μM CPA (similar experiments n = 5). (B) Short stretches of the traces in (A) shown at a higher time resolution. In the left panel dashed lines indicate several distinct conductance levels. (C) Amplitude histogram of the recording at − 100 mV in control solution and in the presence of 50 μM CPA. Dashed lines are fit curves superimposed. In the presence of CPA the dominant current level has an amplitude of 0.66 pA. There is no clear correspondence between the peaks in control and in CPA.
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Fig. 11. NFA affinity of N257A CLC-K1 is comparable to that of WT CLC-K1. Comparison of normalized currents of WT CLC-K1/barttin (n = 5), N257A/barttin (n = 3), and N257A (n = 5) at 200 μM NFA normalized to the current recorded in control condition. On top of the figure typical current traces of WT/barttin, N257A/barttin, and N257A CLC-K1 (from left to right).
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