
Graphical Abstract


Fig. 1. Localization of F256 and N257 and of the Ca2 + binding site on the homology model of CLCKa. (A) Surface representation of the CLCKa 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 CLCKa, Kb, K1, CLC0, and EcCLC.


Fig. 2. F256A CLCKa is hugely activated by NFA. (A–C) Effect of NFA on F256A and WT CLCKa in Xenopus oocytes. Typical currents of F256A (A) and WT CLCKa (B) in response to the IVprotocol (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 CLCKa. 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).


Fig. 3. Nonstationary noise analysis of F256A CLCKa. (A) Typical current traces recorded by patch clamp in configuration insideout from different oocytes without NFA (left) and with 200 μM NFAext (right) evoked by the stimulation protocol (bottom). (B–C) Examples of nonstationary noise analysis of F256A CLCKa 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 ttest) (background variance and leak currents were subtracted).


Fig. 4. NFA increases the open probability of F256A CLCKa. 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 CLCKa 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).


Fig. 5. WT CLCK1 is blocked by NFA both with and without barttin. Effect of 200 μM NFA on WT CLCK1/barttin (A) and WT CLCK1 (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 CLCK1 with and without barttin. Currents normalized to those recorded in standard solution are plotted versus [NFA]ext (concentrations tested on CLCK1/barttin: 10, 50, 200, 2000 μM, n ≥ 3; concentrations tested on CLCK1: 10, 200 μM, n ≥ 3).


Fig. 6. Barttin involvement in the potentiation of F256A CLCK1. Voltageclamp traces of F256A CLCK1/barttin (A) and F256A CLCK1 (B) evoked by the IVprotocol in standard solution. (C–E) NFA effect on F256A CLCK1 expressed with barttin or by itself in Xenopus oocytes. Current of F256A CLCK1/barttin (C) and F256A CLCK1 (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 singleexponential function (I1), and the steady state current (I2). (E) Dose–response relationship of NFA potentiation of F256A/barttin CLCK1 and NFA inhibition of F256A CLCK1/barttin and F256A CLCK1. 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).


Fig. 7. NFA potentiation and block of F256A CLCKa in HEK293 cells. (A) Representative current recordings of F256A CLCKa/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 CLCKa. (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).


Fig. 8. Dramatic inhibition of N257A CLCKa by NFA. (A) Typical currents of N257A CLCKa expressed in Xenopus oocytes in control solution (left) and at 5 μM NFA (right). (B) Dose–response relationship of NFA effect on N257A CLCKa. 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 CLCKa 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.


Fig. 9. Nonstationary noise analysis of N257A CLCKa. (A) Typical patch clamp insideout traces of N257A CLCKa 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 nonstationary noise analysis of N257A CLCKa 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 ttest) (background variance and leak currents were subtracted).


Fig. 10. Single channel recordings from N257A CLCKa. (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.


Fig. 11. NFA affinity of N257A CLCK1 is comparable to that of WT CLCK1. Comparison of normalized currents of WT CLCK1/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 CLCK1 (from left to right).
