Accardi A and Pusch M (2003), Conformational changes in the pore of CLC-0.

XB-ART-4868

J Gen Physiol 2003 Sep 01;1223:277-93. doi: 10.1085/jgp.200308834.

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Conformational changes in the pore of CLC-0.

Accardi A , Pusch M .

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The Torpedo Cl- channel, CLC-0, is inhibited by clofibric acid derivatives from the intracellular side. We used the slow gate-deficient mutant CLC-0C212S to investigate the mechanism of block by the clofibric acid-derivative p-chlorophenoxy-acetic acid (CPA). CPA blocks open channels with low affinity (KDO= 45 mM at 0 mV) and shows fast dissociation (koff = 490 s-1 at -140 mV). In contrast, the blocker binds to closed channels with higher affinity and with much slower kinetics. This state-dependent block coupled with the voltage dependence of the gating transitions results in a highly voltage-dependent inhibition of macroscopic currents (KD approximately 1 mM at -140 mV; KD approximately 65 mM at 60 mV). The large difference in CPA affinity of the open and closed state suggests that channel opening involves more than just a local conformational rearrangement. On the other hand, in a recent work (Dutzler, R., E.B. Campbell, and R. MacKinnon. 2003. Science. 300:108-112) it was proposed that the conformational change underlying channel opening is limited to a movement of a single side chain. A prediction of this latter model is that mutations that influence CPA binding to the channel should affect the affinities for an open and closed channel in a similar manner since the general structure of the pore remains largely unchanged. To test this hypothesis we introduced point mutations in four residues (S123, T471, Y512, and K519) that lie close to the intracellular pore mouth or to the putative selectivity filter. Mutation T471S alters CPA binding exclusively to closed channels. Pronounced effects on the open channel block are observed in three other mutants, S123T, Y512A, and K519Q. Together, these results collectively suggest that the structure of the CPA binding site is different in the open and closed state. Finally, replacement of Tyr 512, a residue directly coordinating the central Cl- ion in the crystal structure, with Phe or Ala has very little effect on single channel conductance and selectivity. These observations suggest that channel opening in CLC-0 consists in more than a movement of a side chain and that other parts of the channel and of the selectivity filter are probably involved.

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Species referenced: Xenopus
Genes referenced: cpa1 tbx2

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	Figure 1. . Effect of intracellular CPA on macroscopic currents of the CLC-0 mutant C212S. (A) Currents recorded from an inside out patch in control conditions with a pulse protocol in which, after a 400-ms pulse to â140 mV, to maximally deactivate the channels, the voltage was varied between Vp = â140 and +60 mV, followed by a constant tail pulse to +60 mV. Only the traces for Vp = +60, +20, and â60 are shown. The inset shows a schematic representation of the pulse protocol. (B) Currents recorded from the same patch in the presence of 5 mM CPA in the intracellular solution. The inset shows the chemical structure of CPA. Scale bars in A apply also to B.
	Figure 2. . Open channel block of CPA at positive voltages. Currents recorded with the protocol shown in the top panel in the absence (black line) and in the presence of 20 mM intracellular CPA (gray line). Note the significant inhibition even at +140 mV.
	Figure 3. . Voltage dependence of CPA block. From the mean values of the ratio of the steady-state current in presence and absence of CPA, I(CPA)/I(0), for CPA concentrations of 1, 5, and 20 mM the apparent dissociation constant, KD, was obtained by a fit with Eq. 1 for voltages ranging from â140 to +140 mV. The resulting KD values are plotted as a function of voltage (filled circles). The open squares are the KD values measured from the open channel block of single channels. The open triangles are the KD values were measured at 5 mM extracellular Clâ. The straight, dotted line represents the extrapolation to voltages between â140 and +160 mV of a fit of the KD values for V > 60 mV measured in high (100 mM) extracellular Clâ with Eq. 2. It has a slope corresponding to an apparent electrical distance of 0.21. The dashed straight line is a similar fit to the values measured with 5 mM extracellular Clâ for V â¥ 100 mV with z = 0.35. The solid line represents the fit of Model 1 with H as a single free parameter (see materials and methods), resulting in H = 20. Error bars are SEM.
	Figure 4. . Voltage dependence of the slow time constant measured at 20 mM CPA. (A) Currents recorded immediately after a voltage step to â140 mV from the holding potential of 0 mV without and with 20 mM CPA (see arrows). In control the time course of the deactivating currents is well described with a single exponential function (Ï(control) = 19.5 ms, dashed gray line). In the presence of 20 mM CPA the deactivation is described with the sum of two exponentials (thick solid gray line): a very fast one with a time constant <1 ms and a second one whose time constant is slightly slower that recorded in control condition (Ïf(CPA 20 mM) = 0.9 ms and Ïs(CPA 20 mM) = 35 ms). The capacitive transients were not subtracted. (B) The slow time constants were measured with protocols similar to those described in Fig. 1. Open circles: control; filled circles: 20 mM CPA. The solid line represents the predictions of Model 1 if the simulated relaxations are fitted with a single exponential function (see materials and methods), considering only the slow relaxations and ignoring the faster relaxation.
	Figure 5. . Inhibition of single protopores. A single CLC-0 channel present in an inside-out patch was measured at a holding potential of â80 mV in control conditions (A) and in the presence of 1 mM CPA (B) and 20 mM CPA (C). Next to each trace (DâF) is an amplitude histogram of the complete recording together with a fit of the sum of three Gaussian components (dashed gray lines). The peaks of the Gaussian fits are indicated as dashed lines on the current traces. The respective area of each Gaussian component was used to calculate the relative open probability of each conductance state. GâI show the binomial fits. Gray bars represent the values from the traces in AâC, while the filled circles are the expected values from the binomial fits.
	Figure 6. . Fast open channel block by CPA. (A) The fast open channel block is illustrated at an expanded time scale. Short stretches of a single-channel recording without (left) and with 5 mM CPA in the intracellular solution (right) at â100 mV are shown (digitally filtered at 1 kHz). The solid line represents the zero current level, the dotted line represents the current level associated to one pore open, and the dashed line represents the current level due to the simultaneous opening of two pores. (B) Long events like those shown in Fig. 5 were used to estimate the current reduction caused by the fast flickery block. Mean values of the ratio of the open channel current with and without CPA is plotted as a function of the CPA concentration at â80 mV. The solid line is a fit of Eq. 1. A similar analysis was performed also at â120 and â100 mV and the resulting KD values are shown in Fig. 3 as open squares.
	Figure 7. . Kinetic analysis of a single channel at â100 mV. The long recordings shown in A (control) and B (with 5 mM CPA in the bath solution) were subjected to idealization after filtering at 500 Hz as described in materials and methods. In C is shown the dwell time distribution of the closed state in control conditions. In D are shown in the same graph the dwell time distribution of the closed state in control conditions (circles) (same data as in C; note, however, the different time scale) and in presence of 5 mM CPA (squares). In the absence of CPA dwell times could be well fitted with single exponential function with Ï0 = 13.6 ms (dashed line). In the presence of CPA, the distribution of zero current epochs (empty squares) was fitted with the sum of three exponential functions (solid line) with time constants: Ï01 = 132.9 ms, Ï02 = 30.3 ms, and Ï03 = 3.0 ms. The idealization was performed not accepting transitions lasting <1 ms.
	Figure 8. . Power spectra of a single channel and of macroscopic currents. (A) Power spectrum analysis of a single channel at â80 mV in control conditions (gray circles) and in presence of 20 mM CPA (black squares). The control spectrum is well fitted with a single Lorentzian function with a corner frequency of 14 Hz (solid gray line). The sum of two Lorentzians (see materials and methods) with corner frequencies of 2.3 and 920 Hz was needed to fit the spectrum in presence of 20 mM CPA. Baseline spectrum was subtracted from both spectra. The spectra were calculated as described in materials and methods. (B) A representative spectrum of macroscopic currents measured with 20 mM CPA at â140 mV (circles) fitted with the sum of two Lorentzian functions (see materials and methods) with corner frequencies of 5.1 Hz and 231 Hz, respectively. Spectra of macroscopic currents in the absence of CPA presented a major component at 14 Hz and a small component above 2 kHz (unpublished data).
	Figure 9. . Effect of [Clâ]ext on the open channel KD at negative voltages. (A) Open channel block with 5 mM [Clâ]ext. Stretches of a single-channel recording without (left) and with 5 mM CPA in the intracellular solution (right) at â80 mV are shown. The dotted line represents the zero current level, the dashed line the current level associated to one pore open. (B) Comparison of the open channel KD in high (black bars) and low (gray bars) [Clâ]ext at â80 and â100 mV. Errors bars are SEM.
	MODEL 1.
	Figure 10. . Location of mutated residues. The residues of StCLC that correspond to the ones mutated here (correspondence according to the alignment of (Dutzler et al., 2002): S123(CLC-0) â S107(StCLC); E166(CLC-0)->E148(StCLC); T471(CLC-0) â V402(StCLC); Y512(CLC-0) â Y445(StCLC); K519(CLC-0) â T452 (StCLC)) and the highly conserved Glut148 are highlighted in a view of part of the structure of StCLC (Dutzler et al., 2002). Several helices and loops are shown as cartoon. The green sphere corresponds to the Clâ ion seen in the crystal structure. The figure was prepared with the program VMD (Humphrey et al., 1996).
	Figure 11. . Properties of mutants. (A) Macroscopic traces of WT CLC-0, Y512A, Y512F, and T471S mutants. After a prepulse to 60 mV the voltage was varied between â140 and +60 mV followed by a constant tail pulse to â140 mV. Pulse duration was adjusted depending on the characteristics of the different mutants. Only the traces at â140 and +60 mV are shown for clarity's sake. The dotted line represents zero current level. (B) Single-channel traces recorded at â100 mV. The traces were acquired at 1 KHz and digitally filtered at 500 Hz for display. The three dotted lines represent the three conductance levels found in CLC-0 single-channel recordings. (C) Activation curves for the WT (circles), T471S (inverted triangles), Y512A (squares), and Y512F (triangles). Solid lines represent fits obtained with the following equation: f(V)=P0 + (1 â P0)/(1 + exp(zF(V1/2 â V)/RT)) where P0 is the residual open probability at most negative voltages, V1/2 is the half activation potential, and z is the apparent gating charge. (D) Voltage dependence of the time constants of the WT and mutant channels. Symbols are the same as in C. Solid lines represent fits obtained with Eq. 4 for V > â60 mV.
	Figure 12. . Apparent KD of the mutants. The apparent KD of the WT (filled circles), S123T (filled squares), T471S (triangles), Y512A (open squares), Y512F (inverted triangles), K519E (diamonds), and K519Q (open circles) is shown as a function of voltage. For clarity only the values at â140 mV and at positive voltages are shown. Solid lines are fits with Eq. 2. Error bars are SEM.

References [+] :

Accardi, Drastic reduction of the slow gate of human muscle chloride channel (ClC-1) by mutation C277S. 2001, Pubmed, Xenbase