From these pictures it is apparent that those amino acids that strongly affected channel inhibition define a small “pocket” close to the chloride binding site. To give an idea of the binding pocket with the relevant amino acid side chains, Figure 6C shows the same region in a homology model of ClC-1 based on the StClC structure. In addition to the high-impact amino acids already shown in panel A, Figure 6B shows residues corresponding to amino acids in ClC-1 that had only intermediate effects on K d when mutated (medium-impact residues, in pink) and those with minor effects (low-impact residues, in blue). These residues cluster in the region of V402 (the equivalent to ClC-1 S537) and the chloride ion (yellow) and surround a putative binding pocket, as shown in higher magnification in Figure 6B. As the image is slightly tilted from the symmetry axis, the positions of the amino acids that strongly influence inhibitor binding are shown at slightly different angles in the two subunits. ), with residues corresponding to ClC-1 amino acids strongly influencing 9-AC and/or CPA block being shown in red. When S537 was substituted by K or W, the side chains of which are equal or larger than that of F, the K ds of the mutants were so large that they could not be measured reliably. Inset: K ds for 9-AC for several substitutions at position 537 as a function of side chain volume according to Creighton (1993). Asterisks (*) indicate mutants with currents that were too small for a quantitative analysis of K d, and mutants labeled with “+” resulted in a K d for 9-AC that was too large to be reliably determined. Note that some amino acids that are crucial for 9-AC binding also affect the block by CPA but to quantitatively different degrees (for example, E232 and F484). These colors correspond to the ones used for residues in Figure 6. High-impact residues (arbitrarily defined by |log| ≥ 1) for at least one mutation are shown on red background, medium-impact residues (1 > |log| ≥ 0.5) on pink, and low-impact residues (|log| < 0.5) on blue background. Their importance for inhibitor binding is color coded. The bold letters in the left part of the diagram indicate the helices where the mutated residues are located. These values are shown as the logarithm of ratios over the respective K d values of WT ClC-1 (K d = 13 ± 2 μM for 9-AC and K d = 104 ± 20 μM for CPA). The reason for this difference, which has been noted before (Īpparent inhibitory constants (K ds) for 9-AC (black bars, measured at −40mV) and CPA (yellow bars, measured at −140mV) for mutants of ClC-1 as determined in two-electrode voltage clamp measurements (9-AC) and inside-out patch clamp measurements (CPA) of channels expressed in Xenopus oocytes. By contrast, they almost totally lack voltage-dependent current relaxations when examined by two-electrode voltage clamp measurements of oocytes (Figure 2C) (Gründer et al., 1992). Note that like currents from the N-terminal deletion mutant ClC-2 Δ16-61, ClC-2 Δ16-61,T518S currents show time-dependent activation upon hyperpolarization when measured in excised patches (E and F). The applied CPA concentration (1 mM for the ClC-0 and ClC-1 constructs 5 mM for the ClC-2 constructs) matches approximately the apparent K d at −140mV of the respective channels with a threonine at the position corresponding to 537 in ClC-1. Holding potential in (E) and (F) was +40mV. Similar experiments are shown for the ClC-1 mutant S537T (B), the ClC-0 mutant C212S (C), the ClC-0 double mutant C212S/T471S (D), ClC-2 Δ16-61 (E), and ClC-2 Δ16-61 carrying the T518S mutation (F). From a holding potential of 0mV, the voltage was first stepped to 60mV for 50 ms and then to −140mV. (A) Current traces of ClC-1 before (left) and after (right) application of CPA, measured in an inside-out patch. A Serine between Helix O and P Is Important for 9-AC Binding Taken together, these results demonstrate that the 9-AC binding site was directly accessible from the inside only. Furthermore, the onset of inhibition was considerably faster, with a time constant at 100 μM 9-AC of 38.3 ± 3.4 s (n = 3). In contrast to the two-electrode voltage clamp measurements for which 9-AC inhibition was practically irreversible, washout was nearly complete within minutes in inside-out patch clamp experiments (data not shown). Whereas the compound had no effect in outside-out patches (open circles), it strongly inhibited ClC-1 currents in inside-out patches (filled circles). To address the question of the sidedness of 9-AC binding directly, we used excised patch clamp experiments ( Figure 2E). The slow onset of inhibition upon extracellular application of 9-AC ( Figure 2D) may therefore be caused by a slow intracellular accumulation of membrane-permeable 9-AC. These results strongly suggested that 9-AC binds from the intracellular side.
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