Divergent Cl− and H+ pathways underlie transport coupling and gating in CLC exchangers and channels

The CLC family of anion transporting proteins is comprised of secondary active H+-coupled exchangers and of Cl− channels. Both functional subtypes play key roles in human physiology, and mutations causing their dysfunction lead to numerous genetic disorders. Current models suggest that the CLC exchangers do not utilize a classical ‘ping-pong’ mechanism of antiport, where the transporter sequentially interacts with one substrate at a time. Rather, in the CLC exchangers both substrates bind and translocate simultaneously while moving through partially congruent pathways. How ions of opposite electrical charge bypass each other while moving in opposite directions through a shared permeation pathway remains unknown. Here, we use MD simulations in combination with biochemical and electrophysiological measurements to identify a pair of highly conserved phenylalanine residues that form an aromatic pathway, separate from the Cl− pore, whose dynamic rearrangements enable H+ movement. Mutations of these aromatic residues impair H+ transport and voltage-dependent gating in the CLC exchangers. Remarkably, the role of the aromatic pathway is evolutionarily conserved in CLC channels. Using atomic-scale mutagenesis we show that the electrostatic properties and conformational flexibility of these aromatic residues are essential determinants of channel gating. Our results suggest that Cl− and H+ move through physically distinct and evolutionarily conserved routes through the CLC channels and transporters. We propose a unifying mechanism that describes the gating mechanism of CLC exchangers and channels.


Introduction
The CLC (ChLoride Channel) family is comprised of Clchannels and H + -coupled exchangers 2 whose primary physiological task is to mediate anion transport across biological membranes . 29 The CLC Clpore can adopt at least three conformations, differentiated by the position and   (Fig. 1D). The H + pathway is delimited by 9 two glutamic acids that are conserved in the CLC transporters: Gluin serves as the intracellular 10 proton acceptor and is distal from the Clpermeation pathway, while Gluex is the extracellular 11 intersection between the H + and Clpores (  15 opposite directions through the permeation pathway. These proposals share the critical assumption 16 that protonation of Gluex within the pathway destabilizes its binding to Scen and/or Sext, favoring 17 its exit from the pathway. While this mechanism readily explains outward H + transfer, it predicts 18 that when the transporter is mediating H + influx, a protonated and neutral Glu 0 ex outcompetes the 19 negatively charged Clions bound to the anion-selective Sext and Scen sites. This is an energetically 20 unfavorable transition, which should result in intrinsic rectification of transport. Indeed, free- 21 energy calculations show that a protonated Gluex encounters a high energy barrier to enter the Cl -

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permeation pathway (Kuang, Mahankali, & Beck, 2007). In contrast, the CLC exchangers function of Gluex and of H + through the protein are unknown. While biochemical evidence suggests that ion 28 release is rate-limited by a conformational step (Picollo et al., 2009), no release mechanism has 29 been identified. Therefore, two key mechanistic features at the heart of the H + :Clexchange 30 mechanism of the CLCs, the pathways and coupling mechanism of the substrates, remain 31 unknown. 1 Here we combined molecular dynamics simulations with biochemical and 2 electrophysiological measurements, and atomic mutagenesis to investigate the mechanism of 3 H + /Clexchange. We find that, contrary to previously proposed models, a protonated Gluex does 4 not move through the Clpore. Rather, we identify two highly conserved phenylalanine residues 5 that form an aromatic slide which allows the protonated (neutral) Gluex to move to and from Scen 6 without directly competing with Clions for passage through the anion-selective pathway. Further, 7 we show that the rotational movement of the central phenylalanine residue, that enables the 8 formation of the aromatic slide, regulates ion movement within the pathway, providing the 9 molecular mechanism for the coupled exchange of H + and Clby the CLC exchangers. Mutating 10 these residues in prokaryotic and mammalian CLC exchangers severely impairs transport 11 indicating that the role of these aromatic side chains is evolutionarily conserved. Since these 12 phenylalanine residues are highly conserved throughout the CLC family, we hypothesized the role 13 of the aromatic slide might be evolutionary conserved also between CLC channels and exchangers.
14 Indeed, we found that they are critical determinants of gating of the prototypical CLC-0 channel. 15 Using atomic-scale mutagenesis, we probed how the aromatic slide residues interact with Gluex in 16 CLC-0, and found that the central phenylalanine interacts electrostatically with the gating 17 glutamate, and that its conformational rotation is necessary for channel opening. We propose a 18 novel mechanism for CLC mediated H + :Clexchange, where the Cland H + pathways are distinct 19 and intersect only near the central ion binding site. 20

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Molecular dynamic simulations suggest F357 controls entry and release of Clfrom Scen 23 We used molecular dynamics simulations to probe the energetic landscape of ion movement 24 through the permeation pathway of CLC-ec1 to ask whether it is regulated by conformational 25 rearrangements of the pore. Rotation of F357 enables the formation of an aromatic slide 4 We next asked how Cland Gluex interact along the transport cycle. As in the previous section, we 5 first considered states in which Gluex is protonated (Glu 0 ex) and outside the Clpathway in an 6 E148Q-like conformation (Fig. 3A). Analysis of the conformational sampling of the PMFs 7 presented in Fig. 2 reveals that, in this configuration, Glu 0 ex is stabilized by the carboxylate group 8 of D54 via a water molecule (Fig. 3A) or by a hydrogen bond with the backbone of A189 ( Fig.   9 3B). The simulations also reveal that the carboxylate group of Glu 0 ex rarely visits Sext and rather 10 interacts with the aromatic ring of F190, even if Sext is free of Cl - (Fig. 3C). 11 We then considered states in which Gluex occupies Scen. We calculated the PMF describing  Interestingly, in the case of the protonated Gluex, a free energy well is also observed at the level of  The aromatic slide residues are essential for Cl -:H + coupling and exchange in CLC-ec1 31 Our molecular dynamics simulations suggest that F190 and F357 play a critical role in determining 1 Cl -/H + coupling and control a rate-limiting barrier for ion transport in CLC-ec1. To test these 2 hypotheses, we mutated them to alanine and determined the unitary transport rate and 3 stoichiometry of the Cl -/H + exchange cycle. Both mutations slow the turnover rate and degrade the 4 exchange stoichiometry (Fig. 4). The F190A mutant slows transport ~2-fold ( MD simulations that F190 and F357 play a key role in coupling and transport of CLC-ec1. 17 Remarkably, F190 and F357 are among the highest conserved residues throughout the CLC family, 18 respectively at ~94% and ~76% ( Fig. 4 -Suppl. 2), suggesting that their functional role might be 19 evolutionarily conserved. In the remainder of this work we will refer to these residues across 20 different CLC homologues as Pheex (F190 in CLC-ec1) and Phecen (F357 in CLC-ec1).

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The role of Pheex and Phecen is conserved in mammalian transporters 23 We asked whether the role of Pheex and Phecen is conserved in the mammalian CLC exchangers. 24 The mammalian CLC-5 and CLC-7 exchangers are activated by depolarizing voltages (Fig. 5A, currents of F301A and F514A at +90 mV are reduced by ~25% and ~50%, respectively, compared 1 to WT (Fig. 5D), in line with the reduced transport rates observed for CLC-ec1 (Fig. 4D). The 2 F301A mutant is nearly voltage independent between -80 and +90 mV (Fig. 5B, E) and so are its and out the anion pathway. We hypothesize that these residues interact with the permeating ions 13 and Gluex via direct and electrostatic interactions. 14 15 Pheex and Phecen are key determinants of CLC channel gating 16 To probe whether the aromatic slide also plays a similar role in determining the voltage dependent shows an ~25 mV right-shift of activation and the F214-2,6F2-Phe substitution ~20 mV (Fig. 7B).

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Similarly, substitutions at Phecen also affect common-pore gating (Fig. 7E) while those at Pheex 26 cause only minor alterations (Fig. 7D). Notably, the effect of F418Cha is comparable to that of 27 F418A, suggesting that the electrostatics dominate the interactions of Phecen with Gluex during 28 common-pore gating (Fig. 6H, 7E). Finally, we replaced Phecen with 2,6diMeth-Phe (Fig. 7A), 29 which restricts its interconversion between the 'up' and 'down' rotamers ( Fig. 2E). The kinetics 30 of the common-pore gating process of the F418-2,6diMeth-Phe are accelerated so that they become conformers we identified in the CLC transporters are essential for CLC-0 channel gating, and is 7 necessary to allow the movement of Gluex in and out of the Clpermeation pathway. Therefore, 8 our results show that the aromatic slide forms an evolutionarily conserved structural motif that is 9 necessary to enable Gluex movement in and out of the Clpore during the exchange cycle and in 10 channel opening.  Formation of an aromatic slide is essential to Gluex movement 5 Our data suggest that while the deprotonated and negatively charged Gluex moves through the Cl -6 pathway, the protonated and neutral Glu 0 ex interacts with two highly conserved phenylalanines, 7 Phecen and Pheex. These residues can form an aromatic slide that enables movement of protons  rotamer suggesting this conformation is likely the most stable. However, our combined simulation 14 and functional data suggests that Phecen can adopt two distinct rotamers around its χ1 angle ( Fig.   15 2), and that this rearrangement determines the energy barrier for Clmovement within the pore dependence of channel gating (Fig. 6). We used atomic scale mutagenesis to test the prediction of 23 our MD simulations that formation of the aromatic slide entails a rotation of the side chain of 24 Phecen around its Cα-Cβ bond. When we replace Phecen with 2,6diMet-Phe, a Phe derivative that 25 specifically constrains this rotational rearrangement, we find that opening of the single-and 26 common-pore gates in the CLC-0 channel are severely impaired (Fig. 7, Fig. 7  Our results also suggest that Pheex and Phecen interact differently with Gluex. The aromatic closed state of the single-pore gate (Fig. 7C), while their re-localization to the proximal edge 10 promotes opening (Fig. 7C). These findings are consistent with the location of Phecen within the 11 core of the protein, and with our MD simulations suggesting that Gluex forms a π-dipole interaction 12 with the aromatic ring of Phecen (Fig. 3). Indeed, the interaction between the buried aromatic Phecen  Our finding that two Phe residues form an evolutionarily conserved secondary pathway that 23 enables movement of the protonated Gluex in and out of the ion transport pathway allows us to 24 propose a 7-state mechanism for the CLC transporters that explains the stoichiometry of 2 show ions bound to this site. As a starting configuration, we consider a state where Gluex and Gluin 28 are de-protonated, Gluex occupies Scen, Phecen is in the 'up' position and no Clions are bound to 29 the pathway (Fig. 8, I). After a Clion binds to Sint (Fig. 8, II), the opening of the intracellular gate, to Sext (Fig. 8, III). The binding of a second ion favors the protonation of Gluex, allowing the Cl -1 ions to simultaneously occupy Scen and Sext, accompanied by the displacement of Glu 0 ex out of the 2 pathway and the closure of the internal gate (Fig. 8, IV). The protoned Gluex diffuses toward the 3 aromatic slide and interact with Pheex (Fig. 8, V). Movement of Glu 0 ex along the aromatic slide 4 allows the interdependent rearrangement of Phecen to the 'down' state and release of a Clion, i.e. 5 release from Sext to the extracellular milieu and the transfer of the second ion from Scen to Sext (Fig.   6 8, VI). This conformation, where Glu 0 ex interacts with Phecen on the side of the Clpathway and 7 Sext is occupied, favors the formation of a water wire (Fig. 8, VI), which enables proton transfer 8 from Glu 0 ex to Gluin (Fig. 8, VII). Deprotonation of Gluex allows it to move into Scen, favoring the 9 release of the second Clfrom Sext to the outside, and of the proton from Glu 0 in to the intracellular 10 solution, returning to the starting configuration (Fig. 8, I). In sum, we propose that the Cland H +

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ions bypass each other while moving in opposite directions through a CLC transporter by taking 12 physically distinct routes: the Clions move through the anion selective pore while the H + moves 13 along a pathway comprised of a water-wire and the aromatic slide (Fig. 8B). The distinct routes 14 for Cland H + allow for a complete reversibility of the cycle. 15 Remarkably, our findings show that the role of the aromatic slide residues Phecen and Pheex 16 is evolutionarily conserved between CLC exchangers and channels. We propose that the aromatic 17 slide in CLC-0 could provide a conserved pathway that enables the residual H + transport associated 18 with the common-pore gating process of the CLC-0 channel to occur (Lísal & Maduke, 2008).

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Thus, our proposed exchange mechanism for the CLC transporters also captures the key 20 rearrangements that underlie gating of the CLC channels. In this framework, rapid ion conduction       distinguish single-pore from common-pore gating. During the single-pore gating protocol the 10 voltage was stepped to +80 mV for 50 ms and then a variable voltage from -160 mV to +80 mV 11 increasing in 20 mV steps was applied for 200 ms, followed by a 50 ms pulse at -120 mV for tail 12 current analysis. For CLC-0 common-pore gating, 7 s voltage steps from +20 mV to -140 mV have 13 been applied in -20 mV increments followed by a 2.5 s +60 mV post pulse for tail current analysis. which 50% activation occurs, and k is the slope factor (k=R*T/(z*F) with R as universal gas 23 constant, T as temperature in K, F as Faraday constant, and z as the gating charge). 24 For analysis of the activation kinetics of CLC-7/Ostm1 and its variants, activating voltage pulses 25 (from +20 to +90 mV) were fit to a bi-exponential function of the following form: where I is the current as a function of time; A1, A2 and A0 are fractional amplitudes obtained by 1 normalizing to the total current. While A1 and A2 are time-dependent components, A0 is time-2 independent. τ1 and τ2 are the corresponding time constants. significance Student's t-test (two-tailed distribution; two-sample equal variance) was performed.

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The threshold for significance was set to P=0.05. binding of a Clto the pore when Glu 0 ex is initially bound to Scen (see Figure 3 -Supplement 1). A proton 7 wire is spontaneously formed between Gluex (E148) and Gluin (E203), which are bridged by two water 8 molecules (D). Glu 0 ex can form a dipole-interaction with F357 aromatic side chain, leaving Scen empty 9 (E). Clmoves from Sext to Scen, while the side chain of Glu 0 ex is stabilized outside the ion pathway by its 10 interaction with F357, and in proximity of F190 (F).   Competing interests 13 The authors declare no competing interests. 14 15 Data availability. 16 Data supporting the findings of this manuscript are available from the corresponding authors upon