Binding Of Cl Research Articles

Cytochrome C Binding to Liposomal Surfaces

Cytochrome c is involved in triggering apoptosis in the mitochondria. In order to understand this initiation process of apoptosis, the interactions between cytochrome c and anionic lipid surfaces must be thoroughly characterized. It is well known that cytochrome c adopts partially unfolded conformations on cardiolipin containing liposomes. We examined the binding of cytochrome c to liposomes with different cardiolipin content by measuring the W59 fluorescence as a function of liposome concentration. Surprisingly, the thus obtained binding isotherms indicate a biphasic binding process, which are likely to correspond to different cardiolipin binding sites. Liposome concentrations associated with these binding sites were further investigated by far UV and visible CD as well as by absorption spectroscopy.The initial data obtained indicates that upon binding to cardiolipin complexes, cytochrome c adopts a nonnative state. This conformation is a low spin hexacoordinated state where the heme is ligated to H33 and H18. The far UV CD spectra shows that the protein retains its α-helical content regardless of CL binding, which is characteristic of native cytochrome c. The W59 signal in the near UV CD spectra disappears, indicating a more open protein conformation. This supports our fluorescence data which show an increase in fluorescence intensity as the W59 moves farther away from the heme. Soret band CD simulations also indicate an intermediate state upon protein binding to CL complexes. Data obtained from ultracentrifugation and gel electrophoresis experiments indicate a nonreversible mode of cytochrome c and liposome interactions, which might be due to the insertion of the lipids' hydrophobic tail into the protein crevice.

Photoinduced Proton Release in Proteorhodopsin at Low pH: The Possibility of a Decrease in the pKa of Asp227

Proteorhodopsin (PR) is one of the microbial rhodopsins that are found in marine eubacteria and likely functions as an outward light-driven proton pump. Previously, we [Tamogami, J., et al. (2009) Photochem. Photobiol.85, 578-589] reported the occurrence of a photoinduced proton transfer in PR between pH 5 and 10 using a transparent ITO (indium-tin oxide) or SnO(2) electrode that works as a time-resolving pH electrode. In the study presented here, the proton transfer at low pH (<4) was investigated. Under these conditions, Asp97, the primary counterion to the protonated Schiff base, is protonated. We observed a first proton release that was followed by an uptake; during this process, however, the M intermediate did not form. Through the use of experiments with several PR mutants, we found that Asp227 played an essential role in proton release. This residue corresponds to the Asp212 residue of bacteriorhodopsin, the so-called secondary Schiff base counterion. We estimated the pK(a) of this residue in both the dark and the proton-releasing photoproduct to be ~3.0 and ~2.3, respectively. The pK(a) value of Asp227 in the dark was also estimated spectroscopically and was approximately equal to that determined with the ITO experiments, which may imply the possibility of the release of a proton from Asp227. In the absence of Cl(-), we observed the proton release in D227N and found that Asp97, the primary counterion, played a key role. It is inferred that the negative charge is required to stabilize the photoproducts through the deprotonation of Asp227 (first choice), the binding of Cl(-) (second choice), or the deprotonation of Asp97. The photoinduced proton release (possibly by the decrease in the pK(a) of the secondary counterion) in acidic media was also observed in other microbial rhodopsins with the exception of the Anabaena sensory rhodopsin, which lacks the dissociable residue at the position of Asp212 of BR or Asp227 of PR and halorhodopsin. The implication of this pK(a) decrease is discussed.

Influence of Halide Binding on the Hydrogen Bonding Network in the Active Site of Salinibacter Sensory Rhodopsin I

In nature, organisms are subjected to a variety of environmental stimuli to which they respond and adapt. They can show avoidance or attractive behaviors away from or toward such stimuli in order to survive in the various environments in which they live. One such stimuli is light, to which, for example, the receptor sensory rhodopsin I (SRI) has been found to respond by regulating both negative and positive phototaxis in, e.g., the archaeon Halobacterium salinarum. Interestingly, to date, all organisms having SRI-like proteins live in highly halophilic environments, suggesting that salt significantly influences the properties of SRIs. Taking advantage of the discovery of the highly stable SRI homologue from Salinibacter ruber (SrSRI), which maintains its color even in the absence of salt, the importance of the chloride ion for the color tuning and for the slow M-decay, which is thought to be essential for the phototaxis function of SRIs, has been reported previously [Suzuki, D., et al. (2009) J. Mol. Biol.392, 48-62]. Here the effects of the anion binding on the structure and structural changes of SRI during its photocycle are investigated by means of Fourier transform infrared (FTIR) spectroscopy and electrochemical experiments. Our results reveal that, among other things, the structural change and proton movement of a characteristic amino acid residue, Asp102 in SrSRI, is suppressed by the binding of an anion in its vicinity, both in the K- and M-intermediate. The presence of this anion also effects the extent of chromophore distrotion, and tentative results indicate an influence on the number and/or properties of internal water molecules. In addition, a photoinduced proton transfer could only be observed in the absence of the bound anion. Possible proton movement pathways, including the residues Asp102 and the putative Cl binding site His131, are discussed. In conclusion, the results show that the anion binding to SRI is not only important for the color tuning, and for controlling the photocycle kinetics, but also induces some structural changes which facilitate the observed properties.

Mutations in the GlyT2 Gene (SLC6A5) Are a Second Major Cause of Startle Disease

Hereditary hyperekplexia or startle disease is characterized by an exaggerated startle response, evoked by tactile or auditory stimuli, leading to hypertonia and apnea episodes. Missense, nonsense, frameshift, splice site mutations, and large deletions in the human glycine receptor α1 subunit gene (GLRA1) are the major known cause of this disorder. However, mutations are also found in the genes encoding the glycine receptor β subunit (GLRB) and the presynaptic Na(+)/Cl(-)-dependent glycine transporter GlyT2 (SLC6A5). In this study, systematic DNA sequencing of SLC6A5 in 93 new unrelated human hyperekplexia patients revealed 20 sequence variants in 17 index cases presenting with homozygous or compound heterozygous recessive inheritance. Five apparently unrelated cases had the truncating mutation R439X. Genotype-phenotype analysis revealed a high rate of neonatal apneas and learning difficulties associated with SLC6A5 mutations. From the 20 SLC6A5 sequence variants, we investigated glycine uptake for 16 novel mutations, confirming that all were defective in glycine transport. Although the most common mechanism of disrupting GlyT2 function is protein truncation, new pathogenic mechanisms included splice site mutations and missense mutations affecting residues implicated in Cl(-) binding, conformational changes mediated by extracellular loop 4, and cation-π interactions. Detailed electrophysiology of mutation A275T revealed that this substitution results in a voltage-sensitive decrease in glycine transport caused by lower Na(+) affinity. This study firmly establishes the combination of missense, nonsense, frameshift, and splice site mutations in the GlyT2 gene as the second major cause of startle disease.

On the Mechanism of Gating Charge Movement of ClC-5, a Human Cl−/H+ Antiporter

ClC-5 is a Cl−/H+ antiporter that functions in endosomes and is important for endocytosis in the proximal tubule. The mechanism of transport coupling and voltage dependence in ClC-5 is unclear. Recently, a transport-deficient ClC-5 mutant (E268A) was shown to exhibit transient capacitive currents. Here, we studied the external and internal Cl− and pH dependence of the currents of E268A. Transient currents were almost completely independent of the intracellular pH. Even though the transient currents are modulated by extracellular pH, we could exclude that they are generated by proton-binding/unbinding reactions. In contrast, the charge movement showed a nontrivial dependence on external chloride, strongly supporting a model in which the movement of an intrinsic gating charge is followed by the voltage-dependent low-affinity binding of extracellular chloride ions. Mutation of the external Glu-211 (a residue implicated in the coupling of Cl− and proton transport) to aspartate abolished steady-state transport, but revealed transient currents that were shifted by ∼150 mV to negative voltages compared to E268A. This identifies Gluext as a major component of the gating charge underlying the transient currents of the electrogenic ClC-5 transporter. The molecular events underlying the transient currents of ClC-5 emerging from these results can be explained by an inward movement of the side chain of Gluext, followed by the binding of extracellular Cl− ions.

Synergistic substrate binding determines the stoichiometry of transport of a prokaryotic H+/Cl− exchanger

Active exchangers dissipate the gradient of one substrate to accumulate nutrients, export xenobiotics and maintain cellular homeostasis. Mechanistic studies suggested that all exchangers share two fundamental properties: substrate binding is antagonistic and coupling is maintained by preventing shuttling of the empty transporter. The CLC Cl−: H+ exchangers control the homeostasis of cellular compartments in most living organisms but their transport mechanism remains unclear. We show that substrate binding to CLC-ec1 is synergistic rather than antagonistic: chloride binding induces protonation of a critical glutamate. The simultaneous binding of H+ and Cl− gives rise to a fully-loaded state incompatible with conventional mechanisms. Mutations in the Cl− transport pathway identically alter the stoichiometries of Cl−: H+ exchange and binding. We propose that the thermodynamics of synergistic substrate binding determine the stoichiometry of transport rather than the kinetics of conformational changes and ion binding.

Dissociative electron attachment to the silane derivatives trichlorovinylsilane (SiCl3C2H3), tetravinylsilane (Si(C2H3)4) and trimethylvinylsilane (Si(CH3)3C2H3)

The formation of negative ions following low energy (0–12eV) electron attachment to the title compounds is studied in a crossed electron-molecular beam experiment applying mass spectrometric detection of the anions. All silane derivatives presently studied are weak electron scavengers which also holds for trichlorovinylsilane. The behaviour in the silanes is hence in strong contrast to the hydrocarbons where chlorination usually results in a strong increase of the cross section for dissociative electron attachment (DEA) yielding intense Cl− signals already at very low electron energies (near 0eV). The different behaviour can be explained by the appreciably stronger Si–Cl binding energy compared to that of C–Cl making the DEA reaction leading to Cl− endothermic for trichlorovinylsilane while the corresponding reaction is exothermic for most chlorinated hydrocarbons. Trichlorovinylsilane and tetravinylsilane undergo various further DEA reactions via different resonances at higher energy (in the range between 4 and 10eV) leading to a variety of ionic fragments. These reactions range from simple bond cleavages to considerably more complex unimolecular decompositions involving multiple bond cleavages, hydrogen transfer in the temporary negative ion and formation of new bonds. Trimethylvinylsilane yields only one single fragment due to the loss of a neutral hydrogen atom ((M−H)−).

On the Mechanism of Gating Charge Movement of the Chloride/Proton Antiporter CLC-5

Structural and functional studies identified two critical residues for the transport mechanism of CLC transporters; the so-called gating glutamate (E211 in ClC-5) that controls the access of the anions to the extracellular space and is critical for anion/proton coupling and the proton glutamate (E268 in ClC-5), likely the intracellular entry/exit point for protons. However, the mechanism of voltage-sensitivity of CLC transporters is still poorly understood. Interestingly, it has been recently reported that the E268A mutant of the endosomal Cl-/H+ antiporter ClC-5, beside inhibiting steady-state transports, exhibits transient currents upon voltage steps to large positive voltages. These transient currents may offer the possibility to glean information on the molecular details of transport coupling and voltage-sensitivity. Here we studied the dependence of the transient currents on the extracellular and intracellular pH and Cl- concentration. We conclude that the transient currents represent the movement of an intrinsic gating charge followed by the voltage dependent binding of extracellular Cl- ions. In addition, we find that the gating glutamate mutation E211D abolishes stationary transport but displays transient currents which are shifted by ∼ 150 mV compared to the proton glutamate mutation, identifying E211 as a major component of the voltage sensing mechanism of ClC-5.

Polarized Naphthalimide CH Donors Enhance Cl– Binding within an Aryl-Triazole Receptor

The dipolar character of 1,8-naphthalimide together with polarization of the C(4)-H and C(5)-H donors has been utilized in receptor 1 to effectively bind chloride alongside triazole and phenylene units. The Cl(-) binding strength of 1 shows that the naphthalimide provides greater anion stabilization than an unactivated phenylene, and DFT calculations show that its collinear donor array can be a "urea-like" analog for CH···anion interactions.

Spectral Tuning in Sensory Rhodopsin I from Salinibacter ruber

Organisms utilize light as energy sources and as signals. Rhodopsins, which have seven transmembrane α-helices with retinal covalently linked to a conserved Lys residue, are found in various organisms as distant in evolution as bacteria, archaea, and eukarya. One of the most notable properties of rhodopsin molecules is the large variation in their absorption spectrum. Sensory rhodopsin I (SRI) and sensory rhodopsin II (SRII) function as photosensors and have similar properties (retinal composition, photocycle, structure, and function) except for their λ(max) (SRI, ∼560 nm; SRII, ∼500 nm). An expression system utilizing Escherichia coli and the high protein stability of a newly found SRI-like protein, SrSRI, enables studies of mutant proteins. To determine the residue contributing to the spectral shift from SRI to SRII, we constructed various SRI mutants, in which individual residues were substituted with the corresponding residues of SRII. Three such mutants of SrSRI showed a large spectral blue-shift (>14 nm) without a large alteration of their retinal composition. Two of them, A136Y and A200T, are newly discovered color tuning residues. In the triple mutant, the λ(max) was 525 nm. The inverse mutation of SRII (F134H/Y139A/T204A) generated a spectral-shifted SRII toward longer wavelengths, although the effect is smaller than in the case of SRI, which is probably due to the lack of anion binding in the SRII mutant. Thus, half of the spectral shift from SRI to SRII could be explained by only those three residues taking into account the effect of Cl(-) binding.

Reconciling the Krogh and Ussing interpretations of epithelial chloride transport – presenting a novel hypothesis for the physiological significance of the passive cellular chloride uptake

In 1937, August Krogh discovered a powerful active Cl(-) uptake mechanism in frog skin. After WWII, Hans Ussing continued the studies on the isolated skin and discovered the passive nature of the chloride uptake. The review concludes that the two modes of transport are associated with a minority cell type denoted as the γ-type mitochondria-rich (MR) cell, which is highly specialized for epithelial Cl(-) uptake whether the frog is in the pond of low [NaCl] or the skin is isolated and studied by Ussing chamber technique. One type of apical Cl(-) channels of the γ-MR cell is activated by binding of Cl(-) to an external binding site and by membrane depolarization. This results in a tight coupling of the uptake of Na(+) by principal cells and Cl(-) by MR cells. Another type of Cl(-) channels (probably CFTR) is involved in isotonic fluid uptake. It is suggested that the Cl(-) channels serve passive uptake of Cl(-) from the thin epidermal film of fluid produced by mucosal glands. The hypothesis is evaluated by discussing the turnover of water and ions of the epidermal surface fluid under terrestrial conditions. The apical Cl(-) channels close when the electrodiffusion force is outwardly directed as it is when the animal is in the pond. With the passive fluxes eliminated, the Cl(-) flux is governed by active transport and evidence is discussed that this is brought about by an exchange of cellular HCO(3) (-) with Cl(-) of the outside bath driven by an apical H(+) V-ATPase.

Synergistic Substrate binding in the CLC-Ec1 Exchanger

Secondary active transporters couple the electrochemical gradients of two or more substrates to catalyze their stoichiometric exchange across biological membranes. In canonical models of alternating access exchange binding of different molecules is mutually exclusive or competitive. CLC-ec1 is a structurally known H+/Cl- exchanger of the CLC family and it has served as a guide in understanding the basic functional properties of CLC channels and transporters. Two glutamates (E148, E203) define the external and internal end points of the H+ permeation pathway. However the coupling mechanism of these exchangers is still obscure. We used Isothermal Titration Calorimetry to probe the coupling between H+/Cl- binding and transport by measuring the enthalpy of Cl- binding in different buffers. We found that binding of Cl- and H+ is synergistic: binding of 2 Cl- to CLC-ec1 induces the binding of 1 H+. Thus, the stoichiometries of binding and transport are equal but have opposite sign. Cl- binding induces the uptake of 1 H+ from the buffer in the E203Q mutant despite the fact that this mutant is unable to transport H+. In contrast, Cl- and H+ binding become decoupled in the E148A mutant. Thus, while both glutamates are essential for coupling movement of H+ and Cl- only E148 controls their coupled binding while protonation of E203 takes place in a Cl--independent manner. Decoupling H+ and Cl- fluxes by mutating residues that impair Cl- binding leads to a parallel disruption of their binding synergism, strongly suggesting that this step is critical for the transport process.In conclusion, our results show that during the exchange cycle binding of Cl- at one side of the membrane induces binding of H+ at the other side. This, implies that the transport cycle of CLC-ec1 is drastically different from conventional alternating access mechanisms.

Structure of a Slow CLC Cl−/H+ Antiporter from a Cyanobacterium

X-ray crystal structures have been previously determined for three CLC-type transporter homologues, but the absolute unitary transport rate is known for only one of these. The Escherichia coli Cl(-)/H(+) antiporter (EC) moves ∼2000 Cl(-) ions/s, an exceptionally high rate among membrane-transport proteins. It is not known whether such rapid turnover is characteristic of ClCs in general or if the E. coli homologue represents a functional outlier. Here, we characterize a CLC Cl(-)/H(+) antiporter from the cyanobacterium Synechocystis sp. PCC6803 (SY) and determine its crystal structure at 3.2 Å resolution. The structure of SY is nearly identical to that of EC, with all residues involved in Cl(-) binding and proton coupling structurally similar to their equivalents in EC. SY actively pumps protons into liposomes against a gradient and moves Cl(-) at ∼20 s(-1), 1% of the EC rate. Electrostatic calculations, used to identify residues contributing to ion binding energetics in SY and EC, highlight two residues flanking the external binding site that are destabilizing for Cl(-) binding in SY and stabilizing in EC. Mutation of these two residues in SY to their counterparts in EC accelerates transport to ∼150 s(-1), allowing measurement of Cl(-)/H(+) stoichiometry of 2/1. SY thus shares a similar structure and a common transport mechanism to EC, but it is by comparison slow, a result that refutes the idea that the transport mechanism of CLCs leads to intrinsically high rates.

Computational insights into interactions between Hg species and α-Fe 2O 3 (0 0 1)

First-principle calculations based on Density Functional Theory were performed to investigate the binding mechanisms of mercury species on α-Fe 2O 3 (0 0 1) surface. This is crucial in demonstrating the contribution of α-Fe 2O 3 existing in fly ash for mercury removal. It has been determined that Hg 0 is adsorbed on the α-Fe 2O 3 (0 0 1) surface with physisorption mechanism. The oxidized forms of HgCl and HgCl 2 can be adsorbed on α-Fe 2O 3 (0 0 1) dissociatively or non-dissociatively. In the case of dissociative adsorption, a close examination of the energy diagram indicates that HgCl may be favorable for the adsorption of Cl and desorption of Hg. The dissociation of HgCl 2 with the binding of Cl and HgCl on the surface is possibly the dominant interaction pathway.

Anion and cation binding by a new indole/pyridine/amine-based ion-pair receptor

A new ion-pair receptor bis(3-bromoindol-2-ylmethyl)(2-pyridylmethyl)amine ( 1) was synthesized and studied for its anion and cation binding behavior using ESI-MS and 1H NMR spectroscopy. Among halides, 1 exhibits the strongest binding with Cl − to form a 1:1 adduct ( K a = 1042 ± 21 in CD 3CN). Among alkali metal ions, Li + and Na + showed the strongest binding in the formation of a 1·M + complex. The simultaneous binding of Cl − and Li + to 1 was confirmed by 1H NMR titration of a 1:1 mixture of 1 and Cl − with LiPF 6 in 83:17 v/v mixture of CDCl 3 and DMSO- d 6. DFT-optimized structures of 1·Cl −, 1·Li +, and 1·Li +·Cl − are consistent with the chemical shift changes observed in 1H NMR studies.

Extracellular Chloride Modulates the Desensitization Kinetics of Acid-sensing Ion Channel 1a (ASIC1a)

Acid-sensing ion channels (ASICs) are sodium channels gated by extracellular protons. The recent crystallization of ASIC1a identified potential binding sites for Cl(-) in the extracellular domain that are highly conserved between ASIC isoforms. However, the significance of Cl(-) binding is unknown. We investigated the effect of Cl(-) substitution on heterologously expressed ASIC1a current and H(+)-gated currents from hippocampal neurons recorded by whole-cell patch clamp. Replacement of extracellular Cl(-) with the impermeable and inert anion methanesulfonate (MeSO(3)(-)) caused ASIC1a currents to desensitize at a faster rate and attenuated tachyphylaxis. However, peak current amplitude, pH sensitivity, and selectivity were unchanged. Other anions, including Br(-), I(-), and thiocyanate, also altered the kinetics of desensitization and tachyphylaxis. Mutation of the residues that form the Cl(-)-binding site in ASIC1a abolished the modulatory effects of anions. The results of anion substitution on native ASIC channels in hippocampal neurons mirrored those in heterologously expressed ASIC1a and altered acid-induced neuronal death. Anion modulation of ASICs provides new insight into channel gating and may prove important in pathological brain conditions associated with changes in pH and Cl(-).

Structures of urea/thiourea 1,3-disubstituted thia[4]calixarenes and corresponding monofunctional receptors and their anion recognition properties

Two thiacalix[4]arenes in 1,3-alternate conformation functionalized by two (CH2)2NH(C=X)NHC6H4-NO2-p groups (X = S,O) as well as two related monofunctional receptors MeO(CH2)2NH(C=X)NHC6H4-NO2-p were prepared and characterized by X-ray crystal structures. The thioureido and ureido derivatives have E,Z and E,E conformations respectively both in monofunctional receptors and thiacalixarenes. The thiacalixarene attached thiourea groups are well separated from each other, but respective urea groups are much closer to each other and have mutual parallel orientation making the bisurea derivative a better preorganized receptor as compared to bisthiourea. Binding of Cl−, F−, H2PO4 − and AcO− anions in chloroform and DMSO was studied by spectrophotometric and NMR titrations. In chloroform both bisurea and bisthiourea thiacalix[4]arenes bind anions 3–5 times stronger than corresponding monofunctional compounds in spite of better preorganization of the urea derivative. In DMSO simultaneous deprotonation of ureido NH groups of receptors and hydrogen bonding reactions are observed. Deprotonation by H2PO4 − is accompanied by a strong association between liberated H3PO4 and H2PO4 − (log K = 3.9). For hydrogen bonding associations the binding constants of H2PO4 − and AcO− with bisurea thiacalixarene are up to two orders of magnitude larger than those with corresponding monofunctional receptor, but with bisthiourea thiacalixarene the effect is less than two-fold. Thus in this solvent in contrast to chloroform the preorganization is an important factor.

Spectroscopic Studies of a Sensory Rhodopsin I Homologue from the Archaeon Haloarcula vallismortis

Sensory rhodopsin I (SRI) functions as a dual receptor regulating both negative and positive phototaxis. It transmits light signals through changes in protein-protein interactions with its transducer protein, HtrI. The phototaxis function of Halobacterium salinarum SRI (HsSRI) has been well characterized using genetic and molecular techniques, whereas that of Salinibacter ruber SRI (SrSRI) has not. SrSRI has the advantage of high protein stability compared with HsSRI and, therefore, provided new information about structural changes and Cl(-) binding of SRI. However, nothing is known about the functional role of SrSRI in phototaxis behavior. In this study, we expressed a SRI homologue from the archaeon Haloarcula vallismortis (HvSRI) as a recombinant protein which uses all-trans-retinal as a chromophore. Functionally important residues of HsSRI are completely conserved in HvSRI (unlike in SrSRI), and HvSRI is extremely stable in buffers without Cl(-). Taking advantage of the high stability, we characterized the photochemical properties of HvSRI under acidic and basic conditions and observed the effects of Cl(-) on the protein under both conditions. Fourier transform infrared results revealed that the structural changes in HvSRI were quite similar to those in HsSRI and SrSRI. Thus, HvSRI can become a useful protein model for improving our understanding of the molecular mechanism of the dual photosensing by SRI.

A mathematical model of rat ascending Henle limb. I. Cotransporter function

Kinetic models of Na+-K+-2Cl- costransporter (NKCC2) and K+-Cl- cotransporter (KCC4), two of the key cotransporters of the Henle limb, are fashioned with inclusion of terms representing binding and transport of NH4+. The models are simplified using assumptions of equilibrium ion binding, binding symmetry, and identity of Cl- binding sites. Model parameters are selected to be consistent with flux data from expression of these transporters in oocytes, specifically inwardly directed coupled transport of rubidium. In the analysis of these models, it is found that despite the simplifying assumptions to reduce the number of model parameters, neither model is uniquely determined by the data. For NKCC or KCC there are two- or three-parameter families of "optimal" solutions. As a consequence, one may specify several carrier translocation rates and/or ion affinities before fitting the remaining coefficients to the data, with no loss of fidelity in simulating the experiments. Model calculations suggest that with respect to NKCC2 near its operating point, the curve of ion flux as a function of cell Cl- is steep, and with respect to KCC4, its curve of ion flux as a function of peritubular K+ is also steep. The implication is that the kinetics are suitable for these two transporters in series to act as a sensor for peritubular K+, to modulate AHL Na+ reabsorption, with cytosolic Cl- as the intermediate variable. The models also reveal the potential for luminal NH4+ to be a potent catalyst for NKCC2 Na+ reabsorption, provided suitable exit mechanisms for NH4+ (from cell-to-lumen) are operative. It is found that KCC4 is likely to augment the secretory NH4+ flux, with peritubular NH4+ uptake driven by the cell-to-blood K+ gradient.

Extracellular Chloride Regulates the Epithelial Sodium Channel

The extracellular domain of the epithelial sodium channel ENaC is exposed to a wide range of Cl(-) concentrations in the kidney and in other epithelia. We tested whether Cl(-) alters ENaC activity. In Xenopus oocytes expressing human ENaC, replacement of Cl(-) with SO4(2-), H2PO4(-), or SCN(-) produced a large increase in ENaC current, indicating that extracellular Cl(-) inhibits ENaC. Extracellular Cl(-) also inhibited ENaC in Na+-transporting epithelia. The anion selectivity sequence was SCN(-) < SO4(2-) < H2PO4(-) < F(-) < I(-) < Cl(-) < Br(-). Crystallization of ASIC1a revealed a Cl(-) binding site in the extracellular domain. We found that mutation of corresponding residues in ENaC (alpha(H418A) and beta(R388A)) disrupted the response to Cl(-), suggesting that Cl(-) might regulate ENaC through an analogous binding site. Maneuvers that lock ENaC in an open state (a DEG mutation and trypsin) abolished ENaC regulation by Cl(-). The response to Cl(-) was also modulated by changes in extracellular pH; acidic pH increased and alkaline pH reduced ENaC inhibition by Cl(-). Cl(-) regulated ENaC activity in part through enhanced Na+ self-inhibition, a process by which extracellular Na+ inhibits ENaC. Together, the data indicate that extracellular Cl(-) regulates ENaC activity, providing a potential mechanism by which changes in extracellular Cl(-) might modulate epithelial Na+ absorption.