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KEY POINTS - The ClC-3 2Cl /1H exchanger modulates endosome pH and Cl concentration. We investigated the relationships between ClC-3-mediated ion transport (steady-state transport current, I ), gating charge (Q) and cytoplasmic alkalization. ClC-3 transport is functionally unidirectional. ClC-5 and ClC-3 display indistinguishable exchange ratios, but ClC-3 cycling is less "efficient", as reflected by a large Q/I . An M531A mutation predicted to increase water-wire stability and cytoplasmic proton supply improves efficiency. Protonation (pH 5.0) of the outer glutamate gate (Glu ; E224) reduces Q, inhibits transport, and weakens coupling. Removal of the central tyrosine anion gate (Y572S) greatly increases uncoupled anion current. Tyrosine -OH removal (Y572F) alters anion selectivity and impairs coupling. E224 and Y572 act as anion barriers, and contribute to gating. The Y572 side chain and -OH regulate Q movement kinetics and voltage dependence. E224 and Y572 interact to create a "closed" inner gate conformation that maintains coupling during cycling.
ABSTRACT - We utilized plasma membrane-localized ClC-3 to investigate relationships between steady-state transport current (I ), gating charge (Q) movement, and cytoplasmic alkalization rate. ClC-3 exhibited lower transport efficiency than ClC-5, as reflected by a larger Q/I ratio, but an indistinguishable Cl /H coupling ratio. External SCN reduced H transport rate and uncoupled anion/H exchange by 80-90%. Removal of the external gating glutamate ("Glu ") (E224A mutation) reduced Q and abolished H transport. We hypothesized that Methionine 531 (M531) impedes "water wire" H transfer from the cytoplasm to E224. Accordingly, an M531A mutation decreased the Q/I ratio by 50% and enhanced H transport. External protons (pH 5.0) inhibited I and markedly reduced Q while shifting the Q-voltage (V) relationship positively. The Cl /H coupling ratio at pH 5.0 was significantly increased, consistent with externally protonated Glu adopting an outward/open position. Internal "anion gate" removal (Y572S) dramatically increased I and impaired coupling, without slowing H transport rate. Loss of both gates (Y572S/E224A) resulted in a large "open pore" conductance. Y572F (removing only the phenolic hydroxide) and Y572S shortened Q duration similarly, resulting in faster Q kinetics at all voltages. These data reveal a complex relationship between Q and ion transport. Q/I must be assessed together with coupling ratio to properly interpret efficiency. Coupling and transport rate are influenced by the anion, internal proton supply and external protons. Y572 regulates H coupling as well as anion selectivity, and interacts directly with E224. Disruption of this "closed gate" conformation by internal protons may represent a critical step in the ClC-3 transport cycle.
© 2018 The Authors. The Journal of Physiology © 2018 The Physiological Society.
The single-span membrane protein KCNE3 modulates a variety of voltage-gated ion channels in diverse biological contexts. In epithelial cells, KCNE3 regulates the function of the KCNQ1 potassium ion (K(+)) channel to enable K(+) recycling coupled to transepithelial chloride ion (Cl(-)) secretion, a physiologically critical cellular transport process in various organs and whose malfunction causes diseases, such as cystic fibrosis (CF), cholera, and pulmonary edema. Structural, computational, biochemical, and electrophysiological studies lead to an atomically explicit integrative structural model of the KCNE3-KCNQ1 complex that explains how KCNE3 induces the constitutive activation of KCNQ1 channel activity, a crucial component in K(+) recycling. Central to this mechanism are direct interactions of KCNE3 residues at both ends of its transmembrane domain with residues on the intra- and extracellular ends of the KCNQ1 voltage-sensing domain S4 helix. These interactions appear to stabilize the activated "up" state configuration of S4, a prerequisite for full opening of the KCNQ1 channel gate. In addition, the integrative structural model was used to guide electrophysiological studies that illuminate the molecular basis for how estrogen exacerbates CF lung disease in female patients, a phenomenon known as the "CF gender gap."
Interstitial cells of Cajal (ICC) act as pacemaker cells in the gastrointestinal tract by generating electrical slow waves to regulate rhythmic smooth muscle contractions. Intrinsic Ca(2+) oscillations in ICC appear to produce the slow waves by activating pacemaker currents, currently thought to be carried by the Ca(2+)-activated Cl(-) channel anoctamin 1 (Ano1). In this article we present a novel model of small intestinal ICC pacemaker activity that incorporates store-operated Ca(2+) entry and a new model of Ano1 current. A series of simulations were carried out with the ICC model to investigate current controversies about the reversal potential of the Ano1 Cl(-) current in ICC and to predict the characteristics of the other ion channels that are necessary to generate slow waves. The model results show that Ano1 is a plausible pacemaker channel when coupled to a store-operated Ca(2+) channel but suggest that small cyclical depolarizations may still occur in ICC in Ano1 knockout mice. The results predict that voltage-dependent Ca(2+) current is likely to be negligible during the slow wave plateau phase. The model shows that the Cl(-) equilibrium potential is an important modulator of slow wave morphology, highlighting the need for a better understanding of Cl(-) dynamics in ICC.
Myotonia congenita (MC) is caused by loss-of-function mutations of the muscle ClC-1 chloride channel. Clinical manifestations include the variable association of myotonia and transitory weakness. We recently described a cohort of recessive MC patients showing, at a low rate repetitive nerves stimulation protocol, different values of compound muscle action potential (CMAP) transitory depression, which is considered the neurophysiologic counterpart of transitory weakness. From among this cohort, we studied the chloride currents generated by G190S (associated with pronounced transitory depression), F167L (little or no transitory depression), and A531V (variable transitory depression) hClC-1 mutants in transfected HEK293 cells using patch-clamp. While F167L had no effect on chloride currents, G190S dramatically shifts the voltage dependence of channel activation and A531V reduces channel expression. Such variability in molecular mechanisms observed in the hClC-1 mutants may help to explain the different clinical and neurophysiologic manifestations of each ClCN1 mutation. In addition we examined five different mutations found in compound heterozygosis with F167L, including the novel P558S, and we identified additional molecular defects. Finally, the G190S mutation appeared to impair acetazolamide effects on chloride currents in vitro.
Depletion of interstitial cells of Cajal (ICC) networks is known to occur in several gastrointestinal motility disorders. Although confocal microscopy can effectively image and visualize the spatial distribution of ICC networks, current descriptors of ICC depletion are limited to cell numbers and volume computations. Spatial changes in ICC network structural properties have not been quantified. Given that ICC generate electrical signals, the organization of a network may also affect physiology. In this study, six numerical metrics were formulated to automatically determine complex ICC network structural properties from confocal images: density, thickness, hole size, contact ratio, connectivity and anisotropy. These metrics were validated and applied in proof-of-concept studies to quantitatively determine jejunal ICC network changes in mouse models with decreased (5-HT2B receptor knockout (KO)) and normal (Ano1 KO) ICC numbers, and during post-natal network maturation. Results revealed a novel remodelling phenomenon occurring during ICC depletion, namely a spatial rearrangement of ICC and the preferential longitudinal alignment. In the post-natal networks, an apparent pruning of the ICC network was demonstrated. The metrics developed here enabled the first detailed quantitative analyses of structural changes that may occur in ICC networks during depletion and development.
Migration of vascular smooth muscle cells (VSMCs) into neointima contributes to atherosclerosis and restenosis. This migration requires coordinated plasmalemmal fluxes of water and ions. Here, we show that aortic VSMC migration depends on the regulation of transmembrane Cl(-) flux by ClC-3, a Cl(-) channel/transporter. The contribution of ClC-3 to plasmalemmal Cl(-) current was studied in VSMCs by electrophysiological recordings. Cl(-) current was negligible in cells perfused with 0 [Ca(2+)]. Raising intracellular [Ca(2+)] to 0.5 μM activated a Cl(-) current (I(Cl.Ca)), approximately half of which was eliminated on inhibition by KN-93 of calmodulin-dependent protein kinase II. I(Cl.Ca) was also halved by inositol-3,4,5,6-tetrakisphosphate, a cellular signal with the biological function of specifically preventing calmodulin-dependent protein kinase II from activating I(Cl.Ca). Gene disruption of ClC-3 reduced I(Cl.Ca) by 50%. Moreover, I(Cl.Ca) in the ClC-3 null VSMCs was not affected by either KN-93 or inositol-3,4,5,6-tetrakisphosphate. We conclude that I(Cl.Ca) is composed of 2 components, one is ClC-3 independent whereas the other is ClC-3 dependent, activated by calmodulin-dependent protein kinase II and inhibited by inositol-3,4,5,6-tetrakisphosphate. We also assayed VSMC migration in transwell assays. Migration was halved in ClC-3 null cells versus wild-type cells. In addition, inhibition of ClC-3 by niflumic acid, KN-93, or inositol-3,4,5,6-tetrakisphosphate each reduced cell migration in wild-type cells but not in ClC-3 null cells. These cell-signaling roles of ClC-3 in VSMC migration suggest new therapeutic approaches to vascular remodeling diseases.
OBJECTIVE - We have shown that the chloride-proton antiporter chloride channel-3 (ClC-3) is required for endosome-dependent signaling by the Nox1 NADPH oxidase in SMCs. In this study, we tested the hypothesis that ClC-3 is necessary for proliferation of smooth muscle cells (SMCs) and contributes to neointimal hyperplasia following vascular injury.
METHODS AND RESULTS - Studies were performed in SMCs isolated from the aorta of ClC-3-null and littermate control (wild-type [WT]) mice. Thrombin and tumor necrosis factor-α (TNF-α) each caused activation of both mitogen activated protein kinase extracellular signal-regulated kinases 1 and 2 and the matrix-degrading enzyme matrix metalloproteinase-9 and cell proliferation of WT SMCs. Whereas responses to thrombin were preserved in ClC-3-null SMCs, the responses to TNF-α were markedly impaired. These defects normalized following gene transfer of ClC-3. Carotid injury increased vascular ClC-3 expression, and compared with WT mice, ClC-3-null mice exhibited a reduction in neointimal area of the carotid artery 28 days after injury.
CONCLUSIONS - ClC-3 is necessary for the activation of SMCs by TNF-α but not thrombin. Deficiency of ClC-3 markedly reduces neointimal hyperplasia following vascular injury. In view of our previous findings, this observation is consistent with a role for ClC-3 in endosomal Nox1-dependent signaling. These findings identify ClC-3 as a novel target for the prevention of inflammatory and proliferative vascular diseases.
All eukaryotic and some prokaryotic ClC anion transport proteins have extensive cytoplasmic C-termini containing two cystathionine-β-synthase (CBS) domains. CBS domain secondary structure is highly conserved and consists of two α-helices and three β-strands arranged as β1-α1-β2-β3-α2. ClC CBS domain mutations cause muscle and bone disease and alter ClC gating. However, the precise functional roles of CBS domains and the structural bases by which they regulate ClC function are poorly understood. CLH-3a and CLH-3b are C. elegans ClC anion channel splice variants with strikingly different biophysical properties. Splice variation occurs at cytoplasmic N- and C-termini and includes several amino acids that form α2 of the second CBS domain (CBS2). We demonstrate that interchanging α2 between CLH-3a and CLH-3b interchanges their gating properties. The "R-helix" of ClC proteins forms part of the ion-conducting pore and selectivity filter and is connected to the cytoplasmic C-terminus via a short stretch of cytoplasmic amino acids termed the "R-helix linker". C-terminus conformation changes could cause R-helix structural rearrangements via this linker. X-ray structures of three ClC protein cytoplasmic C-termini suggest that α2 of CBS2 and the R-helix linker could be closely apposed and may therefore interact. We found that mutating apposing amino acids in α2 and the R-helix linker of CLH-3b was sufficient to give rise to CLH-3a-like gating. We postulate that the R-helix linker interacts with CBS2 α2, and that this putative interaction provides a pathway by which cytoplasmic C-terminus conformational changes induce conformational changes in membrane domains that in turn modulate ClC function.
ClC-3 is a Cl(-)/H(+) antiporter required for cytokine-induced intraendosomal reactive oxygen species (ROS) generation by Nox1. ClC-3 current is distinct from the swelling-activated chloride current (ICl(swell)), but overexpression of ClC-3 can activate currents that resemble ICl(swell). Because H(2)O(2) activates ICl(swell) directly, we hypothesized that ClC-3-dependent, endosomal ROS production activates ICl(swell). Whole-cell perforated patch clamp methods were used to record Cl(-) currents in cultured aortic vascular smooth muscle cells from wild type (WT) and ClC-3 null mice. Under isotonic conditions, tumor necrosis factor-alpha (TNF-alpha) (10 ng/ml) activated outwardly rectifying Cl(-) currents with time-dependent inactivation in WT but not ClC-3 null cells. Inhibition by tamoxifen (10 microm) and by hypertonicity (340 mosm) identified them as ICl(swell). ICl(swell) was also activated by H(2)O(2) (500 microm), and the effect of TNF-alpha was completely inhibited by polyethylene glycol-catalase. ClC-3 expression induced ICl(swell) in ClC-3 null cells in the absence of swelling or TNF-alpha, and this effect was also blocked by catalase. ICl(swell) activation by hypotonicity (240 mosm) was only partially inhibited by catalase, and the size of these currents did not differ between WT and ClC-3 null cells. Disruption of endosome trafficking with either mutant Rab5 (S34N) or Rab11 (S25N) inhibited TNF-alpha-mediated activation of ICl(swell). Thrombin also activates ROS production by Nox1 but not in endosomes. Thrombin caused H(2)O(2)-dependent activation of ICl(swell), but this effect was not ClC-3- or Rab5-dependent. Thus, activation of ICl(swell) by TNF-alpha requires ClC-3-dependent endosomal H(2)O(2) production. This demonstrates a functional link between two distinct anion currents, ClC-3 and ICl(swell).
Adenovirus expressing ClC-3 (Ad-ClC-3) induces Cl(-)/H(+) antiport current (I(ClC-3)) in HEK293 cells. The outward rectification and time dependence of I(ClC-3) closely resemble an endogenous HEK293 cell acid-activated Cl(-) current (ICl(acid)) seen at extracellular pH