ClC Family Research Articles

The ClC‐5 N‐terminus confers plasma membrane Cl−/H+ transport capacity to ClC‐3

ClC family Cl−/H+ exchangers are primarily endosomal proteins that support vesicle acidification and reactive O2 production. When expressed in HEK293 cells, WT ClC‐5 localizes to the plasma membrane and produces outwardly rectifying whole‐cell ion currents, whereas WT ClC‐3 localizes intracellularly and ion currents are absent. We utilized N‐terminus mutations and ClC‐5/3 chimeras in combination with confocal imaging and whole‐cell patch clamp recordings, to show that this region regulates ClC‐5 and ‐3 trafficking and functional ion currents in HEK cells. Disruption of an N‐terminal clathrin‐ binding motif (LLDLLD to LLALLA) localized ClC‐3 to the plasma membrane, but yielded no ion current. By contrast, replacing the ClC‐3 N‐terminus (M1‐ E59) with that of ClC‐5 (M1‐A46) resulted in both plasma membrane localization of “N‐ClC‐5/3”, and rapidly activated, strongly rectifying (ClC‐5‐like) ion currents. Cellular pH changes were quantified with BCECF fluorescence and revealed coupled Cl−/H+ transport during both ClC‐5‐ and N‐ClC‐5/3 currents. These findings directly demonstrate for the first time that ClC‐3 is a functional Cl−/H+ exchanger, and support the hypothesis that the N‐terminus of these proteins directs membrane localization and modulates ion current gating.

Functional characterization of a ClC transporter by solid-supported membrane electrophysiology

EcClC, a prokaryotic member of the ClC family of chloride channels and transporters, works as coupled H+/Cl− exchanger. With a known structure and the possibility of investigating its behavior with different biochemical and biophysical techniques, the protein has become an important model system for the family. Although many aspects of its function have been previously characterized, it was difficult to measure transport on the same sample under different environmental conditions. To overcome this experimental limitation, we have studied EcClC by solid-supported membrane electrophysiology. The large transport-related transient currents and a simple way of relating transport rates to the measured signal have allowed a thorough investigation of ion selectivity, inhibition, and the dependence of transport on changes in ion concentration and pH. Our results confirm that the protein transports larger anions with about similar rates, whereas the smaller fluoride is not a substrate. We also show that 4,4′-diisothiocyano-2,2’-stilbenedisulfonic acid (DIDS), a known inhibitor of other anion transport protein, irreversibly inhibits EcClC from the intracellular side. The chloride dependence shows an apparent saturation at millimolar concentrations that resembles a similar behavior in eukaryotic ClC channels. Our experiments have also allowed us to quantify the pH dependence of transport. EcClC shows a strong activation at low pH with an apparent pKa of 4.6. The pronounced pH dependence is lost by the mutation of a conserved glutamate facing the extracellular solution that was previously shown to be an acceptor for transported protons, whereas it is largely retained by the mutation of an equivalent residue at the intracellular side. Our results have provided a quantitative basis for the transport behavior of EcClC, and they will serve as a reference for future investigations of novel electrogenic transporters with still-uncharacterized properties.

A Water-Organizing Residue for H+ Access in a CLC Cl−/H+ Antiporter

Chloride-transporting membrane proteins of the CLC family appear in two distinct mechanistic flavors: H+-gated Cl− channels and Cl−/H+ antiporters. Transmembrane H+ movement is an essential feature of both types of CLC. X-ray crystal structures of CLC antiporters show the Cl− ion pathway through these proteins, but the H+ pathway is known only inferentially by two conserved glutamate residues that act as way-stations for H+ in its path through the protein. The extracellular-facing H+ transfer glutamate becomes directly exposed to aqueous solution during the transport cycle, but the intracellular glutamate E203, Gluin, is buried within the protein. Two regions, denoted “polar” and “interfacial,” at the intracellular surface of the bacterial antiporter CLC-ec1 are examined here as possible pathways by which intracellular aqueous protons gain access to Gluin. Mutations at multiple residues of the polar region have little effect on antiport rates. In contrast, mutation of E202, a conserved glutamate at the protein-water boundary of the interfacial region, leads to severe slowing of the Cl−/H+ antiport rate. The x-ray crystal structure of E202Y, the most severely affected mutant, suggests that the aromatic ring of Y202 makes cross-subunit hydrophobic interaction with an I201 sidechain from neighboring subunit, which physically blocks the interfacial pathway. This mechanism is functionally supported by the minimal effect of E202Y mutation in a monomeric variant of the transporter, which does not have such an interaction. The several lines of experiments presented argue that E202 acts as a water-organizer that creates a proton conduit connecting intracellular solvent with Gluin.

Investigating a Benzofurane Derivative Binding Site on Human CLC-5

Human ClC-5 is a Cl−/H+ antiporter belonging to the CLC family, which includes both Cl− channels and Cl−/H+ antiporters. Small organic molecules have been useful tools for studying ion channels and many commercial drugs target specific ion channel proteins. Some blockers have been found to inhibit the plasma membrane localized CLC channels ClC-0, ClC-1 and ClC-Ka. However, high-affinity ligands for most CLCs are missing.Here, we tested the benzofuran derivative RT-931,2 on ClC-5 and ClC-4. Interestingly RT-93 inhibits ClC-5 (EC50 ∼ 200 μM), but has only a small effect on the highly homologous antiporter ClC-4. Towards our goal to identify the binding site of RT-93 on ClC-5, we constructed 12 chimeras between ClC-4 and ClC-5. Guided by the crystal structure of a bacterial CLC protein and using the alignment between ClC-4 and ClC-5, we identified the sequence stretches with the main differences between these two proteins and substituted in ClC-5 the portions with the corresponding sequence of ClC-4. We expect to remove the RT-93 sensitivity at least in one of these. So far we tested 8 chimeras, none of which, however, showed a significant difference in comparison with ClC-5. If none of the chimeras eliminates the binding site we will proceed with constructing larger chimeras.Discovering the binding site of this blocker might help in creating a more efficient ligand that, apart from being a useful biophysical tool, may provide a way to modulate protein function in vivo.(Supported by PRIN, Telethon (GGP08064; GGP12008) and IIT).1. Liantonio et al. 2008. PNAS2. Liantonio et al. 2012. J Hypertens.

Intracellular Proton Access in a Cl−/H+ Antiporter

Author SummaryChloride-proton antiport proteins of the “CLC” superfamily are transmembrane proteins that form homodimers and are used for myriad physiological purposes, all requiring the coordinated movements of Cl− anions and H+ cations in opposite directions across biological membranes. While the pathway for Cl− ions through CLC antiporters is known, we currently have only indirect glimpses of how protons navigate their way through these membrane-embedded proteins. By combining mechanistic and structural approaches, we identify a proton-access pathway in a bacterial Cl−/H+ antiporter that allows intracellular protons to enter the protein interior and engage in the coupled antiport mechanism. We conclude that E202, a highly conserved glutamate residue, serves to organize water molecules and guide protons to the adjacent glutamate E203 (known as “Gluin”), a critical residue for the antiport mechanism.

Single myotonia mutation strikes multiple mechanisms of a chloride channel

Single myotonia mutation strikes multiple mechanisms of a chloride channel

The N‐terminus of ClC‐3 determines membrane localization

CLC family Cl−/H+ anti‐porters are primarily intracellular (IC) proteins that support endosome acidification. Heterologous ClC‐5 current is readily expressed while ClC‐3 is not. We hypothesized that this difference is due to differential membrane localization directed by N‐terminal signal sequences. ClC‐3 and ‐5 C‐terminal GFP and YFP fusion proteins were localized by confocal microscopy. WT ClC‐3‐GFP (both long or short isoforms) localized to enlarged IC vesicles which were much smaller in H+ transport deficient mutants (E224A). Disruption (D15A, D18A) of a putative clathrin binding motif (LLDLLD) in short ClC‐3 yielded isolated bright dots in the plasma membrane. These dots were dissociated into a smooth membrane signal by methyl‐beta‐cyclodextrin, suggesting localization to lipid rafts. WT ClC‐5‐CFP also localized to bright spots in the membrane. A novel, long‐ClC‐5 isoform (69 aa longer) localized to both diffusely to the plasma membrane and to enlarged IC vesicles. When the N‐terminus of ClC‐5 was replaced by that of long‐ClC‐3 (first 70aa) membrane expression was lost and only enlarged IC vesicles were observed. Thus, ClC‐3 appears to traffic through plasma membrane lipid rafts and the N‐terminus is critical for localization. The first 70aa of long ClC‐3 are sufficient to move ClC‐5 into an exclusively IC localization that is indistinguishable from ClC‐3.

Endoplasmic reticulum potassium–hydrogen exchanger and small conductance calcium-activated potassium channel activities are essential for ER calcium uptake in neurons and cardiomyocytes

Calcium pumping into the endoplasmic reticulum (ER) lumen is thought to be coupled to a countertransport of protons through sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) and the members of the ClC family of chloride channels. However, pH in the ER lumen remains neutral, which suggests a mechanism responsible for proton re-entry. We studied whether cation-proton exchangers could act as routes for such a re-entry. ER Ca(2+) uptake was measured in permeabilized immortalized hypothalamic neurons, primary rat cortical neurons and mouse cardiac fibers. Replacement of K(+) in the uptake solution with Na(+) or tetraethylammonium led to a strong inhibition of Ca(2+) uptake in neurons and cardiomyocytes. Furthermore, inhibitors of the potassium-proton exchanger (quinine or propranolol) but not of the sodium-proton exchanger reduced ER Ca(2+) uptake by 56-82%. Externally added nigericin, a potassium-proton exchanger, attenuated the inhibitory effect of propranolol. Inhibitors of small conductance calcium-sensitive K(+) (SK(Ca)) channels (UCL 1684, dequalinium) blocked the uptake of Ca(2+) by the ER in all preparations by 48-94%, whereas inhibitors of other K(+) channels (IK(Ca), BK(Ca) and K(ATP)) had no effect. Fluorescence microscopy and western blot analysis revealed the presence of both SK(Ca) channels and the potassium-proton exchanger leucine zipper-EF-hand-containing transmembrane protein 1 (LETM1) in ER in situ and in the purified ER fraction. The data obtained demonstrate that SK(Ca) channels and LETM1 reside in the ER membrane and that their activity is essential for ER Ca(2+) uptake.

Lysine 210 Regulates Voltage-Dependent Gating of the Anion-Proton Exchanger CLC-5

The ClC-type Cl-/H+ exchanger ClC-5 displays complex voltage-dependent gating that regulates the number of active transporters and is modified by the concentration of internal and external transport substrates. So far, the molecular basis of ClC transporter gating is insufficiently understood. One residue, a conserved amino acid facing the anion permeation pathway at the external side of the protein, is known to be a crucial determinant of this process. Adjacent to this so-called gating glutamate (E211), there is a positive amino acid (K210) that is also highly conserved among the ClC family. Studies of the corresponding residue in ClC-1 (Fahlke et al. (1997) Nature 390, 529-532) demonstrated the importance of this residue for anion selectivity. Because of the well established connection between anion permeation and voltage-dependent gating in ClC proteins, this residue might also shape the voltage dependence of ClC-5. To test this hypothesis, we introduced the point mutation K210C in the non-conducting E268Q ClC-5. Because the gating process, observed at positive voltages is associated with prominent gating currents, we assessed the effects of K210C by measuring the voltage dependence of the nonlinear capacitances of ClC-5. The charge neutralization K210C shifted the voltage dependence of the bell shaped capacitance curve of ClC-5 by ∼25 mV to the left, which indicates that this residue is part of the voltage sensing machinery of the transporter. Surprisingly, the modification of K210C with the thiol-reactive reagent MTSET that restores the positive charge at this position induced additional 40 mV towards hyperpolarized potentials. Therefore, not only the positive charge of the lysine K210 but also the size and the pK of its side chain contribute to shaping the voltage dependence of ClC-5.

Transport Properties of a Prokaryotic CLC Transporter Assayed by Solid-Supported Membrane Electrophysiology

EcClC (or ClC-Ec1), a prokaryotic member of the ClC family of channels and transporters of known structure, acts as a coupled chloride/proton antiporter. To investigate its electrogenic activity, proteoliposomes containing reconstituted EcClC were adsorbed on a solid-supported membrane (SSM) electrode. The application of concentration jumps of different anions results in robust transient currents with a selectivity sequence that agrees with previously reported measurements on planar lipid bilayers. The transient currents increase at acidic pH with an apparent pKa of 4.6. The pH dependences of three mutations that are known to impair proton translocation while preserving chloride transport (mutations E148A, Y445A, and E203Q) have also been investigated. In agreement with previous results, E148A shows weak pH dependence. The transport impaired Y445A and the uncoupled E203Q present a shift in the apparent pKa towards less acidic values. Mutation E203Q, in addition, presents residual electrogenic activity at neutral pH values. Taken together, our results support the idea that chloride transport in the wild-type varies with pH mainly in response to titration of the external glutamate (E148).

Monitoring Substrate-Driven Conformational Changes of ClC-ec1 by [Methyl-13C] Methionine NMR

ClC-ec1, a bacterial Cl-/H+ antiporter of the CLC family, has been crystallized under many conditions. Yet, only one major conformation of the protein has been observed, leading to the view that the antiport cycle only involves local conformational changes near the chloride binding sites. This view has been challenged in a recent study (Elvington et al, 2009), in which a residue up to 25 A away from the chloride binding sites was shown to undergo substrate-induced, antiport-dependent conformational changes. To gain further understanding of this conformational change, we are using specific 13C-methyl labeling to facilitate the study of the ClC-ec1 structure and dynamics by NMR. Use of a monomeric ClC-ec1 (Robertson et al, 2010) greatly improves spectral quality of this 50-kD (per subunit) membrane protein. Heteronuclear single quantum correlation (HSQC) spectra reveal substrate-dependent spectral changes that may correspond to functionally relevant conformational change. Resonance assignment is in progress.

The CLC-5 2Cl−/H+ exchange transporter in endosomal function and Dent's disease

CLC-5 plays a critical role in the process of endocytosis in the proximal tubule of the kidney and mutations that alter protein function are the cause of Dent's I disease. In this X-linked disorder impaired reabsorption results in the wasting of calcium and low molecular weight protein to the urine, kidney stones, and progressive renal failure. Several different ion-transporting and protein clustering roles have been proposed as the physiological function of CLC-5 in endosomal membranes. At the time of its discovery, nearly 20 years ago, it was understandably assumed to be a chloride channel similar to known members of the CLC family, such as CLC-1, suggesting that chloride transport by CLC-5 was critical for endosomal function. Since then CLC-5 was found instead to be a 2Cl−/H+ exchange transporter with voltage-dependent activity. Recent studies have determined that it is this coupled exchange of protons for chloride, and not just chloride transport, which is critical for endosomal and kidney function. This review discusses the recent ideas that describe how CLC-5 might function in endosomal membranes, the aspects that we still do not understand, and where controversies remain.

Transient Water Chains Connecting the Cytoplasmic and Extracellular Glutamate Gates in CLC

CLC-ec1, a bacterial member of the CLC family, exchanges chloride ions and protons across the cellular membrane. A key step of the transport cycle of CLC- ec1 is the transfer of protons between the extracellular and cytoplasmic gates, E148 and E203, respectively. These residues are 15 A apart, and in dearth of any intermediate titratable groups needed for proton shuttling. Hence, it is an open question as to how protons shuttle between the two gates. Proton hopping through water chains provides a possible mechanism in this regard, having been investigated in numerous computational studies, given the lack of experimental structural data on water. Two possible chains, either involving Y445 or side-chain rotation of E203, have been proposed based on searching algorithms and short molecular dynamics (MD) simulations. We herein propose another water chain characterized through extended (0.42 μs) MD simulations of CLC-ec1 dimer. The water chain forms frequently (once every 50-100 ns) but transiently (lasting for <1–2 ns). Neither Y445 nor the side-chain rotation of E203 is needed for the water chain. The presence of the water chain, however, coincides with significant side-chain conformational changes of F199 and F357 around the chloride-binding site and F208 and F219 at the dimer interface remote from the ion permeation pathway. We further performed a 0.25 μs simulation of monomeric CLC-ec1, which has been shown to be structurally identical to the dimer, but with a halved activity. We show that water chains don’t form as readily in the monomeric simulation as what was observed in the dimer simulation; side-chain conformations of F199, F357, F208 and F219 are also different from the dimer. Our study supports the idea that both local and long-range factors could be important for the CLC-ec1 activity.

Enhanced Selectivity for Sulfatide by Engineered Human Glycolipid Transfer Protein

Human glycolipid transfer protein (GLTP) fold represents a novel structural motif for lipid binding/transfer and reversible membrane translocation. GLTPs transfer glycosphingolipids (GSLs) that are key regulators of cell growth, division, surface adhesion, and neurodevelopment. Herein, we report structure-guided engineering of the lipid binding features of GLTP. New crystal structures of wild-type GLTP and two mutants (D48V and A47D‖D48V), each containing bound N-nervonoyl-sulfatide, reveal the molecular basis for selective anchoring of sulfatide (3-O-sulfo-galactosylceramide) by D48V-GLTP. Directed point mutations of "portal entrance" residues, A47 and D48, reversibly regulate sphingosine access to the hydrophobic pocket via a mechanism that could involve homodimerization. "Door-opening" conformational changes by phenylalanines within the hydrophobic pocket are revealed during lipid encapsulation by new crystal structures of bona fide apo-GLTP and GLTP complexed with N-oleoyl-glucosylceramide. The development of "engineered GLTPs" with enhanced specificity for select GSLs provides a potential new therapeutic approach for targeting GSL-mediated pathologies.

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.

Design, Function and Structure of a Monomeric CLC Transporter

The CLC family of Cl- channels and Cl-/H+ antiporters are homodimeric in structure with each subunit containing a distinct transport pathway. Yet, the role of this conserved dimeric architecture in relation to protein function is not well understood. While certain CLC channels demonstrate dimer-dependent gating through cytosolic domains, the near-normal function of a bacterial CLC transporter strait-jacketed by covalent crosslinks across the dimer interface argues that the transport cycle resides within each subunit and does not require rigid-body rearrangements between subunits. A prediction that follows is that it should be possible to construct a monomeric form of a CLC protein while preserving structural and functional properties. To investigate this, we designed a stable monomeric variant of the E. coli CLC transporter, CLC-ec1, by introducing two tryptophan mutations, I201W and I422W, at the dimer interface. The existence of monomer was confirmed by gel filtration and glutaraldehyde cross-linking in both detergent micelles and PC/PG liposomes. The monomer is functional, exhibiting H+ coupled Cl- transport at rates comparable to the wild-type dimer and with 2:1 stoichiometry. The structure of the monomer was solved to 3.1 A resolution showing a crystal packing arrangement that exposes the previously buried dimer interface. The backbone atoms of this new structure aligns with the original CLC-ec1 crystal structure with 0.4 A RMSD, showing that the intrinsic subunit structure is not affected by these mutations or separation from the dimer state. These results demonstrate that the CLC subunit alone is the basic functional unit for transport and that cross-subunit interaction is not required for Cl-/H+ exchange in CLC transporters.

Identification and Functional Characterization of TMEM16A, a Ca2+-activated Cl− Channel Activated by Extracellular Nucleotides, in Biliary Epithelium

Cl(-) channels in the apical membrane of biliary epithelial cells (BECs) provide the driving force for ductular bile formation. Although a cystic fibrosis transmembrane conductance regulator has been identified in BECs and contributes to secretion via secretin binding basolateral receptors and increasing [cAMP](i), an alternate Cl(-) secretory pathway has been identified that is activated via nucleotides (ATP, UTP) binding apical P2 receptors and increasing [Ca(2+)](i). The molecular identity of this Ca(2+)-activated Cl(-) channel is unknown. The present studies in human, mouse, and rat BECs provide evidence that TMEM16A is the operative channel and contributes to Ca(2+)-activated Cl(-) secretion in response to extracellular nucleotides. Furthermore, Cl(-) currents measured from BECs isolated from distinct areas of intrahepatic bile ducts revealed important functional differences. Large BECs, but not small BECs, exhibit cAMP-stimulated Cl(-) currents. However, both large and small BECs express TMEM16A and exhibit Ca(2+)-activated Cl(-) efflux in response to extracellular nucleotides. Incubation of polarized BEC monolayers with IL-4 increased TMEM16A protein expression, membrane localization, and transepithelial secretion (I(sc)). These studies represent the first molecular identification of an alternate, noncystic fibrosis transmembrane conductance regulator, Cl(-) channel in BECs and suggest that TMEM16A may be a potential target to modulate bile formation in the treatment of cholestatic liver disorders.

Design, function and structure of a monomeric ClC transporter.

Channels and transporters of the CLC family bring about transmembrane movement of inorganic anions in service of a variety of biological tasks, from the arcane - generating the kilowatt pulses by which electric fish parboil their prey - to the quotidian - acidification of endosomes, vacuoles, and lysosomes1. The homodimeric architecture of CLC proteins (Fig 1), initially inferred from single-molecule studies of an elasmobranch Cl− channel2 and later confirmed by crystal structures of bacterial Cl−/H+ antiporters3,4, appears to be universal. Moreover, the basic machinery enabling ion movement through these proteins - the aqueous pores for anion diffusion in the channels and the ion-coupling chambers that coordinate Cl− and H+ antiport in the transporters - are contained wholly within each subunit of the homodimer. The near-normal function of a bacterial CLC transporter strait-jacketed by covalent crosslinks across the dimer interface and the behavior of a concatameric human homologue argue that the transport cycle resides within each subunit and does not require rigid-body rearrangements between subunits5,6. However, this evidence is only inferential, and since examples are known in which quaternary rearrangements of extramembrane CLC domains that contribute to dimerization modulate transport activity7, we cannot declare as definitive a “parallel pathways” picture in which the homodimer consists of two single-subunit transporters operating independently. A strong prediction of such a view is that it should in principle be possible to obtain a monomeric CLC. In this study, we exploit the known structure of a CLC Cl−/H+ exchanger, CLC-ec1 from E. coli, to design mutants that destabilize the dimer interface while preserving both structure and transport function of individual subunits. The results demonstrate that the CLC subunit alone is the basic functional unit for transport and that cross-subunit interaction is not required for Cl−/H+ exchange in CLC transporters.

The LysR-Type Virulence Activator AphB Regulates the Expression of Genes in Vibrio cholerae in Response to Low pH and Anaerobiosis

AphB is a LysR-type activator that initiates the expression of the virulence cascade in Vibrio cholerae by cooperating with the quorum-sensing-regulated activator AphA at the tcpPH promoter on the Vibrio pathogenicity island (VPI). To identify the ancestral chromosomal genes in V. cholerae regulated by AphB, we carried out a microarray analysis and show here that AphB influences the expression of a number of genes that are not associated with the VPI. One gene strongly activated by AphB is cadC, which encodes the ToxR-like transcriptional activator responsible for activating the expression of lysine decarboxylase, which plays an important role in survival at low pH. Other genes activated by AphB encode a Na(+)/H(+) antiporter, a carbonic anhydrase, a member of the ClC family of chloride channels, and a member of the Gpr1/Fun34/YaaH family. AphB influences each of these genes directly by recognizing a conserved binding site within their promoters, as determined by gel mobility shift assays. Transcriptional lacZ fusions indicate that AphB activates the expression of these genes under aerobic conditions in response to low pH and also under anaerobic conditions at neutral pH. Further experiments show that the regulation of cadC by AphB in response to low pH and anaerobiosis is mirrored in the heterologous organism Escherichia coli, is independent of the global regulators Fnr and ArcAB, and depends upon the region of the promoter that contains the AphB binding site. These results raise the possibility that the activity of AphB is influenced by the pH and oxygen tension of the environment.

Expression patterns of ClC-3 mRNA and protein in aortic smooth muscle, kidney and brain in diabetic rats

ClC-3, a member of the ClC family of voltage-gated chloride channels, regulates cell proliferation of cultured rat aortic vascular smooth muscle cells, pathogenesis of allergic rhinitis and tumor cell migration. However, its role in diabetic animals is still unknown. To address this issue, we investigated the expression patterns of ClC-3 in diabetic rats. Five-week-old Sprague-Dawley rats were divided into two groups, 50 non-diabetic control rats (non-DM) and 50 diabetic model rats (DM). ClC-3 mRNA and protein expression in aortic smooth muscle, kidney and brain tissues were examined by fluorimeter-based quantitive RT-PCR assay and Western blot analysis, respectively. ClC-3 mRNA and protein were endogenously expressed in aortic smooth muscle, kidney (cortex and medulla) and brain tissues of both control and streptozotocin-induced diabetic rats. ClC-3 mRNA and protein expression levels were significantly higher in aortic smooth muscle and brain tissues of diabetic rats, but significantly decreased in kidney medulla tissue, relative to non-DM controls. There were no significant differences in ClC-3 mRNA and protein expression in kidney cortex between non-diabetic control and diabetic rats. Furthermore, the altered ClC-3 expression patterns in diabetic rat aortic smooth muscle, brain, and kidney medulla tissues all correlated with the changes in blood glucose levels (p < 0.05). In conclusion, our data show for the first time that diabetes alters both the gene and protein expression of ClC-3 channels. These changes may contribute to the impaired vascular, brain and kidney functions observed in diabetes.