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ABSTRACT: The common gating of CLC-1 has been shown to be inhibited by intracellular adenosine triphosphate (ATP) in acidic pH conditions. Such modulation is thought to be mediated by direct binding of ATP to the cystathionine β-synthase (CBS) domains at the C-terminal cytoplasmic region of CLC-1. Guided by the crystal structure of the C-terminal domain of CLC-5, we constructed a homology model of CLC-1's C terminus and mutated critical amino acid residues lining the potential ATP-binding site. The CLC-1 mutations V634A and E865A completely abolished the ATP inhibition of CLC-1, consistent with the loss of ATP binding seen with the corresponding mutations in CLC-5. Mutating two other residues, V613 and V860, also disrupted the ATP modulation of CLC-1. However, placing aromatic amino acids at position 634 increases the apparent ATP affinity. Mutant cycle analyses showed that the modulation effects of ATP and cytidine triphosphate on wild-type CLC-1 and the V634F mutant were nonadditive, suggesting that the side chain of amino acid at position 634 interacts with the base moiety of the nucleotide. The mutation effects of V634F and V613A on the ATP modulation were also nonadditive, which is consistent with the assertion suggested from the homology model that these two residues may both interact with the bound nucleotide. These results provide evidence for a direct ATP binding for modulating the function of CLC-1 and suggest an overall conserved architecture of the ATP-binding sites in CLC-1 and CLC-5. This study also demonstrates that CLC-1 is a convenient experimental model for studying the interaction of nucleotides/nucleosides with the CBS domain.
The Journal of General Physiology 04/2011; 137(4):357-68. · 3.84 Impact Factor
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William Harley,
Candace Floyd,
Tamara Dunn, Xiao-Dong Zhang,
Tsung-Yu Chen,
Manu Hegde,
Hasan Palandoken,
Michael H Nantz,
Leonardo Leon,
K L Carraway,
Bruce Lyeth,
Fredric A Gorin
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ABSTRACT: Malignant glioma cells maintain an elevated intracellular pH (pH(i)) within hypoxic-ischemic tumor microenvironments through persistent activation of sodium-proton transport (McLean et al., 2000). Amiloride has been reported to selectively kill human malignant glioma cell lines but not primary astrocytes (Hegde et al., 2004). While amiloride reduces pH(i) of malignant gliomas by inhibiting isoform 1 of sodium-proton exchange (NHE1), direct acidification was shown to be cytostatic rather than cytotoxic. At cytotoxic concentrations, amiloride has multiple drug targets including inhibition of NHE1 and sodium-calcium exchange. Amiloride's glioma cytotoxicity can be explained, at least in part, by dual inhibition of NHE1 and of Na(+)-dependent calcium efflux by isoform 1.1 of the sodium-calcium exchanger (NCX1.1), which increases [Ca(2+)](i) and initiates glioma cell demise. As a result of persistent NHE1 activity, cytosolic free levels of sodium ([Na(+)](i)) in U87 and C6 glioma cells are elevated 3-fold, as compared with normal astrocytes. Basal cytosolic free calcium levels ([Ca(2+)](i)) also are increased 5-fold. 2', 4'-dichlorobenzamil (DCB) inhibits the sodium-dependent calcium transporter (NCX1.1) much more potently than NHE1. DCB was employed in a concentration-dependent fashion in glioma cells to selectively inhibit the forward mode of NCX1.1 at ≤1μM, while dually inhibiting both NHE1 and NCX1.1 at ≥20μM. DCB (1μM) was not cytotoxic to glioma cells, while DCB (20μM) further increased basal elevated levels of [Ca(2+)](i) in glioma cells that was followed by cell demise. Cariporide and SEA0400 are more selective inhibitors of NHE1 and NCX1.1 than amiloride or DCB, respectively. Individually, Cariporide and SEA0400 are not cytotoxic, but in combination induced glioma cell death. Like amiloride, the combination of Cariporide and SEA0400 produced glioma cell death in the absence of demonstrable caspase activation.
Brain research 12/2010; 1363:159-69. · 2.46 Impact Factor
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ABSTRACT: Transport of water and electrolytes is critical for corneal clarity. Recent studies indicate another important function of transport of ions and electrolytes - establishing wound electric fields that guide cell migration. We found chloride (Cl(-)) flux is a major component of the corneal wound electric current. In order to elucidate the mechanisms of Cl(-) transport, we studied Cl(-) channels and transporters in human corneal epithelial (HCE) cells. We tested a transformed human corneal epithelial cell line (tHCE), primary cultures of human corneal epithelial cells (pHCE), and human donor corneas. We first used RT-PCR to determine expression levels of mRNA of CLC (Cl(-) channels/transporters of CLC gene family) family members and CFTR (cystic fibrosis transmembrane conductance regulator) in HCE cells. We then confirmed protein expression and distribution of selected CLC family members and CFTR with Western blot and immunofluorescence confocal microscopy. Finally, Cl(-) currents were recorded with electrophysiological techniques. The mRNAs of CLC-2, CLC-3, CLC-4, CLC-5, CLC-6, and CFTR were detected in the HCE cell line. CLC-1 and CLC-7 were not detectable. Western blot and immunostaining confirmed protein expression and distribution of CLC-2, CLC-3, CLC-4, CLC-6 and CFTR in human corneal epithelium. CLC-2 preferentially labeled the apical and basal layers, while CLC-3 and CLC-4 labeled only the superficial layer. CLC-6 and CFTR labeling showed a unique gradient with strong staining in apical layers which gradually decreased towards the basal layers. Corneal endothelium was positive for CLC-2, CLC-3, CLC-4, CLC-6 and possibly CFTR. Human corneal epithelial cells demonstrated voltage dependent Cl(-) currents. HCE cells express functional Cl(-) channels and transporters. CLC-2, CLC-3, CLC-4, CLC-6, and CFTR had distinct expression patterns in human corneal epithelium. Those molecules and their distribution may play important roles in maintaining resting Cl(-) fluxes and in regulating Cl(-) flux at corneal wounds, which may be a major contributor to wound electrical signaling.
Experimental Eye Research 03/2010; 90(6):771-9. · 3.26 Impact Factor
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ABSTRACT: Using the substituted-cysteine-accessibility method, we previously showed that a cysteine residue introduced to the Y512 position of CLC-0 was more rapidly modified by a negatively charged methanethiosulfonate (MTS) reagent, 2-sulfonatoethyl MTS (MTSES), than by the positively charged 2-(trimethylammonium)ethyl MTS (MTSET). This result suggests that a positive intrinsic pore potential attracts the negatively charged MTS molecule. In this study, we further test this hypothesis of a positive pore potential in CLC-0 and find that the preference for the negatively charged MTS is diminished significantly in modifying the substituted cysteine at a deeper pore position, E166. To examine this conundrum, we study the rates of MTS inhibitions of the E166C current and those of the control mutant current from E166A. The results suggest that the inhibition of E166C by intracellularly applied MTS reagents is tainted by the modification of an endogenous cysteine, C229, located at the channel's dimer interface. After this endogenous cysteine is mutated, CLC-0 resumes its preference for selecting MTSES in modifying E166C, reconfirming the idea that the pore of CLC-0 is indeed built with a positive intrinsic potential. These experiments also reveal that MTS modification of C229 can inhibit the current of CLC-0 depending on the amino acid placed at position 166.
Biophysical Journal 02/2010; 98(3):377-85. · 3.65 Impact Factor
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ABSTRACT: The blockade of CLC-0 chloride channels by p-chlorophenoxy acetate (CPA) has been thought to be state dependent; the conformational change of the channel pore during the "fast gating" alters the CPA binding affinity. Here, we examine the mechanism of CPA blocking in pore-open mutants of CLC-0 in which the residue E166 was replaced by various amino acids. We find that the CPA-blocking affinities depend upon the volume and the hydrophobicity of the side chain of the introduced residue; CPA affinity can vary by three orders of magnitude in these mutants. On the other hand, mutations at the intracellular pore entrance, although affecting the association and dissociation rates of the CPA block, generate only a modest effect on the steady-state blocking affinity. In addition, various amphiphilic compounds, including fatty acids and alkyl sulfonates, can also block the pore-open mutants of CLC-0 through a similar mechanism. The blocking affinity of fatty acids and alkyl sulfonates increases with the length of these amphiphilic blockers, a phenomenon similar to the block of the Shaker K(+) channel by long-chain quaternary ammonium (QA) ions. These observations lead us to propose that the CPA block of the open pore of CLC-0 is similar to the blockade of voltage-gated K(+) channels by long-chain QAs or by the inactivation ball peptide: the blocker first uses the hydrophilic end to "dock" at the pore entrance, and the hydrophobic part of the blocker then enters the pore to interact with a more hydrophobic region of the pore. This blocking mechanism appears to be very general because the block does not require a precise structural fit between the blocker and the pore, and the blocking mechanism applies to the cation and anion channels with unrelated pore architectures.
The Journal of General Physiology 01/2009; 133(1):43-58. · 3.84 Impact Factor
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ABSTRACT: Intracellularly applied amphiphilic molecules, such as p-chlorophenoxy acetate (CPA) and octanoate, block various pore-open mutants of CLC-0. The voltage-dependent block of a particular pore-open mutant, E166G, was found to be multiphasic. In symmetrical 140 mM Cl(-), the apparent affinity of the blocker in this mutant increased with a negative membrane potential but, paradoxically, decreased when the negative membrane potential was greater than -80 mV, a phenomenon similar to the blocker "punch-through" shown in many blocker studies of cation channels. To provide further evidence of the punch-through of CPA and octanoate, we studied the dissociation rate of the blocker from the pore by measuring the time constant of relief from the block under various voltage and ionic conditions. Consistent with the voltage dependence of the effect on the steady-state current, the rate of CPA dissociation from the E166G pore reached a minimum at -80 mV in symmetrical 140 mM Cl(-), and the direction of current recovery suggested that the bound CPA in the pore can dissociate into both intracellular and extracellular solutions. Moreover, the CPA dissociation depends upon the Cl(-) reversal potential with a minimal dissociation rate at a voltage 80 mV more negative than the Cl(-) reversal potential. That the shift of the CPA-dissociation rate follows the Cl(-) gradient across the membrane argues that these blockers can indeed punch through the channel pore. Furthermore, a minimal CPA-dissociation rate at a voltage 80 mV more negative than the Cl(-) reversal potential suggests that the outward blocker movement through the CLC-0 pore is more difficult than the inward movement.
The Journal of General Physiology 01/2009; 133(1):59-68. · 3.84 Impact Factor
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ABSTRACT: The effect of intracellular adenosine triphosphate (ATP) on the "common gating" of the CLC-1 chloride channel has been studied by several laboratories with controversial results. Our previous study on the channel expressed in Xenopus oocytes using excised inside-out patch-clamp methods showed a robust effect of ATP in shifting the open probability curve of the common gate toward more depolarizing voltages (Tseng, P.Y., B. Bennetts, and T.Y. Chen. 2007. J. Gen. Physiol. 130:217-221). The results were consistent with those from studying the channel expressed in mammalian cells using whole cell recording methods (Bennetts, B., M.W. Parker, and B.A. Cromer. 2007. J. Biol. Chem. 282:32780-32791). However, a recent study using excised-patch recording methods for channels expressed in Xenopus oocytes reported that ATP had no direct effect on CLC-1 (Zifarelli, G., and M. Pusch. 2008. J. Gen. Physiol. 131:109-116). Here, we report that oxidation of CLC-1 may be the culprit underlying the controversy. When patches were excised from mammalian cells, the sensitivity to ATP was lost quickly--within 2-3 min. This loss of ATP sensitivity could be prevented or reversed by reducing agents. On the other hand, CLC-1 expressed in Xenopus oocytes lost the ATP sensitivity when patches were treated with oxidizing reagents. These results suggest a novel view in muscle physiology that the mechanisms controlling muscle fatigability may include the oxidation of CLC-1.
The Journal of General Physiology 11/2008; 132(4):421-8. · 3.84 Impact Factor
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ABSTRACT: Chloride channels and transporters of the CLC gene family are expressed in virtually all cell types and are crucial in the regulation of membrane potential, chloride homeostasis and intravesicular pH. There are two gating processes that open CLC channels-fast and slow. The fast gating process in CLC channels has recently been linked to a small movement of a glutamate side chain. However, the molecular mechanism underlying the slow gating process is still elusive. Using spectroscopic microscopy, we observed a large backbone movement in the C terminus of the CLC-0 chloride channel that was functionally linked to slow gating. We further showed that the C-terminal movement had a time course similar to slow gating. In addition, a mutation known to lock the slow gate open prevented movement of the C terminus. When combined with recent structural information on the CLC C terminus, our findings provide a structural model for understanding the conformational changes linked to slow gating in CLC transport proteins.
Nature Structural & Molecular Biology 01/2007; 13(12):1115-9. · 12.71 Impact Factor
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ABSTRACT: The ClC family of Cl(-) channels and transporters comprises membrane proteins ubiquitously present in species ranging from prokaryotes to mammals. The recently solved structures of the bacterial ClC proteins have provided a good model to guide the functional experiments for the eukaryotic Cl(-) channels. Theoretical calculations based on the bacterial ClC structures have identified several residues critical for the Cl(-) binding energy in the Cl(-) transport pathway. It was speculated that the corresponding residues in eukaryotic Cl(-) channels might play similar roles for the channel functions. In this study, we made a series of mutations in three such residues in eukaryotic ClC Cl(-) channels (K149, G352, and H401 in ClC-0) and studied the functional consequences on the channel properties. A cysteine modification approach was also employed to evaluate the electrostatic effects of the charge placed at these three positions. The experimental results revealed that among the three residues tested, K149 plays the most important role in controlling both the gating and the permeation functions of ClC-0. On the other hand, mutations of H401 alter the channel conductance but not the gating properties, while mutations of G352 result in very little functional consequence. The mutation of K149 into a neutral residue leucine (K149L) shifts the activation curve and leads to flickery channel openings. The anion permeability ratios derived from bi-ionic experiments are also significantly altered in that the selectivity of Cl(-) over other anions is decreased. Furthermore, removing the positive charge at this position reduces and increases, respectively, the accessibility of the negatively and positively charged methane thiosulfonate reagents to the pore. The control of the accessibility to charged MTS reagents and the regulation of the anion permeation support the idea that K149 exerts an electrostatic effect on the channel function, confirming the prediction from computational studies.
The Journal of General Physiology 05/2006; 127(4):435-47. · 3.84 Impact Factor
