Search

Intracellular levels of adenosine triphosphate (ATP) must remain within normal limits for the sweat ducts to produce large amounts of cystic fibrosis transmembrane conductance regulator (CFTR) the genetic defect of which causes the disease cystic fibrosis. CFTR normally functions as a phosphorylation-induced small-conductance chloride channel. ATP's association with chloride channel activity through CFTR may prevent too great an expenditure of energy from injuring tissue.

CYSTIC FIBROSIS (CF), THE MOST common lethal, recessively inherited disease among Caucasians, affects nearly 1 in 2500 newborns [1]. CF is caused by mutations in the gene encoding CFTR [2-4], [...], The cystic fibrosis transmembrane conductance regulator (CFTR) is associated with expression of a chloride conductance that is defective in cystic fibrosis (CF). Xenopus oocytes injected with RNA coding for CFTR that contained mutations in the first nucleotide binding fold (NBF1) expressed chloride currents in response to raising adenosine 3',5'-monophosphate (cAMP) with forskolin and 3-isobutyl-1-methylxanthine (IBMX). The mutant CFTRs were less sensitive than wild-type CFTR to this activating stimulus, and the reduction in sensitivity correlated with the severity of cystic fibrosis in patients carrying the corresponding mutations. This demonstration provides the basis for detailed analyses of NBF1 function and suggests potential pharmacologic treatments for cystic fibrosis.

Recent work that addresses the mechanistic operations of CLC and CFTR Cl[sup.-] channels and their transporter partners from the same family are reviewed. While investigating the evolutionary connection between ion channels and transporters, mechanistic insights are gained into how these proteins carry out the essential function of membrane transport.

The elucidation of the CLC gene family and their importance for the organism were greatly facilitated by mouse models and by human disease caused mutations in their respective genes. While human mutations in CLC channels are known to cause disease as diverse as mytonia, Bartter syndrome with or without deafness, Dent's disease, osteopetrosis and neurodegeneration, and possibly epilepsy, mouse models revealed blindness and infertility.

The vertebrate CLC structures are predicted to contain two identical but independent pores, whereas bacterial CLC structures could serve as a structural model to explain the gating-permeation coupling mechanism. The molecular basis to explain the differences between vertebrate and bacterial CLCs remains a challenge in structure and function studies for the CLC family.

The assembly of the cystic fibrosis transmembrane regulator (CFTR) chloride channel is of interest for understanding how ion channels and ABC transporters are formed and dealing with the mis-assembly of CFTR in cystic fibrosis. CFTR is functionally distinct from the other ABC transporters because it permits bidirectional permeation of anions rather than vectorial transport of solutes.

Numerous Cl(super -) channels are identified in the kidney using physiological approaches and thus are thought to be involved in a range of physiological processes. The physiological roles of CLC Cl(super -) channels within the kidney, particularly kidney-specific ClC-K Cl(super -) channels and maxi anion channel in macula densa are focused.

The physiological roles of calcium-activated chloride channels (CaCCs), their mechanisms of regulation and activation, and the mechanism of anion selectivity and conduction are discussed. The status of pharmacology and molecular identification of CaCCs is evaluated.

We investigated in detail the mechanism of inhibition by the S(-) enantiomer of 2-(p-chlorophenoxy)butyric acid (CPB) of the Torpedo [Cl.sup.-] channel, ClC-0. The substance has been previously shown to inhibit the homologous skeletal muscle channel, CLC-1. ClC-0 is a homodimer with probably two independently gated protopores that are conductive only if an additional common gate is open. As a simplification, we used a mutant of ClC-0 (C212S) that has the common gate 'locked open' (Lin, Y.W., C.W. Lin, and T.Y. Chen. 1999. J. Gen. Physiol. 114:1-12). CPB inhibits C212S currents only when applied to the cytoplasmic side, and single-channel recordings at voltages (V) between -120 and -80 mV demonstrate that it acts independently on individual protopores by introducing a long-lived nonconductive state with no effect on the conductance and little effect on the lifetime of the open state. Steady-state macroscopic currents at -140 mV are half-inhibited by ~0.5 mM CPB, but the inhibition decreases with V and vanishes for V [is greater than or equal to] 40 mV. Relaxations of CPB inhibition after voltage steps are seen in the current responses as an additional exponential component that is much slower than the gating of drug-free protopores. For V [is less than or equal to] -40 mV, where a significant inhibition is observable for the CPB concentrations used in this study ([is less than or equal to] 10 mM), the concentration dependence of its onset kinetics is consistent with CPB binding according to a bimolecular reaction. At all voltages, only the openings of drug-free protopores appear to contribute significantly to the current observed at any time. Lowering internal [Cl.sup.-] hastens significantly the apparent 'on' rate, suggesting that internal [Cl.sup.-] antagonizes CPB binding to closed pores. Vice versa, lowering external [Cl.sup.-] reduces the apparent rate of CPB dissociation from open pores. We studied also the point mutant K519E (in the context of the C212S mutant) that has altered conduction properties and slower single protopore gating kinetics. In experiments with CPB, the mutant exhibited drastically slowed recovery from CPB inhibition. In addition, in contrast to WT (i.e., C212S), the mutant K519E showed also a significant CPB inhibition at positive voltages ([is greater than or equal to] 60 mV) with an [IC.sub.50] of ~30-40 mM. Altogether, these findings support a model for the mechanism of CPB inhibition in which the drug competes with [Cl.sup.-] for binding to a site of the pore where it blocks permeation. CPB binds preferentially to closed channels, and thereby also strongly alters the gating of the single protopore. Since the affinity of CPB for open WT pores is extremely low, we cannot decide in this case if it acts also as an open pore blocker. However, the experiments with the mutant K519E strongly support this interpretation. CPB block may become a useful tool to study the pore of ClC channels. As a first application, our results provide additional evidence for a double-barreled structure of ClC-0 and ClC-1. KEY WORDS: ClC * slow gate * double barreled * anion channel * clofibric acid

The fast gate of the muscle-type ClC channels (ClC-0 and ClC-1) opens in response to the change of membrane potential (V). This gating process is intimately associated with the binding of external [Cl.sup.-] to the channel pore in a way that the occupancy of [Cl.sup.-] on the binding site increases the channel's open probability ([P.sub.o]). External [H.sup.+] also enhances the fast-gate opening in these channels, prompting a hypothesis that protonation of the binding site may increase the [Cl.sup.-] binding affinity, and this is possibly the underlying mechanism for the [H.sup.+] modulation. However, [Cl.sup.-] and [H.sup.+], modulate the fast-gate [P.sub.o]-V curve in different ways. Varying the external [Cl.sup.-] concentrations ([[[Cl.sup.-]].sub.o]) shifts the [P.sub.o]-V curve in parallel along the voltage axis, whereas reducing external pH mainly increases the minimal [P.sub.o] of the curve. Furthermore, [H.sup.+] modulations at saturating and nonsaturating [[[Cl.sup.-]].sub.o] are similar. Thus, the [H.sup.+] effect on the fast gating appears not to be a consequence of an increase in the [Cl.sup.-] binding affinity. We previously found that a hyperpolarization-favored opening process is important to determine the fast-gate [P.sub.o] of ClC-0 at very negative voltages. This [[[Cl.sup.-]].sub.o]-independent mechanism attracted little attention, but it appears to be the opening process that is modulated by external [H.sup.+]. KEY WORDS: channel gating * ClC-0 * ClC-1 * pH regulation * [Cl.sup.-] dependence

Illek, Beate, Mike E. Lizarzaburu, Vivien Lee, Michael H. Nantz, Mark J. Kurth, and Horst Fischer. Structural determinants for activation and block of CFTR-mediated chloride currents by apigenin. Am J Physiol Cell Physiol 279: C1838-C1846, 2000.--Apigenin (4',5,7-trihydroxyflavone) is an activator of cystic fibrosis transmembrane conductance regulator (CFTR)-mediated [Cl.sup.-] currents across epithelia at low concentrations and a blocker at high concentrations. We determined the roles of structural components of apigenin for both stimulation and block of [Cl.sup.-] currents across Calu-3 epithelia. The half-maximal binding affinity of apigenin for current stimulation ([K.sub.s]) was 9.1 [+ or -] 1.3 [micro]M, and the rank-order of molecular structures was 7-hydroxyl [is greater than] pyrone = 4'-hydroxyl [is greater than] 5-hydroxyl. Both the 7-hydroxyl and the 4'-hydroxyl served as H-bond acceptors, whereas the 5-hydroxyl was an H-bond donor. The half-maximal binding affinity of apigenin during current block was 74 [+ or -] 11 [micro]M. Blocked [Cl.sup.-] currents were structurally determined by 7-hydroxyl = 4'-hydroxyl [is greater than] pyrone [is greater than] 5-hydroxyl. Prestimulation of tissues with forskolin significantly affected activation kinetics and binding characteristics. After forskolin stimulation, [K.sub.s] was 4.1 [+ or -] 0.9 [micro]M, which was structurally determined by pyrone [is greater than] all hydroxyls [is greater than] single hydroxyls. In contrast, block of [Cl.sup.-] current by apigenin was not affected by forskolin stimulation. We conclude that apigenin binds to a stimulatory and an inhibitory binding site, which are distinguished by their affinities and the molecular interactions during binding. flavonoids; resveratrol; binding site; chloride transport; epithelia; cystic fibrosis transmembrane conductance regulator

[Ca.sup.2+]-activated Cl channels ([Cl.sub.Ca]Cs) are an important class of anion channels that are opened by increases in cytosolic [[Ca.sup.2+]]. Here, we examine the mechanisms of anion permeation through [Cl.sub.Ca]Cs from Xenopus oocytes in excised inside-out and outside-out patches. [Cl.sup.Ca]Cs exhibited moderate selectivity for Cl over Na: [P.sub.Na]/[P.sub.Cl] = 0.1. The apparent affinity of [Cl.sub.Ca]Cs for Cl was low: [K.sub.d] = 73 mM. The channel had an estimated pore diameter [is greater than] 0.6 nm. The relative permeabilities measured under bi-ionic conditions by changes in [E.sub.rev] were as follows: C[(CN).sub.3] [is greater than] SCN [is greater than] N[(CN).sub.2] [is greater than] Cl[O.sub.4] [is greater than] I [is greater than] [N.sub.3] [is greater than] Br [is greater than] Cl [is greater than] formate [is greater than] [HCO.sub.3] [is greater than] acetate = F [is greater than] gluconate. The conductance sequence was as follows: [N.sub.3] [is greater than] Br [is greater than] Cl [is greater than] N[(CN).sub.2] [is greater than] I [is greater than] SCN [is greater than] COOH [is greater than] Cl[0.sub.4] [is greater than] acetate [is greater than] [HCO.sub.3] = C[(CN).sub.3] [is greater than] gluconate. Permeant anions block in a voltage-dependent manner with the following affinities: C[(CN).sub.3] SCN = Cl[0.sub.4] [is greater than] N[(CN).sub.2] [is greater than] I [is greater than] [N.sub.3] [is greater than] Br [is greater than] [HCO.sub.3] [is greater than] Cl [is greater than] gluconate [is greater than] formate [is greater than] acetate. Although these data suggest that anionic selectivity is determined by ionic hydration energy, other factors contribute, because the energy barrier for permeation is exponentially related to anion hydration energy. [Cl.sub.Ca]Cs exhibit weak anomalous mole fraction behavior, implying that the channel may be a multi-ion pore, but that ions interact weakly in the pore. The affinity of the channel for [Ca.sup.2+] depended on the permeant anion at low [[Ca.sup.2+]] (100-500 nM). Apparently, occupancy of the pore by a permeant anion increased the affinity of the channel for [Ca.sup.2+]. The current was strongly dependent on pH. Increasing pH on the cytoplasmic side decreased the inward current, whereas increasing pH on the external side decreased the outward current. In both cases, the apparent pKa was voltage-dependent with apparent pKa at 0 mV = ~9.2. The channel may be blocked by [OH.sup.-] ions, or protons may titrate a site in the pore necessary for ion permeation. These data demonstrate that the permeation properties of [Cl.sub.Ca]Cs are different from those of CFTR or ClC-l, and provide insights into the nature of the [Cl.sub.Ca]C pore. KEY WORDS: voltage clamp * chloride current * selectivity * pH * anomalous mole fraction

The ClC channel family consists of chloride channels important for various physiological functions. Two members in this family, ClC-0 and ClC-1, share ~50-60% amino acid identity and show similar gating behaviors. Although they both contain two subunits, the number of pores present in the homodimeric channel is controversial. The double-barrel model proposed for ClC-0 was recently challenged by a one-pore model partly based on experiments with ClC-1 exploiting cysteine mutagenesis followed by modification with methanethiosulfonate (MTS) reagents. To investigate the pore stoichiometry of ClC-0 more rigorously, we applied a similar strategy of MTS modification in an inactivation-suppressed mutant (C212S) of ClC-0. Mutation of lysine 165 to cysteine (K165C) rendered the channel nonfunctional, but modification of the introduced cysteine by 2-aminoethyl MTS (MTSEA) recovered functional channels with altered properties of gating-permeation coupling. The fast gate of the MTSEA-modified K165C homodimer responded to external [Cl.sup.-] less effectively, so the [P.sub.o]-V curve was shifted to a more depolarized potential by ~45 mV. The K165C-K165 heterodimer showed double-barrel-like channel activity after MTSEA modification, with the fast-gating behaviors mimicking a combination of those of the mutant and the wild-type pore, as expected for the two-pore model. Without MTSEA modification, the heterodimer showed only one pore, and was easier to inactivate than the two-pore channel. These results showed that K165 is important for both the fast and slow gating of ClC-0. Therefore, the effects of MTS reagents on channel gating need to be carefully considered when interpreting the apparent modification rate. KEY WORDS: chloride channel * ClC-0 * 2-aminoethyl methanethiosulfonate * double barrel

Opening and closing of a CFTR [Cl.sup.-] channel is controlled by PKA-mediated phosphorylation of its cytoplasmic regulatory (R) domain and by ATP binding, and likely hydrolysis, at its two nucleotide binding domains. Functional interactions between the R domain and the two nucleotide binding domains were probed by characterizing the gating of severed CFTR channels expressed in Xenopus oocytes. Expression levels were assessed using measurements of oocyte conductance, and detailed functional characteristics of the channels were extracted from kinetic analyses of macroscopic current relaxations and of single-channel gating events in membrane patches excised from the oocytes. The kinetic behavior of wild-type (WT) CFTR channels was compared with that of split CFTR channels bearing a single cut (between residues 633 and 634) just before the R domain, of split channels with a single cut (between residues 835 and 837) just after the R domain, and of split channels from which the entire R domain (residues 634-836) between those two cut sites was omitted. The channels cut before the R domain had characteristics almost identical to those of WT channels, except for less than twofold shorter open burst durations in the presence of PKA. Channels cut just after the R domain were characterized by a low level of activity even without phosphorylation, strong stimulation by PKA, enhanced apparent affinity for ATP as assayed by open probability, and a somewhat destabilized binding site for the locking action of the nonhydrolyzable ATP analog AMPPNP. Split channels with no R domain (from coexpression of CFTR segments 1-633 and 837-1480) were highly active without phosphorylation, but otherwise displayed the characteristics of channels cut after the R domain, including higher apparent ATP affinity, and less tight binding of AMPPNP at the locking site, than for WT. Intriguingly, severed channels with no R domain were still noticeably stimulated by PKA, implying that activation of WT CFTR by PKA likely also includes some component unrelated to the R domain. As the maximal opening rates were the same for WT channels and split channels with no R domain, it seems that the phosphorylated R domain does not stimulate opening of CFTR channels; rather, the dephosphorylated R domain inhibits them. KEY WORDS: ATP-binding cassette transporter * chloride ion channel * phosphorylation * gating regulation * kinetic model

It is controversial whether the CFTR chloride channel contains a multi-ion pore. We used channel blockers to explore the anion binding site(s) in the CFTR channel pore. K1250A-CFTR, a mutant CFTR that can stay open for minutes once opened, permits kinetic analysis of the blocking events in isolation of gating transitions. Within the prolonged opening, there are numerous brief closings that are referred to as fast flickers, as seen in wild-type CFTR channels. These intrinsic flickers, likely caused by transient block of the channel from the cytoplasmic side of the membrane, have been shown to be voltage dependent. Surprisingly, our results show that the off rate, but not the on rate, is voltage dependent, and this voltage dependence of off rates can be abolished by removing external permeant anions, suggesting that the binding site of the intrinsic blocker is not deep in the pore. Instead of sensing the transmembrane voltage by the blocker itself, the blocker acquires the voltage-dependent off rate through electrostatic interaction with a neighboring [Cl.sup.-] in the pore. These observations are consistent with a mechanism that can place the unknown blocker and permeant chloride ions in the aqueous pore simultaneously. It is likely that the binding site of the intrinsic blocker is also a [Cl.sup.-] binding site, since the number of flickers decreased dramatically when millimolar [SCN.sup.-] was applied to the cytoplasmic side of the channel, suggesting that [SCN.sup.-], a [Cl.sup.-] surrogate, and the intrinsic blocker may compete for a common binding site. Glibenclamide, a known CFTR blocker, was used to further explore the channel pore. Both off and on rates of glibenclamide block are voltage dependent ([Delta] = ~0.32 and ~0.20, respectively). Removal of external [Cl.sup.-] decreased the off rate, suggesting that internally applied glibenclamide and a [Cl.sup.-] ion from the external entrance can simultaneously occupy the pore. However, the off rate of glibenclamide remained voltage dependent in the absence of external permeant anions ([Delta] = ~0.19), suggesting that the glibenclamide binding site, unlike that for the intrinsic blocker, resides deeper in the channel pore. Our results can be explained by a model that places at least three anion binding sites in the CFTR pore (from cytoplasmic end to extracellular end): a superficial site where the intrinsic blocker binds, a deep site for glibenclamide, and a more external site, [Cl.sup.-] occupancy of which affects glibenclamide binding.

Blaisdell, Carol J., Rebecca D. Edmonds, Xi-Tao Wang, Sandra Guggino, and Pamela L. Zeitlin. pH-regulated chloride secretion in fetal lung epithelia. Am J Physiol Lung Cell Mol Physiol 278: L1248-L1255, 2000.--The fetal lung actively transports chloride across the airway epithelium. ClC-2, a pH-activated chloride channel, is highly expressed in the fetal lung and is located on the apical surface of the developing respiratory epithelium. Our goal was to determine whether acidic pH could stimulate chloride secretion in fetal rat distal lung epithelial cells mounted in Ussing chambers. A series of acidic solutions stimulated equivalent short-circuit current ([I.sub.eq]) from a baseline of 28 [+ or -] 4.8 (pH 7.4) to 70 [+ or -] 5 (pH 6.2), 114 [+ or -] 12.8 (pH 5.0), and 164 [+ or -] 19.2 (pH 3.8) [micro]A/[cm.sup.2]. These changes in [I.sup.eq] were inhibited by 1 mM cadmium chloride and did not result in large changes in [[sup.3]H]mannitol paracellular flux. Immunofluorescent detection by confocal microscopy revealed that ClC-2 is expressed along the luminal surface of polarized fetal distal lung epithelial cells. These data suggest that the acidic environment of the fetal lung fluid could activate chloride channels contributing to fetal lung fluid production and that the changes in [I.sup.eq] seen in these Ussing studies may be due to stimulation of ClC-2. ClC-2; chloride channels; rat

Miyata, Yukio, Shigeaki Muto, Satoru Yanagiba, and Yasushi Asano. Extracellular [Cl.sup.-] modulates shrinkage-induced activation of [Na.sup.+]/[H.sup.+] exchanger in rat mesangial cells. Am J Physiol Cell Physiol 278: C1218-C1229, 2000.--To examine the effect of hyperosmolality on [Na.sup.+]/[H.sup.+] exchanger (NHE) activity in mesangial cells (MCs), we used a pH-sensitive dye, 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-AM, to measure intracellular pH ([pH.sub.i]) in a single MC from rat glomeruli. All the experiments were performed in [CO.sub.2]/[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]-free HEPES solutions. Exposure of MCs to hyperosmotic HEPES solutions (500 mosmol/kg[H.sub.2]O) treated with mannitol caused cell alkalinization. The hyperosmolality-induced cell alkalinization was inhibited by 100 [micro]M ethylisopropylamiloride, a specific NHE inhibitor, and was dependent on extracellular [Na.sup.+]. The hyperosmolality shifted the [Na.sup.+]-dependent acid extrusion rate vs. [pH.sub.i] by 0.15-0.3 pH units in the alkaline direction. Removal of extracellular [Cl.sup.-] by replacement with gluconate completely abolished the rate of cell alkalinization induced by hyperosmolality and inhibited the [Na.sup.+]-dependent acid extrusion rate, whereas, under isosmotic conditions, it caused no effect on [Na.sup.+]-dependent [pH.sub.i] recovery rate or [Na.sup.+]-dependent acid extrusion rate. The [Cl.sup.-]-dependent cell alkalinization rate under hyperosmotic conditions was partially inhibited by pretreatment with 5-nitro-2-(3-phenylpropylamino)benzoic acid, DIDS, and colchicine. We conclude: 1) in MCs, hyperosmolality activates NHE to cause cell alkalinization, 2) the acid extrusion rate via NHE is greater under hyperosmotic conditions than under isosmotic conditions at a wide range of [pH.sub.i], 3) the NHE activation under hyperosmotic conditions, but not under isosmotic conditions, requires extracellular [Cl.sup.-], and 4) the [Cl.sup.-]-dependent NHE activation under hyperosmotic conditions partly occurs via [Cl.sup.-] channel and microtubule-dependent processes. intracellular pH; chloride ion; chloride channel; microtubule

Silva, Ian V., Carol J. Blaisdell, Sandra E. Gugginc., and William B. Guggino. PTH regulates expression of ClC-5 chloride channel in the kidney. Am. J. Physiol. Renal Physiol. 278: F238-F245, 2000.-- Mutations in the chloride channel, ClC-5, have been described in several inherited diseases that result in the formation of kidney stones. To determine whether ClC-5 is also involved in calcium homeostasis, we investigated whether ClC-5 mRNA and protein expression are modulated in rats deficient in 1 [Alpha],25[(OH).sub.2] vitamin [D.sub.3] with and without thyroparathyroidectomy. Parathyroid hormone (PTH) was replaced in some animals. Vitamin D-deficient, thyroparathyrodectomized rats had lower serum and higher urinary calcium concentrations compared with control animals as well as lower serum PTH and calcitonin concentrations. ClC-5 mRNA and protein levels in the cortex decrease in vitamin D-deficient, thyroparathyroidectomized rats compared with both control and vitamin D-deficient animals. ClC-5 mRNA and protein expression increase near to control levels in vitamin D-deficient, thyroparathyroidectomized rats injected with PTH. No significant changes in ClC-5 mRNA and protein expression in the medulla were detected in any experimental group. Our results suggest that PTH modulates the expression of ClC-5 in the kidney cortex and that neither 1 [Alpha],25[(OH).sub.2] vitamin [D.sub.3] nor PTH regulates ClC-5 expression in the medulla. The pattern of expression of ClC-5 varies with urinary calcium. Animals with higher urinary calcium concentrations have lower levels of ClC-5 mRNA and protein expression, suggesting that the ClC-5 chloride channel plays a role in calcium reabsorption. chloride channels; kidney stones; nephrolithiasis

Sakamoto, Hisato, Yoshikazu Sado, Ichiro Naito, TaeHwan Kwon, Shinichi Inoue, Kenichi Endo, Masanobu Kawasaki, Shinichi Uchida, Soren Nielsen, Sei Sasaki, and Fumiaki Marumo. Cellular and subcellular immunolocalization of ClC-5 channel in mouse kidney: colocalization with [H.sup.+]-ATPase. Am. J. Physiol. 277 (Renal Physiol. 46): F957-F965, 1999.--To determine the immunolocalization of ClC-5 in the mouse kidney, we developed a ClC-5-specific rat monoclonal antibody. Immunoblotting demonstrated an 85-kDa band of ClC-5 in the kidney and ClC-5 transfected cells. Immunocytochemistry revealed significant labeling of ClC-5 in brush-border membrane and subapical intracellular vesicles of the proximal tubule. In addition, apical and cytoplasmic staining was observed in the type A intercalated cells in the cortical collecting duct. In contrast, the staining was minimal in the outer and inner medullary collecting ducts and the thick ascending limb. Western blotting of vesicles immunoisolated by the ClC-5 antibody showed the presence of [H.sup.+]-ATPase, strongly indicating that these two proteins were present in the same membranes. Double labeling with antibodies against ClC-5 and [H.sup.+]-ATPase and analysis by confocal images showed that ClC-5 and [H.sup.+]-ATPase colocalized in these ClC-5-positive cells. These findings suggest that ClC-5 might be involved in the endocytosis and/or the [H.sup.+] secretion in the proximal tubule cells and the cortical collecting duct type A intercalated cells in mouse kidney. proximal tubule; endocytosis; proton pump; Dent's disease; chloride channel

Roman, Richard M., Andrew P. Feranchak, Amy K. Davison, Erik M. Schwiebert, and J. Gregory Fitz. Evidence for [Gd.sup.3+] inhibition of membrane ATP permeability and purinergic signaling. Am. J. Physiol. 277 (Gastrointest. Liver Physiol. 40): G1222-G1230, 1999.--Extracellular ATP functions as an important autocrine and paracrine signal that modulates a broad range of cell and organ functions through activation of purinergic receptors in the plasma membrane. Because little is known of the cellular mechanisms involved in ATP release, the purpose of these studies was to evaluate the potential role of the lanthanide [Gd.sup.3+] as an inhibitor of ATP permeability and to assess the physiological implications of impaired purinergic signaling in liver cells. In rat hepatocytes and HTC hepatoma cells, increases in cell volume stimulate ATP release, and the localized increase in extracellular ATP increases membrane [Cl.sup.-] permeability and stimulates cell volume recovery through activation of [P.sub.2] receptors. In cells in culture, spontaneous ATP release, as measured by a luciferin-luciferase-based assay, was always detectable under control conditions, and extracellular ATP concentrations increased 2- to 14-fold after increases in cell volume. [Gd.sup.3+] (200 [micro]M) inhibited volume-sensitive ATP release by [is greater than] 90% (P [is less than] 0.001), inhibited cell volume recovery from swelling (P [is less than] 0.01), and uncoupled cell volume from increases in membrane [Cl.sup.-] permeability (P [is less than] 0.01). Moreover, [Gd.sup.3+] had similar inhibitory effects on ATP release from other liver and epithelial cell models. Together, these findings support an important physiological role for constitutive release of ATP as a signal coordinating cell volume and membrane ion permeability and suggest that [Gd.sup.3+] might prove to be an effective inhibitor of ATP-permeable channels once they are identified. cell volume; liver; chloride channel

Kawasaki, Masanobu, Toshiko Fukuma, Kazushi Yamauchi, Hisato Sakamoto, Fumiaki Marumo, and Sei Sasaki. Identification of an acid-activated [Cl.sup.-] channel from human skeletal muscles. Am. J. Physiol. 277 (Cell Physiol. 46): C948-C954, 1999.--ClC-4 gene was isolated as a putative [Cl.sup.-] channel. Due to a lack of functional expression of ClC-4, its physiological role remains unknown. We isolated a human C1C-4 clone (hC1C-4sk) from human skeletal muscles and stably transfected it to Chinese hamster ovary cells. Whole cell patch-clamp studies showed that the hC1C-4sk channel was activated by external acidic pH and inhibited by DIDS. It passed a strong outward [Cl.sup.-] current with a permeability sequence of [I.sup.-] [is greater than] [Cl.sup.-] [is greater than] [F.sup.-]. The hClC-4sk has consensus sites for phosphorylation by protein kinase A (PKA); however, stimulation of PKA had no effect on the currents, hC1C-4sk mRNA was expressed in excitable tissues, such as heart, brain, and skeletal muscle. These functional characteristics of hC1C-4sk provide a clue to its physiological role in excitable cells. human ClC-4sk; ClCN4; outwardly rectifying chloride channel; acidification

Putney, Luanna K., Cecile Rose T. Vibat, and Martha E. O'Donnell. Intracellular C1 regulates Na-K-C1 cotransport activity in human trabecular meshwork cells. Am. J. Physiol. 277 (Cell Physiol. 46): C373-C383, 1999.--The trabecular meshwork (TM) of the eye plays a central role in modulating intraocular pressure by regulating aqueous humor outflow, although the mechanisms are largely unknown. We and others have shown previously that aqueous humor outflow facility is modulated by conditions that alter TM cell volume. We have also shown that the Na-K-Cl cotransport system is a primary regulator of TM cell volume and that its activity appears to be coordinated with net efflux pathways to maintain steady-state volume. However, the cellular mechanisms that regulate cotransport activity and cell volume in TM cells have yet to be elucidated. The present study was conducted to investigate the hypothesis that intracellular Cl concentration ([[Cl]i.sub.]) acts to regulate TM cell Na-K-Cl cotransport activity, as has been shown previously for some other cell types. We demonstrate here that the human TM cell Na-K-Cl cotransporter is highly sensitive to changes in [[Cl].sub.i]. Our findings reveal a marked stimulation of Na-K-Cl cotransport activity, assessed as ouabain-insensitive, bumetanide-sensitive K influx, in TM cells following preincubation of cells with Cl-free medium as a means of reducing [[Cl].sub.i]. In contrast, preincubation of cells with media containing elevated K concentrations as a means of increasing [[Cl].sub.i] results in inhibition of Na-K-Cl cotransport activity. The effects of reducing [[Cl].sub.i], as well as elevating [[Cl].sub.i], on Na-K-Cl cotransport activity are concentration dependent. Furthermore, the stimulatory effect of reduced [[Cl].sub.i] is additive with cell-shrinkage-induced stimulation of the cotransporter. Our studies also show that TM cell Na-K-Cl cotransport activity is altered by a variety of Cl channel modulators, presumably through changes in [[Cl].sub.i]. These findings support the hypothesis that regulation of Na-K-Cl cotransport activity, and thus cell volume, by [[Cl].sub.i] may participate in modulating outflow facility across the TM. glaucoma; aqueous outflow; intracellular volume; chloride channel; nifiumic acid

Purification and Reconstitution of Chloride Channels from Kidney and Trachea CHLORIDE CHANNELS ARE PRESENT in the plasma membrane of most cells. In epithelia these channels act together with other ion [...]

Several factors influence the difficulties experienced in understanding chloride channels and the outwardly rectifying anion current termed ICl.swell. Aside from the absence of chloride channel sequence resembling a known voltage-gated channel, mutagenesis studies indicate that the chloride pore involves the use of the whole protein. Moreover, the variability of the characteristics displayed by the ICl.swell suggest that it may be composed of several channels.

Researchers have linked Dent's disease, a type of hypercalciuric nephrolithiasis, to a defect in a gene called CLCN5. CLCN5 encodes CLC-5, a putative chloride channel. Dent's disease was originally viewed as a dysfunction in renal calcium transport and not of chloride or fluid volume. Two other types of hypercalciuric nephrolithiasis were found to be associated with mutations in the CLCN5 gene.

Activation of Apical Chloride Channels in the Gastric Oxyntic Cell IN RESPONSE TO THE HORMONES HISTAMINE, acetylcholine, and gastrin, oxyntic cells of the gastric mucosa secrete a solution of hydrochloric [...]

The residue Arg352 surrounding the predicted cytoplasmic end of the M6 segment plays an essential role in charge selectivity in the cystic fibrosis transmembrane conductance regulator chloride channel. An analysis of the Cl(super -) to Na(super +) permeability ratio from the reversal potential measured in a 10-fold NaCl gradient supports this assumption. The positive charge of Arg352 mediates an electrostatic potential in the chloride channel that forms a barrier to cation permeation. Mutation of the residue does not affect the halide selectivity sequence.

The endothelial responses of intracellular Ca2+ concentration ([Ca2+]i) and ionic currents to mechanical stimulation were examined by applying a fluid stream to cultured human aortic endothelial cells via a microtube. The fluid stream was found to increase [Ca2+]i. Results also suggested that the fluid stream-induced current is carried by Cl- and that the Cl- current is responsible in modulating the Ca2+ influx by changing the membrane potential of cells.

A study was conducted on the two distinct swelling-sensitive transport pathways in trout erythrocytes to investigate whether they are also involved in the regulation of other transport pathways. These two transport pathways are a channel of broad specificity and a K+-Cl- cotransporter. Results showed that the two swelling-sensitive transport pathways are regulated differently and can regulate ionic composition of a cell at different volume changes.

The relationship between the pI(sub Cln) protein, volume-activated Cl- current and volume regulation in native bovine nonpigmented ciliary epithelial cells was investigated using the antisense oligonucleotide technique. Results showed that antisense oligonucleotides delayed the activation of volume-activated Cl- current reduced the volume regulation capabilities of the cells and knocked down the pI(sub Cln) protein. These results imply that pI(sub Cln) is involved in the activation of the volume-activated Cl- current and regulatory volume decrease.

RT-PCR, immunohistochemical and immunoprecipitation analyses show that the cystic fibrosis transmembrane conductance regulator (CFTR) is expressed in human primary endothelial cells isolated from umbilical vein (HUVEC) and lung microvasculature (HLMVEC). Cl- efflux and whole cell patch-clamp analyses show that HUVEC and HLMVEC exhibit a cyclic nucleotide-simulated Cl- transport that is blocked by the CFTR.

Research was conducted to examine the hypothesis that pI(sub Cln) is an anion channel-forming protein that is responsible for I(sub Cl, swell) or a channel regulator. Recombinant pI(sub Cln) was reconstituted into artificial and biological membranes. Results indicate that pI(sub Cln) has the capacity to generate channel activity in vitro but the channels formed by the protein are highly cation selective. Findings do not provide evidence to prove that pI(sub Cln) is the I(sub Cl, swell) channel.

Research was conducted to examine the hypothesis that the ClC-O channel can also also be inhibited by Zn2+. Another objective was to show that the inhibition of ClC-O channel by Zn2+ is reversible unlike in the case of ClC-1. Results suggest that the effect of Zn2+ on the kinetics of the fast gate may be attributable to a facilitation of the slow gating. Thus Zn2+ can reversibly inhibit the ClC-O Cl- channel from Torpedo electroplax.

Results of a study on the control of ATP release as modulated by cystic fibrosis transmembrane conductance regulator (CFTR) showed that CFTR expression can result in Cl-sensitive ATP permeability to injected oocytes. Based on mutational analysis, the interaction of extracellular Cl at arginine residues R334 and R347 within the channel pore was found to influence CFTR capacity to control ATP release. The findings point to a novel mechanism by which shifts in concentration of extracellular Cl contribute to the activation of CFTR-modulated ATP release.

The whole cell patch-clamp technique was utilized in the investigation of the chloride conductances of cell cultures derived from proximal and distal convoluted tubule epithelium of the rabbit nephron. Three type of chloride channels were identified in the distal convoluted tubule epithelial cell culture. Only two of these were identified in the cultures of proximal tubule cells. The physiologic roles of the different channels are discussed.

Cystic fibrosis is caused by a defective epithelial Cl- channel, the cystic fibrosis transmembrane regulator (CFTR). The most common mutation on the CFTR gene involves deletion of F508 in one of the two nucleotide-binding domains. Three less common mutations that lead to mild forms of the disease have also been studied. These involve missense mutations each altering a different arginine residue. However, it was found that the clinically severest mutant CFTR gene is much more prevalent than the less harmful variants. It is speculated that a deficiency in epithelial fluid secretion would ameliorate the severity of dehydration due to enterotoxin-slicited diarrhea.