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ClC-3B, a novel ClC-3 splicing variant that interacts with EBP50 and facilitates expression of CFTR-regulated ORCC¹

TAKEHIKO OGURA, TETSUSHI FURUKAWA*², TETSUYA TOYOZAKI, KATSUYA YAMADA*, YA-JUAN ZHENG*, YOSHIFUMI KATAYAMA, HARUAKI NAKAYA and NOBUYA INAGAKI*

Department of Pharmacology, Chiba University Graduate School of Medicine, Chiba, Japan;
Department of Physiology, Akita University School of Medicine, Akita, Japan;
Department of Basic Pathology, Chiba University Graduate School of Medicine, Chiba, Japan;
Department of Autonomic Physiology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan; and
CREST, Japan Science and Technology Corporation, Akita, Japan

²Correspondence: Department of Physiology, Akita University School of Medicine, 1–1-1 Hondo, Akita, 010-8543, Japan. E-mail: tfuru{at}med.akita-u.ac.jp

SPECIFIC AIMS

Cl^- channel activities at the apical membrane determine the rate and direction of fluid and electrolyte transport across polarized epithelia. Defects in the epithelial Cl^- channels lead to serious pathological conditions such as cystic fibrosis (CF), the most common genetic disorder in Caucasians. It is important to determine the molecular entities and precise regulatory mechanisms of epithelial Cl^- channels to better understand epithelial pathophysiology. In the present study, we have characterized the molecular and electrophysiological properties of ClC-3B, a novel Cl^- channel splicing variant expressed predominantly in epithelial cells.

PRINCIPAL FINDINGS

1. Molecular cloning and characterization of ClC-3B
We cloned the novel Cl^- channel splicing variant ClC-3B from a human pancreas cDNA library by homology screening using full-length rat ClC-3 (ClC-3A) as a probe. In ClC-3B, a stretch of 76 bp corresponding to a single exon is inserted in the carboxyl terminus of ClC-3A. As a result, the 29 amino acids of the carboxyl-terminal end of ClC-3A (ClC-3ACT) are replaced with 77 amino acids in the carboxyl terminus of ClC-3B (ClC-3BCT).

Immunostaining using an anti-ClC-3B antibody that recognizes ClC-3B but not ClC-3A revealed that ClC-3B protein is predominantly expressed in epithelial cells of various tissues.

2. Interaction of ClC-3B and EBP50
Alternative splicing newly generates the consensus motif (Ser-Ser/Thr-x-Leu) for interaction with the second PDZ domain (PDZ2) of EBP50 in ClC-3BCT (Ser-Thr-Thr-Leu), but not in ClC-3ACT (Ile-Met-Phe-Asn). Pull-down experiments showed that the carboxyl-terminal 77 amino acids specific to ClC-3B bound to PDZ2 of EBP50. Neither ClC-3BCT(GGGG), in which the last four amino acids had been replaced with four Gly, nor ClC-3ACT bound to PDZ1 or PDZ2, suggesting that the last four amino acids of ClC-3BCT are critical for interaction with EBP50.

To investigate in vivo interaction of ClC-3B with EBP50, coimmunoprecipitation experiments were performed in C127 mouse mammary epithelial cells cotransfected with EBP50 and one of ClC-3B, ClC-3B(GGGG), or ClC-3A. EBP50 was coimmunoprecipitated with ClC-3B but not ClC-3B(GGGG) or ClC-3A. Interaction between ClC-3B and EBP50 was also found in Caco2 cells that express intrinsic ClC-3B and EBP50.

3. Subcellular localization of ClC-3B
C127 cells transfected with ClC-3B alone showed diffuse immunoreactivity for ClC-3B in the cytoplasmic region (Fig. 1 A-a). By cotransfection of EBP50 with ClC-3B, however, the leading edges of membrane ruffles turned strongly immunopositive for ClC-3B (Fig. 1A -b, arrows). No such immunoreactivity for ClC-3A was observed at the leading edges when ClC-3A was cotransfected with EBP50 (Fig. 1A -c). These results suggest that interaction between ClC-3B and EBP50 is required for expression of ClC-3B at the membrane surface. Double immunolabeling for ClC-3B and EBP50 revealed that the distributions of ClC-3B and EBP50 overlap at the leading edges of membrane ruffles (Fig. 1B ).

View larger version (40K): [in this window] [in a new window]   Figure 1. Subcellular localization of ClC-3B in C127 cells. A) C127 cells stably expressing Myc/ClC-3B (a, b) or Myc/ClC-3A (c) were transfected with empty vector (mock; a) or EBP50 (b, c) and immunostained with an anti-Myc antibody. Arrows indicate the expression of ClC-3B at the leading edges of membrane ruffles. Scale bars: 20 µm, upper panel; 10 µm, lower panel. B) C127 cells stably expressing Myc/ClC-3B were transfected with FLAG/EBP50. Distributions of ClC-3B (red; a) and EBP50 (green; b) are labeled with anti-Myc and anti-FLAG antibodies, respectively. c) An overlay of the two immunofluorescences. Scale bars: 10 µm.

4. Electrophysiological studies
We measured membrane currents from 3T3, CHO-K1, HEK293T, and C127 cells transfected with ClC-3B using patch-clamp techniques. There was no significant difference in basal whole cell current amplitude between ClC-3B-transfected and mock-transfected cells. Even when ClC-3B-transfected cells were exposed to a hypotonic solution or to a solution containing Ca²⁺-ionophore A23187 (10 µM), the amplitude of the Cl^- current in response to these stimuli was not different from that observed in mock-transfected cells. These results indicate that ClC-3B does not encode a swelling-activated or a Ca²⁺-regulated Cl^- channel.

Since immunofluorescence data indicate that ClC-3B is expressed at the membrane surface only in membrane ruffles, we excised patches from the leading edges of membrane ruffles. In C127 cells transfected with ClC-3B and EBP50, prolonged depolarization to +60 mV induced channel activities (Fig. 2 A). The electrophysiological properties of depolarization-induced channel activities are similar to those of the outwardly rectifying Cl^- channels (ORCCs) described in native epithelial cells, including outward rectification, anion permeability sequence of I^- > Cl^-, and sensitivity to SITS (Fig. 2B ). The incidence of intrinsic ORCCs in patches from the leading edges of membrane ruffles was <10%. Transfection of either ClC-3B or EBP50 alone or cotransfection of ClC-3A and EBP50 did not increase the incidence of ORCCs. However, cotransfection of ClC-3B and EBP50 markedly increased the incidence of ORCCs to >75%, suggesting that ClC-3B is required in the augmentation of ORCC activity in the presence of EBP50.

View larger version (29K): [in this window] [in a new window]   Figure 2. Single channel currents in C127 cells transfected with ClC-3B and EBP50. A) A representative recording from an inside-out patch excised from the edge of membrane ruffles. Membrane potential was held at -40 mV for 5 min, changed to +60 mV, and returned to -40 mV. Channel activity was not observed at -40 mV initially but was induced by depolarization to +60 mV. Once channel activity had been induced at +60 mV, it persisted even at -40 mV. The timing of voltage clamp at -40 mV and +60 mV is indicated above the current trace. Arrow indicates the timing of patch excision. C = current level with channel closed; O = current level with channel opening. B) Representative current traces of ORCC at various potentials (left panel) and I-V relationships (n=4) (right panel). C) Percentage of appearance of ORCC activities in membrane patches at +60 mV. Combinations of molecules transfected and the region where membrane patches were excised are shown below the graph. Numbers in parentheses are the number of patch trials.

When cystic fibrosis transmembrane conductance regulator (CFTR) was cotransfected with ClC-3B and EBP50, preincubation with forskolin/IBMX induced ORCC activities without depolarization. A deletion mutation of Phe at position 508 (CFTRF508), the most prevalent mutation in CF patients, did not activate ORCCs with forskolin/IBMX incubation. These data indicate the involvement of CFTR in protein kinase A (PKA)-dependent activation of ClC-3B-induced ORCCs.

CONCLUSIONS AND SIGNIFICANCE

ClC-3B, a novel splicing variant of ClC-3A, has a different, slightly longer carboxyl-terminal end than ClC-3A and is expressed predominantly in epithelial cells. Alternative splicing generates a PDZ-interacting motif in ClC-3B. In vitro and in vivo interaction assays both indicate that ClC-3BCT interacts with PDZ2 of EBP50.

Various data have been reported on the localization and function of ClC-3A. Some investigators have reported ClC-3A to mediate Ca²⁺-regulated Cl^- currents or swelling-activated Cl^- currents, whereas others were unable to record ClC-3A currents in plasma membrane and have reported that ClC-3A protein is found primarily in intracellular vesicles. Our data indicate that in the absence of EBP50, ClC-3A and ClC-3B are expressed only intracellularly. In the presence of EBP50, ClC-3B but not ClC-3A is expressed at the surface of specific membrane regions, for which interaction of ClC-3B with EBP50 is required (Fig. 3 ). In the present study, ClC-3B did not mediate Ca²⁺-regulated or swelling-activated Cl^- currents. Activity of the ORCC is important, as cotransfection of ClC-3B and EBP50 markedly increased ORCC activity. These findings suggest that ClC-3B is a candidate for the ORCC molecule. Alternatively, ClC-3B, interacting with EBP50 may be required to activate ORCCs endogenously present in C127 cells.

View larger version (38K): [in this window] [in a new window]   Figure 3. Schematic diagram. The role of interaction with EBP50 in localization of ClC-3B and activation of ORCC. EBP50 underlies the cytosolic face of specific surface membranes such as the ruffling membranes and epithelial apical membranes, which results from protein–protein interactions between EBP50 and ezrin and between ezrin and actin. ClC-3BCT interacts with PDZ2 of EBP50, causing ClC-3B to be expressed at the surface membrane. Since CFTR is known to interact with PDZ1 of EBP50, ClC-3B may locate closely to CFTR and thereby play an important role in activation of ORCCs. It is not known whether ClC-3B is the ORCC molecule itself or its activator or whether ClC-3B and CFTR can bind to the same EBP50 molecule.

In CF epithelia, PKA-dependent activation of ORCCs by CFTR is defective, but normal ORCC activities can be induced by alternative measures independent of CFTR and PKA, such as depolarization, extracellular ATP, and an src-like kinase, each of which represents a potential therapeutic strategy for the treatment of CF. Our findings provide important clues to better understand epithelial Cl^- channels and the pathophysiology of CF.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0845fje; to cite this article, use FASEB J. (April 10, 2002) 10.1096/fj.01-0845fje.

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