CLIC-1 functions as a chloride channel when expressed and purified from bacteria.

CLIC-1 is a member of a family of proteins related to the bovine intracellular chloride channel p64 which has been proposed to function as a chloride channel. We expressed CLIC-1 as a glutathione S-transferase fusion protein in bacteria. The fusion protein was purified by glutathione affinity, and CLIC-1 was released from its fusion partner by digestion with thrombin. After further purification, CLIC-1 was reconstituted into phospholipid vesicles by detergent dialysis. Chloride permeability of reconstituted vesicles was assessed using a valinomycin dependent chloride efflux assay, demonstrating increased vesicular chloride permeability with CLIC-1 compared with control. CLIC-1-dependent chloride permeability was inhibited by indanyloxyacetic acid-94 with an apparent IC(50) of 8.6 micrometer. The single channel properties of CLIC-1 were determined using the planar lipid bilayer technique. We found that CLIC-1 forms a voltage-dependent, Cl-selective channel with a rectifying current-voltage relationship and single channel conductances of 161 +/- 7.9 and 67.5 +/- 6.9 picosiemens in symmetric 300 and 150 mm KCl, respectively. The anion selectivity of this activity is Br approximately Cl > I. The open probability of CLIC-1 channels in planar bilayers was decreased by indanyloxyacetic acid-94 with an apparent IC(50) of 86 micrometer at 50 mV. These data convincingly demonstrate that CLIC-1 is capable of forming a novel, chloride-selective channel in the absence of other subunits or proteins.

NCC27, a homolog of intracellular Cl- channel p64, is expressed in brush border of renal proximal tubule.

NCC27, a 27-kDa homolog of the intracellular chloride channel p64, was recently described as a chloride channel in nuclear membrane. We probed human Northern blots for NCC27 and found an approximately 1.7-kb message in all tissues examined, including kidney, the transcript being most abundant in heart and skeletal muscle.NCC27-specific antisera was raised to a COOH-terminal peptide derived from the NCC27 coding region. Using this antisera, we find NCC27 is expressed in an intracellular vesicular compartment in HeLa cells, PancI cells, and macrophages. In human and mouse kidney, NCC27 is expressed at low levels in most cells of the kidney. NCC27 is highly expressed in glomeruli, in periarterial smooth muscle, and in the apical membrane of a subset of cortical tubule cells. Double staining with nephron segment-specific lectins indicates that the NCC27-expressing cells are proximal tubule cells.

Functional expression of p64, an intracellular chloride channel protein.

p64 is a protein identified as a chloride channel by biochemical purification from kidney microsomes. We expressed p64 in HeLa cells using a recombinant vaccinia virus/T7 RNA polymerase driven system. Total cell membranes were prepared from infected/transfected cells and fused to a planar lipid bilayer. A novel chloride channel activity was found in cells expressing p64 and not in control cells. The p64-associated activity shows strong anion over cation selectivity. Single channels show prominent outward rectification with single channel conductance at positive potentials of 42 pS. The chloride channel activity is activated by treatment of the membranes with alkaline phosphatase and inhibited by DNDS and by TS-TM calix(4)arene. Whole membrane anion permeability was determined by a chloride efflux assay, revealing that membranes from cells expressing p64 showed a small but highly significant increase in chloride permeability, consistent with expression of a novel chloride channel activity.

Expression and characterization of the NBD1-R domain region of CFTR: evidence for subunit-subunit interactions.

To study interactions between the contiguous NBD1 and R domains of CFTR, wild-type and DeltaF508 NBD1-R (amino acids 404-830, in fusion with His6 tag) were expressed as single proteins in Escherichia coli. NBD1-R (10-25 mg/L culture) was purified from inclusion bodies in 8 M urea by Ni-affinity chromatography, and renatured by rapid dilution at pH 5. In vitro phosphorylation by protein kinase A increased the apparent size of NBD1-R from approximately 52 to approximately 56 kDa by SDS-PAGE. The fluorescent ATP analogue TNP-ATP bound to renatured NBD1-R with of 0.81 +/- 0.1 microM (wild-type), 0.93 +/- 0.1 microM (wild-type, phosphorylated), 0.75 +/- 0.1 microM (DeltaF508 NBD1-R), and 0.72 +/- 0.1 microM (DeltaF508 NBD1-R, phosphorylated) with a stoichiometry of approximately 1 TNP-ATP site per NBD1-R molecule; TNP-ATP binding was reversed by ATP, AMP-PCP, and AMP-PNP with KIs of approximately 3.2, 4.2, and 4.6 mM, respectively. Secondary structure analysis by circular dichroism gave 19% alpha-helix, 43% beta-sheet and turn, and 38% "other" structure. To determine if nucleotide binding to NBD1 influenced R domain phosphorylation, NBD1-R was in vitro phosphorylated with protein kinase A and [gamma-32P]ATP in the presence of AMP-PCP, AMP-PNP, or TNP-ATP. Whereas the nucleotide analogues did not affect 32P-incorporation in control proteins (Kemptide, GST-R domain), phosphorylation of NBD1-R was reduced >75% by AMP-PNP or AMP-PCP (0.25 mM) and >50% by TNP-ATP (0.25 microM). Analysis of phosphorylation sites indicated that inhibition involved multiple sites in NBD1-R, including serines 660, 712, 737, 795, and 813. These results establish the conditions for NBD1-R expression, purification, and renaturation. The inhibition of R domain phosphorylation by nucleotide binding to the NBD1 domain indicates significant domain-domain interactions and suggests a novel mechanism for regulation of CFTR phosphorylation.

Identification of protein kinase A phosphorylation sites on NBD1 and R domains of CFTR using electrospray mass spectrometry with selective phosphate ion monitoring.

HPLC-electrospray mass spectrometry was used to identify the phosphorylated sites on a bacterially expressed cystic fibrosis transmembrane conductance regulator (CFTR) fragment containing the first nucleotide binding domain (NBD1) and the regulatory domain (R). Tryptic digests of NBD1-R (CFTR residues 404-830) were analyzed after protein kinase A (PKA) treatment for all possible peptides and phosphopeptides (a total of 118 species) containing Ser residues within "high-probability" PKA consensus sequences: R-R/K-X-S/T, R-X-X-S/T, and R-X-S/T. Three criteria were used to assign phosphorylated sites: (1) an 80-Da increase in the predicted average molecular weight of the tryptic peptides; (2) co-elution with the PO3- ion induced by stepped energy collision; and (3) the relative elution positions of the phosphorylated and unmodified peptides. Ser residues within the eight dibasic sites in the NBD1 and R domains (positions 422, 660, 700, 712, 737, 768, 795, and 813) were phosphorylated, a pattern similar to that observed for full-length CFTR. The serine at position 753, which in CFTR is phosphorylated in vivo, was not phosphorylated. The remaining potential PKA sites, Ser489, Ser519, Ser557, Ser670, and Thr788, were not phosphorylated. The "low-probability" PKA sites (those not containing an Arg residue) were not phosphorylated. The results suggest that isolated domains of CFTR developed useful models for investigating the biochemical and structural effects of phosphorylation within CFTR. The mass spectrometry approach in this study should prove useful for defining phosphorylation sites of CFTR in vitro and in vivo.

Long-wavelength chloride-sensitive fluorescent indicators.

The goal of this study was to develop long-wavelength fluorescent Cl indicators that have improved optical properties over quinolinium compounds. A series of quaternized tricyclic heterocycles was screened. We found that N,N-dimethyl-9,9-bisacridinium (lucigenin) had very high halide sensitivity with Stern-Volmer constants for collisional quenching of 390 M-1 (Cl), 585 M-1 (Br), 750 M-1 (I), and 590 M-1 (SCN), much higher than those for the reference compound 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ) (118 M-1 (Cl)); lucigenin fluorescence was insensitive to phosphate, sulfate, and nitrate. Lucigenin fluorescence at 505 nm was excited with molar absorbances of 34,000 M-1 cm-1 (368 nm) and 7500 M-1 cm-1 (455 nm). To examine structure-activity relationships, five 9-substituted N-methylacridinium compounds were synthesized (9-amino, 9-carboxaldehyde, 9-carboxamide, 9-N,N-dimethylcarboxamide, and 9-methylcarboxylate), of which N-methylacridinium-9-carboxamide (MACA) had the best optical properties. MACA had excitation and emission maxima at 424 and 500 nm, and Stern-Volmer constants of 225 M-1 (Cl), 480 M-1 (Br), 550 M-1 (I), and 480 M-1 (SCN). The quantum yields of lucigenin and MACA were 0.6-0.7. The acridinium compounds are useful as Cl indicators in liposomes and membrane vesicles, but are not stable in cell cytoplasm.

Modulation of coated vesicle chloride channel activity and acidification by reversible protein kinase A-dependent phosphorylation.

We have previously shown that activity of a Cl- channel is required for acidification of clathrin-coated vesicles by the coated vesicle (H+)-ATPase (Arai, H., Pink, S. and Forgac, M. (1989) Biochemistry 28, 3075-3082). We demonstrate that activity of the coated vesicle Cl- channel is modulated by phosphorylation. Cl- conductance was measured in a reconstituted preparation of coated vesicle membrane proteins using the Cl(-)-sensitive fluorescence probe, 6-methoxy-N-(3-sulfopropyl)quinolinium. Treatment of coated vesicle membranes with alkaline phosphatase resulted in a 25 +/- 5% decrease in Cl- channel activity. A parallel decrease in ATP-dependent acidification of coated vesicles was also observed. The decrease in Cl- conductance and ATP-dependent acidification was reversed by treatment with protein kinase A and MgATP; the alkaline phosphatase inhibitor, sodium orthovanadate, blocked the inhibition of acidification. These results indicate that Cl- conductance in coated vesicles is modulated by a protein kinase A-dependent phosphorylation and that this modulation in turn affects ATP-dependent acidification.