A monomer is the minimum functional unit required for channel and ATPase activity of the cystic fibrosis transmembrane conductance regulator.

The cystic fibrosis transmembrane conductance regulator (CFTR) normally functions as a phosphorylation-regulated chloride channel on the apical surface of epithelial cells, and lack of this function is the primary cause for the fatal disease cystic fibrosis (CF). Previous studies showed that purified, reconstituted CFTR can function as a chloride channel and, further, that its intrinsic ATPase activity is required to regulate opening and closing of the channel gate. However, these previous studies did not identify the quaternary structure required to mediate conduction and catalysis. Our present studies show that CFTR molecules may self-associate in CHO and Sf9 membranes, as complexes close to the predicted size of CFTR dimers can be captured by chemical cross-linking reagents and detected using nondissociative PAGE. However, CFTR function does not require a multimeric complex for function as we determined that purified, reconstituted CFTR monomers are sufficient to mediate regulated chloride conduction and ATPase activity.

Perturbation of the pore of the cystic fibrosis transmembrane conductance regulator (CFTR) inhibits its atpase activity.

Mutations in the cystic fibrosis gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR) lead to altered chloride (Cl(-)) flux in affected epithelial tissues. CFTR is a Cl(-) channel that is regulated by phosphorylation, nucleotide binding, and hydrolysis. However, the molecular basis for the functional regulation of wild type and mutant CFTR remains poorly understood. CFTR possesses two nucleotide binding domains, a phosphorylation-dependent regulatory domain, and two transmembrane domains that comprise the pore through which Cl(-) permeates. Mutations of residues lining the channel pore (e.g. R347D) are typically thought to cause disease by altering the interaction of Cl(-) with the pore. However, in the present study we show that the R347D mutation and diphenylamine-2-carboxylate (an open pore inhibitor) also inhibit CFTR ATPase activity, revealing a novel mechanism for cross-talk from the pore to the catalytic domains. In both cases, the reduction in ATPase correlates with a decrease in nucleotide turnover rather than affinity. Finally, we demonstrate that glutathione (GSH) inhibits CFTR ATPase and that this inhibition is altered in the CFTR-R347D variant. These findings suggest that cross-talk between the pore and nucleotide binding domains of CFTR may be important in the in vivo regulation of CFTR in health and disease.

Chloride channel activity of ClC-2 is modified by the actin cytoskeleton.

The chloride channel ClC-2 has been implicated in essential physiological functions, including cell-volume regulation and fluid secretion by specific epithelial tissues. Although ClC-2 is known to be activated by hyperpolarization and hypo-osmotic shock, the molecular basis for the regulation of this channel remains unclear. Here we show in the Xenopus oocyte expression system that the chloride-channel activity of ClC-2 is enhanced after treatment with the actin-disrupting agents cytochalasin and latrunkulin. These findings suggest that the actin cytoskeleton normally exerts an inhibitory effect on ClC-2 activity. An inhibitory domain was previously defined in the N-terminus of ClC-2, so we sought to determine whether this domain might interact directly with actin in binding assays in vitro. We found that a glutathione S-transferase fusion protein containing the inhibitory domain was capable of binding actin in overlay and co-sedimentation assays. Further, the binding of actin to this relatively basic peptide (pI 8.4) might be mediated through electrostatic interactions because binding was inhibited at high concentrations of NaCl with a half-maximal decrease in signal at 180 mM NaCl. This work suggests that electrostatic interactions between the N-terminus of ClC-2 and the actin cytoskeleton might have a role in the regulation of this channel.

Quaternary structure of the chloride channel ClC-2.

The chloride channel ClC-2 is thought to be essential for chloride homeostasis in neurons and critical for chloride secretion by the developing respiratory tract. In the present work, we investigated the quaternary structure of ClC-2 required to mediate chloride conduction. We found using chemical cross-linking and a novel PAGE system that tagged ClC-2 expressed in Sf9 cells exists as oligomers. Fusion of membranes from Sf9 cells expressing this protein confers double-barreled channel activity, with each pore exhibiting a unitary conductance of 32 pS. Polyhistidine-tagged ClC-2 from Sf9 cells can be purified as monomers, dimers, and tetramers. Purified, reconstituted ClC-2 monomers do not possess channel function whereas both purified ClC-2 dimers and tetramers do mediate chloride flux. In planar bilayers, reconstitution of dimeric ClC-2 leads to the appearance of a single, anion selective 32 pS pore, and tetrameric ClC-2 confers double-barreled channel activity similar to that observed in Sf9 membranes. These reconstitution studies suggest that a ClC-2 dimer is the minimum functional structure and that ClC-2 tetramers likely mediate double-barreled channel function.

Novel method for evaluation of the oligomeric structure of membrane proteins.

Assessment of the quaternary structure of membrane proteins by PAGE has been problematic owing to their relatively poor solubility in non-dissociative detergents. Here we report that several membrane proteins can be readily solubilized in their native quaternary structure with the use of the detergent perfluoro-octanoic acid (PFO). Further, PFO can be used with PAGE, thereby providing a novel, accessible tool with which to assess the molecular mass of homo-multimeric protein complexes.

ClC-2 activation modulates regulatory volume decrease.

ClC-2 belongs to a large family of chloride channels and its expression in certain cell types is associated with the appearance of swelling-activated chloride (Cl-) currents. In the present report, we examined the hypothesis that ClC-2 plays a role in regulatory volume decrease by expressing ClC-2 in Sf9 cells using the baculovirus system. First, we showed that ClC-2 protein expression is associated with appearance of a Cl- conductance which is activated by hypo-osmotic shock and can be distinguished from swelling-activated chloride currents endogenous to Sf9 cells on the basis of its pharmacology and specific inhibition by an anti-ClC-2 antibody. Second, we show that the rate of regulatory volume decrease is significantly enhanced in Sf9 cells expressing ClC-2 protein. Hence, our data support the hypothesis that ClC-2 is capable of mediating regulatory volume decrease.

Walker mutations reveal loose relationship between catalytic and channel-gating activities of purified CFTR (cystic fibrosis transmembrane conductance regulator).

The cystic fibrosis transmembrane conductance regulator (CFTR) functions as an ATPase and as a chloride channel. It has been hypothesized, on the basis of electrophysiological findings, that the catalytic activity of CFTR is tightly coupled to the opening and closing of the channel gate. In the present study, to determine the structural basis for the ATPase activity of CFTR, we assessed the effect of mutations within the "Walker A" consensus motifs on ATP hydrolysis by the purified, intact protein. Mutation of the lysine residue in the "Walker A" motif of either the first nucleotide binding fold (CFTRK464A) or the second nucleotide binding fold (CFTRK1250A) inhibited the ATPase activity of the purified intact CFTR protein significantly, by greater than 50%. This finding suggests that the two nucleotide binding folds of CFTR are functioning cooperatively in catalysis. However, the rate of channel gating was only significantly inhibited in one of these purified mutants, CFTRK1250A, suggesting that ATPase activity may not be tightly coupled to channel gating as previously hypothesized.

Assessment of the efficacy of in vivo CFTR protein replacement therapy in CF mice.

Cystic Fibrosis (CF) is caused by mutations in the CF gene that lead, for the most part, to mislocalization of the protein product, the cystic fibrosis transmembrane conductance regulatory (CFTR). CFTR is a chloride channel normally situated in the apical membrane of epithelial cells where it contributes to transepithelial ion transport. In this study we demonstrated the feasibility of in vivo transfer of purified CFTR protein via phospholipid liposomes into the apical membrane of nasal epithelia of CFTR knockout mice. Membrane incorporation of immunogold-labeled CFTR could be visualized by electron microscopy and correction of CF-related defects in ion transport measured by nasal potential difference (PD) measurements in about one-third of the animals treated. Although these initial results are promising, effectiveness of this therapeutic approach appears to be limited by the inefficient incorporation of CFTR into the apical epithelial cell membrane.

A novel procedure for the efficient purification of the cystic fibrosis transmembrane conductance regulator (CFTR).

This report describes a novel, single-step strategy for the purification of the cystic fibrosis transmembrane conductance regulator from Sf9 cells, which will facilitate studies of the structure-function relationships of this clinically important molecule. The new method combines the use of the novel detergent sodium pentadecafluoro-octanoate with metal-affinity chromatography to produce a high yield of purified protein which can be functionally reconstituted as a chloride channel and an ATPase.

Coupling of ATP hydrolysis with channel gating by purified, reconstituted CFTR.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel situated on the apical membrane of epithelial cells. Our recent studies of purified, reconstituted CFTR revealed that it also functions as an ATPase and that there may be coupling between ATP hydrolysis and channel gating. Both the ATP turnover rate and channel gating are slow, in the range of 0.2 to 1 s(-1), and both activities are suppressed in a disease-causing mutation situated in a putative nucleotide binding motif. Our future studies using purified protein will be directed toward understanding the structural basis and mechanism for coupling between hydrolysis and channel function.

ATPase activity of the cystic fibrosis transmembrane conductance regulator.

The gene mutated in cystic fibrosis codes for the cystic fibrosis transmembrane conductance regulator (CFTR), a cyclic AMP-activated chloride channel thought to be critical for salt and water transport by epithelial cells. Plausible models exist to describe a role for ATP hydrolysis in CFTR channel activity; however, biochemical evidence that CFTR possesses intrinsic ATPase activity is lacking. In this study, we report the first measurements of the rate of ATP hydrolysis by purified, reconstituted CFTR. The mutation CFTRG551D resides within a motif conserved in many nucleotidases and is known to cause severe human disease. Following reconstitution the mutant protein exhibited both defective ATP hydrolysis and channel gating, providing direct evidence that CFTR utilizes ATP to gate its channel activity.

Purified cystic fibrosis transmembrane conductance regulator (CFTR) does not function as an ATP channel.

The gene mutated in cystic fibrosis codes for the cystic fibrosis transmembrane conductance regulator (CFTR). Previously, we provided definitive evidence that CFTR functions as a phosphorylation-regulated chloride channel in our planar lipid bilayer studies of the purified, reconstituted protein. Recent patch-clamp studies have lead to the suggestion that CFTR may also be capable of conducting ATP or inducing this function in neighboring channels. In the present study, we assessed the ATP channel activity of purified CFTR and found that the purified protein does not function as an ATP channel in planar bilayer studies of single channel activity nor in ATP flux measurements in proteoliposomes. Hence, CFTR does not possess intrinsic ATP channel activity and its putative role in cellular ATP transport may be indirect.

Engineering resistance to trypsin inactivation into L-asparaginase through the production of a chimeric protein between the enzyme and a protective single-chain antibody.

We have demonstrated that a trypsin sensitive enzyme such as L-asparaginase can be rendered trypsin resistant by genetically fusing its gene with that of a single-chain antibody derived from a preselected monoclonal antibody capable of providing protection against trypsin. The chimeric L-asparaginase retained 75% of its original activity upon exposure to trypsin, whereas the native unprotected L-asparaginase control was totally inactivated.

The cystic fibrosis mutation (delta F508) does not influence the chloride channel activity of CFTR.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a phosphorylation-regulated Cl- channel. In most mammalian cells, the functional consequences of the most common CF mutation, delta F508-CFTR, cannot be assessed as the mutant protein undergoes biosynthetic arrest. However, function can be studied in the baculovirus-insect cell expression system where delta F508-CFTR does not appear to undergo such arrest. Our results show that phosphorylation-regulated Cl- channel activity of delta F508-CFTR is similar to that of wild-type CFTR. This observation was confirmed in comparative studies of purified delta F508-CFTR and CFTR reconstituted in planar lipid bilayers. Therefore, we suggest that this common mutation does not result in a significant alteration in CFTR function.

Monoclonal antibodies can protect L-asparaginase against inactivation by trypsin.

We show that a non-inhibitory monoclonal antibody (MAB) can be selected that provides substantial and sustained protection against proteolytic inactivation of L-asparaginase by trypsin. Of six non-inhibitory, high affinity, monoclonal antibodies to L-asparaginase, one afforded approximately 70% protection. Inactivation of L-asparaginase is associated with a single cleavage adjacent to lysine-29 that results in loss of an N-terminal fragment with a calculated MW of 2,647. The protective MAB prevented this trypsin cleavage. The products of gene fusions of "humanized" fragments of such antibodies and L-asparaginase could have increased clinical utility.

Purification and functional reconstitution of the cystic fibrosis transmembrane conductance regulator (CFTR).

Circumstantial evidence has accumulated suggesting that CFTR is a regulated low-conductance Cl- channel. To test this postulate directly, we have purified to homogeneity a recombinant CFTR protein from a high-level baculovirus-infected insect cell line. Evidence of purity included one- and two-dimensional gel electrophoresis, N-terminal peptide sequence, and quantitative amino acid analysis. Reconstitution into proteoliposomes at less than one molecule per vesicle was accomplished by established procedures. Nystatin and ergosterol were included in these vesicles, so that nystatin conductance could serve as a quantitative marker of vesicle fusion with a planar lipid bilayer. Upon incorporation, purified CFTR exhibited regulated chloride channel activity, providing evidence that the protein itself is the channel. This activity exhibited the basic biophysical and regulatory properties of the type of Cl- channel found exclusively in CFTR-expressing cell types and believed to underlie cAMP-evoked secretion in epithelial cells.

Stabilization of enzymes by their specific antibodies.

In nature, increased stability of enzymes has often been found to be associated with noncovalent protein-protein interactions. Specific antibodies should be suitable for this purpose. To test this hypothesis, we used a number of model enzymes, complexed them with their specific antibodies, and exposed them and the free enzymes to low and high temperature, lyophilization, oxidation, and alcohol. The retained activity of the antibody-complexed enzymes was substantially, and in some cases dramatically, higher. In general mechanistic terms, stabilization may have been accomplished either by noncovalent antibody crosslinking of discontinuous oligopeptide chains on the surface of the enzyme, thereby increasing resistance to unfolding of the enzyme, or by physical shielding by the antibodies of vulnerable sites on the surface of the enzyme.

Antigen protection of monoclonal antibodies undergoing labelling.

The effectiveness of a methodology designed to protect the antigen binding capacity of monoclonal antibodies undergoing labelling with a number of reagents was examined. The antigen binding sites of monoclonal antibodies were protected by complexing them with their antigen. Chemical modification with 6 mM of the water soluble Bolton-Hunter reagent of site protected monoclonal antibodies to glucoamylase resulted in antibodies that could tolerate a four-fold increase in reagent incorporation, without any loss of antigen binding capacity. Iodination of these antibodies (modified under site protected conditions) yielded over 70% increase in radioactivity incorporated in the active antibody fraction, compared with the incorporation into unprotected antibodies. Site protected labeling was found to be effective in retaining the antigen binding capacity of monoclonal antibodies modified with all reagents tested with the exception of chloramine-T.