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S Devidas

Publications and source records attributed to S Devidas.

8 recordsLinked to original sources
PubMed2000

The two halves of CFTR form a dual-pore ion channel.

The cystic fibrosis transmembrane conductance regulator (CFTR) exhibits two conductance states, 9 picosiemens (pS) and 3 pS. To investigate the origin of these two distinct conductance states, we measured the single-channel activity of three truncated forms of CFTR. These include: TNR, which contains the first transmembrane domain, the first nucleotide binding domain, and the R domain; RT2N2, which contains the R domain, the second transmembrane domain, and the second nucleotide-binding domain; and T2N2, which contains only the second transmembrane domain and the second nucleotide-binding domain. The results show that TNR exhibits only the large conductance of 9.2 pS, whereas RT2N2 and T2N2 exhibit only the small conductance (3.8-4.0 pS). Co-expression of TNR with T2N2 resulted in a mixed pattern of two conductance states, which is similar to that observed in wild-type CFTR. In further studies, a "dual-R mutant," R334W and R347P in the transmembrane segment 6 of the first half of CFTR, severely impaired the large conductance channel without affecting the small conductance channel. The ion selectivity and gating behavior of the two conductance channels are different regardless of whether they are measured in wild-type CFTR or in truncated CFTRs. The ion selectivity of the large conductance channel is Br(-) > Cl(-) > I(-), whereas the ion selectivity of the small conductance channel is Br(-) = Cl(-) = I(-). The open probability (P(o)) of the large conductance is about 4-fold higher than that of the small conductance. Transition from closed to open states of the small conductance is not dependent upon the open or closed states of the large conductance. The independent behaviors of the two conductances in CFTR strongly suggest that CFTR may have two distinct pores. Thus, like ClC0, CFTR is likely to be a double-barreled ion channel, with the first half of CFTR forming the large conductance and the second half forming the small conductance.

Adenosine Triphosphate↗
PubMed1998

The second half of the cystic fibrosis transmembrane conductance regulator forms a functional chloride channel.

The cystic fibrosis transmembrane conductance regulator (CFTR) consists of two transmembrane domains (TMDs), TMD1 and TMD2, two cytoplasmic nucleotide binding domains (NBDs), NBD1 and NBD2, and a regulatory domain. To elucidate the complex function of the CFTR, deletion constructs encompassing the second half of the CFTR distal to the first transmembrane domain were expressed in Xenopus oocytes and IB3 cells (a cystic fibrosis cell line). Constructs containing the regulatory domain, the second transmembrane domain, and the second nucleotide binding domain formed constitutively active channels, which were further stimulated upon the addition of cAMP. On the other hand, a construct encompassing the second transmembrane domain and the second nucleotide binding domain was stimulated to a small but noticeable extent upon the addition of cAMP. The selectivity of the second-half construct was the same for iodide and chloride, in contrast to the selectivity of wild-type CFTR, which is Cl- > I-. However, both constructs displayed single-channel conductances that were significantly smaller than those displayed by the first half of the CFTR. We conclude that regions of the second transmembrane domain may contribute to the overall channel of the pore, although the first half of the CFTR may confer its selectivity.

Animals↗
PubMed1998

Cystic fibrosis transmembrane conductance regulator-associated ATP release is controlled by a chloride sensor.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that is defective in cystic fibrosis, and has also been closely associated with ATP permeability in cells. Using a Xenopus oocyte cRNA expression system, we have evaluated the molecular mechanisms that control CFTR-modulated ATP release. CFTR-modulated ATP release was dependent on both cAMP activation and a gradient change in the extracellular chloride concentration. Activation of ATP release occurred within a narrow concentration range of external Cl- that was similar to that reported in airway surface fluid. Mutagenesis of CFTR demonstrated that Cl- conductance and ATP release regulatory properties could be dissociated to different regions of the CFTR protein. Despite the lack of a need for Cl- conductance through CFTR to modulate ATP release, alterations in channel pore residues R347 and R334 caused changes in the relative ability of different halides to activate ATP efflux (wtCFTR, Cl >> Br; R347P, Cl >> Br; R347E, Br >> Cl; R334W, Cl = Br). We hypothesize that residues R347 and R334 may contribute a Cl- binding site within the CFTR channel pore that is necessary for activation of ATP efflux in response to increases of extracellular Cl-. In summary, these findings suggest a novel chloride sensor mechanism by which CFTR is capable of responding to changes in the extracellular chloride concentration by modulating the activity of an unidentified ATP efflux pathway. This pathway may play an important role in maintaining fluid and electrolyte balance in the airway through purinergic regulation of epithelial cells. Insight into these molecular mechanisms enhances our understanding of pathogenesis in the cystic fibrosis lung.

1-Methyl-3-isobutylxanthine↗
PubMed1998

Molecular characterization of a broad selectivity neutral solute channel.

In all living cells, coordination of solute and water movement across cell membranes is of critical importance for osmotic balance. The current concept is that these processes are of distinct biophysical nature. Here we report the expression cloning of a liver cDNA encoding a unique promiscuous solute channel (AQP9) that confers high permeability for both solutes and water. AQP9 mediates passage of a wide variety of non-charged solutes including carbamides, polyols, purines, and pyrimidines in a phloretin- and mercury-sensitive manner, whereas amino acids, cyclic sugars, Na+, K+, Cl-, and deprotonated monocarboxylates are excluded. The properties of AQP9 define a new evolutionary branch of the major intrinsic protein family of aquaporin proteins and describe a previously unknown mechanism by which a large variety of solutes and water can pass through a single pore, enabling rapid cellular uptake or exit of metabolites with minimal osmotic perturbation.

Amino Acid Sequence↗
PubMed1998

Chloride channel and chloride conductance regulator domains of CFTR, the cystic fibrosis transmembrane conductance regulator.

CFTR is a cyclic AMP (cAMP)-activated chloride (Cl-) channel and a regulator of outwardly rectifying Cl- channels (ORCCs) in airway epithelia. CFTR regulates ORCCs by facilitating the release of ATP out of cells. Once released from cells, ATP stimulates ORCCs by means of a purinergic receptor. To define the domains of CFTR important for Cl- channel function and/or ORCC regulator function, mutant CFTRs with N- and C-terminal truncations and selected individual amino acid substitutions were created and studied by transfection into a line of human airway epithelial cells from a cystic fibrosis patient (IB3-1) or by injection of in vitro transcribed complementary RNAs (cRNAs) into Xenopus oocytes. Two-electrode voltage clamp recordings, 36Cl- efflux assays, and whole cell patch-clamp recordings were used to assay for the Cl- channel function of CFTR and for its ability to regulate ORCCs. The data showed that the first transmembrane domain (TMD-1) of CFTR, especially predicted alpha-helices 5 and 6, forms an essential part of the Cl- channel pore, whereas the first nucleotide-binding and regulatory domains (NBD1/R domain) are essential for its ability to regulate ORCCs. Finally, the data show that the ability of CFTR to function as a Cl- channel and a conductance regulator are not mutually exclusive; one function could be eliminated while the other was preserved.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1997

The cystic fibrosis transmembrane conductance regulator and ATP.

A controversy in the field of cystic fibrosis (CF) research has arisen concerning the role of the cystic fibrosis transmembrane conductance regulator (CFTR) in the transport of ATP. Does the CFTR actually conduct ATP or does it regulate the conductance of ATP? Recent findings either support or reject the hypothesis that the CFTR can transport ATP. In addition, recent research from several laboratories has suggested that ATP mediates its effects after traversing the plasma membrane and reaching the extracellular surface. The current model suggests that the released ATP exerts its various influences via a purinergic receptor to regulate outwardly rectifying chloride channels and epithelial sodium channels.

Adenosine Triphosphate↗
PubMed1997

CFTR: domains, structure, and function.

Mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) cause cystic fibrosis (CF) (Collins, 1992). Over 500 naturally occurring mutations have been identified in CF gene which are located in all of the domains of the protein (Kerem et al., 1990; Mercier et al., 1993; Ghanem et al., 1994; Fanen et al., 1992; Ferec et al., 1992; Cutting et al., 1990). Early studies by several investigators characterized CFTR as a chloride channel (Anderson et al.; 1991b,c; Bear et al., 1991). The complex secondary structure of the protein suggested that CFTR might possess other functions in addition to being a chloride channel. Studies have established that the CFTR functions not only as a chloride channel but is indeed a regulator of sodium channels (Stutts et al., 1995), outwardly rectifying chloride channels (ORCC) (Gray et al., 1989; Garber et al., 1992; Egan et al., 1992; Hwang et al., 1989; Schwiebert et al., 1995) and also the transport of ATP (Schwiebert et al., 1995; Reisin et al., 1994). This mini-review deals with the studies which elucidate the functions of the various domains of CFTR, namely the transmembrane domains, TMD1 and TMD2, the two cytoplasmic nucleotide binding domains, NBD1 and NBD2, and the regulatory, R, domain.

Animals↗