Membrane chloride transport measured using a chloride-sensitive fluorescent probe

Illsley, Nicholas P.; Verkman, A. S.

doi:10.1021/bi00379a002

Cited by 223 publications

(122 citation statements)

References 15 publications

Supporting

Mentioning

112

Contrasting

Unclassified

Order By: Relevance

“…Blank control vesicles without protein were prepared as above except solubilization buffer without protein was applied to the gel filtration column. External quenching of the fluorescent indicator by iodide was complete within the instrumental dead time, similar to that observed for chloride (less than 2 ms) (41). Given the rapid quenching, external fluorophores were not removed after vesicle loading due to the large reduction in fluorescent signal observed after the additional gel filtration required to remove external MQAA or SPQ (data not shown).…”

Section: Methodssupporting

confidence: 63%

Functional Reconstitution and Characterization of Recombinant Human α1-Glycine Receptors

Cascio

Shenkel

Grodzicki

et al. 2001

Journal of Biological Chemistry

View full text Add to dashboard Cite

By utilizing a baculoviral expression system described previously (Cascio, M., Schoppa, N. E., Grodzicki, R. L., Sigworth, F. J., and Fox, R. O. (1993) J. Biol. Chem. 268, 22135-22142), functional recombinant homomeric human ␣ 1 -glycine receptors (GlyR) were overexpressed in insect cell culture, solubilized, purified, and reconstituted into lipid vesicles via gel filtration. Reconstituted GlyR channels were observed to retain native-like activity in single channel recordings of planar bilayers and in flux assays of small unilamellar vesicles, providing evidence that the recombinant homomeric receptor may be functionally reconstituted. This reconstitution is significant in that it indicates that the overexpressed homomeric receptor is an appropriate substrate for subsequent biophysical characterization aimed at the general elucidation of structure-function. Circular dichroism spectroscopy of reconstituted GlyR indicated a low ␣-helical content and a significant fraction of polyproline structure. The small fraction of observed ␣-helix is insufficient to accommodate the four helical transmembrane domains proposed in models for this receptor. By inference, other members of the homologous ligand-gated channel superfamily, which include the ionotropic ␥-aminobutyric acid, acetylcholine, and serotonin receptors, may also be erroneously modeled, and alternate models should be considered.Ligand-gated channels act in mediating signal transduction rapidly at the synapse. Members of this family of channels include receptors for both inhibitory neurotransmitters such as glycine and ␥-aminobutyric acid (GABA) 1 and excitatory neurotransmitters such as acetylcholine and serotonin (1). These membrane protein channels are amphiphilic molecules which, in response to neurotransmitter binding, transiently form pores through the lipid membrane where they are embedded, allowing the passive movement of small ions down their concentration gradient. This flux changes the electrical potential across the membrane, thus affecting the probability of the opening of voltage-gated channels.Upon binding of glycine, the glycine receptor (GlyR) channel becomes permeable to small anions (Cl Ϫ ), whose passive flux causes hyperpolarization of the cell. Strychnine, a convulsive alkaloid whose neurotoxic effect is attributed to blocking of the glycinergic transmission in the central nervous system, is the most potent known specific antagonist to the GlyR. By exploiting the strong antagonistic binding of strychnine (K d ϳ 5 nM), this ligand-gated channel was the first channel isolated and purified from mammalian nervous tissue via affinity chromatography on an aminostrychnine matrix (2). Cross-linking studies indicate that the native channel is a pentameric assembly of ␣ (48 kDa) and ␤ (58 kDa) subunits (3). These receptors copurify with gephyrin, an associated 93-kDa peripheral polypeptide that is essential for GlyR clustering (4 -6). Receptor heterogeneity arises from variable subunit subtypes (7, 8) as well as from alternative RNA splicing (9).Onl...

show abstract

Section: Methodssupporting

confidence: 63%

Functional Reconstitution and Characterization of Recombinant Human α1-Glycine Receptors

Cascio

Shenkel

Grodzicki

et al. 2001

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…1B). Channel-mediated halide efflux was estimated in Cos-7 cells using the SPQ fluorescence assay (12). SPQ fluorescence measured 240 s after cAMP-dependent stimulation of CFTR channels was almost fivefold higher than CFTRV232D (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Macroscopic function of CFTR and of the indicated mutants was evaluated in Cos-7 cells using the 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ) fluorescence assay (12). Cells were seeded into a six-well plate at a density of 2 ϫ 10 6 cells/well and transfected with 1 g of CFTR pcDNA3.1 constructs using Effectene transfection reagents.…”

Section: Methodsmentioning

confidence: 99%

Increased folding and channel activity of a rare cystic fibrosis mutant with CFTR modulators

Caldwell

Grove

Houck

et al. 2011

American Journal of Physiology-Lung Cellular and Molecular Phys

View full text Add to dashboard Cite

Cystic fibrosis (CF) is a lethal recessive genetic disease caused by mutations in the CFTR gene. The gene product is a PKA-regulated anion channel that is important for fluid and electrolyte transport in the epithelia of lung, gut, and ducts of the pancreas and sweat glands. The most common CFTR mutation, ΔF508, causes a severe, but correctable, folding defect and gating abnormality, resulting in negligible CFTR function and disease. There are also a large number of rare CF-related mutations where disease is caused by CFTR misfolding. Yet the extent to which defective biogenesis of these CFTR mutants can be corrected is not clear. CFTRV232D is one such mutant that exhibits defective folding and trafficking. CFTRΔF508 misfolding is difficult to correct, but defective biogenesis of CFTRV232D is corrected to near wild-type levels by small-molecule folding correctors in development as CF therapeutics. To determine if CFTRV232D protein is competent as a Cl− channel, we utilized single-channel recordings from transfected human embryonic kidney (HEK-293) cells. After PKA stimulation, CFTRV232D channels were detected in patches with a unitary Cl− conductance indistinguishable from that of CFTR. Yet the frequency of detecting CFTRV232D channels was reduced to ∼20% of patches compared with 60% for CFTR. The folding corrector Corr-4a increased the CFTRV232D channel detection rate and activity to levels similar to CFTR. CFTRV232D-corrected channels were inhibited with CFTRinh-172 and stimulated fourfold by the CFTR channel potentiator VRT-532. These data suggest that CF patients with rare mutations that cause CFTR misfolding, such as CFTRV232D, may benefit from treatment with folding correctors and channel potentiators in development to restore CFTRΔF508 function.

show abstract

“…In Vitro Assay of Channel Activity-Voltage-independent chloride efflux from SPQ-loaded vesicles was measured as described by Illsley and Verkman (25). Dye-loaded vesicles were diluted to 0.3 mg/ml in 20 mM DMG, 100 mM NaNO 3 buffer, at the desired pH.…”

Section: Methodsmentioning

confidence: 99%

The Molecular Basis for the pH-activation Mechanism in the Channel-forming Bacterial Colicin E1

Musse

Merrill

2003

Journal of Biological Chemistry

View full text Add to dashboard Cite

The colicins are a family of antimicrobial proteins that are secreted by Escherichia coli strains under environmental stress, due to nutrient depletion or overcrowding, and these proteins often target-sensitive bacterial strains (1). The lethal action of colicins against their target cells is manifested in a number of different modes that include the following: (i) formation of depolarizing ion channels in the cytoplasmic membrane, (ii) inhibition of protein and peptidoglycan synthesis, and (iii) degradation of cellular nucleic acids (1-7). In this context, the bacterial machinery responsible for colicin biological activity features important mechanisms that are fundamental to various biological processes. These mechanisms include protein receptor binding, membrane translocation, membrane binding and protein unfolding, membrane insertion, voltage-gated ion channel formation, catalysis, and inhibition of enzymes.Colicin E1 is a member of the channel-forming subfamily of colicins and is secreted by E. coli that possesses the naturally occurring colE1 plasmid; the holoprotein consists of three functional segments, the translocation, receptor-binding, and channel-forming domains. Initially, the receptor-binding domain (8) interacts with the vitamin B 12 receptor of target cells. Following recognition, the translocation domain associates with the tolA gene product, which permits the translocation of colicin E1 across the outer membrane and into the periplasm (9). In the periplasm, the channel domain undergoes a conformational change to an insertion-competent state and then inserts spontaneously into the cytoplasmic membrane of the host cell, forming an ion channel. The channel allows the passage of monovalent ions, resulting in the dissipation of the cationic gradients (H ϩ , K ϩ , and Na ϩ ) of the target cell, causing depolarization of the cytoplasmic membrane. In an effort to compensate for the membrane depolarization effected by the colicin E1 channel, Na ϩ /K ϩ -ATPase activity is increased in the host cell, resulting in the consumption of ATP reserves, without concomitant replenishment (4). The final outcome is host cell death.In addition to structural similarities between colicin E1 and several membrane-targeting proteins, an intriguing characteristic shared by colicin E1 and many membrane-active proteins is the observed requirement of an acidic environment for activation of the soluble structure in order to bind and insert into target cell membranes. The important effect of an acidic environment in conferring an optimum activity in vitro, and perhaps in vivo, for colicin E1 has been attributed to an onset of acid-induced protein unfolding events, resulting in an increased structural mobility/flexibility that may potentiate the complex sequence of structural changes of the channel peptide observed at the surface of a membrane (10 -12).An important contribution to our understanding of the mechanism of colicin E1 low pH activation was conducted by Merrill et al. (13) in which a precise pH-sensitive region with...

show abstract

Membrane chloride transport measured using a chloride-sensitive fluorescent probe

Cited by 223 publications

References 15 publications

Functional Reconstitution and Characterization of Recombinant Human α1-Glycine Receptors

Functional Reconstitution and Characterization of Recombinant Human α1-Glycine Receptors

Increased folding and channel activity of a rare cystic fibrosis mutant with CFTR modulators

The Molecular Basis for the pH-activation Mechanism in the Channel-forming Bacterial Colicin E1

Contact Info

Product

Resources

About