Cl- channel activity in Xenopus oocytes expressing the cystic fibrosis gene.

Bear, Christine E.; Duguay, F; Naismith, Angela; Kartner, Norbert; Hanrahan, John W.; Riordan, John R.

doi:10.1016/s0021-9258(18)54971-1

Cited by 185 publications

(28 citation statements)

References 15 publications

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“…In agreement with the reduced MTND4 found in CF cells, reduced activity of mCx-I was also found [9]. We soon realized that these results were in consonance with an earlier mitochondrial theory for CF postulated by Burton Shapiro in the late 80 s, which was completely disregarded (erroneously) when the CFTR was cloned and found to be a chloride channel, located in the plasma membrane and not in the mitochondria [10][11][12]. The work of Shapiro's laboratory was discussed in detail elsewhere [13].…”

Section: Introductionsupporting

confidence: 82%

NLR family pyrin domain containing 3 (NLRP3) and caspase 1 (CASP1) modulation by intracellular Cl^– concentration

et al. 2021

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The impairment of the cystic fibrosis transmembrane conductance regulator (CFTR) activity induces intracellular chloride (Cl -) accumulation. The anion Cl -, acting as a second messenger, stimulates the secretion of interleukin-1β (IL-1β), which starts an autocrine positive feedback loop. Here, we show that NLR family pyrin domain containing 3 (NLRP3) and caspase 1 (CASP1) are indirectly modulated by the intracellular Clconcentration, showing maximal expression and activity at 75 mM Cl -, in the presence of the ionophores nigericin and tributyltin. The expression of PYD and CARD domain containing (PYCARD/ASC) remained constant from 0 to 125 mM Cl -. The CASP1 inhibitor VX-765 and the NLRP3 inflammasome inhibitor MCC950 completely blocked the Cl -stimulated IL-1β mRNA expression and partially the IL-1β secretion. DCF fluorescence (cellular reactive oxygen species, cROS) and MitoSOX fluorescence (mitochondrial ROS, mtROS) also showed maximal ROS levels at 75 mM Cl -, a response strongly inhibited by the ROS scavenger N-acetyl-L-cysteine (NAC) or the NADPH oxidase (NOX) inhibitor GKT137831. These inhibitors also affected CASP1 and NLRP3 mRNA and protein expression. More importantly, the serum/glucocorticoid regulated kinase 1 (SGK1) inhibitor GSK650394, or its shRNAs, completely abrogated the IL-1β mRNA response to Cland the IL-1β secretion, interrupting the autocrine IL-1β loop. The results suggest that Cleffects are mediated by SGK1, in which under Clmodulation stimulates the secretion of mature IL-1β, in turn, responsible for the upregulation of ROS, CASP1, NLRP3 and IL-1β itself, through autocrine signalling. K E Y W O R D Sautocrine signalling, CFTR, chloride anion, chloride channel, inflammasome, interleukin, NLRP3, ROS, second messenger, SGK1 Protein extractionCells were incubated as above indicated, washed twice with cold PBS, scraped with cold extraction buffer (10 mM Tris pH 7•4, 100 mM NaCl, 0•1% SDS, 0•5% sodium deoxycholate, 1% Triton X-100, 10% glycerol) containing the protease inhibitor cocktail (5 ml of cocktail/20 g of cell extract) plus phosphatase inhibitors (2 mM Na 3 VO 4 , 1 mM NaF and 10 mM Na 2 PO 7 ), and centrifuged at 14000 × g for 20 min

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Section: Introductionsupporting

confidence: 82%

NLR family pyrin domain containing 3 (NLRP3) and caspase 1 (CASP1) modulation by intracellular Cl^– concentration

et al. 2021

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“…To test whether the expressed β2-AR was functioning well, we used cystic fibrosis transmembrane conductance regulator (CFTR) channel, a chloride channel activated by PKA phosphorylation (51). CFTR is robustly activated by cAMP and PKA-CS injection in Xenopus oocytes (52,53). Coexpressing CFTR with a range of β2-AR doses (RNA range, 5 to 50 pg/oocyte) was associated with significantly higher basal chloride currents (I CFTR ) compared with cells expressing CFTR alone (P < 0.001), and Iso did not further increase I CFTR in β2-AR-expressing oocytes (SI Appendix, Fig.…”

Section: Resultsmentioning

confidence: 99%

Reconstitution of β-adrenergic regulation of Ca _V 1.2: Rad-dependent and Rad-independent protein kinase A mechanisms

Katz

Subramaniam

Chomsky-Hecht

et al. 2021

Proc. Natl. Acad. Sci. U.S.A.

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L-type voltage-gated CaV1.2 channels crucially regulate cardiac muscle contraction. Activation of β-adrenergic receptors (β-AR) augments contraction via protein kinase A (PKA)–induced increase of calcium influx through CaV1.2 channels. To date, the full β-AR cascade has never been heterologously reconstituted. A recent study identified Rad, a CaV1.2 inhibitory protein, as essential for PKA regulation of CaV1.2. We corroborated this finding and reconstituted the complete pathway with agonist activation of β1-AR or β2-AR in Xenopus oocytes. We found, and distinguished between, two distinct pathways of PKA modulation of CaV1.2: Rad dependent (∼80% of total) and Rad independent. The reconstituted system reproduces the known features of β-AR regulation in cardiomyocytes and reveals several aspects: the differential regulation of posttranslationally modified CaV1.2 variants and the distinct features of β1-AR versus β2-AR activity. This system allows for the addressing of central unresolved issues in the β-AR–CaV1.2 cascade and will facilitate the development of therapies for catecholamine-induced cardiac pathologies.

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“…Xenopus oocytes have proven to be a convenient expression system for functional and biochemical analysis of a variety of ion channels, including CFTR (e.g., Bear et al, 1991;Smit et al, 1993;Wilkinson et al, 1996;Naren et al, 1999), and were adopted for this study. Fig.…”

Section: Wild-type Cftr Channels Display Basal Activity In Resting Xenopus Oocytesmentioning

confidence: 99%

Severed Molecules Functionally Define the Boundaries of the Cystic Fibrosis Transmembrane Conductance Regulator's Nh2-Terminal Nucleotide Binding Domain

Chan

Csanády

Seto-Young

et al. 2000

The Journal of General Physiology

122

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The cystic fibrosis transmembrane conductance regulator is a Cl− channel that belongs to the family of ATP-binding cassette proteins. The CFTR polypeptide comprises two transmembrane domains, two nucleotide binding domains (NBD1 and NBD2), and a regulatory (R) domain. Gating of the channel is controlled by kinase-mediated phosphorylation of the R domain and by ATP binding, and, likely, hydrolysis at the NBDs. Exon 13 of the CFTR gene encodes amino acids (aa's) 590–830, which were originally ascribed to the R domain. In this study, CFTR channels were severed near likely NH2- or COOH-terminal boundaries of NBD1. CFTR channel activity, assayed using two-microelectrode voltage clamp and excised patch recordings, provided a sensitive measure of successful assembly of each pair of channel segments as the sever point was systematically shifted along the primary sequence. Substantial channel activity was taken as an indication that NBD1 was functionally intact. This approach revealed that the COOH terminus of NBD1 extends beyond aa 590 and lies between aa's 622 and 634, while the NH2 terminus of NBD1 lies between aa's 432 and 449. To facilitate biochemical studies of the expressed proteins, a Flag epitope was added to the NH2 termini of full length CFTR, and of CFTR segments truncated before the normal COOH terminus (aa 1480). The functionally identified NBD1 boundaries are supported by Western blotting, coimmunoprecipitation, and deglycosylation studies, which showed that an NH2-terminal segment representing aa's 3–622 (Flag3-622) or 3–633 (Flag3-633) could physically associate with a COOH-terminal fragment representing aa's 634–1480 (634-1480); however, the latter fragment was glycosylated to the mature form only in the presence of Flag3-633. Similarly, 433-1480 could physically associate with Flag3-432 and was glycosylated to the mature form; however, 449-1480 protein seemed unstable and could hardly be detected even when expressed with Flag3-432. In excised-patch recordings, all functional severed CFTR channels displayed the hallmark characteristics of CFTR, including the requirement of phosphorylation and exposure to MgATP for gating, ability to be locked open by pyrophosphate or AMP-PNP, small single channel conductances, and high apparent affinity of channel opening by MgATP. Our definitions of the boundaries of the NBD1 domain in CFTR are supported by comparison with the solved NBD structures of HisP and RbsA.

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Cl- channel activity in Xenopus oocytes expressing the cystic fibrosis gene.

Cited by 185 publications

References 15 publications

NLR family pyrin domain containing 3 (NLRP3) and caspase 1 (CASP1) modulation by intracellular Cl^– concentration

NLR family pyrin domain containing 3 (NLRP3) and caspase 1 (CASP1) modulation by intracellular Cl^– concentration

Reconstitution of β-adrenergic regulation of Ca _V 1.2: Rad-dependent and Rad-independent protein kinase A mechanisms

Severed Molecules Functionally Define the Boundaries of the Cystic Fibrosis Transmembrane Conductance Regulator's Nh2-Terminal Nucleotide Binding Domain

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Cl- channel activity in Xenopus oocytes expressing the cystic fibrosis gene.

Cited by 185 publications

References 15 publications

NLR family pyrin domain containing 3 (NLRP3) and caspase 1 (CASP1) modulation by intracellular Cl– concentration

NLR family pyrin domain containing 3 (NLRP3) and caspase 1 (CASP1) modulation by intracellular Cl– concentration

Reconstitution of β-adrenergic regulation of Ca V 1.2: Rad-dependent and Rad-independent protein kinase A mechanisms

Severed Molecules Functionally Define the Boundaries of the Cystic Fibrosis Transmembrane Conductance Regulator's Nh2-Terminal Nucleotide Binding Domain

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Product

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NLR family pyrin domain containing 3 (NLRP3) and caspase 1 (CASP1) modulation by intracellular Cl^– concentration

NLR family pyrin domain containing 3 (NLRP3) and caspase 1 (CASP1) modulation by intracellular Cl^– concentration

Reconstitution of β-adrenergic regulation of Ca _V 1.2: Rad-dependent and Rad-independent protein kinase A mechanisms