Use of a bicistronic GFP-expression vector to characterise ion channels after transfection in mammalian cells

Trouet, Dominique; Nilius, Bernd; Voets, Thomas; Droogmans, Guillaume; Eggermont, Jan

doi:10.1007/s004240050445

Cited by 68 publications

(56 citation statements)

References 19 publications

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“…Molecular Biology-The open reading frame from rbECaC was cloned as a PvuII-BamHI fragment in the pCINeo/IRES-GFP vector (2,4,13). We used this bicistronic expression vector, pCINeo/IRES-GFP/ rbECaC, to coexpress rbECaC and enhanced GFP.…”

Section: Methodsmentioning

confidence: 99%

The Single Pore Residue Asp542 Determines Ca2+ Permeation and Mg2+ Block of the Epithelial Ca2+ Channel

Nilius¹,

Vennekens²,

Prenen³

et al. 2001

Journal of Biological Chemistry

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Section: Methodsmentioning

confidence: 99%

The Single Pore Residue Asp542 Determines Ca2+ Permeation and Mg2+ Block of the Epithelial Ca2+ Channel

Nilius¹,

Vennekens²,

Prenen³

et al. 2001

Journal of Biological Chemistry

Self Cite

180

136

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“…In these experiments, we employed a bi-cistronic vector expression system where both LRRC8 gene products and fluorescent reporter gene products are translated from a single mRNA ensuring identification of target protein-expressing cells with 100% efficiency. 51 Figure 5. Hypothetical model of the VSOR pore domain depicted by presuming a heteromeric hexamer structure before (A) and after (B, C) repletion of LRRC8A.…”

Section: Cell Culturementioning

confidence: 99%

Specific and essential but not sufficient roles of LRRC8A in the activity of volume-sensitive outwardly rectifying anion channel (VSOR)

et al. 2016

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The broadly expressed volume-sensitive outwardly rectifying anion channel (VSOR, also called VRAC) plays essential roles in cell survival and death. Recent findings have suggested that LRRC8A is a core component of VSOR in human cells. In the present study, VSOR currents were found to be largely reduced by siRNA against LRRC8A in mouse C127 cells as well. In contrast, LRRC8A knockdown never affected activities of 4 other types of anion channel activated by acid, Ca 2C , patch excision or cAMP. While cisplatin-resistant KCP-4 cells poorly expressed endogenous VSOR activity, molecular expression levels of LRRC8A, LRRC8D and LRRC8E were indistinguishable between VSOR-deficient KCP-4 cells and the parental VSOR-rich KB cells. Furthermore, overexpression of LRRC8A alone or together with LRRC8D or LRRC8E in KCP-4 cells failed to restore VSOR activity. These results show that deficiency of VSOR currents in KCP-4 cells is not due to insufficient expression of the LRRC8A/D/ E gene, suggesting an essential involvement of some other factor(s), and indicate that further study is required to better understand the complexities of the molecular determinants of VSOR, including the precise role of LRRC8 proteins.

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“…23 Wild-type (WT) TRPM6 in the pCINeo/IRES-GFP vector was HA-tagged at the N-terminal tail as described previously. 13 Template pCINeo/ IRES-GFP without TRPM6 was used as mock.…”

Section: Dna Constructsmentioning

confidence: 99%

New TRPM6 missense mutations linked to hypomagnesemia with secondary hypocalcemia

et al. 2013

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Despite recent progress in our understanding of renal magnesium (Mg 2 þ ) handling, the molecular mechanisms accounting for transepithelial Mg 2 þ transport are still poorly understood. Mutations in the TRPM6 gene, encoding the epithelial Mg 2 þ channel TRPM6 (transient receptor potential melastatin 6), have been proven to be the molecular cause of hypomagnesemia with secondary hypocalcemia (HSH; OMIM 602014). HSH manifests in the newborn period being characterized by very low serum Mg 2 þ levels (o0.4 mmol/l) accompanied by low serum calcium (Ca 2 þ ) concentrations. A proportion of previously described TRPM6 mutations lead to a truncated TRPM6 protein resulting in a complete loss-of-function of the ion channel. In addition, five-point mutations have been previously described. The aim of this study was to complement the current clinical picture by adding the molecular data from five new missense mutations found in five patients with HSH. To this end, patch-clamp analysis and cell surface measurements were performed to assess the effect of the various mutations on TRPM6 channel function. All mutant channels, expressed in HEK293 cells, showed loss-of-function, whereas no severe trafficking impairment to the plasma membrane surface was observed. We conclude that the new TRPM6 missense mutations lead to dysregulated intestinal/renal Mg 2 þ (re)absorption as a consequence of loss of TRPM6 channel function.

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Use of a bicistronic GFP-expression vector to characterise ion channels after transfection in mammalian cells

Cited by 68 publications

References 19 publications

The Single Pore Residue Asp542 Determines Ca2+ Permeation and Mg2+ Block of the Epithelial Ca2+ Channel

The Single Pore Residue Asp542 Determines Ca2+ Permeation and Mg2+ Block of the Epithelial Ca2+ Channel

Specific and essential but not sufficient roles of LRRC8A in the activity of volume-sensitive outwardly rectifying anion channel (VSOR)

New TRPM6 missense mutations linked to hypomagnesemia with secondary hypocalcemia

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