ADP-ribose gating of the calcium-permeable LTRPC2 channel revealed by Nudix motif homology (original) (raw)

Accession codes

Accessions

GenBank/EMBL/DDBJ

Data deposits

The GenBank accession number for NUDT9 cDNA and protein sequences is AY026252.

References

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Acknowledgements

We thank D. Tani and M. Monteilh-Zoller for technical assistance. This work was funded in part by a Beth Israel Pathology Foundation grant, a BIDMC Fireman Fellowship award to A.M.S. and NIH grants to A.M.S. and M.J.B. A.L.P. is supported by the German Academic Exchange Service (DAAD). In memory of Q. Zhu.

Author information

Author notes

1. Andrew M. Scharenberg
   Present address: Department of Pediatrics and Immunology, University of Washington and Children's Hospital and Medical Center, Seattle, Washington, 98195-6320, USA
2. Anne-Laure Perraud, Andrea Fleig, Christopher A. Dunn and Maurice J. Bessman: These authors contributed equally to this work.

Authors and Affiliations

1. Department of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, 02215, Massachusetts, USA
   Anne-Laure Perraud, Pierre Launay, Carsten Schmitz, Alexander J. Stokes, Qiqin Zhu, Jean-Pierre Kinet & Andrew M. Scharenberg
2. Laboratory of Cell and Molecular Signaling, Center for Biomedical Research at The Queen's Medical Center and John A. Burns School of Medicine at the University of Hawaii, Honolulu, 96813, Hawaii, USA
   Andrea Fleig, Leigh Ann Bagley & Reinhold Penner
3. Department of Biology, Johns Hopkins University, Baltimore, 21218, Maryland, USA
   Christopher A. Dunn & Maurice J. Bessman

Authors

1. Anne-Laure Perraud
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2. Andrea Fleig
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3. Christopher A. Dunn
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4. Leigh Ann Bagley
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5. Pierre Launay
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6. Carsten Schmitz
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7. Alexander J. Stokes
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8. Qiqin Zhu
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9. Maurice J. Bessman
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10. Reinhold Penner
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11. Jean-Pierre Kinet
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12. Andrew M. Scharenberg
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Corresponding author

Correspondence toAndrew M. Scharenberg.

Supplementary information

Cloning and sequence analysis of LTRPC2 and NUDT9.

The genetrapper II solution hybridization method (Life Technologies) was used to isolate both LTRPC2 and NUDT9 cDNA’s. For LTRPC2, five PCR positive colonies were obtained from the leukocyte library that was positive for LTRPC2 expression by RT-PCR, and the longest of these (4.0 kb) was sequenced. For NUDT9, 35 colonies were obtained from the spleen library that was positive for NUDT9 expression by RT-PCR. Eight of these were end-sequenced to confirm that they represented the same transcript and one was fully sequenced in both directions.

Construction of a FLAG-tagged LTRPC2 expression construct.

Brain cDNA was purchased from Clontech and used to obtain by RT-PCR the LTRPC2 coding sequence not present in the 4.0 kb fragment isolated above. This piece was ligated onto the 4.0 kb fragment, and the resulting full length LTRPC2 sequence was used to construct a full length LTRPC2 cDNA with an N-terminal FLAG epitope tag in a modified pCDNA4/TO vector. The correct sequence of the full-length FLAG-LTRPC2 expression construct was confirmed by DNA sequencing. The FLAG-LTRPC2 cDNA in pCDNA4/T0 was electroporated into HEK-293 cells previously transfected with the pCDNA6/TR construct. Cells were placed under zeocin selection, and zeocin-resistant clones were screened for inducible expression of the FLAG-tagged LTRPC2 protein.

Expression and purification of NUDT9 and NUDT9-H in E. coli

A full-length coding sequence for NUDT9 was produced by PCR to place an NcoI site at the 5' end and an NotI site at the 3' end. For the LTRPC2 NUDT9 homology region, a construct was made by PCR to include an NcoI site, an artificial start codon, amino acids 1197-1503, a stop codon, and a 3' NotI site. These were both subcloned into pET-24d (Novagen). BL21 (DE3) cells containing the respective expression plasmids were grown at 37 °C in LB broth on a shaker to an A600 of about 0.6 and induced by the addition of isopropyl-b?D-thiogalactopyranoside to a concentration of 1 mM. The cells were grown for an additional 4 h, harvested, washed by suspension in isotonic saline, and the packed cells were stored at –80 °C. The expressed protein leaked out of the frozen and thawed cells when washing them in 50 mM Tris, pH 7.5, 1 mM EDTA, 0.1 mM dithiothreitol. In the case of NUDT9, enzyme was extracted in the freeze-thaw fraction and ammonium sulfate was added to 35% final concentration. The precipitate was discarded after centrifugation and ammonium sulfate was added to the supernatant to a final concentration of 50%. The precipitate was collected by centrifugation, dissolved, then chromatographed on a gel filtration column (Sephadex G-100). The active fractions containing the majority of the enzyme were pooled, concentrated, dialyzed, and rechromatographed on DEAE-sepharose. The purified enzyme was concentrated from the pooled active fractions again using an Amicon Centriprep30. For the NUDT9 homology region of LTRPC2, the protein was extracted in the freeze-thaw fraction and ammonium sulfate was added to 35% final concentration and centrifuged. The precipitate was dissolved, dialyzed, and chromatographed on DEAE-sepharose. The purified enzyme was concentrated from the pooled active fractions by precipitation with 70% ammonium sulfate.

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Perraud, AL., Fleig, A., Dunn, C. et al. ADP-ribose gating of the calcium-permeable LTRPC2 channel revealed by Nudix motif homology.Nature 411, 595–599 (2001). https://doi.org/10.1038/35079100

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• Received: 05 December 2000
• Accepted: 16 March 2001
• Issue Date: 31 May 2001
• DOI: https://doi.org/10.1038/35079100